Close
About
FAQ
Home
Collections
Login
USC Login
Register
0
Selected
Invert selection
Deselect all
Deselect all
Click here to refresh results
Click here to refresh results
USC
/
Digital Library
/
University of Southern California Dissertations and Theses
/
The effect of tumor-mediated immune suppression on prostate cancer immunotherapy
(USC Thesis Other)
The effect of tumor-mediated immune suppression on prostate cancer immunotherapy
PDF
Download
Share
Open document
Flip pages
Contact Us
Contact Us
Copy asset link
Request this asset
Transcript (if available)
Content
THE EFFECT OF TUMOR-MEDIATED IMMUNE SUPPRESSION ON PROSTATE
CANCER IMMUNOTHERAPY
by
Andrew Gray
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR AND CELLULAR BIOLOGY)
December 2011
Copyright 2011 Andrew Gray
ii
DEDICATION
I’d like to dedicate this thesis to all those who helped make it possible. My family, who
are always close despite the distance. My friends, who have stood with me through it all,
and have made the last few years outrageously entertaining. My labmates, past and
present, who have taught me much more than experimental techniques and have made
daily lab life a joy. My thesis committee, for their invaluable guidance. My other mentors
amongst USC’s faculty, always ready with good advice and new perspectives. My friends
in the PIBBS office, who have helped me navigate USC’s infamous (cardinal) red tape.
Last but by no means least, my mentor W. Martin Kast. His wisdom, patience and
friendship have been priceless.
In gratitude, A.G.
iii
ACKNOWLEDGEMENTS
The United States Department of Defense for partially funding these studies via Prostate
Cancer Research Program Predoctoral Fellowship number DAMD PC073417.
The National Institutes of Health for partially funding these studies via Predoctoral
Fellowship number NIH T32 GM 067587.
Profs. Timothy Sparwasser, Nunzio Botinni, Pradip Roy-Burman, Uli von Andrian and
Andrew Chan for supplying the transgenic mice used throughout these studies.
Profs. Ian Hutchinson, Si Yi Chen, W. Martin Kast, Nunzio Botinni, Pradip Roy-Burman
and Gerry Coetzee for current and past service on my thesis committee.
All of the members of the Kast Lab, current and past, for their invaluable assistance,
advice and friendship. Prof. Diane Da Silva deserves special mention for being
extraordinarily helpful and a true friend. Dr. Maria de la Luz Garcia-Hernandez for
helping with the initiation of this project and for supplying the long-term survival data
from vaccination studies.
My mentor, Prof. W. Martin Kast, for his invaluable advice, patience and guidance over
the years. I can only hope to be half the scientist, teacher and leader he is.
iv
TABLE OF CONTENTS
DEDICATION................................................................................................................... ii
ACKNOWLEDGEMENTS ............................................................................................ iii
LIST OF TABLES ........................................................................................................... vi
LIST OF FIGURES ........................................................................................................ vii
ABSTRACT ....................................................................................................................... x
CHAPTER 1: PROSTATE CANCER IMMUNOTHERAPY ..................................... 1
The Prostate and Prostate Cancer ............................................................................... 1
Demographics of Prostate Cancer Patients ................................................................. 1
Prostate Cancer Screening, Diagnosis and Staging .................................................... 2
Current Therapeutic Approaches to Prostate Cancer .................................................. 6
Cancer Immunotherapy ............................................................................................. 10
Prostate Cancer Stem Cells ....................................................................................... 15
Overall Research Aims ............................................................................................. 17
CHAPTER 2: EARLY VACCINATION YIELDS SUPERIOR EFFICACY IN
PROSTATE CANCER IMMUNOTHERAPY ............................................................ 18
INTRODUCTION ........................................................................................................ 18
The Limitations of Current Cancer Immunotherapy Clinical Trial Design .............. 19
Large tumors fundamentally alter the immune systems of patients and limit their
ability to mount an anti-tumor immune response ..................................................... 21
Therapeutic Vaccination in the Adjuvant Setting ..................................................... 24
Interplay between Cancer Immunotherapy and Conventional Treatments ............... 26
Therapeutic Cancer Vaccination in the Preventive Setting ...................................... 29
A Novel Approach to Prostate Cancer Immunotherapy ........................................... 34
The TRAMP Mouse .................................................................................................. 34
Heterologous Prime-Boost Vaccination ................................................................... 35
Prostate Stem Cell Antigen ....................................................................................... 37
Heterologous DNA Prime/VRP Boost Confers Excellent Immunity Against Prostate
Cancer ....................................................................................................................... 37
MATERIALS AND METHODS .................................................................................. 39
RESULTS ..................................................................................................................... 48
DISCUSSION ............................................................................................................... 70
Future Clinical Trials of Therapeutic Cancer Vaccines ............................................ 80
Conclusion ................................................................................................................ 91
v
CHAPTER 3: ABROGATING REGULATORY T CELL FUNCTION TO
ENHANCE THERAPEUTIC PROSTATE CANCER VACCINE EFFICACY ...... 92
INTRODUCTION ........................................................................................................ 92
Immune Regulation in Health and Disease ............................................................... 92
Natural Regulatory T Cells ....................................................................................... 95
Inducible Regulatory T Cells .................................................................................... 96
Tregs in Human Cancer and Efforts to Attenuate Their Function ............................ 96
Transgenic Mouse Models with Altered Treg Function ........................................... 98
PEST-enriched Phosphatase Knockout and Gain of Function Mice ........................ 98
Depletion of Treg (DEREG) Mice ............................................................................ 99
Hypothesis and Aims .............................................................................................. 101
MATERIALS AND METHODS ................................................................................ 102
RESULTS ................................................................................................................... 110
DISCUSSION ............................................................................................................. 132
Conclusion .............................................................................................................. 142
CHAPTER 4: NOTCH SIGNALING IN PROSTATE CANCER STEM CELL
DIFFERENTIATION ................................................................................................... 143
INTRODUCTION ...................................................................................................... 143
Stem Cells ............................................................................................................... 143
Cancer Stem Cells ................................................................................................... 145
Identification of Cancer Stem Cells ........................................................................ 147
Prostate Cancer Stem Cells ..................................................................................... 148
Notch Signaling ...................................................................................................... 151
Hypotheses and Aims ............................................................................................. 152
MATERIALS AND METHODS ................................................................................ 154
RESULTS ................................................................................................................... 158
DISCUSSION ............................................................................................................. 186
Conclusion .............................................................................................................. 194
RESEARCH SUMMARY ............................................................................................ 196
RELATED PUBLICATIONS BY THE AUTHOR ................................................... 199
REFERENCES .............................................................................................................. 200
vi
LIST OF TABLES
1 Prostate tumor staging and grading........................................................................5
2 Summary of recent clinical trials of therapeutic prostate cancer vaccines ..........13
vii
LIST OF FIGURES
2.1 Therapeutic vaccination against prostate tumor-associated antigens
results in long term survival in TRAMP mice. ....................................................49
2.2 Therapeutic vaccination against mPSCA results in reduced prostate
tumor burden in TRAMP mice. ...........................................................................50
2.3 Therapeutic vaccination against prostate tumor-associated antigens
results in superior survival when administered to TRAMP mice with PIN
lesions. .................................................................................................................53
2.4 The proportion of CD4+ T cells and CD4+FOXP3+ Tregs decrease in the
periphery of TRAMP mice with increasing age. .................................................56
2.5 The fraction of the total splenic CD4+ T cell population that are
CD4+FOXP3+ Tregs increases with age in TRAMP mice. ................................57
2.6 The proportion of CD8+ T cells decreases in the periphery of TRAMP
mice in old age. ....................................................................................................59
2.7 Increased infiltration of CD4+ T cells and CD4+FOXP3+ Treg into the
prostate tumor with increasing age. .....................................................................62
2.8 CD4+CD25+ T cells isolated from prostate tumor draining lymph nodes
of younger mice are more are functionally suppressive. .....................................64
2.9 The cytokine/chemokine expression profile of spontaneous TRAMP
prostate tumors changes over time. ......................................................................66
2.10 Increased expression of immunosuppressive molecules in prostate
cancer. ..................................................................................................................69
2.11 Schematic representation of prostate cancer immunotherapy trial
timelines. ..............................................................................................................85
3.1 There is no difference in the numbers of peripheral Treg in PEP
KO(LYP-W620) mice compared to wild type littermate controls. ....................111
3.2 CD4+FOXP3+ Tregs are specifically depleted in TD mice but not
TRAMP mice upon diphtheria toxin treatment. ................................................114
viii
3.3 mPSCA vaccination inhibits prostate growth in TD and TRAMP mice. ..........117
3.4 Induction of mPSCA-specific IFNγ-expressing CD8+ T cells in
vaccinated TD mice. ..........................................................................................120
3.5 Treg depletion acts additively with mPSCA vaccination to increase
peripheral CD8+ T cell numbers........................................................................123
3.6 Treg depletion acts additively with mPSCA vaccination to increase
peripheral CD4+ T cell numbers........................................................................125
3.7 Vaccination against mPSCA increases the proportion of Treg cells in the
periphery, regardless of prior Treg depletion. ...................................................127
3.8 Treg depletion enhances the increase in the CD8+ T cell compartment of
lymphocytes in response to mPSCA vaccination. .............................................129
3.9 Treg depletion persists in the lymph nodes of TD mice for up to four
weeks..................................................................................................................131
4.1 Cell surface markers identifying prostate/prostate cancer cells as they
proceed down their differentiation pathway. .....................................................150
4.2 TRAMP-C2 cells express Notch1, the activation of which can be
inhibited by a γ-secretase inhibitor. ...................................................................159
4.3 Treatment of TRAMP-C2 cells with γ-secretase inhibitors inhibits
transcription of Hes1, a critical downstream mediator of Notch signaling. ......162
4.4 Treatment of TRAMP-C2 cells with a γ-secretase inhibitor causes them
to express a marker profile associated with a more differentiated state. ...........165
4.5 Treatment of TRAMP-C2 cells with a γ-secretase inhibitor results in
formation of fewer and smaller prostaspheres. ..................................................168
4.6 Transient siRNA-mediated knockdown of Notch partially recapitulates
the γ-secretase-mediated effect on prostasphere formation. ..............................171
4.7 Treatment of primary prostate cancer cells from TRAMP mice with γ-
secretase inhibitor results in formation of fewer and smaller
prostaspheres. .....................................................................................................174
ix
4.8 Growth of prostaspheres in the presence of a γ-secretase inhibitor causes
them to express a marker profile associated with a more differentiated
state. ...................................................................................................................177
4.9 Notch signaling is not required for the maintenance of the founding
prostate cancer stem cell in prostaspheres. ........................................................181
4.10 Treatment with γ-secretase inhibitor cannot attenuate TRAMP-C2 tumor
growth in vivo and inhibits the antitumor immune response elicited by
vaccination against mPSCA. ..............................................................................184
4.11 Proposed scheme of the role of Notch signaling in prostate cancer stem
cell differentiation and proliferation. .................................................................192
x
ABSTRACT
Immunotherapy has long been proposed as a novel method of specifically, safely and
inexpensively treating cancer. Despite decades of research and hundreds of clinical trials,
only one therapeutic cancer vaccine has been approved for human use. The vast majority
of these clinical trials have been carried out in terminally ill patients with advanced
cancer. These patients are severely immune compromised. The studies herein
demonstrate that multiple immunosuppressive mechanisms develop within prostate
tumors as they progress. These mechanisms include the accumulation of regulatory T
cells in the prostate tumor, and increased expression of TGFβ and indoleamine 2,3
dioxygenase in the prostate tumor. Vaccination of mice with spontaneously arising
prostate cancer against tumor-associated antigens is vastly superior to immunization at
later stages of disease, when tumor microenvironments are more suppressive. A novel
murine model of prostate cancer in which major mediators of immune suppression –
FOXP3-expressing regulatory T cells – can be specifically depleted was developed. This
model was termed the TRAMP DEREG mouse. Depletion of regulatory T cells improved
the response to therapeutic cancer vaccinination in TRAMP DEREG mice. This was the
case even though Treg depletion was carried out at a phase of carcinogenesis prior to the
development of serious immune suppression, suggesting that even better results can be
obtained if Tregs are inhibited when they are most prevalent in immunosuppressive
prostate tumors. Prostate cancer stem cells are responsible for driving tumor growth and
regeneration. Additionally, they do not express the tumor-associated antigens that are
targeted in prostate cancer immunotherapy, thus rendering them resistant to immune-
xi
medidated eradication. An effort was made to eliminate this population by inhibition of
Notch signaling, but it was found that Notch signaling is not involved in the maintenance
of the prostate cancer stem cell phenotype. In addition, Notch inhibition severely limited
the efficacy of concomitant therapeutic cancer vaccination. However, the novel finding
that Notch signaling is critical to the differentiation of prostate cancer cells, and thus to
tumor growth and regeneration, is extremely important. Taken together, these studies
show that immune-mediated tumor eradication is extremely difficult to achieve in
advanced cancer. Therefore it is proposed that clinical trials of cancer immunotherapies
be carried out in the preventative setting as soon as possible.
1
CHAPTER 1: PROSTATE CANCER IMMUNOTHERAPY
The Prostate and Prostate Cancer
The human prostate is a small, walnut-sized organ of the male genitourinary system that
is primarily responsible for secreting the fluid components of semen. It is located
immediately below the urinary bladder, surrounding approximately the first four
centimeters of the urethra. Structurally, the prostate is made up of three zones. The
transition zone – sometimes referred to as the “inner prostate” – is normally the smallest
section, forming only 5% of the volume of the organ. The central zone and the peripheral
zone – collectively the “outer prostate” – form 25% and 70% of the organ, respectively.
The ejaculatory ducts of the prostate are found in the central zone. The peripheral zone
largely consists of small, round acini that secrete seminal fluids, surrounded by smooth
muscle and collagen. The majority of prostatic intraepithelial neoplasia (PIN) lesions and
prostate adenocarcinoma occur in the peripheral zone acini.
Demographics of Prostate Cancer Patients
In the United States, prostate cancer is the most frequently diagnosed cancer in men, with
217,730 new cases in 2010 (Jemal et al.). It was the second-leading cause of cancer
mortality in that year, causing 32,050. The probability of a man being diagnosed with
invasive prostate cancer increases as he ages, with an incidence rate of 2.44% (1 in 41)
between the ages of 40-59 years, rising to 6.45% (1 in 16) between 60-69 years and
12.48% (1 in 8) at 70 years and older. The overall probability of a man developing
2
invasive prostate cancer in the United States from birth to death is 15.90% (1 in 6) (Jemal
et al.). Significant risk factors are a family history of prostate cancer amongst first degree
relatives (father, brother, son) and race. Notably, the incidence rate of prostate cancer in
African American men (231.9 per 100,000) is significantly higher than that of whites
(146.3 per 100,000) and hispanics (131.1 per 100,000), whilst men of Native American
(82.7 per 100,000) and Asian American (82.3 per 100,000) descent have the lowest
incidence rates.
Prostate Cancer Screening, Diagnosis and Staging
Starting at 40 years of age, men are advised to undergo prostate cancer screening in the
United States. This involves digital rectal examination (DRE) of the prostate by a
physician in conjunction with blood testing to assess serum levels of prostate serum
antigen (PSA). In most cases, a PSA serum level below 4 nanograms per milliliter of
blood is considered normal, while a level of 4 – 10 nanograms per milliliter of blood is
considered borderline for increased risk of prostate cancer. A PSA level above 10
nanograms per milliliter of blood is associated with a 50% likelihood of prostate cancer
being present. Despite the wide use of these methods in screening for prostate cancer,
neither has especially good sensitivity and specificity. Small tumors, particularly those
that are not in the peripheral zone of the prostate (i.e. those that are furthest away from
the rectum) are frequently missed by DRE. In addition, benign prostate hyperplasia
(BPH) – in which the transitional zone of the prostate can become dramatically enlarged
– confounds the usefulness of DRE as a diagnostic tool for prostate cancer. Some men
3
normally have serum PSA levels that would be considered suspicious for prostate cancer
despite being completely healthy, whilst small tumors in men may not produce enough
PSA to push their serum levels of the protein to “dangerous” concentrations.
Furthermore, the increase in the numbers of benign prostate cells can push PSA levels
above 4 nanograms per milliliter of blood in men with BPH and no prostate cancer.
Though PSA density (the PSA level divided by the volume of the prostate as estimated
by transrectal ultrasound (TRUS)) can be calculated in an effort to distinguish between
men with raised PSA levels due to BPH (low PSA density) and those in whom elevated
serum PSA concentrations are due to prostate cancer (high PSA density), this method is
by no means definitive. Thus, authoritative diagnosis of prostate cancer relies on
microscopic analysis of biopsies taken when DRE and/or PSA screening suggest the
possible presence of a prostate tumor. Typically, a minimum of twelve biopsy cores are
taken from the prostate, guided by TRUS.
Upon histological analysis, normal prostate acini are round with smooth contours, and are
arranged in regular structures. In contrast, the acini in malignant prostate tumors are
typically smaller, with irregular contours and chaotic arrangement. Approximately 95%
of human prostate malignancies are adenocarcinomas. The diagnosing pathologist will
assign a grade to the tumor as a Gleason score (a measure of how histologically different
the prostate cancer tissue is compared to normal prostate tissue) and stage it (a measure
of how invasive the tumor is). Gleason scores are obtained by giving an individual biopsy
core section a score from one to five (where one is normal and five is radically different
4
from normal tissue). The scores from the worst two biopsies are added together to give an
overall score for the tumor. Generally, tumors with an overall score over seven are
considered high risk. A summary of prostate cancer grading and staging is shown in
Table 1 (Hoedemaeker et al., 2000; Humphrey, 2004). If prostate cancer is diagnosed,
there are a number of therapeutic options available, depending on the grade and stage of
disease.
5
TABLE 1: Prostate tumor staging and grading
6
Current Therapeutic Approaches to Prostate Cancer
Active Surveillance
A significant strategic consideration in prostate cancer therapy is that the treatments that
are currently available can have devastating, life-changing side effects, whilst prostate
cancer designated very low risk (Stage T1C and Gleason score <6 and PSA <10 ng/ml
and <3 positive biopsy cores with <50% cancer tissue in each and PSA density <0.15) or
low risk (Stage T1 to T2a and Gleason score <6 and PSA <10 ng/ml) can be tolerated for
years without significant detriment to the health or overall survival odds of the patient.
Thus, it is common for men diagnosed with very low or low risk prostate cancer to
undergo a period of active surveillance, a modification of the older “watchful waiting”
treatment protocol. Active surveillance is also recommended in cases where the life
expectancy of the patient (notwithstanding his prostate cancer diagnosis) is less than ten
years. During active surveillance, the patient undergoes ongoing evaluation by DRE
(every 6-12 months), PSA level assessment (every 3-6 months) and repeat biopsy if there
is any suspicion that the tumor is growing. Active surveillance can be continued until the
disease progresses to a stage of intermediate risk (Stage T2b to T2c or Gleason score 7 or
PSA 10-20 ng/ml) or high risk (Stage T3a or Gleason score 8-10 or PSA >20 ng/ml), or if
the patient wishes to proceed to more aggressive treatment (for example if he is
experiencing anxiety due to the possibility of cancer progressing rapidly during the
period of active surveillance).
7
Surgical Treatment of Prostate Cancer
The most common surgical intervention for prostate cancer is radical prostatectomy, in
which the entire prostate gland and some surrounding tissue are physically removed. It is
an option for young patients with localized, high grade prostate cancer (stage T1a and
Gleason score 8-10) with a long life expectancy (>10 years) and is the standard of care of
men with intermediate risk prostate cancer (stage T1b-T2b). The variants of this
procedure include radical retropubic prostatectomy, robotic prostatectomy (in which the
surgeon conducts the operation via a robotic proxy rather than with his own hands,
allowing him to make finer incisions at the cost of haptic feedback) and radical perineal
prostatectomy. The success rate of radical prostatectomy in curing prostate cancer is
excellent. Biochemical recurrence (BR, reemergence of detectable PSA levels) after
radical prostatectomy are generally below 30% in most large long-term follow up studies
(Roberts and Han, 2009). Even of the men who do experience biochemical recurrence,
not all will suffer prostate cancer-specific mortality (PCSM). Only 34% of patients
progress to metastatic disease at a median of 8 years after diagnosis of BR, and of them
only 43% actually suffer PCSM (Pound et al., 1999).
Though radical prostatectomy is very successful in treating prostate cancer, it is not
without its costs. Regardless of the surgical technique used, impotence and incontinence
are common side effects. Bladder function returns to normal a few weeks or months after
surgery, though stress incontinence (caused by sneezing, laughing or exercising, for
example) persists in up to 35% of patients. The majority of men undergoing radical
8
prostatectomy will experience erectile dysfunction for 3 to 18 months after surgery, even
if nerve sparing radical retropubic prostatectomy was performed (in which the surgeon
attempts not to damage the nerves responsible for erectile function that run on either side
of the prostate, provided that they have not been invaded by the prostate tumor). Though
erectile dysfunction can be treated medically, it can be a permanent side effect of radical
prostatectomy for many men that has a dramatic impact on their quality of life.
Radiation Therapy of Prostate Cancer
Radiotherapy is an option for young patients with localized, high grade prostate cancer
(stage T1a and Gleason score 8-10) with a long life expectancy, and is the standard of
care for patients with intermediate stage prostate cancer (stage T1b-T2b) who are
unsuitable candidates for surgery and for patients with high risk prostate cancer (stage
T3) with a long life expectancy. Radiation therapy is also recommended for men who
have not seen a complete or near-complete reduction of serum PSA levels post radical
prostatectomy, indicative of remaining prostate/prostate cancer tissue that was missed
during the surgery. Radiotherapeutic approaches for prostate cancer treatment include
external beam radiation therapy (EBRT) (in which a focused beam of radiation from an
external source directly targets the prostate tumor and sentinel lymph nodes) and
brachytherapy (in which radioactive pellets are implanted directly into the
prostate/prostate tumor). The side effects of EBRT are comparable to those of radical
prostatectomy, except that erectile function is maintained in the short term but can
progressively worsen over time. Brachytherapy generally has less severe side effects than
9
EBRT due to the lower doses of radiation used, but alone is only suitable for treatment of
patients with low risk prostate cancer. Brachytherapy can be used in combination with a
shorter series of EBRT treatments in men with higher risk disease. Systemic radiation
therapy, involving direct venous injection of strontium-89 and/or samarium-153, can also
be used palliatively reduce bone pain by reducing the size of metastatic prostate cancer
bone lesions.
Hormone Treatment
Most prostate and prostate cancer cells are dependent on androgens for survival
(Arnoldussen et al.). Hormone therapy is designed to block the production and/or activity
of androgens, thus killing androgen-dependent prostate and prostate cancer cells. It is an
option for men initially diagnosed with prostate cancer metastatic to the lymphatic system
(usually determined by histological analysis of sentinel lymph nodes removed during
radical prostatectomy or by biopsy of those lymph nodes during radiotherapy) and for
men with recurrent prostate cancer following radical prostatectomy and/or radiotherapy.
Hormone therapy usually involves the administration of luteinizing hormone releasing
hormone (LHRH) antagonists that ultimately prevent the production of testosterone by
the testes. This can be combined with direct antagonists of the androgen receptor, a
strategy termed combined androgen blockade. The initial response to hormone therapy is
very good in the majority of prostate cancer patients, the notable exception being the 5%
or so who have neuroendocrine tumors rather than adenocarcinomas. However, prostate
tumors will inevitably adapt to the low-androgen milieu that results from hormone
10
therapy. This leads to the development of a condition termed hormone refractory prostate
cancer (HRPC). This is something of a misnomer given that prostate cancer cells in
HRPC actually become hypersensitive to the remaining low levels of androgen that are
available after prostate cancer hormone therapy, thus allowing them to proliferate and
regenerate the tumor (Kawata et al.). Therapeutic options for patients that have developed
HRPC are very limited and are essentially palliative. Chemotherapy can be used,
commonly docetaxel or mitoxantrone combined with a steroid such as prednisone.
Docetaxel regimens have been demonstrated to slightly improve survival. However, those
involving mitoxantrone have only been demonstrated to improve quality of life, for
example by temporarily shrinking bone metastases and thus transiently relieving pain
associated with them.
Cancer Immunotherapy
Given the limitations of conventional prostate cancer treatments, there is constant
research into developing novel therapies that improve survival and that have less severe
side effects. Immunotherapy has long been proposed as a method of treating cancer. The
overall aim of prostate cancer immunotherapy is to induce a specific immune response
against one or more prostate tumor-associated antigens (TAA) in an effort to precisely
target and eradicate cancer cells expressing those antigens. Hypothetically, a strong,
specific immune response directed against a properly selected TAA should be capable of
destroying any cancer cell expressing that TAA at sufficiently high levels. This should be
true even if the primary tumor has metastasized, as the immune cells stimulated by
11
therapeutic cancer vaccines should theoretically be able to home in on and destroy tumor
cells wherever they end up in the body. The severe side effects that characterize
conventional, systemic cancer therapies are largely caused by the collateral damage done
to the patient’s body as a result of their non-specific nature. In contrast, successful tumor
immunotherapy should result in minimal side effects due to the exceptional ability of the
immune system to eradicate cells expressing specific molecular markers whilst leaving
their healthy neighbors intact. Hundreds of animal studies have shown that myriad
cancers can be treated specifically, safely and effectively using a wide variety of
immunotherapeutic strategies. The extraordinary successes in eliciting immune responses
against tumor-associated antigens that are capable of mediating eradication of tumors in
animal models of cancer have led to an explosion in clinical trials designed to test the
viability of translating similar strategies to treating patients. This is particularly true in the
case of prostate cancer, in which a preponderance of TAAs, the devastating side effects
of conventional treatments and the lack of therapeutic options when those treatments fail
combine to make an ideal candidate for immunotherapy.
An extraordinary variety of tumor immunotherapies have undergone clinical trials in
recent years. These have included vaccines based on tumor cell lysates, peptides of
tumor-associated antigens, recombinant proteins, dendritic cells (mature and immature)
loaded with all of the above, dendritic cells transfected with tumor RNA or with DNA
encoding TAA, whole tumor cells, recombinant viral vectors expressing TAA and direct
immunization with plasmid DNA encoding TAA. In addition, many of these strategies
12
have been combined with each other and with administration of other immunomodulatory
molecules including, but not limited to, IL-2, GM-CSF and co-stimulatory molecules. All
of these methodologies have been used to target an astonishing variety of TAA. In the
case of prostate cancer, several TAA have been targeted in clinical trials (Gray et al.,
2008), including Prostate Specific Antigen (PSA) (Noguchi et al., 2003), Six-
Transmembrane Epithelial Antigen of the Prostate (STEAP) (Garcia-Hernandez Mde et
al., 2007), Prostate Stem Cell Antigen (PSCA) (Ross et al., 2002), Prostate Specific
Membrane Antigen (PSMA) (Zhu et al., 1999) and Prostatic Acid Phosphatase (PAP)
(Wang et al., 2005c). A summary of some recent clinical trials of therapeutic prostate
cancer vaccines is shown in Table 2 (Gray et al., 2008). Despite these many and varied
attempts at eliciting a clinically beneficial antitumor immune response, very few are
capable of doing so. Thus far none have proven consistently capable of eradicating an
existing tumor.
13
14
To date only one such immunotherapy – sipuleucel-T for metastatic prostate cancer – has
been successful enough to win approval by the Food and Drug Administration (FDA) for
clinical use (Small et al., 2006). Sipuleucel-T, known by the brand name Provenge, is an
active cellular vaccine which is based on autologous peripheral blood mononucleocytes
(PBMC) harvested from an individual patient by leukapheresis and treated ex vivo with a
recombinant fusion protein consisting of human prostatic acid phosphatase (hPAP) fused
to granulocyte-macrophage colony stimulating factor (GM-CSF). The PAP protein is a
normal human antigen that is frequently overexpressed by prostate cancer cells, whilst
GM-CSF acts to stimulate the antigen presenting cells (APC) present within the PBMC
isolated from the patient. Upon in vitro stimulation with sipuleucel-T, these APCs
become activated and present peptide fragments (epitopes) of PAP via major
histocompatibility complex (MHC) molecules at their cell surface, thereby allowing them
to stimulate PAP-specific T cells by interacting with their T cell receptors (TCR) when
transferred back into the patient. To assess the efficacy of sipuleucel-T, 225 men with
metastatic hormone refractory prostate cancer were recruited into two identical
randomized, double-blind, placebo controlled phase 3 clinical trials. In total, 147 men
were randomized to the sipuleucel-T arm and 78 received the placebo. Three infusions of
sipuleucel-T or placebo were administered to each trial subject at intervals of
approximately two weeks. The integrated results of these studies demonstrated an
improved median overall survival time of 4.3 months in patients that received Sipuleucel-
T (23.2 months) compared to those that received a placebo (18.9 months) (Higano et al.,
2009). This unprecedented improvement in overall survival was achieved in the absence
15
of severe side effects, and thus sipuleucel-T became the first therapeutic cancer vaccine
to be approved for human use by the FDA.
Though sipuleucel-T has demonstrated the proof of principle that immunotherapy can
safely and effectively be used to treat cancer, it has its limitations. The fact that the
vaccine is manufactured from the patient’s own cells is its first serious drawback. Not
only is the patient required to be healthy enough to undergo one or more leukapheresis
procedures, it is enormously expensive. Each course of sipuleucel-T treatment costs
approximately $100,000 at the time of writing. The second disadvantage of sipuleucel-T
is that although it improves survival, it is incapable of shrinking existing prostate tumors.
It most certainly has not been shown to be able to eradicate them completely, as is the
ultimate goal of prostate cancer immunotherapy.
Prostate Cancer Stem Cells
The cancer stem cell hypothesis has rapidly gained traction in recent years. Traditionally,
it was hypothesized that tumors originate from the malignant transformation of single
cells randomly acquiring multiple mutations that confer upon them the phenotypic
characteristics that are the hallmarks of cancer cells. In contrast, the cancer stem cell
hypothesis posits that the cell of origin for cancer is actually a somatic stem cell which
acquires mutations causing it to adopt a malignant phenotype whilst maintaining the
hallmarks of “stemness”, i.e. the ability to self-renew and produce differentiated daughter
cells. Like normal stem cells, these cancer stem cells are relatively rare compared to the
16
population of terminally differentiated tumor cells. Crucially, cancer stem cells, like their
healthy counterparts, are capable of regenerating tissues after damage, in this case a
tumor. Thus, such cells are responsible for the common recurrence of tumors after
effective primary cancer treatment that frequently results in patient deaths. Stem cells, as
suits their physiological role, are naturally resistant to DNA damage and toxins. Thus,
they are resistant to most anticancer strategies that are currently available. Due to their
critical role in tumor development and recurrence, cancer stem cells are a focus of intense
research in oncology.
Prostate tumors are largely androgen dependent, and regress upon castration and/or
pharmacological androgen ablation. However, both the prostate and prostate tumors will
regenerate if androgen is made available again. In addition, hormone refractory prostate
cancer – in which tumors adapt to the low androgen milieu in castrate conditions –
inevitably occurs in both humans and mouse models. Thus, there must exist an androgen-
independent cell type with stem-like properties which can regenerate the prostate/prostate
tumor under the correct conditions. Great advances in understanding the biology of
prostate and prostate cancer stem cells have been made in recent years. Markers
identifying populations of prostate/prostate cancer cells at various stages of
differentiation have been identified. Both prostate and prostate cancer stem cells share
many features with the cells of the prostate basal layer, including expression of
cytokeratins 5 and 14, CD44, p63 and Notch1 (Tran et al., 2002). It was demonstrated
very recently that the cell of origin in human prostate cancer is in the basal layer
17
(Goldstein et al.). Critically, prostate cancer stem cells have not yet been demonstrated to
express known prostate tumor associated antigens. Thus, they may represent a pool of
cells that is resistant to prostate immunotherapy and which can contribute to prostate
tumor immune escape.
Overall Research Aims
The central theme of this thesis is an investigation of why prostate cancer
immunotherapies – even successful ones such as sipuleucel-T – are incapable of
eradicating tumors. Chapter Two will demonstrate there are immunological mechanisms
that inhibit the efficacy of prostate cancer immunotherapies, giving insight into their
relatively poor performance in clinical trials. Chapter Three will focus on the inhibition
of one such immunosuppressive mechanism, the action of regulatory T cells, and will
attempt to improve vaccine efficacy by depletion of these cells in vivo. Finally, Chapter
Four will investigate whether prostate cancer stem cells can be eliminated or their
function attenuated in vivo, and if so whether this can help improve the effectiveness of
prostate immunotherapy.
18
CHAPTER 2: EARLY VACCINATION YIELDS SUPERIOR EFFICACY IN
PROSTATE CANCER IMMUNOTHERAPY
INTRODUCTION
Immunotherapy has long been regarded as a potential strategy for treating cancer. Tumor
cells frequently express unique antigenic targets and/or overexpress normal antigens that
are usually expressed only at low levels on healthy cells, allowing the immune system to
differentiate between cancerous and normal cells. Due to the exquisite specificity of the
immune system, cancer cells can potentially be eradicated with minimal collateral
damage to surrounding tissues. This is an enormous advantage over conventional cancer
therapies that are characterized by dramatic and overwhelming side effects that result
from their non-specific modes of action. Further, if a therapeutic cancer vaccine is
successful in eliciting a strong immune response, very few treatments are needed.
Perhaps only an initial priming immunization followed by a few booster shots is all that
will be necessary. Combined with relatively cheap off-the-shelf cancer vaccines based on
DNA, peptides, proteins and/or viral vectors, this means that immunotherapy may be a
comparatively inexpensive treatment regimen for cancer treatment when compared to
multiple inpatient chemotherapeutic, radiological and surgical interventions in
conventional cancer therapy.
Recently, immunotherapy was validated as a viable strategy for treating cancer with the
FDA approval of the first true therapeutic cancer vaccine sipuleucel-T. Despite this
19
extraordinary success, there is still much work to be done. The field is still a long way
away from realizing the ambition of immune-mediated tumor eradication. There have
been far more failures than successes in clinical trials of cancer vaccines. In decades of
research, the standard response of the tumor immunotherapy field to a failed clinical trial
has been to attempt to enhance the immunogenicity of the next candidate vaccine. This
has involved the identification and targeting of novel tumor-specific antigens, use of
different vaccine vectors or types (DNA, protein, peptide, tumor cell lysate and so on),
adding adjuvants or immunostimulatory cytokines to the vaccine formulation, or a
combination thereof. Despite decades of tweaking, the number of cancer
immunotherapies that have failed in clinical trials vastly outweighs the single success
story to date.
The Limitations of Current Cancer Immunotherapy Clinical Trial Design
Given the almost uniform failure of cancer immunotherapies – despite the enormous
variety of strategies and targets that have been investigated – it is logical to assess what
they all have in common when attempting to determine why they are ineffective. It is
striking that virtually all therapeutic cancer vaccines that have undergone clinical trials to
date have been tested in patients with advanced or metastatic cancer who have failed all
conventional treatment options. This philosophy of clinical trial design is exemplified in
the case of prostate cancer immunotherapy. Some recent trials of therapeutic prostate
cancer vaccines have been summarized in Table 2 (Burch et al., 2004; DiPaola et al.,
2006; Eder et al., 2000; Fong et al., 2001; Fuessel et al., 2006; Heiser et al., 2002;
20
Kaufman et al., 2004; Murphy et al., 1999; Noguchi et al., 2007; Small et al., 2000; Small
et al., 2006; Su et al., 2005; Thomas-Kaskel et al., 2006; Tjoa et al., 1998; Waeckerle-
Men et al., 2006). The majority of these trials has been conducted in patients with
metastatic hormone-refractory prostate cancer, for whom only palliative treatment is
available. From an ethical standpoint, these individuals make good candidates for
experimental therapies. However, these trials have produced very little positive data.
Measurable immunological responses to vaccination, as measured by ELISPOT, tetramer
staining, cytotoxicity assay for cellular responses and ELISA for the development of
humoral immunity, are usually limited within these studies. Moreover, objective clinical
responses are even rarer.
Interestingly, there have been several prostate cancer immunotherapy clinical trials in
which a subset of patients that did mount either cellular or humoral immune responses
upon administration of the vaccine being trialed demonstrated improved survival and/or
time to progression compared to the vaccinated patients that did not mount an antigen-
specific response. This implies that at least some of the vaccines that have been trialed
are actually capable of eliciting a clinically beneficial immune response, but only in
patients who are healthy enough to mount such an immune response (Gray et al., 2008).
Similar results have been observed in trials of therapeutic vaccines for renal cell
carcinoma (Berntsen et al., 2006; Uemura et al., 2006), melanoma (Berd et al., 2001;
Markovic et al., 2006), pancreatic adenocarcinoma (Gjertsen et al., 2001) and breast
cancer (Vonderheide et al., 2004). Thus, it is hypothesized that it most likely that the
21
failure to date of immunotherapeutic agents to mediate tumor clearance is mostly due to
the immuno-compromised status of advanced cancer patients in which they are tested.
Several studies have attempted to assess the general immune competence of test subjects,
for example by measuring their ability to mount recall responses to common antigens.
However, the ability of the immune system to respond to an antigen to which it has
already been exposed is not necessarily a measure of its ability to mount a completely
new immune response upon vaccination. In recent years, the cellular and molecular
mechanisms underlying immune suppression, and their implications for therapeutic
cancer vaccination, have been the subject of intense interest. These studies have revealed
multiple mechanisms of immune suppression in advanced cancer patients that render
them poor candidates for immunotherapeutic intervention.
Large tumors fundamentally alter the immune systems of patients and limit their
ability to mount an anti-tumor immune response
Advanced tumors have been shown to have multiple, often redundant pathways of
immune escape. It has become apparent that there are myriad immunosuppressive
mechanisms that can be subverted by tumors in order to blunt patient immune responses
mounted against them. The tumor immunology field is currently focused on the activities
and functional significance of tumor-associated suppressive immune cells such as
regulatory T cells (Treg) (Curiel, 2008) and myeloid-derived suppressor cells (MDSC)
(Gabrilovich and Nagaraj, 2009). In addition, suppression of T cell activity can be
mediated by tryptophan depletion due to increased expression of indoleamine-2,3-
22
dioxygenase within the tumor (Katz et al., 2008), or by increased arginine metabolism
due to upregulation of arginase and/or inducible nitrous oxide synthase (iNOS)
expression within the tumor (Bronte and Zanovello, 2005). Finally, increased expression
of suppressive cytokines such as interleukin(IL)-10 or tumor growth factor β (TGFβ) can
result in an immunosuppressive tumor microenvironment (Kretschmer et al., 2006;
Wrzesinski et al., 2007).
Immunosuppressive tumor microenvironments inhibit the local antitumor immune
response, both natural and in response to vaccination, but they can also produce
suppressive cells of multiple phenotypes that migrate from the tumor to lymphoid organs
where they can mediate systemic immunosuppression. These cells can inhibit immune
responses even after the original suppressive network has been eliminated, for example
by tumor resection. Moreover, metastasis of tumor cells to a sentinel lymph node, the
first lymph node away from the primary tumor in the lymphatic drainage pathway, leads
to local immunosuppression within that lymph node (Shu et al., 2006). This is significant
as the sentinel node is the first lymphoid organ in which antigenic stimulation occurs as
the first stage in the development of a systemic immune response. Though the local
immunosuppression mediated by metastatic tumor cells within lymph nodes does not
normally lead to complete systemic immunosuppression, this process nevertheless
represents a mechanism by which the presence of tumors can compromise the immune
response of the patient.
23
A recent example of systemic tumor-mediated immune dysfunction was recently
demonstrated in the patients enrolled in a failed Phase II clinical trial of a melanoma
vaccine based on the glycoprotein MPS160 (Celis, 2007). Though many patients
demonstrated an increase in vaccine specific CTL numbers, as measured by tetramer
analysis, these cells were broadly incapable of producing IFNγ when stimulated with the
relevant MPS160 peptides in vitro. This indicates that the antigen-specific T cells
induced by administration of the vaccine were functionally inactive. Repeated
immunizations simply caused an increase in the numbers of these antigen-specific cells
that failed to produce IFNγ. Furthermore, expansions in this non-responsive CTL
population were correlated with tumor progression events. Analysis of the peripheral
blood of vaccinated patients and healthy controls indicated that the profile of plasma
cytokines was skewed toward immunosuppressive molecules. The authors hypothesized
that these suppressive cytokines were being produced within the tumor microenvironment
and were “spilling over” into the plasma, resulting in a failure of systemic immune
competence.
Observable alterations in the plasma cytokine profiles of cancer patients compared to
healthy subjects and those with benign tumors have also been noted in other cancers. For
example, a recent study profiled the serum levels of fourteen cytokines in 187 ovarian
cancer patients and compared them to those of 45 patients with benign ovarian tumors
and 50 healthy controls (Lambeck et al., 2007). New multiplex ELISA bead array
technology has allowed the simultaneous analysis of multiple cytokines present in serum
24
samples. Univariate analyses demonstrated that serum IL-6, IL-7 and MCP-1 were
increased in ovarian cancer patients compared to healthy controls and patients with
benign ovarian tumors (p < 0.05 in all cases). Many other studies have highlighted
differences in serum levels of cytokines in prostate cancer patients compared to controls
(Miller and Pisa, 2007). Altered serum levels of IL-4, IL-6, IL-10 and TGF-β have been
noted in prostate cancer patients, with several studies demonstrating that increased IL-6
levels are a negative predictor of disease outcome.
Therapeutic Vaccination in the Adjuvant Setting
Given the apparent role of heavy tumor burdens in altering the immune systems of
terminally ill patients, a new paradigm of conducting clinical trials patients with minimal
residual disease was investigated. A commonly held belief in the tumor immunotherapy
field was that tumor growth is frequently too rapid for the immune system to contain,
despite the induction of a robust anti-tumor response (Emens and Jaffee, 2005). Given
that immunotherapy can in many cases be successfully combined with certain
conventional therapies, protocols were investigated in which therapeutic cancer vaccines
are administered in the adjuvant setting, where minimal residual disease is present.
Clinical trials in which patients have been vaccinated after their tumors have been
debulked by surgical, chemotherapeutic or radiological means have been conducted for
several cancers, most notably in melanoma (Berd et al., 2004; Chung et al., 2003; Hsueh
et al., 2002; Hsueh et al., 2004; Kirkwood et al., 2001; Livingston et al., 1994; Sanderson
et al., 2005; Sosman et al., 2002; Tagawa et al., 2006; Takeuchi et al., 2005; Wallack et
25
al., 1997; Wang et al., 1999; Weber et al., 2003), and lymphoma (Hsu et al., 1997; Weng
et al., 2004). Other examples of therapeutic cancer vaccines being used in the adjuvant
setting include those directed against ovarian cancer (Berek et al., 2004; Reinartz et al.,
2004), lung cancer (Giaccone et al., 2005; Kimura and Yamaguchi, 1997), pancreatic
carcinoma (Gjertsen et al., 2001; Jaffee et al., 2001) and breast cancer (Sportes et al.,
2005).
Most clinical trials of therapeutic cancer vaccines in the adjuvant setting have had
positive but limited results. While disease-free survival (DFS) and/or overall survival
(OS) are often improved (sometimes very significantly so), therapeutic vaccination in the
adjuvant setting is rarely capable of eradicating the residual tumor cells that are present
after surgery and eliciting a complete response. This is probably because debulking
tumors removes only one component of the systemic immune suppression that they
establish, namely the intratumoral suppressive milieu. One mouse study has demonstrated
that immune competence of the host can be restored upon surgical removal of the primary
tumor, despite the continued presence of metastases in a mouse model of mammary
carcinoma (Danna et al., 2004). However, the limited results of human clinical trials
suggest that this effect is abrogated in humans, indicating that suppressive T cells/DCs
which have already escaped the tumor may be active in the LN and the
immunosuppressive cytokine profile present in the peripheral blood of the patient are
capable of limiting vaccine efficacy even in the absence of the immunosuppressive tumor
microenvironment. Nevertheless, the improvements in DFS and OS observed in many
26
studies of therapeutic vaccination in the adjuvant setting indicate that tumor-mediated
immunosuppression is a major factor in the limited efficacy of therapeutic cancer
vaccines, and that by partially disrupting that immunosuppression their effectiveness can
be increased.
Interplay between Cancer Immunotherapy and Conventional Treatments
Another important factor that might limit the success of immunotherapy clinical trials
conducted in patients who have advanced cancer is that current conventional anticancer
therapies have significant effects on the immune system. Cancer treatments such as
chemotherapy and radiotherapy are well known to have potent immunomodulatory
effects that can either inhibit or enhance the anti-tumor immune response elicited by
vaccination. Under certain circumstances, radiation therapy and many chemotherapeutic
agents are immunosuppressive, particularly at high dosages. Lymphocytes responding to
antigens, including those stimulated by vaccination against TAAs, rapidly proliferate.
Therefore they are susceptible to anticancer therapies that preferentially kill proliferating
cells (Mitchell, 2003).
Despite the traditionally held view that radiotherapy and chemotherapy are generally
immunosuppressive, the effects of conventional anticancer treatments on the immune
system are extremely complex. As a result, chemotherapy and radiotherapy can be
combined with vaccination strategies to improve their effectiveness so long as due
attention is paid to dosages and the relative timing of each treatment. For example,
27
Doxorubicin and Melphalan were shown not to impede the efficacy of two separate
vaccination strategies in mice when they were administered shortly before immunization
(Casati et al., 2005). An excellent recent review has covered scenarios in which cytotoxic
chemotherapies have been combined with immunotherapy to yield enhanced responses
(Emens and Jaffee, 2005). Radiotherapy has also been combined with immunotherapy.
For example, a course of radiotherapy was recently integrated into a vaccination schedule
in prostate cancer patients (Gulley et al., 2005). Thirty patients were either given
radiotherapy alone, or radiotherapy plus vaccination with a recombinant vaccinia-PSA
vaccine followed by monthly boosting with recombinant fowlpox-PSA. In the
combination therapy arm, patients received radiotherapy between the third and fifth
boosts. Thirteen out of seventeen patients in the combination arm developed PSA-
specific cellular responses, versus none in the radiotherapy-only arm (p < 0.0005). Given
that there was no immunotherapy-only treatment arm in this study, it cannot be concluded
that radiotherapy enhanced the immune response to vaccination. However, the study ably
demonstrates that local radiotherapy does not inhibit antigen-specific responses elicited
by vaccination. Finally, we demonstrated that a peptide vaccine directed against HPV
was capable of eliciting complete protection against challenge with HPV-16-expressing
tumors in mice despite prior treatment with pelvic radiation or cisplatin. This was true
even though the radiation- and cisplatin-pretreated mice had measurably lower peptide
specific immune responses to vaccination than untreated controls, as measured by IFN-γ
ELISPOT (Small et al., 2001).
28
Despite the success of combining conventional anticancer treatments with
immunotherapy in certain instances, this approach may not always be feasible. In many
cases, the optimal chemotherapeutic agents and/or radiotherapeutic schedules available to
physicians are dictated by the nature of the cancer they are treating. Not all of these
treatment options will be suitable for combination with immunotherapeutic strategies,
and therefore not all cancers will be viable targets for developing combination treatment
strategies. Furthermore, cancer patients are most commonly enrolled in clinical trials for
therapeutic vaccines as a treatment of last resort. Most clinical immunotherapy trials
exclude patients that have received prior treatments that may affect vaccine efficacy, but
this is usually limited to treatment received less than one month before the start of the
trial. As a result, patients enrolled in cancer immunotherapy clinical trials have already
received a variety of prior treatments that may have had long-lasting effects on their
immune competence. The complexity of the interactions between these various
treatments and the immune system makes accurate assessment of the results of clinical
trials – and indeed comparisons between otherwise similar trials – exceedingly difficult.
The reality of cancer immunotherapy clinical trials is that patients who already have
cancer must be given the standard of care. It is an absolute ethical requirement that
patients be given the best possible chance of cure, or at least the best possible quality of
life for the longest possible time. However, it is equally true that the current paradigm of
performing clinical trials of therapeutic cancer vaccines in extremely ill patients who
have severely compromised immune systems is not yielding the result that is ultimately
desired: safe, effective immune-mediated tumor eradication. These considerations have
29
led to the hypothesis that immunization with a therapeutic cancer vaccine at the earliest
stages of carcinogenesis – before local and systemic immunosuppressive environments
are established by the tumor and before any other treatment would normally be
administered – will yield the best immune responses to vaccination and therefore confer
excellent protection against the progression of cancer development.
Therapeutic Cancer Vaccination in the Preventive Setting
A number of studies highlight the potential of employing cancer vaccines in the
preventive setting. Such an approach was elegantly demonstrated very recently by Jaini et
al (Jaini et al.). The authors showed that vaccination against α-lactalbumin, a protein
normally only expressed in lactating breast tissue but strongly upregulated by breast
cancer cells, yielded outstanding long term protection against the autochthonous
development of breast cancer in a transgenic mouse model of breast cancer. In these
transgenic animals, overexpression of the neu receptor protooncogene is driven by the
glucocorticoid response element found in the long terminal repeat sequence of mouse
mammary tumor virus (MMTV) (Guy et al., 1992). This recapitulates the overexpression
of Her2/neu observed in 25-30 percent of invasive human breast cancers (Lohrisch and
Piccart, 2001), and is a widely used model of the disease. Normally, 50 percent of
MMTV-neu mice would develop spontaneous mammary tumors by 205 days. MMTV-
neu mice were vaccinated using either recombinant α-lactalbumin with complete
Freund’s adjuvant (CFA) or CFA alone. At 10 months of age, all animals were
euthanized. None of the α-lactalbumin vaccinated mice developed mammary tumors, in
30
stark contrast to the CFA-only control group which had a 100% incidence rate. In
addition, the vaccination strategy was extremely successful in prophylactically inhibiting
the growth of implanted 4T1 breast cancer cells. The immune response elicited was
characterized a dramatic influx of tumor infiltrating lymphocytes (TIL) into the tumor
(predominantly CD4
+
T cells but also CD8
+
T cells) that produced large amounts of
IFNγ. The CD8
+
T cells were capable of directly killing 4T1 cells in vitro. Vaccination
against α-lactalbumin was also capable of eliciting less significant therapeutic protection
against implanted 4T1 tumors at five and 13 days post inoculation, but not at 21 days post
inoculation. This is consistent with the majority of animal studies of cancer vaccines,
which frequently show better prophylactic protection than therapeutic efficacy. However,
it is exceedingly rare to achieve complete, long-term protection via a prophylactic
vaccination as was achieved in this study.
Prevention of the spontaneous development of tumors by vaccination at an early stage of
carcinogenesis has also been demonstrated in BALB-neuT mice that spontaneously
develop mammary tumors. Nava-Parada et al demonstrated that a single vaccination of
BALB-neuT mice with a peptide vaccine derived from the RNEU TAA with concomitant
administration of the Toll-like receptor agonist CpG can significantly delay spontaneous
tumor development (Nava-Parada et al., 2007). BALB-neuT mice remained completely
tumor free until approximately 23 weeks of age when they were vaccinated at 8 weeks of
age, at which time they display diffuse atypical hyperplasia but not overt carcinoma.
Interestingly, the authors report that peptide vaccination at later stages of carcinogenesis
31
was less effective in eliciting anti-tumor immune responses and controlling tumor
growth.
There is evidence from human studies that therapeutic cancer vaccination is very
effective if it is administered when patients have only precancerous lesions. Several
studies have demonstrated that vaccination of women with premalignant cervical
intraepithelial neoplastic lesions (CIN) can cause their complete eradication or partial
regression to a lower-grade lesion. Muderspach et al demonstrated three complete
responses and six partial responses in twelve patients with grade II/III CIN that were
vaccinated with a vaccine directed against HPV E7 peptides (Muderspach et al., 2000).
Immunization with a recombinant protein (SGN-00101) consisting of bacterial heat shock
protein (M. bovis Hsp65) fused to the complete HPV-16 E7 sequence was recently shown
by Roman et al to confer clinical benefit to patients with high-grade CIN lesions. Of the
twenty women enrolled in the study, seven showed complete regression of their CIN
lesions, one had a partial regression to a low-grade lesion, eleven had stable disease and
one patient progressed (Roman et al., 2007). A phase III randomized study of the same
vaccine conducted by Einstein et al yielded very similar results. Of the 58 patients that
had completed the full vaccination protocol, thirteen had a complete response, 32 had
partial responses, eleven had stable disease and two progressed (Einstein et al., 2007). A
vaccine based on a vaccinia virus vector expressing recombinant MVA E2 has also been
demonstrated to be effective in treating high grade CIN lesions (Corona Gutierrez et al.,
2004). Of the 34 women immunized in a recent phase II clinical trial of the vaccine,
32
nineteen showed complete regression and fifteen showed partial regression (Garcia-
Hernandez et al., 2006). Early trials of other therapeutic cervical cancer vaccines have
demonstrated the generation of excellent immune responses in patients with CIN lesions,
though no clinical responses were assessed (Brinkman et al., 2007; Kanodia et al., 2007).
Generally, vaccination of CIN patients elicits the development of very robust
immunological responses. This indicates that these patients are generally immune
competent, as is expected at the early stages of carcinogenesis (Ressing et al., 1996).
Similarly, robust CD4
+
and CD8
+
immune responses were also observed in patients with
vulvar intraepithelial neoplasia (VIN) lesions who were vaccinated with a mixture of long
peptides derived from the HPV-16 E6 and E7 oncoproteins (Kenter et al., 2009). This led
to a startling clinical response rate of 79% (15 out of 19 patients), including a complete
response rate of 47% (9 out of 19 patients) that was maintained for at least two years.
These studies highlight that excellent immune responses can be elicited in patients who
are vaccinated at an early stage of carcinogenesis, and that these responses can mediate
excellent long-term protection against the development of cancer. Given the availability
of efficacious therapeutic vaccines and the accessibility and reliability of current
screening methods, cervical cancer represents an excellent candidate for a treatment
modality in which premalignant lesions are treated by therapeutic vaccination.
Though the circumvention of immunosuppression mediated by tumors and their treatment
is the primary justification for vaccination against premalignant lesions, in some cases
administration of therapeutic cancer vaccines has had unexpected beneficial effects on
the outcomes of subsequent conventional treatments. As has been recently discussed
33
(Schlom et al., 2007), superior responses to standard therapies have been observed in
patients who have initially received a therapeutic vaccine and have subsequently been
given conventional treatments upon disease progression. This fascinating phenomenon
has been observed in trials of vaccines directed against small-cell lung cancer (Antonia et
al., 2006) and prostate cancer (Arlen et al., 2006; Arlen et al., 2005). These observations
are particularly significant in the case of prostate cancer. The trials in question were
conducted in patients with hormone-refractory prostate cancer who were probably
immuno-compromised given that prostate cancer patients with metastatic disease are less
able to mount immune responses than patients that have less advanced disease (Salgaller
et al., 1998). Early detection of prostate cancer is commonplace as a result of PSA
screening. The potential side effects of conventional prostate cancer therapies are more
severe than the consequences of living with contained, low grade prostate cancer. As a
result, men with rising PSA levels frequently undergo very long periods of active
surveillance and only elect to undergo curative surgery when it becomes apparent that the
disease has begun to progress. It is conceivable that therapeutic vaccination during this
“watchful waiting” phase may clear premalignant prostate intraepithelial lesions or delay
their progression to prostate adenocarcinoma. If and when this occurs, prior vaccination
may then help enhance the efficacy of standard prostate cancer therapies that the patient
receives. In summary, early vaccination of premalignant prostate lesions is an attractive
proposition because it may yield clinical benefit to patients at multiple stages of disease
progression.
34
A Novel Approach to Prostate Cancer Immunotherapy
The overall hypothesis of this chapter is that administration of a therapeutic prostate
cancer vaccine at an early stage of disease yields superior immune responses and survival
benefits than administration at a later stage in which tumor-mediated immune
suppression has become established. In order to test this hypothesis, a combination of an
excellent animal model of prostate cancer, a novel prostate cancer immunotherapy and an
outstanding prostate tumor associated antigen was employed.
The TRAMP Mouse
In previous studies, the efficacy of therapeutic cancer vaccines has been investigated in
the TRAMP (transgenic adenocarcinoma mouse prostate) autochthonous model prostate
cancer. In these animals, expression of the simian virus 40 (SV40) oncogenic large T
antigen is driven by the prostate-specific rat probasin promoter. This results in a line of
transgenic mice that develop prostate cancer in a manner that recapitulates the course of
disease in humans (Kaplan-Lefko et al., 2003). Typically, the development of
precancerous PIN lesions can be observed at eight weeks of age. Progression to invasive
prostate adenocarcinoma or neuroendocrine tumors occurs in TRAMP mice between 12
and 16 weeks of age. Finally, TRAMP mice develop metastatic disease starting at
approximately 24 weeks of age (Greenberg et al., 1995). Untreated, virtually all TRAMP
mice will die of prostate cancer by 40 weeks of age. The TRAMP mice that are available
are all fully on the C56BL/6 background. Thus, any concerns regarding variability in
immune responses due to genetic diversity are eliminated since all the mice have
35
identical major histocompatibility complex (MHC) haplotypes. Overall, the TRAMP
model is an ideal system in which to investigate the effects of therapeutic vaccination at
different stages of prostate cancer progression.
Heterologous Prime-Boost Vaccination
In order to investigate whether immunization at an earlier stage of disease is more
effective, a state-of-the-art therapeutic prostate cancer vaccine was employed. One of the
latest advances in vaccinology is the development of heterologous prime-boost
vaccination regimes that involve sequential vaccination with different antigen delivery
systems encoding the same antigen (Wang et al., 2005b). For example, Goldberg et al
showed that priming first with DNA and boosting with recombinant alphavirus replicon
particles expressing the melanoma antigen tyrosinase resulted in a better immune
response and tumor protection than vaccinating with plasmid DNA alone (Goldberg et
al., 2005). DNA prime/virus boost heterologous vaccination regimes are the most
commonly used to increase immunogenicity (Mwau et al., 2004). In order to make use of
this immunotherapy strategy, collaboration was established with the developer of a
unique viral vector system based on the Venezuelan Equine Encephalitis (VEE) virus,
Alphavax. VEE and other enveloped, positive-stranded RNA alpha viruses (AV)
such as
Sindbis and Semliki Forest Virus have been engineered as replication-incompetent
viral-
delivery vectors or replicons (RP) (Velders et al., 2001). Replicon vectors
are generated
by removing the structural genes of the virus
and replacing them with a foreign gene; they
contain AV replicase
genes that mediate RNA replication and high-level protein
36
expression but produce no progeny virus. The replicon-recombinant
RNA that encodes
the foreign gene of interest in lieu of the VEE
structural genes can be packaged into VEE
replicons (VRP) on provision of the
structural RNA in trans (Velders et al., 2001). A
VRP in which the packaged foreign gene encodes the tumor-associated antigen being
targeted can elicit potent cellular immune responses and protective immunity against
implanted tumors (Cassetti et al., 2004). This system has many advantages, including
high-level of expression of heterologous genes and vector amplification through double-
stranded RNA intermediates that avoids integration of genetic material into the host
DNA. In addition, it stimulates innate immunity, through activation of the IFNγ cascade,
thereby inducing apoptosis of infected cells which may increase its immunogenicity via
antigen cross-priming (Leitner et al., 2003). The surface glycoproteins of the VEE virus
confer tropism to dendritic cells (DC), thereby allowing efficient delivery of TAA genes
to these professional antigen presenting cells (APC) (MacDonald and Johnston, 2000).
VEE virus-like replicon particles (VRP) can break immune tolerance in rats vaccinated
against the neu molecule and can induce efficient immune responses to tumors (Wang et
al., 2005a). Given that VRP induce potent and protective immune responses in primates
(Davis et al., 2000) and that humans generally have no preexisting humoral immunity to
the VEE virus, they may be the ideal tool for vaccine delivery in patients.
37
Prostate Stem Cell Antigen
Prostate stem cell antigen (PSCA) is a 123 amino acid protein. Analysis of its amino acid
sequence predicts that it is a membrane-bound protein related to the Ly-6/Thy-1 gene
family (Reiter et al., 1998). Human PSCA is expressed in esophagus, stomach, bladder
and prostate basal cells (Bahrenberg et al., 2000). It is overexpressed in 33% of primary
prostate tumors and always present in samples from patients with bone metastasis.
Higher levels of PSCA correlate with increasing tumor grade, stage and the progression
to androgen independence (Gu et al., 2000). Recently, a murine homologue (mPSCA)
was detected with 65% similarity to human PSCA
at the nucleotide and amino acid
levels. Mouse PSCA is expressed in the kidneys,
colon, and testes, as well as in the
normal prostate and in prostate tumor cells in the TRAMP mouse (Yang et al., 2001).
Heterologous DNA Prime/VRP Boost Confers Excellent Immunity Against Prostate
Cancer
The outstanding efficacy of the DNA prime/VRP boost protocol outlined above in
treating prostate cancer has previously been demonstrated in TRAMP mice. Dramatic
anti-tumor immunity was elicited when the prostate TAA STEAP (six transmembrane
epithelial antigen of the prostate) was targeted (Garcia-Hernandez Mde et al., 2007). This
resulted in very significant improvement in the survival of C57BL/6 mice implanted with
TRAMP-C2 cells, a tumorigenic prostate cancer cell line derived from TRAMP mice. By
far the best improvement in overall TRAMP mouse survival ever demonstrated was
38
achieved using the DNA prime/VRP boost strategy targeting PSCA (Garcia-Hernandez
Mde et al., 2008). This yielded outstanding results, with 90% of vaccinated TRAMP mice
still alive and healthy at 340 days, compared to just 10% of control animals (Garcia-
Hernandez Mde et al., 2008). Protection against the spontaneous development of prostate
cancer was mediated by tumor-infiltrating CD4
+
and CD8
+
T cells. Based on the
spectacular results of this previous study, prostate stem cell antigen was selected as the
target TAA for the experiments described in this chapter.
In this study, it is explored whether superior efficacy of therapeutic vaccination in
TRAMP mice can be elicited if it is administered to animals that have only developed
PIN lesions compared to when vaccination of these mice occurs after invasive
carcinomas or neuroendocrine tumors have developed, and if so what
immunosuppressive mechanisms may be involved in hampering therapeutic vaccination
effectiveness at the later stages of disease.
39
MATERIALS AND METHODS
Mice
C57BL/6 mice were obtained from Taconic farms (Germantown, NY). TRAMP mice
(Greenberg et al., 1995) on the C57BL/6 background were bred at USC. Research was
conducted in compliance with the Institutional Animal Care and Use Committee
guidelines.
Plasmid DNA constructs and VRP generation
For gene gun-mediated DNA priming, a plasmid DNA construct encoding mouse PSCA
was used as has previously been described (Garcia-Hernandez Mde et al., 2008). A DNA
fragment encoding mouse PSCA was obtained from pCR-mPSCA (Yang et al., 2001).
For the generation of PSCA-expressing plasmid (pcDNA3-PSCA), mPSCA DNA was
amplified using two specific primers. Primer sequences were designed to include either a
HindIII or a BamHI site: 5’-CCCAAGCTTATGACTCACAGG-3’ and 5’-
TGTGAGGAGTGCACA-3’. Amplification was performed for 30s at 94°C, 30s at 55°C,
30s at 72°C. An additional extension step was performed for 10 min at 72°C. The PCR
product was then cloned into the pcDNA3 expression vector (Invitrogen, Carlsbad, CA).
DNA sequencing was performed to confirm that the pcDNA3-PSCA construct had the
desired sequence and open reading frame. pcDNA3-PSCA or empty vector (pcDNA3)
were transformed into TOP10 competent E. coli (Stratagene, La Jolla, CA). Plasmid
DNA copies were amplified in liquid culture and purified using a plasmid maxi kit
40
(Qiagen Sciences, MD). The DNA that was used to coat gold particles in the making of
gene gun bullets for vaccination had a minimum OD
260
:OD
280
ratio =1.9. For boosting,
VRP were custom made and supplied on demand by Alphavax, Inc (Research Triangle
Park, NC). For VRP production, mPSCA DNA was amplified by using specific primers:
5'-GACTCACAGGACTACTACGTGGGCAAGAA-3' and 5’-TTAATTAAGGCGAGC-
TCCTACAACC-3. A Pac-1 site was added to the antisense primer. Amplification was
performed for 30s at 94°C, 30s at 58°C, 30s at 72°C. An additional extension step was
performed for 10 min at 72°C. The PCR product was digested with the appropriate 3’
enzyme and ligated into pERK (AlphaVax replicon vector plasmid) that had been
digested with EcoRV and the matching 3’ enzyme. mPSCA presence in the construct was
verified by DNA sequencing. VRP were made using a two-helper system (Pushko et al.,
1997).
Immunization
Male C57BL/6 or TRAMP mice were anesthetized by intraperitoneal injection of 80-100
mg/kg ketamine (Phoenix Pharmaceutical Inc, St Joseph, MO) and 10 mg/kg xylazine
(Phoenix). DNA-gold particles were delivered to a shaved area on the abdomen using a
helium-driven gene gun (BioRad) with a discharge pressure of 300 – 400 psi. Each
mouse received 2 μg of mPSCA cDNA vaccine. Ten days after gene gun vaccination
mice were subcutaneously boosted 1 cm from the tail base with 10
6
infectious units (IU)
mPSCA-VRP. As control groups, C57BL/6 or TRAMP mice were vaccinated with
41
pcDNA3 and boosted with 10
6
IU GFP-VRP. Ten days after boosting, mice were
euthanized in order to carry out assays as required.
Peptide synthesis
The peptide mPSCA
(83-91)
(amino acid sequence NITCCYSDL) was synthesized
according to standard methods used at the Norris Comprehensive Cancer Center facility
at the University of Southern California. Peptide was dissolved at 50 mg/ml in DMSO
(Sigma) and stored at -80°C until further use.
Cell culture medium
Splenocytes and lymphocytes were stored and cultured in IMDM supplemented with
10% fetal calf serum (Gemini) and 50 μg/ml kanamycin (Sigma). Cells were cultured at
37
o
C, 5% CO
2
and 100% relative humidity.
Isolation of splenocytes
Mice were euthanized and dissected using aseptic techniques in a biosafety hood. The
spleen was isolated from each mouse and immediately stored in a sterile 15 ml conical
tube (Falcon) in 3 ml complete IMDM. A 70 μm nylon cell strainer (BD Falcon, ref. #
352350) was placed in a 50 ml conical tube (BD Falcon) and the spleen and media
poured into it. The spleen was ground through the strainer with the rubber end of a 5 ml
syringe plunger (Becton Dickinson), wiping any tissue/cells stuck to the end of the
plunger being wiped on the inside edge of the strainer. The plunger was discarded and the
42
strainer rinsed with 10 ml complete IMDM. The cells were spun down at 300 g for 5
minutes in a tabletop centrifuge. The supernatant was discarded and the cells resuspended
in 1 ml Red Blood Cell Lysing Buffer (Sigma). The cells were incubated for 1 minute at
room temperature, then the tube was topped up with sterile PBS and spun at 300 g for 5
minutes in a tabletop centrifuge. The supernatant was discarded, and the cells suspended
in 25 ml PBS and run through a new 70 μl cell strainer into a new 50 ml conical tube in
order to remove clumps of red cell ghosts. After the final spin (300 g for 5 minutes), the
remaining splenocytes were resuspended in 10 ml complete IMDM for counting.
Isolation of lymphocytes
Mice were euthanized and dissected using aseptic techniques in a biosafety hood. The
tumor draining lymph nodes were isolated from each mouse and immediately stored in a
sterile 15 ml conical tube (Falcon) in 3 ml complete IMDM. A 70 μm nylon cell strainer
(BD Falcon, ref. # 352350) was placed in a sterile 60 mm Petri dish (Falcon) and the
lymph nodes and media poured into it. The lymph nodes were ground through the strainer
with the plastic end of a 5 ml syringe plunger (Becton Dickinson). The plunger end and
the strainer were lifted out of the Petri dish and rinsed with 5 ml complete IMDM. The
suspended cells in the Petri dish were transferred to a sterile 15 ml conical tube using a
10 ml serological pipette. The Petri dish was then held at a 45 degree angle and rinsed
again with 5 ml of fresh complete IMDM to collect any remaining cells which were
added to the same 15 ml conical tube. The cells were spun down at 300 g for 5 minutes in
a tabletop centrifuge. The supernatant was discarded, and the cells washed in 10 ml PBS.
43
After the final spin (300 g for 5 minutes), the lymphocytes were resuspended in 10 ml
complete IMDM for counting.
Isolation of tumor infiltrating lymphocytes (TIL)
Tumor infiltrating lymphocytes were obtained by enzymatic digestion of fresh prostate
and prostate tumor tissue from C57BL/6 and TRAMP mice, respectively. Mice were
euthanized and dissected using aseptic techniques in a biosafety hood. The entire
genitourinary tract was removed from each mouse and the testes, seminal vesicles and
penis were dissected away and discarded. In TRAMP mice in which prostate tumors had
invaded the seminal vesicles, solid tumor from these tissues was retained whilst normal
seminal vesicle tissue was dissected away. Tumor tissue isolated from seminal vesicle
was washed by rinsing in sterile PBS in order to remove any remaining semen. All
prostate and prostate tumor tissue obtained from each animal was weighed and placed in
a sterile 50 ml conical tube (Falcon) containing 5 ml IMDM (Lonza). A sterile
disposable scalpel (Becton Dickinson) was then used to mince the tissue in the tube. For
samples weighing 100 micrograms or less, 0.71 ml of 0.2% dispase (Invitrogen) in
IMDM was added to the tube, for a final concentration of 0.025% dispase in IMDM. For
samples of weighing more than 100 micrograms, 5 ml IMDM was added per additional
100 micrograms (or part thereof) of tissue, then 0.71 ml of 0.25% dispase was added for a
final concentration of 0.025% dispase in IMDM. Samples were then shaken at 37
o
C at
250 rpm for two hours and strained through a sterile cell strainer (Falcon) into a fresh 50
ml conical tube. Isolated cells were centrifuged at 300g in a desktop centrifuge for five
44
minutes and the supernatant removed. Cells were washed twice in 25 ml sterile PBS,
before being resuspended in 10 ml PBS for counting.
Cell staining and Fluorescence Activated Cell Scanning
Splenocytes, lymphocytes and TIL isolated as described above were counted using a
hemocytometer. For each sample to be stained, 10
6
cells were transferred to two wells of
a 96-well V-bottom plate (VWR) in a maximum volume of 200 μl media/PBS. The plate
was then spun at 300 g for 5 minutes in a floor centrifuge ((Beckman Coulter). The
supernatant removed by flicking into a sink and the plate gently dabbed dry using paper
towels. The cells were washed with 150 μl per well ice cold FACS buffer, spun again, the
supernatant removed and the plate dabbed dry. This was repeated twice. The cells were
then resuspended in 50 μl per well ice cold FACS buffer containing 1 μl FcBlock
antibody (RD Systems) and incubated for 15 minutes on ice. Each sample was then
incubated on ice for 30 minutes in the dark with either 50 μl ice cold FACS buffer
containing a cocktail of cell surface marker antibodies (1 μl FITC-conjugated anti-mouse
CD3 antibody (Becton Dickinson), 1 μl PE-Cy5-conjugated anti-mouse CD4 antibody
(Becton Dickinson) and 1 μl PE-Cy7-conjugated anti-mouse CD8a antibody) or 50 μl ice
cold FACS buffer containing a cocktail of the appropriate isotype controls for the above
antibodies (1 μl FITC-conjugated hamster IgG1 antibody (Becton Dickinson), 1 μl PE-
Cy5-conjugated rat IgG2a antibody (Becton Dickinson) and 1 μl PE-Cy7-conjugated rat
IgG1 antibody). The cells were washed three times with 150 μl per well ice cold FACS
buffer as described above. The cell pellets were then resuspended in 100 μl per well
45
fixation/permeablization buffer (eBioscience) and incubated overnight in the dark at 4
o
C.
The plate was then spun at 300 g for 5 minutes in a floor centrifuge. The supernatant
removed by flicking into a sink and the plate gently dabbed dry using paper towels. The
cells were washed twice with 100 μl per well ice cold 1X permeablization buffer
(eBioscience) as described above. The cell pellets were then resuspended in 50 μl per
well 1X permeablization buffer containing 1 μl FcBlock and incubated for 15 minutes on
ice in the dark. For identification of FOXP3
+
regulatory T cells, each sample was then
incubated on ice for 30 minutes in the dark with either 50 μl ice cold permeablization
buffer containing either 1 μl PE-conjugated anti-mouse FOXP3 antibody (eBioscience) or
1 μl of the appropriate PE conjugated isotype control antibody (Becton Dickinson). After
staining with PE-conjugated antibodies, each well was washed twice with 100 ul
Permeablization buffer as described above. After the final spin, the cell pellets were
resuspended in 100 μl ice cold FACS buffer. Cells were analyzed using a Becton
Dickinson FC500 flow cytometer.
Measurement of intratumoral cytokine expression and polymerase chain reaction
Tumors were collected, weighed and homogenized in PBS containing 2x Halt Protease
Inhibitor Cocktail (Pierce, Rockford, IL). 1% BSA (Sigma) was added and the
supernatants were collected by centrifugation at 4
o
C for 20 min. The cytokine levels in
the supernatants were quantified using the Bio-Plex mouse cytokine Th1/Th2 assay (Bio-
Rad) and a Bio-Plex HTF system, following the manufacturer’s instructions. For
quantitative PCR total RNA was isolated using an RNeasy kit (Qiagen). DNase-treated
46
RNA was reverse transcribed with random hexamer primers and SuperScript III
(Invitrogen). Quantitative PCR was performed using SensiMix SYBR QPCR Master
Mix, following the manufacturer’s protocol (Bioline, Tauton, MA). Primer sequences for
GAPDH (Forward TCAATGAAGGGGTCGTTGAT, Reverse
CGTCCCGTAGACAAAATGGT), TGFβ (Forward AAGTTGGCATGGTAGCCCTT,
Reverse GGAGAGCCCTGGATACCAAC), indoleamine 2,3 dioxygenase (Forward
GTGGGCAGCTTTTCAACTTC, Reverse GGGCTTTGCTCTACCACATC), arginase 2
(Forward AGGGATCATCTTGTGGGACA, Reverse AGAAGCTGGCTTGCTGAAGA)
and FOXP3 (Forward TCCAAGTCTCGTCTGAAGGC, Reverse
GCGAAAGTGGCAGAGAGGTA) were obtained from qPrimerDepot
(primerdepot.nci.nih.gov). Quantitative PCRs were performed using a CFX system (BIO-
RAD, Carlsbad, CA). The relative level of mRNA expression for each gene in TRAMP-
C2 cell/prostasphere treatment group was first normalized to the expression of GAPDH
RNA in that tumor and then normalized to the level of mRNA expression in
cells/prostaspheres treated with DMSO vector control only. The relative level of mRNA
expression for each gene in each tumor was first normalized to the expression of GAPDH
RNA in that tumor and then normalized to the level of mRNA expression in tumor from
control mice.
Statistical analysis
Flow cytometry and real time quantitative PCR data were analyzed by two tailed,
independent t tests. Multiplex ELISA data were analyzed by two tailed independent t
47
tests followed by Bonferroni corrections applied for multiple analyses. Survival rates
were analyzed by the log rank test for survival. Delta survival data were analyzed by
linear regression analysis, the results of which were then further analyzed using an F test
to compare the slopes.
48
RESULTS
To evaluate whether the stage of prostate cancer progression at which therapeutic
vaccination is applied affects the efficacy of the vaccine, the long-term survival rates of
TRAMP mice that were vaccinated at two distinct stages of prostate cancer
carcinogenesis were compared. TRAMP mice were vaccinated at 8 weeks of age, at
which point they have developed precancerous prostate intraepithelial neoplastic (PIN)
lesions, or at 16 weeks of age when prostate neuroendocrine carcinomas or
adenocarcinomas have developed. The groups of mice were vaccinated using an identical
heterologous DNA prime/VRP boost scheme, though the antigens targeted were different
(mPSCA at 8 weeks and mSTEAP at 16 weeks). However, it has been previously
demonstrated that vaccination against both mPSCA (Garcia-Hernandez Mde et al., 2008)
and mSTEAP (Garcia-Hernandez Mde et al., 2007) using this immunization scheme
induce strong and comparable immune responses in mice. Survival in mice vaccinated at
8 weeks and at 16 weeks was statistically significantly improved compared to age
matched controls (p < 0.0001 and p = 0.0001, respectively), indicating that vaccination at
both time points yielded excellent protection from prostate cancer development (Figure
2.1). In the case of TRAMP mice vaccinated at 8 weeks, there was a dramatic difference
in tumor burden between mPSCA-vaccinated mice and negative controls euthanized at
340 days (Figure 2.2).
49
Figure 2.1
Figure 2.1: Therapeutic vaccination against prostate tumor-associated antigens
results in long term survival in TRAMP mice. Groups of 20 male 8 week old TRAMP
mice were vaccinated by helium-driven gene gun at day 0 with either 2 ug mPSCA-
pcDNA or 2 ug empty vector and boosted at days 15 and 60 with 10
6
IU mPSCA-VRP
and 10
6
IU GFP-VRP, respectively. Groups of 20 male 16 week old mice were
vaccinated by helium-driven gene gun with either 2 ug mSTEAP-pcDNA or 2 ug empty
vector and boosted at days 15 and 60 with 10
6
IU mSTEAP-VRP and 10
6
IU GFP-VRP,
respectively.
50
Figure 2.2
Figure 2.2: Therapeutic vaccination against mPSCA results in reduced prostate
tumor burden in TRAMP mice. All surviving mPSCA-vaccinated TRAMP mice and
their age-matched controls were euthanized at day 340 and necropsy performed.
Representative images of a prostate tumor isolated from a negative control animal (left,
“Control”) and that of an mPSCA-vaccinated mouse (right, “mPSCA”) are shown.
51
The survival of mice vaccinated at 8 weeks and at 16 weeks could not be directly
compared because the mice vaccinated at 16 weeks were castrated prior to immunization.
The effect of androgen ablation on long-term TRAMP mouse survival is seen in the
statistically significant difference in survival between the non-castrated 8 week negative
control group and the castrated 16 week negative control group (p = 0.0074). Both of
these groups were immunized with an empty DNA vector and boosted with a GFP-VRP,
so the difference in survival between the two groups is due to castration in the 16 week
group. It has been demonstrated in a previous study that castration only affects the results
of prostate cancer immunotherapy when it is carried out three weeks after vaccination
(Koh et al., 2009). Therefore, the effect of castration on survival in TRAMP mice that
were vaccinated at 16 weeks is most likely due to the retardation of androgen-dependent
prostate tumor growth rather than any immunological effect. To remove this confounding
factor from the analysis, the difference between the cumulative survival of the vaccinated
groups and the cumulative survival of their age- and castration-matched control groups
(which was termed “delta survival”) was calculated at each time point. Scatter plots of
delta survival versus survival time were plotted, and linear regression analysis performed
(Figure 1c). There was a very strong correlation between delta survival and survival time
in groups of mice vaccinated at 8 weeks (R
2
= 0.856) and a weaker correlation at 16
weeks (R
2
= 0.695). This indicated that, as expected, the difference in survival probability
between vaccinated mice and negative controls steadily increases over time. To
determine whether the improvement in survival in mice vaccinated at 8 weeks was better
than that of mice vaccinated at 16 weeks, the slopes of the regressions were compared via
52
F-test. The linear regression slope of the 8 week vaccination groups was statistically
significantly higher than that of the 16 week groups (F = 5.398, p = 0.0346). This
demonstrates that the survival of the group vaccinated at 8 weeks improved statistically
significantly more than did the survival of the group vaccinated at 16 weeks when each
was compared to their age- and castration-matched control groups.
53
Figure 2.3
Figure 2.3: Therapeutic vaccination against prostate tumor-associated antigens
results in superior survival when administered to TRAMP mice with PIN lesions.
The difference between the cumulative survival of each group of vaccinated mice and the
cumulative survival of their age-matched controls was calculated at each time point at
which one or more mice died. This value was termed “delta survival” and was plotted
against survival time. Linear regression analysis was performed on each data set, and the
slopes of the regression lines compared.
54
In an effort to understand the improvement of survival in mice vaccinated earlier in
carcinogenesis, it was hypothesized that one or more prostate tumor-mediated
immunosuppressive mechanisms become established at later stages of carcinogenesis
which are responsible for the reduction in therapeutic vaccine efficacy at later stages of
disease. Several immunosuppressive mechanisms have been identified as having a
possible role in prostate cancer. Given the significantly worse response to therapeutic
vaccination at later stages of prostate cancer progression, it was investigated whether any
of these mechanisms either become active or are more prevalent at later stages of disease.
First assessed was whether regulatory T cells (Treg) are more prevalent in either the
periphery or prostate tumors of TRAMP mice with more advanced disease. Splenocytes
and tumor infiltrating lymphocytes (TIL) were isolated from young (≤ 8 weeks old, n =
3), middle-aged (16-20 weeks old, n = 4) and old (≥ 24 weeks old, n = 3) TRAMP mice
and analyzed by flow cytometry. The mean percentage of splenocytes that were
CD3
+
CD4
+
FOXP3
-
non-regulatory T cells was strongly statistically significantly
different between groups (Figure 2.4, ANOVA, p < 0.0001). Specifically, the mean
percentage of splenocytes that were CD3
+
CD4
+
FOXP3
-
non-regulatory T cells in young
TRAMP mice (9.57 ± 0.49%) was statistically significantly higher than in middle-aged
(7.65 ± 0.52%, p < 0.01, Tukey’s multiple comparison test) and in old (5.47 ± 0.29%, p <
0.001, Tukey’s multiple comparison test) TRAMP mice (Figure 2.4). In addition, the
mean percentage of splenic CD3
+
CD4
+
FOXP3
-
T cells was statistically significantly
decreased in old compared to middle-aged TRAMP mice (p < 0.01, Tukey’s multiple
55
comparison test) (Figure 2.4). There was a trend towards a decrease in the mean
percentage of splenocytes that were CD3
+
CD4
+
FOXP3
+
Tregs with age, but it was
marginally outside of statistical significance (Figure 2.4, p = 0.0511, ANOVA).
Regulatory T cells suppress the function of effector cells more efficiently when the ratio
of Tregs to effector cells is higher. As a result of the decrease in CD3
+
CD4
+
FOXP3
+
Tregs being less pronounced than that of CD3
+
CD4
+
FOXP3
-
T cells with increasing age,
the ratio of mean CD3
+
CD4
+
FOXP3
+
splenocyte percentage to mean CD3
+
CD4
+
splenocyte percentage in TRAMP mice statistically significantly increased with age
(Figure 2.5, p = 0.0215, ANOVA). Specifically, ratio of mean CD3
+
CD4
+
FOXP3
+
splenocyte percentage to mean CD3
+
CD4
+
splenocyte percentage in TRAMP mice was
statistically significantly increased in old (0.42 ± 0.02) compared to young (0.34 ± 0.03)
TRAMP mice (Figure 2.5, p < 0.05, Tukey’s multiple comparison test). This indicates an
increased accumulation of Treg in the spleens of TRAMP mice the course of prostate
cancer progression.
56
Figure 2.4
Figure 2.4: The proportion of CD4+ T cells and CD4+FOXP3+ Tregs decrease in
the periphery of TRAMP mice with increasing age. Splenocytes isolated from groups
of young (≤ 8 weeks old, n = 3), middle-aged (16-20 weeks old, n = 4) and old (≥ 24
weeks old, n = 3) TRAMP mice were washed and stained with anti-mouse CD3-FITC
and anti-mouse CD4-PE/Cy7. Cells were fixed and permeablized overnight, then stained
with anti-mouse FOXP3-PE and analysed by flow cytometry. Events were collected
gated on live, CD3
+
cells and then further gated on the CD4
+
FOXP3
-
fraction and the
CD4
+
FOXP3
+
fraction. Bars indicate mean percentage of splenocytes ± SEM. The ** and
*** symbols indicate p < 0.01 and p < 0.001, respectively.
57
Figure 2.5
Young Middle-A ged Old
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
*
TRA M P M ouse A ge Group
CD4+FOXP3+:Total CD4+
Fraction
Figure 2.5: The fraction of the total splenic CD4+ T cell population that are
CD4+FOXP3+ Tregs increases with age in TRAMP mice. The fraction of splenocytes
of the total CD4
+
population that were Tregs in each individual TRAMP mouse was
calculated with the formula (n = (CD4
+
FOXP3
+
%)/( CD4
+
FOXP3
-
%+ CD4
+
FOXP3
+
%)).
The mean fraction of CD4
+
FOXP3
+
cells was then calculated per age group. Bars
represent the mean fraction of CD4
+
FOXP3
+
cells ± SEM. The * symbol indicates p <
0.05.
58
The increased prevalence of Tregs in the spleens of older TRAMP mice was correlated
with a concomitant decrease in the proportion of splenic CD3
+
CD8
+
T cells. The mean
percentage of splenocytes that were CD3
+
CD8
+
T cells was strongly statistically
significantly different between groups (Figure 2.6, ANOVA, p < 0.0012). Specifically,
the mean percentage of splenocytes that were CD3
+
CD8
+
T cells in old TRAMP mice
(6.47 ± 0.40%) was statistically significantly decreased compared to both young (9.63 ±
1.15%,) and middle-aged (10.4 ± 0.52% p < 0.01, Tukey’s multiple comparison test)
TRAMP mice (Figure 2.6).
59
Figure 2.6
Young Middle-Aged Old
0
1
2
3
4
5
6
7
8
9
10
11
**
**
TRAMP Mouse Age Group
CD8+ Proportion of
Total Splenocytes (%)
Figure 2.6: The proportion of CD8+ T cells decreases in the periphery of TRAMP
mice in old age. Splenocytes isolated from groups of young (≤ 8 weeks old, n = 3),
middle-aged (16-20 weeks old, n = 4) and old (≥ 24 weeks old, n = 3) TRAMP mice were
washed and stained with anti-mouse CD3-FITC and anti-mouse CD8-PE/Cy5. Cells were
fixed and analysed by flow cytometry. Events were collected gated on live, CD3
+
cells
and then further gated on the CD8
+
population. Bars indicate mean percentage of
splenocytes ± SEM. The ** symbol indicates p < 0.01.
60
A previous study demonstrated that TRAMP tumors are infiltrated by CD3
+
CD4
+
T cells
even in the absence of vaccination and that DNA prime/VRP boost immunization against
mPSCA statistically significantly increased this infiltration (Garcia-Hernandez Mde et
al., 2008). To investigate whether tumor infiltration by CD4
+
T cells varies as TRAMP
mice age, flow cytometric analysis of tumor infiltrating lymphocytes was performed.
These data revealed a statistically significant increase in the mean number of tumor
infiltrating CD3
+
CD4
+
FOXP3
-
cells in old (35844 ± 7963 cells/g tumor) compared to
middle-aged (7182 ± 3792 cells/g tumor) TRAMP mice (Figure 2.7, Independent t test, p
< 0.001). In addition, the mean number of tumor infiltrating CD3
+
CD4
+
FOXP3
+
Treg
cells in old (5962 ± 4642 cells/g tumor) increased compared to middle-aged (1008 ± 291
cells/g tumor) TRAMP mice, though this was not statistically significantly different
(Figure 2.7, Independent t test, p = 0.1388). In contrast to the situation in the periphery,
the proportion of tumor infiltrating CD3
+
CD4
+
FOXP3
-
cells that were
CD3
+
CD4
+
FOXP3
+
Treg cells remained constant (15.6% versus 15.4% in middle-aged
and old mice, respectively). However, the absolute number of Treg per gram of tumor
increased over the course of disease progression. No data were available from young
TRAMP mice because insufficient TIL for flow cytometry could be isolated from the
prostates of these animals. Given the critical role of cytotoxic T cells in killing tumor
cells, an attempt was made to analyze the numbers of CD3
+
CD8
+
cells that infiltrated the
tumors of the same TRAMP mice. In both middle-aged and old TRAMP mice there was
no detectable CD3
+
CD8
+
TIL (data not shown). This is consistent with previous data
61
indicating that efficient tumor infiltration by CD3
+
CD8
+
cells only occurs in vaccinated
TRAMP mice (Garcia-Hernandez Mde et al., 2008).
62
Figure 2.7
Middle-Aged Old
0
5,000
10,000
15,000
20,000
25,000
30,000
35,000
40,000
45,000
CD4+
CD4+FOXP3+
TRAMP Mouse Age Group
Tumor Infiltrating
Lymphocytes
(cells/g tumor)
**
Figure 2.7: Increased infiltration of CD4+ T cells and CD4+FOXP3+ Treg into the
prostate tumor with increasing age. Tumor infiltrating lymphocytes isolated from
groups of young (≤ 8 weeks old, n = 3), middle-aged (16-20 weeks old, n = 4) and old (≥
24 weeks old, n = 3) TRAMP mice were washed and stained with anti-mouse CD3-FITC,
anti-mouse CD8-PE/Cy5 and anti-mouse CD4-PE/Cy7. Cells were fixed and
permeablised overnight, then stained with anti-mouse FOXP3-PE and analysed by flow
cytometry. Events were collected gated on live, CD3
+
CD8
-
cells and then further gated
on the total CD4
+
FOXP3
-
fraction and the CD4
+
FOXP3
+
fraction. Bars represent mean
number of cells per gram of tumor ± SEM. The ** symbol indicates p < 0.01.
63
To assess the whether suppressive capacity of Tregs altered over the course of prostate
tumor development, lymphocytes were isolated from the prostate tumor draining lymph
nodes of young and old TRAMP mice and divided into CD4
+
CD25
-
effector and
CD4
+
CD25
+
regulatory T cell populations by magnetic activated cell sorting (MACS,
Miltenyi Biotec, Bergisch Gladbach, Germany). The proliferation of the CD4
+
CD25
-
responder cells steadily decreased when cocultured with increasing numbers of
CD4
+
CD25
+
regulatory T cells isolated from both middle-aged and old mice, indicating
that this population is functionally suppressive (Figure 2.8). Tregs isolated from old mice
could only suppress proliferation of responder cells when cocultured at a ratio of 1:2
Treg:Tresp. In contrast, Tregs isolated from young mice were capable of suppressing the
proliferation of responder cells even when cocultured at ratios of 1:4 and 1:8 Treg:Tresp.
This indicates that Tregs isolated from the prostate tumor draining lymph nodes of old
TRAMP mice are less functionally suppressive than Tregs isolated from their young
counterparts (because each Treg isolated from young TRAMP mice is capable of
suppressing the proliferation of more responder cells than each Treg isolated from old
TRAMP mice). This difference in functional suppressive capacity was statistically
significantly different at the 1:4 Treg:Tresponder ratio (Figure 2.8, Independent t test, p =
0.0087)
64
Figure 2.8
Figure 2.8: CD4+CD25+ T cells isolated from prostate tumor draining lymph nodes
of younger mice are more are functionally suppressive. T cells from tumor-draining
lymph nodes isolated from middle-aged (≤ 16-20 weeks old, n = 3) and old (≥ 24 weeks
old, n = 3) TRAMP mice were purified into CD4
+
CD25
–
(responders, Tresp) and
CD4
+
CD25
+
(Treg) populations by magnetic separation. 5x10
4
CD4
+
CD25
+
T cells were
cocultured for 72 hours with 5x10
4
allogenic T cell-depleted splenocytes from C3H mice
in complete IMDM supplemented with 1 ug/ml activating anti-mouse CD3 antibody,
either alone or with autologous CD4
+
CD25
–
Tresp cells in 1:2, 1:4, 1:8 and 1:16 ratios.
As positive controls for maximal proliferation, 5x10
4
CD4
+
CD25
–
Tresp cells were
cocultured with 5x10
4
allogenic T cell-depleted splenocytes from C3H mice in complete
IMDM supplemented with 1 ug/ml activating anti-mouse CD3 antibody.
3
H-thymidine (1
μCi/well) was added in the last 8 hours of culture. Tresp cell proliferation was measured
by
3
H-thymidine incorporation. The relative proliferation index of responder cells for
each mouse at each Treg:Tresp ratio was calculated by dividing the mean Tresp
proliferation at each ratio by the maximal proliferation (Tresp cultured in the absence of
Treg, Tresp(Max)) of Tresp in that animal. Points represent mean relative proliferation ±
SD. The ** symbol indicates p < 0.01.
65
To determine whether the cytokine profile of the tumor microenvironment becomes
immunosuppressive or inhibitory over the course of prostate cancer development,
expression of a panel of cytokines and chemokines was measured by multiplex
immunoassay (Figure 2.9). The expression of several cytokines and chemokines,
including IL-1a, IL-2, IL-4, IL-5, IL-9, IL-10, IL-12(p40 and p70), IL-13, IL-15, IFNγ,
G-CSF, GM-CSF, LIX, MIP-1a, MIP-2 and TNFα was reduced in the prostate tumors of
middle-aged TRAMP mice compared to the prostates of young TRAMP mice.
Conversely, there were increases in the expression of M-CSF, MIG, RANTES and
VEGF. After Bonferroni adjustments for multiple comparisons, none of these changes
were statistically significant. Overall, there was a trend towards a reduction in both Th1
and Th2 function in middle-aged mice compared to young mice, while expression of
angiogenic factors (KC and RANTES) increased with age.
66
Figure 2.9: The cytokine/chemokine expression profile of spontaneous TRAMP
prostate tumors changes over time. Spontaneously arising prostate tumors harvested
from groups of young (≤ 8 weeks old, n = 5) and old (≥ 24 weeks old, n = 4) were
homogenized at 4
o
C in 10 μl/mg sterile PBS containing 1x protease inhibitors. Cytokine
levels were quantified with a custom 32-plex Milliplex MAP mouse cytokine
immunoassay (Millipore, Billerica MA) using the Bio-Plex multiplex system (Bio-Rad,
Hercules, CA).
67
Figure 2.9 continued
68
Expression of several immunosuppressive factors, including TGFβ and indoleamine 2,3
dioxygenase has been shown to be increased in multiple tumor types. To assess whether
their expression increases with prostate tumor progression, their expression in the
prostate tumors of TRAMP mice was quantified by quantitative real-time PCR. Relative
expression of both TGFβ and indoleamine 2,3-dioxygenase mRNA normalized to
GAPDH were increased in middle-aged mice compared to young mice (Figure 2.10). The
relative expression of FOXP3 mRNA normalized to GAPDH was also increased in the
prostates of middle-aged mice compared to young mice, as was expected given the
increase in the number of FOXP3-expressing Treg per gram of prostate tumor with
increasing age (Figure 2.7). Taken together, these data suggest a reduction in pro-
inflammatory cytokines combined with an increase in the expression of
immunosuppressive factors during disease progression may lead to an inhibition of
prostate cancer-specific immunity and reduced efficacy of vaccination in the later stages
of prostate cancer. These data support the hypothesis that immunosuppressive
mechanisms become active over the course of prostate cancer progression, and that
immunization at early stages of disease may yield superior vaccine efficacy by avoiding
their effects.
69
Figure 2.10
FOXP3 INDO ARG2 TGFb
0
1
2
Young
Old
5
25
45
65
85
105
125
Analyte
Expression fold change
Figure 2.10: Increased expression of immunosuppressive molecules in prostate
cancer. Spontaneously arising prostate tumors harvested from groups of young (≤ 8
weeks old, n = 3) and old (≥ 24 weeks old, n = 3) mice, fixed in RNAlater and were
homogenized at 4
o
C in 10 μl/mg buffer RLT with β-mercaptoethanol. Total RNA was
isolated using a QIAGEN RNeasy kit according to the manufacturer’s instructions.
Complementary DNA was generated using this RNA as a template, then quantitative
real-time PCR performed.
70
DISCUSSION
Here it is demonstrated that a heterologous DNA prime/VRP boost prostate cancer
therapeutic vaccination strategy yields superior long term protection when it is applied at
a precancerous stage of disease compared to when it is administered after invasive cancer
has developed in the TRAMP mouse model of human prostate cancer. In addition,
several immunosuppressive mechanisms that may be involved in the differential efficacy
of a therapeutic vaccine at different stages of prostate carcinogenesis in TRAMP mice
have been identified.
A wide variety of immunotherapeutic strategies for treating prostate cancer have
undergone clinical trials, but so far only sipuleucel-T has been approved for clinical use.
A fundamental problem with these clinical trials is that they have been carried out almost
exclusively in terminally ill patients who have failed all other therapeutic options. These
patients are frequently immunocompromised, and are therefore poor candidates in whom
to test immunotherapies. With this in mind, it has been proposed by this group that
therapeutic prostate cancer vaccines should be applied in the preventive setting (Gray et
al., 2008). Prior to seeking approval for clinical trials that involve immunizing early-stage
prostate cancer patients, it is vital to demonstrate that application of a therapeutic cancer
vaccine at the early stages of disease is likely to yield superior results than when it is
administered in the advanced stages of disease. Therefore, it was decided to evaluate
whether therapeutic vaccination confers superior protection in the TRAMP mouse model
of human prostate cancer when it is applied at a precancerous stage of cancer
71
development (PIN lesions at 8 weeks) compared to when the mice have more advanced
disease (adenocarcinomas or neuroendocrine tumors at 16 weeks) (Gingrich et al., 1997).
To evaluate this, the long-term survival rates of TRAMP mice that were vaccinated at 16
weeks of age were compared to the survival rates of TRAMP mice that had been
vaccinated at 8 weeks of age as part of a previous study (Garcia-Hernandez Mde et al.,
2008).
The two long-term survival studies evaluated here were not originally designed to be
compared to each other. Most notably, the antigens targeted were different (mPSCA at 8
weeks and mSTEAP at 16 weeks). However, we have previously demonstrated that
vaccination elicits strong and comparable immune responses against both mPSCA
(Garcia-Hernandez Mde et al., 2008) and mSTEAP (Garcia-Hernandez Mde et al., 2007)
using this immunization scheme. Here it is shown that vaccination of TRAMP mice
against either mPSCA and mSTEAP yields very strongly statistically significant
improvements in survival compared to age-matched negative controls (Figure 2.1). It
should be noted that by priming with an empty DNA vector and boosting with GFP-VRP
as negative controls, strictly speaking a secondary response against TAA is being
compared to a primary response against GFP in these experiments. However, previous
studies have demonstrated that priming and boosting against GFP makes no difference to
TRAMP tumor growth. The survival of mice DNA primed with empty pcDNA3 vector
and boosted with GFP-VRP is identical to that of mice primed and boosted against GFP
(Garcia-Hernandez Mde et al., 2007). A key difference in the design of the two studies is
72
that the mice vaccinated at 16 weeks were castrated prior to vaccination. In the time since
this study was initiated, this group has demonstrated that castration only elicits an
immunological effect on prostate cancer vaccine efficacy when it is carried out three
weeks after vaccination and not before (Koh et al., 2009). Therefore, it was concluded
that the apparent improvement in long-term TRAMP mouse survival due to castration
(Figure 2.1, compare 8 weeks control curve to 16 weeks control curve) is a direct result
of androgen ablation on prostate tumor growth rather than an immunological effect. In
order to analyze the effect on survival of vaccination at 8 weeks and at 16 weeks without
the confounding factor of castration, the difference between the survival of the vaccinated
groups and their age- and castration-matched control groups (which was termed delta
survival) was calculated for each time point. Linear regression analysis revealed that the
improvement in survival of mice vaccinated at 8 weeks compared to their age-matched
controls was statistically significantly better than that of mice vaccinated at 16 weeks
compared to their age- and castration-matched controls (Figure 2.3). It should also be
noted that of the mice vaccinated at 8 weeks with mPSCA, 90% had not yet reached the
survival endpoint at 340 days post-vaccination compared to 0% in age-matched controls.
Unfortunately, despite not reaching their survival endpoint the mPSCA vaccinated mice
were euthanized at 340 days, according to the study design that was then being followed.
At the time of their euthanasia, the mPSCA-vaccinated mice were in outstanding
condition. They looked outwardly healthy, were eating and behaving exactly as would be
expected of one year old wild type C57BL/6 mice and showed no signs of pain or
distress. Upon necropsy, the prostates of these animals were outwardly normal. This was
73
in stark contrast with the few animals of the control group that reached the survival
endpoint on the same day and were euthanized. These mice were weak, obviously
unhealthy and had very large, palpable tumors. Figure 2.2 shows the dramatic difference
in tumor burden in mPSCA-vaccinated TRAMP mice and controls euthanized at 340
days. Overall, it was concluded that therapeutic vaccination against prostate cancer TAAs
confers superior survival benefits if it is applied when PIN lesions are present but
invasive adenocarcinomas or neuroendocrine tumors have not yet developed.
In an effort to explain the difference in survival between TRAMP mice vaccinated at
different stages of disease, it investigated whether several different tumor-mediated
immunosuppressive mechanisms become present or are more active at later points of
prostate cancer development. There are conflicting data regarding the role of
CD4
+
CD25
+
FOXP3
+
regulatory T cells in prostate cancer. It has been concluded that
these cells are more prevalent in the blood of prostate cancer patients (Miller et al., 2006)
and more active (Yokokawa et al., 2008), while a conflicting study asserted that
peripheral tolerance in prostate cancer was not mediated by Treg (Degl'Innocenti et al.,
2008). Consistent with the first two of these studies, the data presented here indicate a
relative increase in the numbers of Tregs in periphery of TRAMP mice with increasing
age (Figure 2.4). The percentage of splenic CD4
+
FOXP3
-
T cells decreased with
increasing TRAMP mouse age, but the percentage of splenic CD4
+
FOXP3
+
Tregs stays
relatively constant. As a result, the ratio of Tregs to other CD4
+
T cells increases with
TRAMP mouse age, suggesting progressively increasing systemic immunosuppression.
74
This is supported by a significant decrease in the number of CD8
+
T cells in the splenic
population of the oldest TRAMP mice (Figure 2.5). It is not clear from these experiments
whether these phenomena are due to the presence of an advancing tumor in the older
mice, or are merely changes normally associated with aging. Ideally, this experiment
should be repeated with age-matched normal C57BL/6 mice as negative controls. Despite
this limitation, the data do still indicate that therapeutic prostate cancer vaccination is
likely to be more successful when mice are younger due to the presence of more
favorable numbers of CD8
+
cells and a better CD4
+
:CD4
+
FOXP3
+
cell ratio. If this is also
true in humans (regardless of whether the less favorable immune environment is due to
the presence of a tumor or is simply due to aging), then these data argue in favor of
vaccinating men as early as possible after diagnosis.
Next was assessed whether Treg also accumulate within the prostate tumors of TRAMP
mice. It was found that though the number of Tregs per gram of prostate tumor did
indeed increase over time, it was part of a general accumulation of CD4
+
T cells within
the prostate tumor as it grew (Figure 2.6). The constant presence of Treg within the tumor
infiltrating lymphocyte population coupled with the fact that the prostate tumors of
unvaccinated TRAMP mice will grow rapidly and inevitably kill the animal suggests that
despite increased immune infiltration of the prostate, the constant presence of Treg
impedes the ability of the attempted immune response to control tumor growth. This is
supported by the observation that in unvaccinated TRAMP mice, CD8
+
T cells seem to
be incapable of infiltrating the prostate tumor in detectable numbers at any stage of
75
carcinogenesis. What is not clear from these data is whether the Treg that infiltrate the
tumor do so as part of a general accumulation of lymphocytes within the tumor, whether
they are attracted there specifically and independently of the mechanism attracting
effector T cells, or whether they are induced in situ from tumor infiltrating effector T
cells. Resolving this question will be crucial in the development of mechanisms to
prevent Treg infiltration to or induction within the prostate tumor.
Though Treg are capable of infiltrating prostate tumors, it was unknown whether they
were functionally active. Given the difficulty in isolating sufficient numbers of live Treg
from prostate tumors, the suppressive capacities of CD4
+
CD25
+
Treg isolated from the
tumor-draining lymph nodes of TRAMP mice was investigated (Figure 2.7). The data
show that there are functionally suppressive Treg in the draining lymph nodes of
prostates/prostate tumors of both young and old TRAMP mice. However, the suppressive
capacities of Tregs isolated from older TRAMP mice seemed to be lower than those
isolated from younger mice. This result was somewhat surprising; given the apparent
skewing in the periphery towards greater suppression, it might have been expected that
Tregs isolated from older TRAMP mice with more advanced tumors would be more
suppressive. This experiment needs to be repeated in order to verify this result.
It is significant that Tregs isolated from the tumor draining lymph nodes are functionally
suppressive even when isolated from young mice with only PIN lesions. This is the stage
at which the best improvements in survival are obtained with therapeutic vaccination
76
against prostate TAA, suggesting that these highly suppressive Tregs alone are not
capable of inhibiting a potent anti-tumor immune response. Understanding whether there
are differences between the microenvironment of the prostate tumor as disease progresses
may shed some light on the dramatic differences between the antitumor immune
responses elicited by vaccines at different time points. To investigate this, the expression
of a panel of cytokines and chemokines within TRAMP prostate tumors was quantified
by multiplex immunoassay (Figure 2.9). The data indicate a general reduction of Th1 and
Th2 type cytokines in more advanced tumors, suggesting that the microenvironment is
indeed become less permissive to immune responses as the tumor develops. There were
decreases in the expression of GM-CSF, IFNγ, IL-2, IL-4, IL-5, IL-9, IL-10, IL-12, IL-
13, IL-15 and TNFα in the prostate/prostate tumor tissues of young mice versus those of
old mice (Figure 2.9a). Conversely, there were increases in the expression of M-CSF,
MIG, RANTES and VEGF (Figure 2.9b). Though these differences were not statistically
significant after Bonferroni correction for multiple analyses, they were large and
indicated a trend towards differential expression of these cytokines and chemokines at
different stages of prostate cancer development. Most striking was the reduction of IFNγ
expression from 69.8 ± 40.7 pg/ml/g tumor in the prostates of young TRAMP mice to
completely undetectable levels in the prostate tumors of old TRAMP mice. This indicates
that despite the heavy infiltration of advanced prostate tumors by CD4
+
T cells, they are
not activated. IFNγ expression is upregulated via the p38 MAP kinase pathway which is
driven by TNFα, GM-CSF and IL-3, all of which are reduced in more advanced tumors.
77
Therefore, the observed lack of IFNγ expression is consistent with the cytokine milieu
observed in more advanced tumors.
Of particular interest was the somewhat counterintuitive reduction in IL-10 expression in
more advanced tumors. Though it is generally considered and anti-inflammatory
cytokine, several studies have indicated that IL-10 has strong anti-tumor effects
(Mocellin et al., 2003). Thus, its downregulation in more advanced tumors is not
unexpected. Interestingly, there was a decrease in the expression of macrophage
inflammatory protein-1α (MIP-1α) but an increase in MIP-1β expression. A previous
study has demonstrated that MIP-1α stimulated the release of IL-1 and TNFα by the
peripheral blood monocytes of women with breast cancer but not those of healthy women
(Nath et al., 2006). In contrast, the same study showed that MIP-1β could stimulate
release of these cytokines only healthy women and not in breast cancer patients. These
findings are consistent with our own results, and suggest that downregulation of MIP-1α
that we observed is a mechanism of prostate tumor immune escape that may be
responsible for the decreases in IL-1a and TNFα that occur in more advanced prostate
tumors. A large increase in the expression of monocyte chemoattractant protein 1
(MCP1) was observed in more advanced prostate tumors, which given that this
chemokine normally drives Th2 responses was initially puzzling (Matsukawa et al.,
2000). However, it has been shown that vaccine-induced eradication of r-p185 carcinoma
was dramatically increased in MCP1 knockout mice (Curcio et al., 2003). This result
would be consistent with our own, and suggests an important role for this protein in
78
tumor progression. In addition, a statistically significant increase in keratinocyte-derived
cytokine (KC, CXCL1) was observed. This cytokine is driven by prostaglandin E2 and
promotes angiogenesis (Wang et al., 2006). The upregulation of KC coupled with that of
VEGF may result in an increase in prostate tumor vasculature, which might explain the
observed general increase in tumor infiltrating lymphocytes in mice with more advanced
disease.
It is not clear whether these phenomena are due to the tumor becoming more advanced,
or whether it is due to the increased age of the mice tested. Ideally, the cytokine profiles
of the prostates of age-matched C57BL/6 mice should also have been analyzed. However,
previous experiments had indicated that there was no detectable infiltration of normal
mouse prostates by immune cells in the absence of vaccination. Given that such cells are
the primary source of the Th1/Th2 cytokines detected in the prostate tumors of TRAMP
mice, it was not expected that expression of these cytokines would be detectable in the
prostates of normal mice. Nevertheless, these controls should be included when this
experiment is repeated.
Finally, it was investigated whether other immunosuppressive mechanisms thought to be
involved in tumor immune evasion are upregulated in more advanced prostate tumors.
Quantitative real-time PCR data revealed increases in FOXP3, TGFβ and indoleamine-
2,3-dioxygenase in the prostate tumors of older TRAMP mice (Figure 2.10). An
approximately tenfold increase in FOXP3 expression between the prostate tumors of
79
young and old mice was observed. This is consistent with the sixfold increase in tumor-
infiltrating Tregs between middle-aged and old TRAMP mice, and suggests that the
accumulation of FOXP3
+
Tregs occurs throughout prostate tumor progression from its
earliest development. TGFβ is critical for the conversion of CD4
+
effector cells into
FOXP3
+
Tregs. The increase in TGFβ thus lends credence to the idea that the increase in
the numbers of regulatory T cells observed within advanced prostate tumors is at least
partially due to in situ Treg induction, but this requires further study. The increase in
indoleamine-2,3-dioxygenase indicates that advanced prostate tumors may be directly
capable of downregulating the activity of tumor infiltrating lymphocytes. In addition,
indoleamine-2,3-dioxygenase has a role in the generation of inducible Treg (Chen et al.,
2008; Chung et al., 2009) and in preventing their further conversion in to
proinflammatory Th17 cells (Sharma et al., 2009). This is consistent with the observation
that there was no change in the amount of IL-17 in the prostate tumors of older mice
versus those of younger mice (Figure 2.9b). These data suggest that increased expression
of immunosuppressive molecules involved in the induction and maintenance of inducible
Treg is an important event in the establishment of an immunosuppressive prostate tumor
microenvironment in TRAMP mice. It is not clear from these data whether the tumor
cells upregulate expression of these molecules themselves, or whether they are produced
by infiltrating immune cells. Further studies will be required to establish this. No change
in arginase 2 expression was observed in the prostate tumors of older compared to
younger mice, suggesting that the loss of vaccine efficacy at this stage of prostate tumor
80
development is not a result of loss of T cell function due to decreased availability of
arginine.
Overall, the data indicate that therapeutic vaccination against prostate TAA confers
superior protection on TRAMP mice in terms of survival when they are vaccinated at the
earliest possible stage of prostate cancer development. These data are consistent with
another recent study, in which the authors showed that vaccination against α-lactalbumin,
a protein normally only expressed in lactating breast tissue but strongly upregulated by
breast cancer cells, yielded outstanding long term protection against the autochthonous
development of breast cancer in a transgenic mouse model of breast cancer (Jaini et al.).
In addition, this study has identified multiple potential mechanisms of prostate tumor
mediated immunosuppression that may be responsible for the reduction in efficacy of
therapeutic prostate cancer immunotherapy at the later stages of disease progression.
Future Clinical Trials of Therapeutic Cancer Vaccines
The data presented here have significant implications for the design and execution of
clinical trials of therapeutic prostate cancer vaccines. The animal studies described above
demonstrate very aptly the challenges facing tumor immunotherapy researchers:
Excellent cancer vaccines have been developed that have been demonstrated to work
most effectively in the preventive setting, but which can only be tested in clinical trials in
gravely ill patients with advanced disease. These individuals are the very same
demographic in which the preclinical studies suggest cancer vaccines will be least
81
effective. From this perspective it seems illogical, but there are a number of good reasons
why this patient cohort is chosen for clinical trials of cancer vaccines. The first is ethical;
the patients recruited to these trials have failed all other therapies. Since they would no
longer receive anything other than palliative treatment, it is ethically acceptable to assign
some of them to the active arm of the clinical trial and the remainder to a placebo arm.
Those who are given the placebo continue to receive the standard of care, whilst in a
successful trial those in the active arm might gain a few extra months as in the sipuleucel-
T trials. The second reason this cohort is selected for clinical trials of cancer
immunotherapies is cost. By selecting patients with more advanced disease, shorter
clinical trials can be conducted using events such as death or time to progression as the
primary endpoints of the study. Though this approach has served the oncology research
community well for novel chemotherapeutic agents – where the overall health of the
patient has less effect on the efficacy of the therapeutic agent – the evidence is mounting
that the approach is simply impractical for the assessment of new cancer
immunotherapies.
As a result of the ethical and financial considerations discussed above, to date there have
been no clinical trials of cancer vaccines targeting autoantigens in the prophylactic
setting. However, there is a precedent for large scale, long term clinical trials aimed at the
prevention of cancer, as opposed to its cure. Several chemoprevention trials have been
carried out or are in progress in patients at risk for developing breast cancer or prostate
cancer. For example, there is currently a randomized, placebo-controlled, double-blind
82
clinical trial to examine the effect of administering oral anastrozole in women at
increased risk of developing breast cancer (NCT00078832). Anastrozole is an aromatase
inhibitor that prevents the synthesis of estrogen, thereby limiting the growth of estrogen-
dependent breast cancer cells (Howell et al., 2005). An estimated total of 6000 women
between 40-70 years of age will be recruited and randomized into two arms, one of which
will receive anastrozole daily for five years and one of which will receive a placebo.
Subjects in both arms will be followed until the primary outcome measure (the
development of histologically confirmed breast cancer, including ductal carcinoman in
situ, DCIS) is reached, with an estimated median follow up of five years. The secondary
outcome measure is breast cancer mortality, with an estimated median follow-up of 10
years. Recruitment for this trial started in 2003 and it is ongoing.
In the case of prostate cancer, there have been several large scale, long term
interventional chemoprevention trials (Thompson et al., 2005). One of these, the Prostate
Cancer Prevention Trial was a randomized, placebo-controlled, double-blind study with
the aim of determining the effect of daily finasteride treatment on the prevalence of
prostate cancer over seven years (Thompson et al., 2003). A total of 18,882 men aged 55
years or older were randomized into two arms, one of which received finasteride daily for
seven years, and the other of which received a placebo. At the time of recruitment, the
subjects had not been diagnosed with prostate cancer, had normal digital rectal
examination (DRE) results and prostate-specific antigen (PSA) levels lower than 3.0
milligrams per liter. A dramatic 24.8 percent reduction in the prevalence of prostate
83
cancer was observed in the finasteride arm compared to the control arm (p < 0.001).
Initially, this success was tempered by an apparent increase of high grade prostate
cancers (Gleason grade 7, 8, 9 or 10) in the finasteride arm, but this was later
demonstrated to be incorrect and that high grade prostate cancers were also less prevalent
in the finasteride treated subjects (Thompson et al., 2009). Though the PCPT was
expensive, costing an estimated $73 million, it reached its primary objective of
demonstrating that daily finasteride treatment can reduce the prevalence of prostate
cancer in men. Indeed, the PCPT was so successful in reaching its primary objective that
it was terminated 15 months early. In addition, it continues to yield data indicating that
finasteride can significantly reduce the risk of high-grade prostatic intraepithelial
neoplasia (HGPIN, a precancerous prostate lesion), reduce the risk of radical
prostatectomy or radiotherapy, reduce the risk of benign prostate hyperplasia (BPH)
related complications and progression, improve the sensitivity and specificity of DRE and
PSA screening and improve the odds of successful diagnosis if prostate cancer is present.
Considering the proven feasibility of long term, large scale preventive cancer clinical
trials and the increasing evidence that cancer vaccines are likely to be far more successful
in the prophylactic setting, the tumor immunotherapy field has an opportunity to adopt a
new paradigm regarding how it assesses the efficacy of cancer vaccines. Rather than
continue to test cancer immunotherapy strategies in end-stage patients in whom there is
little chance of a successful outcome, the field can and should begin studies of cancer
vaccines in healthy subjects who are at increased risk of cancer. Current and proposed
84
timelines for clinical trials of cancer vaccines targeting prostate cancer are presented in
Figure 2.11. The current timeline involves clinical trials of cancer vaccines in men with
metastatic prostate cancer that is resistant to immunotherapy. This is contrasted with the
proposed timeline of randomized controlled clinical trials designed to test the hypothesis
that therapeutic prostate cancer vaccines used in the prophylactic setting can improve
time to progression and mortality in immunocompetent men with precancerous HGPIN
lesions.
85
Figure 2.11: Schematic representation of prostate cancer immunotherapy trial
timelines. The timelines of screening, diagnostic, therapeutic and disease progression
events are compared between the current paradigm of therapeutic prostate cancer
immunotherapy trials and the proposed structure of studies designed to test the efficacy
of prophylactic cancer vaccination. The current timeline shows the structure of cancer
vaccine clinical trials performed to date in men with metastatic prostate cancer that is
resistant to immunotherapy. It is contrasted with the proposed timeline of randomized,
controlled clinical trials of cancer vaccines in the prophylactic setting. These would test
the hypothesis that cancer immunotherapies administered prophylactically can increase
the time to progression to adenocarcinoma in immunocompetent men who have
precancerous PIN lesions. Standard prostate cancer therapies would be administered upon
progression to adenocarcinoma; precisely when this occurs will depend on the efficacy of
the prophylactic cancer vaccination strategy in delaying time to progression. This figure
was originally published in one of the author’s reviews (Gray et al.).
Figure 2.11 continued
86
87
There are many challenges facing the adoption of this approach. Not least of these is the
high cost incurred by the nature of these trials. They will require very large numbers of
subjects, all of whom will need to be followed for many years. These individuals must
undergo screening prior to the trials, and undergo comprehensive followup at regular
intervals. In the case of breast cancer, this may involve mammogram plus MRI screening
as is currently recommended for women at increased risk for breast cancer (Saslow et al.,
2007). For prostate cancer studies, DRE and PSA level screening will be necessary. In
both cases, biopsy will be required when indicated to confirm progression to cancer, and
in the case of prostate cancer trials an optional end-of-study biopsy should be requested
of all subjects regardless of progression. Given that the subjects taking part in the trials
are healthy upon enrollment, it will take many years for significant numbers to reach the
primary endpoint (time to progression) and even longer to reach the secondary endpoint
(mortality). As a result of the large numbers of subjects and the very lengthy and detailed
followup that is required, large scale clinical trials of prophylactic cancer vaccines will be
expensive. Another challenge is that the only currently FDA approved cancer vaccine –
sipuleucel-T – is unsuitable for use in an intervention trial. It is prohibitively expensive
for use in a clinical trial involving thousands of subjects. In addition, it is not feasible to
require healthy men to undergo leukapheresis procedures for something that may not
benefit them. In the case of the cancer chemoprevention trials discussed above, the agents
in question – anastrozole and finasteride – were previously approved for use in women
with breast cancer after surgery and in men with benign prostate hyperplasia and male
pattern baldness, respectively. The chemoprevention trials aimed to establish new
88
indications for these drugs. The fact that there are no suitable approved cancer vaccines
to trial for prophylactic use, and that suitable cancer vaccines are not likely to be
approved unless they are trialed in the preventive setting represents something of a
Catch-22 situation to the field. It is possible that a suitable therapeutic cancer vaccine will
be approved in the relatively near future that can then be trialed in the preventive setting.
Recently, a successful phase 2 clinical trial indicated that PROSTVAC-VF may represent
a candidate for this (Kantoff et al.). PROSTVAC-VF is an off-the-shelf vaccine
consisting of viral vectors that can be used in any individual. Thus, it is a far more
feasible candidate for use in a large scale preventive trial than sipuleucel-T, which must
be tailor made for each subject. However, approval of PROSTVAC-VF is still years
away at best, meaning that the results of additional clinical trials of this vaccine in the
preventive setting will not be available for decades.
The fundamental question that faces the tumor immunotherapy field now is this: Given
the lack of an approved cancer vaccine that might be retasked for prophylactic use, do the
putative risks of testing a currently unapproved cancer vaccine in the preventive setting
outweigh the potential benefits of successful prophylactic cancer immunotherapy? The
biggest potential risk associated with cancer immunotherapy stems from the fact that self
antigens (autoantigens) that are expressed on normal healthy cells in addition to cancer
cells are almost invariably targeted. Thus, when administered to immune competent
subjects, successful induction of an immune response to those autoantigens by
vaccination may result in the autoimmune destruction of any healthy organs that express
89
those autoantigens. The avoidance of harmful autoimmunity must be the top priority of
any cancer immunotherapy trial. Early trials should be carried out targeting tumor-
associated antigens that are only expressed by non-essential organs. For example, Jaini et
al targeted α-lactalbumin, an autoantigen that is overexpressed by breast cancer cells but
is normally only expressed during lactation (Jaini et al.). No inflammation was observed
in the normal mammary tissue of prophylactically vaccinated mice, except when they
were lactating. This study demonstrates that clinical trials of breast cancer vaccines in the
preventive setting should only be carried out in women who, at the time of enrollment,
have no intention of having children in the future.
The prostate is also a non-essential organ. Therefore, it also represents a good candidate
for prophylactic cancer immunotherapy trials that pose minimal risk to the test subjects in
the event of harmful autoimmune reactions. When this group studied the effect of
vaccination against the autoantigen PSCA in TRAMP mice prior to the development of
prostate cancer, there was little evidence of harmful autoimmunity (Garcia-Hernandez
Mde et al., 2008). Of the 20 PSCA vaccinated TRAMP mice, 18 survived to 340 days, at
which point all but two control TRAMP mice had died of prostate cancer. Of the 18
surviving mice, none produced autoantibodies that could react to other tissues that
express PSCA at low levels, including kidney, colon and normal prostate. Only two of
the 18 produced autoantibodies that could detectably bind to PSCA expressed at the
levels found on testis tissue. No evidence of inflammatory responses was found in any of
these organs in PSCA vaccinated TRAMP mice (Garcia-Hernandez Mde et al., 2008).
90
Similar results were observed when TRAMP mice were vaccinated against another
prostate tumor associated autoantigen, six-transmembrane epithelial antigen of the
prostate (STEAP) (Garcia-Hernandez Mde et al., 2007). Despite the lack of harmful
inflammatory responses, a cellular anti-PSCA response was elicited by vaccination which
prevented the outgrowth of tumor cells expressing high levels of PSCA, whilst sparing
healthy prostate tissue expressing normal levels of PSCA (Gray et al., 2009). These data
suggest that in healthy men expressing normal levels of prostate tumor-associated
antigens, harmful autoimmune reactions induced by prophylactic prostate cancer
immunotherapy are unlikely to occur. Care must still be taken however, as the worst-case
scenario is the complete autoimmune destruction of the prostate, whereupon the patient
would be rendered infertile. As with studies involving prophylactic breast cancer
vaccination, any initial safety trials of preventive prostate cancer immunotherapy that are
proposed should be limited to individuals who do not wish to have further children, and
who could tolerate the potential loss of prostate function. It should be noted that even if
the prostate is eradicated by a harmful autoimmune response, the subject involved would
be no worse off than he would be after radical prostatectomy. If prophylactic prostate
cancer trials are carried out in men who are at increased risk of progressing to prostate
cancer, and thus are more likely to have to undergo radical prostatectomy anyway, the
subjects involved might be more likely to accept of the low risk of autoimmune prostate
destruction. Indeed, in this eventuality they would probably not suffer many of the
significant side effects associated with radical prostatectomy due to collateral damage
during this procedure, due to the extraordinary specificity of the immune system. Overall,
91
with careful consideration of the antigens targeted and study cohort selected, it is very
likely that harmful autoimmunity can be avoided during clinical trials of prophylactic
cancer vaccines.
Conclusion
The data presented here and by others (Jaini et al.) have demonstrated that prophylactic
vaccination targeting autoantigens can be a powerful method of preventing cancer
development. This work has highlighted the painful fact that eliciting therapeutic immune
responses to established tumors is extraordinarily difficult, and of limited benefit even
when successful. Tumor- and treatment-mediated immune suppression in cancer patients
may be avoided by vaccinating them early in disease, before other treatments are
dispensed and prior to the establishment of systemic immune failure by the tumor.
Clinical trials taking this preventive approach will be expensive and are potentially risky
even with careful planning. However, they may prove to be the more economical and
ethical option given that of the clinical trials conducted under the current paradigm, only
one has yielded an approved therapeutic agent with any potential public health benefit or
return on investment.
92
CHAPTER 3: ABROGATING REGULATORY T CELL FUNCTION TO
ENHANCE THERAPEUTIC PROSTATE CANCER VACCINE EFFICACY
INTRODUCTION
Immune Regulation in Health and Disease
A functional immune system that is capable of controlling and eradicating the myriad
pathogens - including bacteria, viruses, fungi and parasites - that cause infectious disease
is vital for human survival. A minority of the dizzying array of molecular structures
expressed by pathogens has remained largely unchanged in the course of the evolution of
these organisms. These structures are termed pathogen associated molecular patterns
(PAMPs), and the human immune system has evolved an innate arm with which to
quickly target and eliminate microbes expressing them. However, bacteria and viruses are
capable of extremely rapid evolution, allowing them to evade innate immunity. Generally
speaking, there is no way for the immune system to predict what pathogenic threats it will
face during the course of life. By necessity, the human immune system must be able to
recognize and respond to novel molecular targets expressed by pathogens. As a result, the
adaptive arm of the immune system has evolved. Adaptive immunity is one of the most
complex biological systems known, and our understanding of it is far from complete.
Briefly, adaptive immunity is principally mediated by B cells, which produce antibodies,
and T cells, which are responsible for maximizing the efficacy of the immune response
(T helper cells) and for directly killing infected and/or cancerous cells (cytotoxic T cells).
93
The absolute necessity for a functional adaptive immune system aptly demonstrated by
severe combined immunodeficiency (SCID), in which patients have one or more rare
genetic mutations that result in the virtual absence of B and T cells. SCID can be
corrected via a transplant of bone marrow stem cell from a healthy donor, provided that
the transplant occurs in the first 3.5 months of the recipient’s life. However, if this
treatment is not available or if it fails, the patient will be required to live in an aseptic
environment to ensure survival. Another instance in which compromised adaptive
immune function leads to death is observed in untreated patients with acquired immune
deficiency syndrome (AIDS), in which CD4 T cell function is lost. Prior to the
introduction of HAART (highly active antiretroviral therapy), death due to AIDS was a
virtual certainty in patients infected with the human immunodeficiency virus (HIV).
Despite the crucial function of the human immune system, it is a double-edged sword. T
cells recognize and bind to specific antigens via the T cell receptor (TCR). Each T cell
clone recognizes a single epitope when it is presented by the major histocompatibility
complex (MHC) expressed by mammalian cells. Since the adaptive immune system must
theoretically be able to respond to any antigen, it must be able to make a potentially
infinite number of TCR molecules. By definition, some of these TCR molecules will
recognize and bind to epitopes of self-antigens. When these autoreactive T cells are
allowed to respond to self antigens, autoimmune diseases result. A wide spectrum of
autoimmune diseases exist, the symptoms of which depend upon the cell type in the body
that is under attack. For example, autoimmune reactions to the Schwann cells that form
94
the myelin sheaths of neurons result in multiple sclerosis. In mammals, the immune
system has evolved to balance the threat of autoimmunity with the benefit of being able
to respond to the widest possible of pathogens. The first means by which this is achieved
is by thymic selection, resulting in central tolerance. All T cells initially develop in the
thymus. During this phase, they undergo two rounds of selection based on the affinity of
their specific TCR with particular targets. First, T cells undergo positive selection. In this
process, T cells that are incapable of binding to any MHC/self antigen epitope complex
expressed in the thymus will die due to a lack of stimulation. Such T cells, if released into
the periphery, would be essentially useless since they could never interact with epitopes
presented by MHC. Second, T cells undergo negative selection. Within the thymus, only
epitopes derived from self antigens are presented to the nascent T cells. If a T cell binds
with high affinity to an MHC presenting a self-epitope in the thymus, it is deleted. If such
a cell were to be released into the periphery it could potentially cause an autoimmune
disease. As a result of the combination of thymic positive and negative selection, in
principle the only T cells that are released into the periphery are those that can bind to an
MHC reasonably well (and may be able to bind significantly more strongly if that MHC
undergoes a particular conformational change by presenting a specific epitope peptide)
but which will not bind strongly to MHC presenting any of the self epitopes that were
present in the thymus.
Despite the effectiveness of central tolerance, potentially autoreactive T cells escape into
and are detectable within the circulatory and lymphatic systems of all humans. In order to
95
prevent autoimmunity, a second means of controlling their autoimmune responses is
required – peripheral tolerance. Though there are several mechanisms by which
peripheral tolerance is maintained, key players in this process are regulatory T cells
(Treg) (Curiel, 2008). Though it is becoming apparent that there are many subtypes of
Tregs that have a variety of phenotypes, functions and localizations (Campbell and
Koch), they can broadly be divided into natural Tregs (nTreg) and inducible or adaptive
Tregs (iTreg).
Natural Regulatory T Cells
Natural Tregs develop in the thymus and are functionally suppressive as soon as they
enter the periphery. They are the primary means by which peripheral self-tolerance is
maintained, a function that they carry out in an antigen-independent manner (Curiel,
2008). Expression of the transcription factor forkhead-box P3 (FOXP3) has been
accepted as being essential for natural thymic regulatory T cell (nTreg) development and
function (Fontenot et al., 2003). Both mice and humans with Foxp3/FOXP3 loss of
function mutations suffer catastrophic loss of Treg activity in the periphery. This causes
the lethal autoimmune disorders scurfy and IPEX (immune dysregulation,
polyendocrinopathy, enteropathy, X-linked) syndrome in mice and humans, respectively
(Bennett et al., 2001; Brunkow et al., 2001). Ectopic expression of Foxp3 in CD4
+
CD25
-
cells is sufficient for them to adopt a CD4
+
CD25
+
Treg phenotype (Fontenot et al., 2003).
Thymically-derived CD4
+
CD25
+
FOXP3
+
naturally occurring Tregs are currently the best
characterized class of suppressor T cell. The nTreg population accounts for
96
approximately 5-10% of all peripheral CD4
+
T cells. Interestingly, naturally occurring
CD4
+
CD25
+
FOXP3
+
Tregs can induce naïve CD4
+
CD25
-
FOXP3
-
to adopt a
CD4
+
CD25
+
FOXP3
+
suppressive phenotype, i.e. become iTregs (Zheng et al., 2004).
Inducible Regulatory T Cells
Inducible Treg cells develop in the periphery in response to stimulation by self antigens
and the presence of TGFβ and IL-2 (Flavell et al.). They suppress the responses of both
naïve and memory T cells by producing the immunosuppressive cytokines IL-10 and
TGFβ, thereby inducing anergy in these target cells. Several tumors – including prostate
tumors as demonstrated in Chapter Two – produce TGFβ themselves or induce
neighboring cells to do so, suggesting that iTreg cells may be actively involved in the
suppression of an immune response mounted against a self antigen as part of a tumor
immunotherapy protocol.
Tregs in Human Cancer and Efforts to Attenuate Their Function
As mechanisms of tumor-mediated immune suppression have been elucidated over recent
years, a determined research drive has been concentrated on eliminating or inhibiting
them in order to enhance the efficacy of therapeutic cancer vaccines. FOXP3-expressing
Tregs have been identified as potentially having a role in several human cancers (Lizee et
al., 2006). Naturally occurring CD4
+
CD25
+
Tregs have been the focus of particularly
intense study in this regard, though they do not always seem to be involved in mediating
97
the suppression of immune responses elicited by cancer vaccines (Zimmermann et al.,
2007). Nevertheless, the accumulation of larger numbers of Tregs has been associated
with poorer clinical outcomes in both breast (Merlo et al., 2009) and ovarian cancer
(Curiel et al., 2004). Natural Tregs have been hypothesized as central players in tumor-
mediated immune suppression in many cancers and several methods to deplete them or
limit their suppressive activity have been developed (Curiel, 2007). For example,
inhibition of CD4
+
CD25
+
Tregs by cyclophosphamide pretreatment before immunization
with a vaccine directed against HER-2/neu in mice allowed the activation of latent
antigen-specific CD8
+
T cells that were capable of mounting an anti-tumor response
(Ercolini et al., 2005). Efforts to abrogate natural Treg activity to boost vaccine efficacy
have also included depletion via treatment with a monoclonal anti-CD25 antibody (Viehl
et al., 2006b) and administration of denileukin diftitox (Ontak) (Dannull et al., 2005;
Litzinger et al., 2007). Ontak is an engineered fusion protein consisting of interleukin-2
fused to diphtheria toxin. Tregs are heavily dependent on IL-2 signalling and highly
express the IL-2 receptor, the alpha chain of which is CD25. Thus, it was hypothesized
that administration of either anti-CD25 antibodies or Ontak would deplete Tregs from the
periphery, improving anti-tumor immune responses elicited by therapeutic vaccines.
Improved anti-tumor immune responses have been observed when these Tregs have been
depleted from the T cell pool using an anti-CD25 monoclonal antibody in several animal
models of human cancer (Comes et al., 2006; Dannull et al., 2005; El Andaloussi et al.,
2006; Nagai et al., 2004; Prasad et al., 2005; Tanaka et al., 2002; Viehl et al., 2006a).
However, Treg depletion in these cases occurred prior to tumor challenge, raising the
98
question of whether this strategy would be beneficial in humans with established tumors.
Of greatest concern is that CD25 is expressed on the surface of recently activated CD4
+
and CD8
+
effector cells, the concomitant depletion of which may actually hinder
antitumor vaccine efficacy rather than improve it.
Transgenic Mouse Models with Altered Treg Function
To investigate the role of Tregs in the anti-tumor immune response, it is important to
develop a model system in which Treg activity is modulated prior to tumorigenesis in
order to prevent the development of the intratumoral suppressive milieu. This must be
achieved without interfering with the function of effector T cells that will mediate the
antitumor immune response elicited by therapeutic vaccination. The development and
characterization of such a system is a primary focus of this chapter. Two different
transgenic mouse models have been used in this effort.
PEST-enriched Phosphatase Knockout and Gain of Function Mice
The strength of TCR signaling in the thymus has a critical role in the development of
regulatory T cells. During thymic negative selection, T cells with high affinity TCRs are
normally deleted to prevent autoimmunity (Apostolou et al., 2002). Natural regulatory T
cells develop from nascent T cells that have TCRs with high affinities that are just below
the upper limit to avoid negative selection. Lymphoid tyrosine phosphatase (LYP) and its
murine homolog PEST-enriched phosphatase (PEP) are critical negative regulators of
99
TCR signaling (Apostolou et al., 2002). A collaborator at USC was the first to identify a
single-nucleotide polymorphism in the gene encoding LYP (PTPN22) that results in
arginine 620 (R620) being changed to tryptophan (W620) in the coding region. The
R620W polymorphism produces a gain-of-function version of the phosphatase that
predisposes individuals carrying it to autoimmunity (Vang et al., 2005). It was
hypothesized that the increased activity of the gain-of-function LYP/PEP proteins leads
to weaker TCR signaling and therefore insufficient generation and/or activity of Tregs. In
contrast, PEP knockout (KO) mice [B6.Cg-Ptpn8
tm1Gne
] (Hasegawa et al., 2004) have
increased numbers of peripheral Tregs (Andrew Chan, Genentech, CA, personal
communication). Mice that over-express LYP-W620 on a PEP KO background (PEP
KO(LYP-W620) in the thymus were developed in order to determine whether they
display reduced numbers of Tregs in the periphery and are thus more susceptible to
developing autoimmunity. The availability of PEP gain-of-function mutant mice provided
an opportunity to study the effect decreased numbers of Tregs on the efficacy of
therapeutic prostate cancer vaccines.
Depletion of Treg (DEREG) Mice
The depletion of Treg (DEREG) mouse a transgenic animal model in which a fusion
protein consisting of the diphtheria toxin receptor (DTR) and green fluorescent protein is
expressed under the control of the promoter of the Treg-specific transcription factor
FOXP3 (Lahl et al., 2007). In these animals, FOXP3
+
Treg develop normally, but
universally express the DTR at their cell surface. The advantage of this is that FOXP3
+
100
Treg in DEREG mice are highly sensitive to diphtheria toxin, and thus can be specifically
depleted at any time by the administration of a low dose of the DT. Since no other tissue
or cell type expresses the DTR in DEREG mice, depletion is Treg-specific. Provided that
a sufficiently low dose of DT is used, Treg depletion can be achieved in the absence of
toxicity in the animals involved. It has already been demonstrated that Treg depletion in
the DEREG mouse can inhibit carcinogenesis (Teng et al.). Thus, the DEREG model is
ideal for determining the role of regulatory T cells in responses to therapeutic prostate
cancer vaccines.
101
Hypothesis and Aims
Hypothesis: Regulatory T cells suppress the antitumor immune response elicited by
therapeutic prostate cancer vaccines, and their depletion will improve vaccine efficacy.
Objective: Develop strains of mice which spontaneously develop prostate cancer and
which have altered numbers of regulatory T cells in order to assess their effects on the
anti-tumor response induced by vaccination against a prostate cancer-associated antigen.
Specific Aims:
1.) Generate mice that spontaneously develop prostate cancer and which either
release fewer Tregs from the Thymus or in which Tregs can be specifically
depleted.
2.) Determine whether the efficacy of vaccination against mPSCA is improved by
depleting Tregs immediately prior to immunization.
102
MATERIALS AND METHODS
Mice
TRAMP mice (Greenberg et al., 1995) on the C57BL/6 background were bred at USC.
PEP KO(LYP-R620W) mice were kindly donated by Dr. Nunzio Bottini (La Jolla
Allergy and Immunology Institute, La Jolla, CA). DEREG mice on the C57BL/6
background were kindly donated by Dr. Uli von Andrian (Harvard University, Boston,
MA) with the permission of their creator, Dr. Timothy Sparwasser (TWINCORE,
Hanover, Germany). TRAMP DEREG mice were bred at USC by crossing male TRAMP
mice with female DEREG mice, then backcrossing until mice with the desired genotype
(homozygous for the TRAMP transgene and hemizygous for the DEREG transgene) were
obtained. Research was conducted in compliance with the Institutional Animal Care and
Use Committee guidelines.
Plasmid DNA constructs and VRP generation
For gene gun-mediated DNA priming, a plasmid DNA construct encoding mouse PSCA
was used as has previously been described (Garcia-Hernandez Mde et al., 2008). A DNA
fragment encoding mouse PSCA was obtained from pCR-mPSCA (Yang et al., 2001).
For the generation of PSCA-expressing plasmid (pcDNA3-PSCA), mPSCA DNA was
amplified using two specific primers. Primer sequences were designed to include either a
HindIII or a BamHI site: 5’-CCCAAGCTTATGACTCACAGG-3’ and 5’-
TGTGAGGAGTGCACA-3’. Amplification was performed for 30s at 94°C, 30s at 55°C,
30s at 72°C. An additional extension step was performed for 10 min at 72°C. The PCR
103
product was then cloned into the pcDNA3 expression vector (Invitrogen, Carlsbad, CA).
DNA sequencing was performed to confirm that the pcDNA3-PSCA construct had the
desired sequence and open reading frame. pcDNA3-PSCA or empty vector (pcDNA3)
were transformed into TOP10 competent E. coli (Stratagene, La Jolla, CA). Plasmid
DNA copies were amplified in liquid culture and purified using a plasmid maxi kit
(Qiagen Sciences, MD). The DNA that was used to coat gold particles in the making of
gene gun bullets for vaccination had a minimum OD
260
:OD
280
ratio =1.9. For boosting,
VRP were custom made and supplied on demand by Alphavax, Inc (Research Triangle
Park, NC). For VRP production, mPSCA DNA was amplified by using specific primers:
5'-GACTCACAGGACTACTACGTGGGCAAGAA-3' and 5’-TTAATTAAGGCGAGC-
TCCTACAACC-3. A Pac-1 site was added to the antisense primer. Amplification was
performed for 30s at 94°C, 30s at 58°C, 30s at 72°C. An additional extension step was
performed for 10 min at 72°C. The PCR product was digested with the appropriate 3’
enzyme and ligated into pERK (AlphaVax replicon vector plasmid) that had been
digested with EcoRV and the matching 3’ enzyme. mPSCA presence in the construct was
verified by DNA sequencing. VRP were made using a two-helper system (Pushko et al.,
1997).
Treg depletion in TRAMP DEREG mice
FOXP3
+
Tregs were depleted from TRAMP DEREG mice based on their expression of a
fusion protein consisting of the DTR and GFP. Depletion was achieved by intraperitoneal
injection of 1 μg DT (Sigma) in 50 μl sterile PBS per mouse per day. The optimal
104
number of consecutive DT treatments required to mediate good depletion of Tregs
without toxicity caused by excessive DT was empirically determined to be two treatments
twenty four hours apart. Lethal toxicity was caused by DT treatment regimens of four and
six daily doses. This was the case in TRAMP DEREG mice and in their TRAMP
littermate controls, indicating that the lethality was caused by direct DT effects and not
by effects associated with the specific depletion of Tregs. Maximal depletion of Tregs
occurred three days after the first DT injection, consistent with the literature (Lahl and
Sparwasser), thus immunizations were carried out at this point.
Immunization
Male TRAMP and TRAMP DEREG mice (previously treated with DT as described
above) were anesthetized by intraperitoneal injection of 80-100 mg/kg ketamine (Phoenix
Pharmaceutical Inc, St Joseph, MO) and 10 mg/kg xylazine (Phoenix). DNA-gold
particles were delivered to a shaved area on the abdomen using a helium-driven gene gun
(BioRad) with a discharge pressure of 300 – 400 psi. Each mouse received 2 μg of
mPSCA cDNA vaccine. Fourteen days after gene gun vaccination mice were
subcutaneously boosted 1 cm from the tail base with 10
6
infectious units (IU) mPSCA-
VRP. As control groups, TRAMP and TRAMP DEREG mice were vaccinated with
pcDNA3 and boosted with 10
6
IU GFP-VRP. Fourteen days after boosting, mice were
euthanized in order to carry out assays as required.
105
Peptide synthesis
The peptide mPSCA
(83-91)
(amino acid sequence NITCCYSDL) was synthesized
according to standard methods used at the Norris Comprehensive Cancer Center facility
at the University of Southern California. Peptide was dissolved at 50 mg/ml in DMSO
(Sigma) and stored at -80°C until further use.
Cell culture medium
Splenocytes and lymphocytes were stored and cultured in IMDM supplemented with
10% fetal calf serum (Gemini) and 50 μg/ml kanamycin (Sigma). Cells were cultured at
37
o
C, 5% CO
2
and 100% relative humidity.
Isolation of splenocytes
Mice were euthanized and dissected using aseptic techniques in a biosafety hood. The
spleen was isolated from each mouse and immediately stored in a sterile 15 ml conical
tube (Falcon) in 3 ml complete IMDM. A 70 μm nylon cell strainer (BD Falcon, ref. #
352350) was placed in a 50 ml conical tube (BD Falcon) and the spleen and media
poured into it. The spleen was ground through the strainer with the rubber end of a 5ml
syringe plunger (Becton Dickinson), wiping any tissue/cells stuck to the end of the
plunger being wiped on the inside edge of the strainer. The plunger was discarded and the
strainer rinsed with 10 ml complete IMDM. The cells were spun down at 300 g for 5
minutes in a tabletop centrifuge. The supernatant was discarded and the cells resuspended
in 1 ml Red Blood Cell Lysing Buffer (Sigma). The cells were incubated for 1 minute at
106
room temperature, then the tube was topped up with sterile PBS and spun at 300 g for 5
minutes in a tabletop centrifuge. The supernatant was discarded, and the cells suspended
in 25 ml PBS and run through a new 70 μl cell strainer into a new 50 ml conical tube in
order to remove clumps of red cell ghosts. After the final spin (300 g for 5 minutes), the
remaining splenocytes were resuspended in 10 ml complete IMDM for counting.
Isolation of lymphocytes
Mice were euthanized and dissected using aseptic techniques in a biosafety hood. The
tumor draining lymph nodes were isolated from each mouse and immediately stored in a
sterile 15 ml conical tube (Falcon) in 3 ml complete IMDM. A 70 μm nylon cell strainer
(BD Falcon, ref. # 352350) was placed in a sterile 60 mm Petri dish (Falcon) and the
lymph nodes and media poured into it. The lymph nodes were ground through the strainer
with the plastic end of a 5 ml syringe plunger (Becton Dickinson). The plunger end and
the strainer were lifted out of the Petri dish and rinsed with 5 ml complete IMDM. The
suspended cells in the Petri dish were transferred to a sterile 15 ml conical tube using a
10 ml serological pipette. The Petri dish was then held at a 45 degree angle and rinsed
again with 5 ml of fresh complete IMDM to collect any remaining cells which were
added to the same 15 ml conical tube. The cells were spun down at 300 g for 5 minutes in
a tabletop centrifuge. The supernatant was discarded, and the cells washed in 10 ml PBS.
After the final spin (300 g for 5 minutes), the lymphocytes were resuspended in 10 ml
complete IMDM for counting.
107
Isolation of prostate/prostate tumor tissue
Mice were euthanized and dissected under aseptic conditions. The entire genitourinary
tract was removed from the abdominal cavity of each mouse, including the
prostate/prostate tumor, seminal vesicles, penis, urinary bladder and testes. All other
tissue was dissected away from the prostate/prostate tumor using sterile forceps and
scalpels. The total mass of the prostate/prostate tumor tissue was determined using a
digital scale that had previously been wiped with 70% ethanol. Tumor tissue was then
snap frozen in glass scintillation vials using liquid nitrogen, and stored at -80
o
C for
further processing.
Identification of mPSCA-specific CD8
+
T cells
Splenocytes were isolated as described above and counted. Cells were plated out in sterile
24-well tissue culture plates (SUPPLIER) at 5x10
6
cells per well, six wells per animal in
1 ml complete IMDM. To all wells, 0.5 ml complete IMDM containing IL-2 at 80 IU/ml
were added. In addition, to half of the wells for each mouse, 0.5 ml complete IMDM
containing 4 μg/ml mPSCA
(83-91)
was added. To the remainder, 0.5 ml complete IMDM
containing the same amount of DMSO used as a vector for the mPSCA peptide was
added. This gave final concentrations of 20 IU/ml IL-20 and 1 μg/ml peptide, where
applicable. Cells were cultured in an incubator for 24 hours at 37
o
C, 5% CO
2
. In the last
six hours of culture, 5 μl Brefeldin A was added per well in order to arrest the Golgi
apparatus of the cells. Cells were then harvested, washed twice in complete IMDM and
live cells counted via trypan blue exclusion using a haemocytometer. A total of 1x10
6
108
cells per well were stained for CD3, CD4, CD8 and IFNγ as described below. The total
number of CD8
+
IFNγ
+
cells per million splenocytes was calculated using the formula (n
= (CD8
+
IFNγ
+
events/total events)*1,000,000).
Cell staining and Fluorescence Activated Cell Scanning
Splenocytes, lymphocytes and TIL isolated as described above were counted using a
hemocytometer. For each sample to be stained, 10
6
cells were transferred to two wells of
a 96-well V-bottom plate (VWR) in a maximum volume of 200 μl media/PBS. The plate
was then spun at 300 g for 5 minutes in a floor centrifuge ((Beckman Coulter). The
supernatant removed by flicking into a sink and the plate gently dabbed dry using paper
towels. The cells were washed with 150 μl per well ice cold FACS buffer, spun again, the
supernatant removed and the plate dabbed dry. This was repeated twice. The cells were
then resuspended in 50 μl per well ice cold FACS buffer containing 1 μl FcBlock
antibody (RD Systems) and incubated for 15 minutes on ice. Each sample was then
incubated on ice for 30 minutes in the dark with either 50 μl ice cold FACS buffer
containing a cocktail of cell surface marker antibodies (1 μl FITC-conjugated anti-mouse
CD3 antibody (Becton Dickinson), 1 μl PE-Cy5-conjugated anti-mouse CD4 antibody
(Becton Dickinson) and 1 μl PE-Cy7-conjugated anti-mouse CD8a antibody) or 50 μl ice
cold FACS buffer containing a cocktail of the appropriate isotype controls for the above
antibodies (1 μl FITC-conjugated hamster IgG1 antibody (Becton Dickinson), 1 μl PE-
Cy5-conjugated rat IgG2a antibody (Becton Dickinson) and 1 μl PE-Cy7-conjugated rat
IgG1 antibody). The cells were washed three times with 150 μl per well ice cold FACS
109
buffer as described above. The cell pellets were then resuspended in 100 μl per well
fixation/permeablization buffer (eBioscience) and incubated overnight in the dark at 4
o
C.
The plate was then spun at 300 g for 5 minutes in a floor centrifuge. The supernatant
removed by flicking into a sink and the plate gently dabbed dry using paper towels. The
cells were washed twice with 100 μl per well ice cold 1X permeablization buffer
(eBioscience) as described above. The cell pellets were then resuspended in 50 μl per
well 1X permeablization buffer containing 1 μl FcBlock and incubated for 15 minutes on
ice in the dark. For identification of FOXP3
+
regulatory T cells, each sample was then
incubated on ice for 30 minutes in the dark with either 50 μl ice cold permeablization
buffer containing either 1 μl PE-conjugated anti-mouse FOXP3 antibody (eBioscience) or
1 μl of the appropriate PE conjugated isotype control antibody (Becton Dickinson). For
identification of CD8
+
IFNγ
+
activated T cells, each sample was then incubated on ice for
30 minutes in the dark with either 50 μl ice cold permeablization buffer containing either
1 μl PE-conjugated anti-mouse IFNγ antibody (Becton Dickinson) or 1 μl of the
appropriate PE conjugated isotype control antibody (Becton Dickinson). After staining
with PE-conjugated antibodies, each well was washed twice with 100 ul Permeablization
buffer as described above. After the final spin, the cell pellets were resuspended in 100 μl
ice cold FACS buffer. Cells were analyzed using a Becton Dickinson FC500 flow
cytometer.
Statistical analysis
Flow cytometry data were analyzed by two tailed, independent t tests.
110
RESULTS
To begin investigating the role of Tregs in prostate cancer, PEP KO(LYP-W620) mice
were obtained. As discussed above, these mice harbor a gain-of function variant of LYP,
the human ortholog of the PEP protein. Given that PEP knockout mice have increased
numbers of peripheral Treg, it was hypothesized thatPEP KO(LYP-W620) mice would
have decreased numbers of peripheral Treg. It was intended that the PEP KO(LYP-
W620) mice would be crossed with TRAMP mice, thus generating an animal model that
should spontaneously develop prostate cancer and have reduced numbers of peripheral
Treg. Prior to beginning this time-consuming process, it was deemed wise to check
whether PEP KO(LYP-W620) mice did indeed have reduced numbers of Treg as was
predicted. To determine this, the numbers of Treg in the thymuses and spleens of PEP
KO(LYP-W620) mice and wild type littermate controls were analyzed by flow
cytometry. There was a difference in the number of thymic CD4
+
FOXP3
-
T cells but not
CD4
+
FOXP3
+
Treg in PEP KO(LYP-W620) mice. There was no difference in the
numbers of peripheral CD4
+
FOXP3
-
T cells or CD4
+
FOXP3
+
Treg in the spleens of PEP
KO(LYP-W620) mice compared to those of their wild type littermates (Figure 3.1).
111
Figure 3.1
THY CD4+FoxP3-
THY CD4+FoxP3+
SPL CD4+FoxP3-
SPL CD4+FoxP3+
0
1
2
3
4
5
WT Control
PEP KO(LYP-W620)
Cell Type
Proportion of Cells
(%)
Figure 3.1: There is no difference in the numbers of peripheral Treg in PEP
KO(LYP-W620) mice compared to wild type littermate controls. Splenocytes and
thymocytes isolated from four week old PEP KO(LYP-W620) mice and wild type
littermate controls were were washed and stained with anti-mouse CD8-PE/Cy5 and anti-
mouse CD4-PE/Cy7. Cells were fixed and permeablized overnight, then stained with
anti-mouse FOXP3-PE and analysed by flow cytometry. Events were collected gated on
either live CD4
+
cells (spleens) or single-positive CD4
+
CD8
-
cells (thymuses) and then
further gated on the CD4
+
FOXP3
-
fraction and the CD4
+
FOXP3
+
fraction. THY =
thymus. SPL = spleen.
112
Given that the PEP KO(LYP-W620) did not have the phenotype that was necessary to
test the hypothesis, an alternative research strategy was devised. DEREG mice were
obtained. Female mice hemizygous for the DEREG transgene were crossed with male
TRAMP mice. Female mice from the F1 generation that were demonstrated to be
hemizygous for both the DEREG transgene and the TRAMP transgene (SV40 Large T
antigen driven by the prostate-specific probasin promoter) were crossed with TRAMP
males. In the F2 generation, one half of the females were demonstrated to be hemizygous
for the DEREG transgene and homozygous for the TRAMP transgene. In all subsequent
breeding, these females were crossed with TRAMP males. In the F3 generation and
beyond, all animals were full TRAMP mice, and fifty percent possessed the DEREG
transgene. This was advantageous for the experiments to be carried out, because in a
given litter fifty percent of the males would have the desired TRAMP DEREG (TD)
phenotype (that spontaneously developed prostate cancer and in which Tregs could be
specifically depleted) whilst the remainder would be TRAMP mice. These littermates
make ideal negative controls for the TD mice, since they can be treated with DT in
exactly the same manner but their Treg population would be unaffected. This controls for
the possibility of off-target effects of diphtheria toxin treatment on immunological
responses to vaccination. In order to verify that the desired phenotype had been obtained,
the numbers of FOXP3
+
Tregs in the lymph nodes of these animals was assessed by flow
cytometry after DT treatment (Figure 3.2). As expected, the mean percentage of
CD4
+
FOXP3
+
Tregs was statistically significantly reduced in the lymph nodes of TD
mice (4.4 ± 2.4%) compared to their TRAMP littermate controls (8.7 ± 2.4%) (Figure
113
3.2, Independent t test, p = 0.0047). Importantly, the mean percentage of CD4
+
lymphocytes was unchanged in TD mice (23.9 ± 3.9%) compared to TRAMP littermate
controls (23.5 ± 5.1%) (Figure 3.2, Independent t test, p = 0.8928), indicating that the
dosage of DT used had no deleterious effect on CD4
+
T effector cells that did not express
FOXP3.
114
Figure 3.2
Figure 3.2: CD4+FOXP3+ Tregs are specifically depleted in TD mice but not
TRAMP mice upon diphtheria toxin treatment. Ten week old TD mice and standard
TRAMP littermate controls were treated IP with 1 μg DT daily for six days.
Lymphocytes were isolated two days later and were washed and stained with anti-mouse
CD3-FITC, anti-mouse CD8-PE/Cy5 and anti-mouse CD4-PE/Cy7. Cells were fixed and
permeablised overnight, then stained with anti-mouse FOXP3-PE and analysed by flow
cytometry. Events were collected gated on live, CD3
+
CD8
-
cells and then further gated
on the total CD4
+
FOXP3
-
fraction (CD4
+
) and the CD4
+
FOXP3
+
fraction. The * symbol
indicates p < 0.01.
CD4+ CD4+FOXP3+
0
10
20
30
T R AM P
T D
Cell Type
Proportion of
Lymphocytes (% )
*
115
The central hypothesis of this chapter is that that FOXP3
+
regulatory T cells inhibit the
efficacy of therapeutic prostate cancer vaccines and that their depletion prior to
immunization will enhance vaccine efficacy. In Chapter One, data were presented
showing the presence of functionally suppressive Tregs within the lymph nodes of
TRAMP mice at 8 weeks of age. Therefore, it was hypothesized that depletion of Tregs
in TD mice at this stage of carcinogenesis would enhance the anti-tumor immune
response that was shown to be elicited in TRAMP mice immunized at the same time
point. In order to test this, groups of 8-10 week old TD mice were depleted of Tregs by
treating them with 1 μg DT daily for two consecutive days. It had previously been
determined that this treatment protocol would efficiently deplete Tregs without the
toxicity that was observed when treating the animals for six consecutive days with DT
(data not shown). Treg-depleted TD mice were DNA vaccinated against mPSCA two
days after the last DT injection via helium gene gun. A negative control group of Treg-
depleted TD mice were DNA vaccinated using the empty vector plasmid. As additional
controls, groups of TRAMP littermates (lacking the DEREG transgene) were treated with
DT in the same manner and then DNA vaccinated with 2 μg plasmid encoding mPSCA
or with 2 μg empty plasmid vector. Mice that had been DNA primed against mPSCA
were boosted fourteen days later using 10
6
VRP encoding mPSCA, whilst those that had
received empty vector plasmids were boosted at the same time using 10
6
VRP encoding
GFP. In order to assess whether vaccination against mPSCA has functional anti-tumor
effects in these animals, the mass of prostate/prostate tumor tissue in each animal was
assessed (Figure 3.3). There was a strong trend towards reduced mean prostate/prostate
116
tumor mass in mPSCA vaccinated TRAMP mice (42 ± 12 mg) compared to control
vaccinated TRAMP mice (64 mg ± 19 mg), though this did not quite reach statistical
significance (Figure 3.3, Independent t test, p = 0.0852). However, the mean mass of the
prostate/prostate tumor tissue isolated from mPSCA vaccinated TD mice (73 ± 31 mg)
was statistically significantly lower than that of control vaccinated TD mice (34 ± 11)
(Figure 3.3, Independent t test, p = 0.0157). The rapid inhibition of prostate growth in TD
and TRAMP mice (53% and 34%, respectively) due to mPSCA vaccination is dramatic
but consistent with the spectacular improvement in the survival of TRAMP mice when
they are vaccinated against mPSCA at this time point (Figures 2.1 – 2.3). The
prostate/prostate tumor tissue isolated from control vaccinated animals had tumor lesions
visible to the naked eye, and obviously enlarged seminal vesicles. In contrast, the
prostate/prostate tumor tissue isolated from PSCA vaccinated animals had no such visible
lesions, and seminal vesicles that were the normal size for 12 week old C57BL/6 mice.
Taken together, these data indicate that even at this early stage of carcinogenesis,
depletion of Tregs seems to enhance the inhibition of tumor growth.
117
Figure 3.3
TRAMP Control
TRAMP mPSCA
TD Control
TD mPSCA
0
10
20
30
40
50
60
70
80
90
*
Mean Tumor Mass (mg)
Figure 3.3: mPSCA vaccination inhibits prostate growth in TD and TRAMP mice.
Eight to ten week old TD mice and standard TRAMP littermate controls were treated IP
with 1 μg DT daily for two days. TRAMP mice and Treg-depleted TD mice were
vaccinated three days later by helium-driven gene gun with either 2 ug mPSCA-pcDNA
or 2 ug empty vector and boosted at day 14 with 10
6
IU mPSCA-VRP and 10
6
IU GFP-
VRP, respectively. The mice were euthanized fourteen days after boosting and the entire
genitourinary (GU) tract harvested. The prostate/prostate tumor tissue was dissected
away from the remainder of the GU tract and weighed. Bars represent mean mass of
prostate/prostate tumor tissue ± SEM. The * symbol indicates p < 0.05.
118
To determine whether vaccination against mPSCA was eliciting a specific anti-tumor
immune response, the ability of CD8
+
cytotoxic T cells to produce interferon-gamma
(IFNγ) in response to an mPSCA peptide was assessed. Candidate mPSCA peptides that
could potentially bind to MHC class I molecules H-2D
b
or H-2K
b
had previously been
identified. Two peptides were selected based on the highest score of peptide/MHC class I
half life of dissociation generated using prediction programs (D'Amaro et al., 1995;
Parker et al., 1994). The capacities of these peptides to stabilize MHC class I on RMA-S
cells was also evaluated (Garcia-Hernandez Mde et al., 2008). The peptide mPSCA
83-91
had the highest predicted binding affinity and has since been used in order to quantify
mPSCA-specific responses elicited by vaccination in TRAMP mice (Garcia-Hernandez
Mde et al., 2008). This peptide was loaded onto spleen cells to evaluate antigen-specific
MHC class
I-restricted CD8 T-cell responses in terms of IFNγ production by flow
cytometry. Splenocytes were isolated from mPSCA vaccinated TD (n = 6), control
vaccinated TD (n = 6), mPSCA vaccinated TRAMP (n = 4) and control vaccinated
TRAMP (n = 5) mice. The splenocytes were treated for twenty four hours with mPSCA
83-
91
and IL-2, with Brefeldin-A being added in the last six hours in order to arrest the Golgi
apparatus of the cells, thus allowing detectable levels of IFNγ to build up in the
cytoplasm of activated T cells. The cells were then analyzed by flow cytometry to
determine the number of CD8
+
IFNγ
+
cells per million splenocytes (Figure 3.4). In
TRAMP mice, there was no statistically change in the mean number of CD8
+
IFNγ
+
T
cells per million splenocytes in mPSCA vaccinated mice (86.2 cells per million
splenocytes) compared to control vaccinated mice (65.1 cells per million splenocytes)
119
(Figure 3.5, independent t test, p = 0.8914). In contrast, there was a large increase in the
mean number of CD8
+
IFNγ
+
T cells per million splenocytes in mPSCA vaccinated TD
mice (436.8 cells per million splenocytes) compared to control vaccinated TD mice
(163.6 cells per million splenocytes) (Figure 3.4, independent t test, p = 0.3017). This
increase was not statistically significant due to large variations in the number of
CD8
+
IFNγ
+
T cells in each mouse. Indeed, the splenocytes obtained from approximately
half of the mice in each group failed to respond to mPSCA
83-91
stimulation at all, which is
surprising given that they are all genetically identical at their MHC loci. Overall, there
were more IFN-γ producing splenocytes in TD mice than in TRAMP mice, regardless of
vaccination status, indicating that prior depletion of Tregs can result in an increase in
antigen-specific immune responses even in the absence of vaccination.
120
Figure 3.4
TRAMP Control
TRAMP mPSCA
TD Control
TD mPSCA
0
250
500
750
1000
1250
1500
1750
CD8+IFNg+ Cells
per Million Splenocytes
Figure 3.4: Induction of mPSCA-specific IFNγ-expressing CD8+ T cells in
vaccinated TD mice. Eight to ten week old TD mice and standard TRAMP littermate
controls were treated IP with 1 μg DT daily for two days. TRAMP mice and Treg-
depleted TD mice were vaccinated three days later by helium-driven gene gun with either
2 μg mPSCA-pcDNA or 2 μg empty vector and boosted at day 14 with 10
6
IU mPSCA-
VRP and 10
6
IU GFP-VRP, respectively. The mice were euthanized fourteen days after
boosting and the spleens harvested. Single cell suspensions of splenocytes were treated
for twenty four hours with mPSCA
83-91
and IL-2, with Brefeldin-A being added in the last
six hours in order to arrest the Golgi apparatus of the cells. Cells were then harvested,
washed and stained with anti-mouse CD3-FITC and anti-mouse CD8-PE/Cy5 and fixed
and permeablized overnight. Cells were washed, and intracellularly stained with anti-
mouse IFNγ-PE, then fixed and analyzed by flow cytometry. Events were collected gated
on live, CD3
+
cells and then further gated on the CD8
+
IFNγ
+
fraction.
121
In an effort to determine the immunological mechanisms responsible for the inhibition of
tumor growth in mPSCA vaccinated TD mice, the effects of vaccination on the splenic
populations of CD4
+
, CD8
+
and CD4
+
FOXP3
+
T cells were first assessed. Splenocytes
were isolated from mPSCA vaccinated TD (n = 6), control vaccinated TD (n = 6),
mPSCA vaccinated TRAMP (n = 4) and control vaccinated TRAMP (n = 5) mice and
analyzed by flow cytometry. There was no statistically significant difference in the mean
percentages of splenocytes that were CD3
+
CD8
+
T cells between control vaccinated TD
mice (14.8 ± 5.6%) and control vaccinated TRAMP mice (12.9 ± 2.2%), indicating that
Treg depletion alone is incapable of boosting peripheral numbers of CD3
+
CD8
+
T cells
(Figure 3.5, Independent t test, p = 0.4961). There was a trend towards an increase in the
mean percentage of splenocytes that were CD3
+
CD8
+
T cells in mPSCA vaccinated TD
compared to control vaccinated TD mice (Figure 3.5, Independent t test, p = 0.1626) and
in mPSCA vaccinated TRAMP mice (16.1 ± 3.3%) compared to control vaccinated
TRAMP mice (12.9 ± 2.2%) (Figure 3.5, Independent t test, p = 0.1236). There was no
statistically significant difference in the mean percentages of splenocytes that were
CD3
+
CD8
+
T cells between mPSCA vaccinated TD mice (19.5 ± 4.0%) and mPSCA
vaccinated TRAMP mice (16.1 ± 3.3%), indicating that Treg depletion does not enhance
the numbers of these cells produced in response to vaccination (Figure 3.5, Independent t
test, p = 0.2577). However, there was a statistically significant difference in the mean
percentages of splenocytes that were CD3
+
CD8
+
T cells between mPSCA vaccinated TD
(19.5 ± 4.0%) and control vaccinated TRAMP mice (12.9 ± 2.2%), indicating that the
122
combination of Treg depletion and vaccination against mPSCA acts additively to increase
the numbers of these cells in vivo (Figure 3.5, Independent t test, p = 0.0120).
123
Figure 3.5
TRAMP Control
TRAMP mPSCA
TD Control
TD mPSCA
0
5
10
15
20
25
CD8+ Proportion of
Total Splenocytes (%)
*
Figure 3.5: Treg depletion acts additively with mPSCA vaccination to increase
peripheral CD8+ T cell numbers. Eight to ten week old TD mice and standard TRAMP
littermate controls were treated IP with 1 μg DT daily for two days. TRAMP mice and
Treg-depleted TD mice were vaccinated three days later by helium-driven gene gun with
either 2 μg mPSCA-pcDNA or 2 μg empty vector and boosted at day 14 with 10
6
IU
mPSCA-VRP and 10
6
IU GFP-VRP, respectively. The mice were euthanized fourteen
days after boosting and the spleens harvested. Single cell suspensions of splenocytes
were washed and stained with anti-mouse CD3-FITC and anti-mouse CD8-PE/Cy5, then
analysed by flow cytometry. Events were collected gated on live, CD3
+
cells and then
further gated on the CD8
+
fraction. The * symbol indicates p < 0.05.
124
Next, the mean percentage of splenocytes that were CD3
+
CD4
+
FOXP3
-
non-regulatory T
cells was assessed. There was no statistically significant change in the mean percentages
of splenocytes that were CD3
+
CD4
+
FOXP3
-
T cells between control vaccinated TRAMP
mice (8.1 ± 2.2%) and control vaccinated TD mice (10.1 ± 6.0%) (Figure 3.6,
Independent t test, p = 0.5006). There was a statistically significant increase in the mean
percentage of splenocytes that were CD3
+
CD4
+
FOXP3
-
T cells in mPSCA vaccinated
TRAMP mice compared (11.8 ± 1.5%) to control vaccinated TRAMP mice (8.1 ± 2.2%)
(Figure 3.6, Independent t test, p = 0.0245). There was no statistically significant
difference between the mean percentage of splenocytes that were CD3
+
CD4
+
FOXP3
-
T
cells in mPSCA vaccinated TD mice (13.9 ± 4.1%) compared to control vaccinated TD
mice (10.1 ± 6.0%) (Figure 3.6, Independent t test, p = 0.2618). There was no statistically
significant difference in the mean percentages of splenocytes that were CD3
+
CD4
+
FOXP3
-
T cells between mPSCA vaccinated TRAMP (11.8 ± 1.5 %) and TD mice (13.9
± 4.1%), indicating that Treg depletion does not enhance the numbers of these cells
produced in response to vaccination (Figure 3.6, Independent t test, p = 0.3677). Despite
this, there was a statistically significant difference in the mean percentages of splenocytes
that were CD3
+
CD4
+
FOXP3
-
T cells between mPSCA vaccinated TD mice (13.9 ±
4.1%) compared to control vaccinated TRAMP mice (8.1 ± 2.2%), again indicating that
the combination of Treg depletion and vaccination against mPSCA acts additively to
increase the numbers of these cells in vivo (Figure 3.6, Independent t test, p = 0.0237).
125
Figure 3.6
TRAMP Control
TRAMP mPSCA
TD Control
TD mPSCA
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
CD4+FOXP3-
Proportion of Total
Splenocytes (%)
*
Figure 3.6: Treg depletion acts additively with mPSCA vaccination to increase
peripheral CD4+ T cell numbers. Eight to ten week old TD mice and standard TRAMP
littermate controls were treated IP with 1 μg DT daily for two days. TRAMP mice and
Treg-depleted TD mice were vaccinated three days later by helium-driven gene gun with
either 2 μg mPSCA-pcDNA or 2 μg empty vector and boosted at day 14 with 10
6
IU
mPSCA-VRP and 10
6
IU GFP-VRP, respectively. The mice were euthanized fourteen
days after boosting and the spleens harvested harvested. Single cell suspensions of
splenocytes were washed and stained with anti-mouse CD3-FITC and anti-mouse CD4-
PE/Cy7, then analysed by flow cytometry. Events were collected gated on live, CD3
+
cells and then further gated on the CD4
+
FOXP3
-
fraction. The * symbol indicates p <
0.05.
126
It is known that the FOXP3
+
population rebounds in DEREG mice between two and three
weeks after depletion. Therefore, it was next investigated whether this was the case in
TRAMP DEREG mice, and if so whether mPSCA vaccination could prevent or attenuate
Treg repopulation in these animals. There was no statistically significant difference in the
mean percentages of splenocytes that were CD3
+
CD4
+
FOXP3
+
Tregs between control
vaccinated TRAMP (1.59% +/- 1.02%) and control vaccinated TD mice (1.4% +/-
0.46%) (Figure 3.7, Independent t test, p = 0.5006). This indicates that the Treg
population of TD mice had completely rebounded to pre-depletion levels within the time
course of this experiment (approximately four weeks), exactly as would be observed with
DEREG mice. Vaccination against mPSCA increased the mean percentage of spenocytes
that were CD3
+
CD4
+
FOXP3
+
Tregs in both TRAMP and TD mice. This increase was
statistically significant in the mean percentage of splenocytes that were CD3
+
CD4
+
FOXP3
+
Tregs in mPSCA vaccinated TRAMP mice compared to control vaccinated
TRAMP mice (Figure 3.7, Independent t test, p = 0.0277), but not in mPSCA vaccinated
TD compared to control vaccinated TD mice (Figure 3.7, Independent t test, p = 0.1314).
There was no statistically significant difference in the mean percentages of splenocytes
that were CD3
+
CD4
+
FOXP3
+
Tregs between mPSCA vaccinated TRAMP and TD mice,
indicating that prior Treg depletion does not change the numbers of these cells produced
in response to vaccination (Figure 3.7, Independent t test, p = 0.8182).
127
Figure 3.7
TRAMP Control
TRAMP mPSCA
TD Control
TD mPSCA
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
CD4+FOXP3+
Proportion
of Splenocytes (%)
*
Figure 3.7: Vaccination against mPSCA increases the proportion of Treg cells in the
periphery, regardless of prior Treg depletion. Eight to ten week old TD mice and
standard TRAMP littermate controls were treated IP with 1 μg DT daily for two days.
TRAMP mice and Treg-depleted TD mice were vaccinated three days later by helium-
driven gene gun with either 2 μg mPSCA-pcDNA or 2 μg empty vector and boosted at
day 14 with 10
6
IU mPSCA-VRP and 10
6
IU GFP-VRP, respectively. The mice were
euthanized fourteen days after boosting and the spleens harvested harvested. Single cell
suspensions of splenocytes were washed and stained with anti-mouse CD3-FITC and
anti-mouse CD4-PE/Cy7, fixed and permeablised overnight, then stained with anti-mouse
FOXP3-PE and analysed by flow cytometry. Events were collected gated on live, CD3
+
cells and then further gated on the CD4
+
FOXP3
+
fraction. The * symbol indicates p <
0.05.
128
Given the critical role of lymph nodes in effector T cell activation, the effects of
vaccination on the populations of CD4
+
, CD8
+
and CD4
+
FOXP3
+
T cells were assessed
in these organs. Lymphocytes were isolated from mPSCA vaccinated TD (n = 6), control
vaccinated TD (n = 6), mPSCA vaccinated TRAMP (n = 4) and control vaccinated
TRAMP (n = 5) mice and analyzed by flow cytometry. There was a striking, statistically
significant decrease in the mean percentages of lymphocytes that were CD3
+
CD8
+
T cells
in control vaccinated TD mice (26.0 ± 5.3%) compared to control vaccinated TRAMP
(38.6 ± 7.7%) mice (Figure 3.8, Independent t test, p = 0.0106). It is not clear from these
data whether this effect of prior Treg depletion is due to a reduction of trafficking of
CD8
+
T cells to the lymph nodes, or whether it is due to an increase in activation of CD8
+
T cells and a subsequent increase in their trafficking out of the lymph nodes and back into
the periphery. There was a statistically significant increase in the mean percentage of
lymphocytes that were CD3
+
CD8
+
T cells in mPSCA vaccinated TD mice (36.8 ± 8.5%)
compared to control vaccinated TD mice (26.0 ± 5.3%) (Figure 3.8, Independent t test, p
= 0.0257). Though the mean percentages of lymphocytes that were CD3
+
CD8
+
T cells
were generally higher in TRAMP mice than in TD mice, the enhancement in CD3
+
CD8
+
lymphocytes in TRAMP mice due to mPSCA vaccination was much less pronounced
than in their TD littermates. Indeed, the increase in the mean percentage of lymphocytes
that were that were CD3
+
CD8
+
T cells in mPSCA vaccinated TRAMP mice compared to
control vaccinated TRAMP mice was not statistically significant (Figure 3.8, Independent
t test, p = 0.4886).
129
Figure 3.8
TRAMP Control
TRAMP mPSCA
TD Control
TD mPSCA
0
10
20
30
40
50
*
*
CD8+ Proportion of
Lymphocytes (%)
Figure 3.8: Treg depletion enhances the increase in the CD8+ T cell compartment of
lymphocytes in response to mPSCA vaccination. Eight to ten week old TD mice and
standard TRAMP littermate controls were treated IP with 1 μg DT daily for two days.
TRAMP mice and Treg-depleted TD mice were vaccinated three days later by helium-
driven gene gun with either 2 μg mPSCA-pcDNA or 2 μg empty vector and boosted at
day 14 with 10
6
IU mPSCA-VRP and 10
6
IU GFP-VRP, respectively. The mice were
euthanized fourteen days after boosting and the lymph nodes harvested. Single cell
suspensions of lymphocytes were washed and stained with anti-mouse CD3-FITC and
anti-mouse CD8-PE/Cy5, then analysed by flow cytometry. Events were collected gated
on live, CD3
+
cells and then further gated on the CD8
+
fraction. The * symbol indicates p
< 0.05.
130
Regulatory T cells are known to exert their suppressive function within lymph nodes.
Therefore, whether there was any effect of prior depletion of FOXP3
+
regulatory T cells
and concomitant mPSCA vaccination on the Treg populations of TRAMP DEREG lymph
nodes was investigated. In stark contrast to the striking increase in the percentage of
CD3
+
CD4
+
FOXP3
+
Tregs that was associated with mPSCA vaccination in the spleens of
TD and TRAMP mice, there was no change in the percentage of Tregs in the lymph
nodes of both TD and TRAMP mice when they were vaccinated against mPSCA. Treg
depletion seemed to persist longer in the lymph nodes of TD mice compared to in their
spleens. There was a small but statistically nonsignificant decrease in the mean
percentages of lymphocytes that were CD3
+
CD4
+
FOXP3
+
Tregs between control
vaccinated TD (1.92% ± 0.35%) and control vaccinated TRAMP (2.28% ± 0.71%) mice
(Figure 3.9, Independent t test, p = 0.2634). The decrease in the mean percentage of
lymphocytes that were CD3
+
CD4
+
FOXP3
+
Tregs between mPSCA vaccinated TD
(1.99% ± 0.12%) and control vaccinated TRAMP (2.42% ± 0.30%) mice was statistically
significant (Figure 3.9, Independent t test, p = 0.0147).
131
Figure 3.9
TRAMP Control
TRAMP mPSCA
TD Control
TD mPSCA
0.0
0.5
1.0
1.5
2.0
2.5
3.0
*
CD4+FOXP3+
Proportion
of Lymphocytes (%)
Figure 3.9: Treg depletion persists in the lymph nodes of TD mice for up to four
weeks. Eight to ten week old TD mice and standard TRAMP littermate controls were
treated IP with 1 μg DT daily for two days. TRAMP mice and Treg-depleted TD mice
were vaccinated three days later by helium-driven gene gun with either 2 μg mPSCA-
pcDNA or 2 μg empty vector and boosted at day 14 with 10
6
IU mPSCA-VRP and 10
6
IU GFP-VRP, respectively. The mice were euthanized fourteen days after boosting and
the lymph nodes harvested. Single cell suspensions of lymphocytes were washed and
stained with anti-mouse CD3-FITC and anti-mouse CD4-PE/Cy7, fixed and permeablised
overnight, then stained with anti-mouse FOXP3-PE and analysed by flow cytometry.
Events were collected gated on live, CD3
+
cells and then further gated on the
CD4
+
FOXP3
+
fraction. The * symbol indicates p < 0.05.
132
DISCUSSION
In this study, a murine model of human prostate cancer in which regulatory T cells can be
specifically depleted at any time has been developed, named the TRAMP DEREG
mouse. In addition, the immunological responses to therapeutic prostate cancer
vaccination in these mice have been characterized and compared to those of the widely-
used TRAMP mouse model of human prostate cancer.
Research efforts to confirm the role of Tregs in suppressing antitumor immune responses
have been hampered by the lack of a specific means to deplete and/or inhibit them.
Protocols involving reagents such as Ontak and PC61 have largely been unsuccessful
since they also inhibit the activity of immune effector cells in addition to regulatory T
cells. In recent years, two transgenic mouse models that have effects on Treg numbers –
the PEP KO(LYP-R620W) mouse and the DEREG mouse – have been developed. In
principle availability of a mouse line with fewer Tregs – and thus reduced peripheral
tolerance – gave an opportunity to test the hypothesis that Tregs are responsible for
inhibiting the antitumor immune response elicited by therapeutic prostate cancer
vaccines. It was intended that if PEP KO(LYP-R620W) mice did have the expected
phenotype of having fewer Tregs, they could be crossed with TRAMP mice in order to
generate a mouse model in which superior immune responses to therapeutic cancer
vaccines might result in improved survival. Unfortunately, the data presented here
demonstrate that the PEP KO(LYP-R620W) mice do not have the desired Treg
phenotype, either in the thymus or in the periphery (Figure 3.1).
133
As these disappointing data were obtained, a novel transgenic mouse became available
that allowed the specific depletion of FOXP3
+
Tregs at any time. This depletion of Treg
(DEREG) mouse was the ideal tool with which to investigate the hypothesized inhibitory
effects of Tregs on therapeutic prostate cancer vaccine efficacy. Indeed, the use of the
DEREG mouse has a number of advantages over the use of the PEP KO(LYP-W620)
mouse as was originally planned. The model is fully developed and the ability to
selectively deplete Treg has already been shown in published literature (Lahl and
Sparwasser). Like the TRAMP mouse that is used by this group as a model of human
prostate cancer, the DEREG mouse is fully on the C57BL/6 background. This negates the
need for multiple generations of back-crossing in order to yield animals in which
immunological responses can be measured easily. In addition, there is no manipulation of
TCR signaling in the DEREG mouse, and thus no possibility of inadvertently affecting
effector T cells that do not express FOXP3. Finally, the DEREG mouse system allows the
selective depletion of FOXP3-expressing Tregs at any time during the development of
prostate cancer. If the PEP KO(LYP-W620) mouse has the desired phenotype, it will
have fewer Treg cells throughout life. This may lead to unintended consequences (such as
long-term autoimmune disease) and potentially confound study results. Using the
DEREG model, Treg can be specifically depleted immediately prior to and during
vaccination. This scenario mimics the treatment protocol envisioned for future human
therapy where Treg would be depleted by from the patient’s system prior to and during
therapeutic prostate cancer immunotherapy. For example, small molecule inhibitors of
134
FOXP3 could be developed (perhaps which prevent FOXP3 homodimerization, which is
critical to its function as a transcription factor) in order to abrogate Treg function in
humans. However, developing a completely novel drug like this would cost hundreds of
millions of dollars. Therefore, it is vital to demonstrate in relatively inexpensive
preclinical models (such as the DEREG mouse) that inhibition of Tregs prior to
immunization actually enhances the efficacy of therapeutic cancer vaccines.
DEREG mice were obtained with the permission of their developer (Dr. Timothy
Sparwasser, TWINCORE, Hanover, Germany) and crossed with TRAMP mice. This
yielded the first autochthonous murine model of human prostate cancer in which FOXP3
+
Tregs can be specifically depleted at the discretion of the investigator. As anticipated,
administration of a short course of diphtheria toxin at a low dose (1 μg DT
intraperitoneally for two consecutive days) resulted in significant depletion of Tregs from
these mice (Figure 3.2). Importantly, the remainder of the CD4
+
T cell population (i.e.
non-regulatory cells that do not express FOXP3) was unaffected by DT treatment. As
expected, neither the CD4
+
FOXP3
-
nor the CD4
+
FOXP3
+
T cell populations were
affected by DT treatment in TRAMP littermate control mice that lacked the DEREG
transgene.
To directly demonstrate the efficacy of the DNA prime/VRP boost therapeutic prostate
cancer vaccine protocol used here in eliciting a strong antitumor response, and whether
depletion of Tregs could enhance this response, the mass of prostate/prostate tumor tissue
135
was measured in groups of vaccinated TRAMP and Treg-depleted TD mice. In TRAMP
mice, a 34 percent decrease in prostate/prostate tumor size was observed in mice
vaccinated against mPSCA at 8 weeks versus age matched mock vaccinated controls.
This decrease, though not statistically significant, was large considering that the time
between initial DNA priming and euthanasia was only four weeks. Strikingly, the
prostate/prostate tumor tissue isolated from control animals had tumor lesions that were
visible to the naked eye, whilst there were no such visible lesions in mice that had been
vaccinated against PSCA. In addition, the seminal vesicles of control vaccinated animals
were enlarged, whilst those of mice vaccinated against PSCA appeared normal. It is
likely that although outwardly normal, the prostates of the mice vaccinated against PSCA
still contain microscopic foci of tumor tissue, as has been previously observed by this
group in TRAMP mice vaccinated against PSCA at 8 weeks of age (Garcia-Hernandez
Mde et al., 2008). Histological analysis of the prostate/prostate tumor tissue obtained
from these mice is required to confirm whether this is the case. Overall, these data are
completely consistent with the findings presented in Chapter Two and previous studies
(Garcia-Hernandez Mde et al., 2008) which clearly demonstrate that the most dramatic
results using this therapeutic prostate cancer vaccine are obtained at 8 weeks of age.
TRAMP mice at 8 weeks of age all have PIN lesions, and by 12 weeks of age (the time at
which they were euthanized in this experiment) most have progressed to invasive cancer.
Vaccination applied at this time point apparently prevents this early tumor outgrowth,
explaining why such dramatic improvements in survival are demonstrated in TRAMP
mice immunized at 8 weeks of age (Figure 2.1). The data presented in Chapter Two
136
demonstrated that even at 8 weeks of age, functionally suppressive Tregs were present in
the prostate/prostate tumor draining lymph nodes (Figure 2.8). Thus, it was hypothesized
that depletion of Tregs in TD mice at this age might enhance antitumor response shown
in their TRAMP littermates. This was indeed the case, with a statistically significant
reduction in prostate/prostate tumor mass of 53 percent in mPSCA vaccinated TD mice
compared to mock vaccinated TD controls (Figure 3.3).
To determine what immunological mechanisms might be responsible for the retardation
of tumor growth in vaccinated TD mice, attention was turned to the effects of
concomitant Treg depletion and vaccination against mPSCA on the splenic populations
of immune cells in mice. An improvement in the number of CD8
+
T cells that produced
IFNγ specifically in response to stimulation with an mPSCA peptide was observed in the
splenic population of some mPSCA vaccinated TD mice (Figure 3.4). Cytotoxic CD8
+
T
cells that respond to TAA are critical mediators of successful antitumor immune
responses in both mice and humans. Considering that the TRAMP and TD mice used in
this experiment are identical at their MHC loci, it is not clear why some mice responded
well to mPSCA stimulation whilst others did not. Splenocytes from all of the animals
used in this experiment responded strongly to stimulation by the superantigen PHA,
ruling out the possibility that the cells from individual mice were generally incapable of
mounting a response. This experiment must be repeated to ascertain whether this pattern
is repeatable, or whether it was simply a technical issue. Interestingly, there was a general
increase in the number of IFNγ-producing CD8
+
T cells in Treg-depleted TD mice
137
compared to TRAMP mice, regardless of vaccination status. It is possible that the
increase in IFNγ-producing CD8
+
cells in control vaccinated TD mice (which are boosted
with VRP encoding GFP) might be due to the expression of GFP in TD mice. However,
this is unlikely since there was no DNA priming step with GFP, and immunological
responses have not been observed from a single VRP prime in the experience of this
group. Rather, this result suggests a loss of peripheral tolerance upon depletion of Tregs.
If so, then protocols developed to deplete or inhibit Treg function in order to enhance
vaccine efficacy must be applied with care in human studies. Participants must be
carefully monitored for the development of autoimmunity directed against self antigens
other than the specific TAA that is being targeted.
It was next determined that Treg depletion affected the numbers of peripheral CD8
+
and
CD4
+
cells produced in response to vaccination (Figures 3.5 and 3.6, respectively).
Vaccination against mPSCA increased the numbers of these cell types in both TRAMP
and Treg-depleted TD mice, as expected. Treg depletion alone seemed to slightly
increase the proportion of these cell types expressed as a percentage of the total splenic
population. There was an additive effect of vaccination and Treg depletion, with mPSCA
vaccinated Treg-depleted TD mice having statistically significantly more of both CD4
+
and CD8
+
cells in the periphery compared to control vaccinated TRAMP mice. This
suggested a microenvironment more permissive to immune responses. However, this was
tempered somewhat by the surprising observation that the numbers of splenic
CD4
+
FOXP3
+
Tregs increased in response to mPSCA vaccination in both TRAMP and
138
Treg-depleted TD mice (Figure 3.7). Though FOXP3 is transiently expressed in recently
activated T cells in humans, this is not the case in mice. It is possible that the observed
increase in CD4
+
FOXP3
+
represents the beginning of CD4
+
Th cells conversion into
iTregs in response to the strong stimulation from the vaccine. These cells are apparently
not capable of inhibiting the immune response elicited by vaccination at this stage, at
least not enough to allow tumor outgrowth. If this was so, then the dramatic improvement
in the survival of TRAMP mice shown in Figure 2.1 would not have been observed. The
lack of a similar increase in CD4
+
FOXP3
+
T cells in the lymph nodes of TRAMP and
Treg-depleted TD mice in response to vaccination may offer an explanation for this
phenomenon: Transient conversion of some CD4
+
T cells to the iTreg phenotype in
response to vaccination may be irrelevant if these cells do not (or cannot) traffic to an
environment where they are exposed to TGFβ, IL-2 and self antigen, and thus receive the
signals necessary to stably express FOXP3 and become permanent iTregs. If the
peripheral CD4
+
FOXP3
+
T cells induced in response to vaccination did so, it would be
expected that they would ultimately accumulate in the tumor/tumor draining lymph nodes
and exert immune suppression. This is apparently not the case in TRAMP mice that are
vaccinated at 8 weeks of age.
Finally, a statistically significant increase in CD8
+
T cells was observed in the lymph
nodes of Treg-depleted TD mice vaccinated against mPSCA, but not in vaccinated
TRAMP mice (Figure 3.8). However, this result is currently difficult to interpret as it was
in the context of a general decrease in the number of these cells in the lymph nodes of TD
139
mice compared to their TRAMP littermates. It is conceivable that in the absence of Treg
suppression, CD8
+
cells are more likely to become activated within the lymph nodes.
Thus, they can traffic more readily to the periphery, perhaps being responsible for the
increase in the numbers of these cells observed in Figure 3.5. Excitingly, an increased
number of these CD8
+
T cells might have trafficked to the prostate tumors of these mice,
resulting in the retardation of tumor growth that was observed in vaccinated TD mice
(Figure 3.3). However, no data are currently available to confirm or refute this
possibility. Frozen tumor tissue samples exist for all of the mice investigated here. They
may be relatively easily used to determine relative CD8
+
T cell infiltration, for example
by performing real-time quantitative PCR to quantify the relative levels of intratumoral
CD8 mRNA transcripts, for example.
Though these data are extremely encouraging, further studies are required. The findings
of Chapter Two raised a fundamental question: What causes the difference between the
improvements in survival of mice vaccinated at 8 weeks versus those vaccinated at 16
weeks? It would be very instructive to repeat this experiment in TRAMP and Treg-
depleted TD mice at 16 weeks of age. Two important facts could be ascertained. Firstly,
it could be determined whether a rapid reduction in TRAMP tumor mass occurs in the
first month post vaccination, as it does with mice vaccinated at 8 weeks. If it does not,
this would suggest that an important shift in the tumor microenvironment occurs between
8 and 16 weeks that prevents the swift immune-mediated killing of tumor cells that is
observed when mice are vaccinated early. Secondly, it could be learned whether the
140
accumulation of Tregs that is observed in TRAMP prostate tumors as they progress
(Figure 2.7) is responsible for the lack antitumor activity at this stage. If so, it would be
expected that depletion of Tregs in TD mice prior to their vaccination at 16 weeks of age
would at least partially restore the immune-mediated retardation of tumor growth. If
antitumor immunity is not improved by depleting Tregs at this stage, important
information will still be gleaned from these studies. Such a result may indicated that
Tregs play no role whatsoever in limiting the efficacy of therapeutic prostate cancer
vaccines, or that they only play a role in the initial development of a suppressive tumor
microenvironment. It is also possible that Treg depletion will only mediate a short-term
improvement in antitumor immunity, but then outgrowth of the tumor continues as it
develops and/or subverts other mechanisms of immune tolerance to suppress the
antitumor immune response. If this is the case, it would be extremely important for the
field as it would suggest that targeting individual immunosuppressive mechanisms in the
dynamic tumor microenvironment may not be capable of improving the efficacy of
therapeutic cancer vaccines in the long term. If so, it could be inferred that applying
therapeutic cancer vaccines prior to the development of any tumor-mediated immune
suppression (for example, in men with PIN lesions) is the best way of establishing long-
term antitumor immunity.
Another additional study that is required is to compare the survival of mPSCA vaccinated
TRAMP and TD mice, both at 8 weeks and 16 weeks of age. The survival data obtained
from the 16 week old mice would unequivocally answer the question whether Tregs
141
reduce the efficacy of this therapeutic prostate cancer vaccine. The results obtained from
TRAMP mice vaccinated at 16 weeks of age compared to mock vaccinated controls
should be similar to those that were observed in Figure 2.1. However, if Tregs are
responsible for the difference in survival observed between mice vaccinated at 8 weeks
versus 16 weeks, then the difference in survival between Treg-depleted TD mice
vaccinated at 16 weeks and their mock vaccinated controls should be greater than that
observed between TRAMP mice and their controls. In addition, there should be a
measurable improvement in survival between vaccinated Treg depleted TD mice and
vaccinated TRAMP mice at 16 weeks. A study comparing the survival of Treg-depleted
TD mice vaccinated at 8 weeks compared to TRAMP mice vaccinated at this time may
also show improvements in survival due to Treg depletion. However, given the already
outstanding survival of mice vaccinated at this stage of carcinogenesis (Figure 2.1), such
a study would by necessity be very long if it is to detect any differences in survival
resulting from Treg depletion. Nevertheless, it is an important experiment to carry out. If
Treg depletion at 8 weeks does not improve survival, then it may be concluded that Treg
mediated suppression of the antitumor immune responses elicited by vaccines only
becomes established after the PIN lesion stage of carcinogenesis. This has important
implications for the tumor immunotherapy field, as it lends weight to the idea that
conducting clinical trials of prostate cancer vaccines in men that only have PIN lesions
would be an effective means of circumventing the tumor-mediated immune suppression
that has plagued the outcomes clinical trials conducted in men with advanced cancer.
142
Conclusion
The data presented in this chapter indicate that regulatory T cells have some role in
modulating the immune response elicited by therapeutic prostate cancer vaccines even at
the earliest stage of carcinogenesis. Given the observation in Chapter Two that Tregs
progressively accumulate in prostate tumors as they become more advanced, it is
expected that the benefit incurred by depleting regulatory T cells prior to therapeutic
vaccination will be even more profound when applied at later stages of carcinogenesis.
The development of the TRAMP DEREG mouse model allows the unambiguous testing
of this hypothesis. As discussed in Chapter Two, it is likely that the optimal time to
administer therapeutic cancer vaccines is at the earliest stage of carcinogenesis, prior to
the development of a tumor-mediated immunosuppressive network. However, in order
for this to become the standard of care, a radical paradigm shift in how tumor
immunotherapy clinical trials are carried out will be required. In the meantime, the
unequivocal demonstration that regulatory T cells do indeed play a major role in limiting
the efficacy of therapeutic cancer vaccines will provide significant impetus to the search
for specific pharmacological inhibitors of these cells. The application of such reagents in
order to disrupt the immunosuppressive tumor microenvironment may allow tumor
immunotherapy in very advanced, even metastatic, cases, finally allowing therapeutic
cancer vaccines to live up to their full potential.
143
CHAPTER 4: NOTCH SIGNALING IN PROSTATE CANCER STEM CELL
DIFFERENTIATION
INTRODUCTION
Stem Cells
The term stem cell covers several broad classes of cells that share the common features of
self-renewal and the ability to undergo asymmetric division in order to give rise to
differentiated daughter cells. There are three main types of stem cells. The first type is the
totipotent stem cell. These have the unique capacity to form any type of cell, i.e. they are
able to generate an entire organism. These stem cells only occur during very early
development, and lose their totipotent capacity at the four to eight cell stage of
embryogenesis. As the embryo develops, its stem cells undergo repeated divisions and
become progressively more differentiated. As the organism progresses to adulthood, most
of its constituent cells are terminally differentiated. Only a tiny minority retains stem cell
properties. These adult or somatic stem cells maintain the ability to self-renew, but can
only form a small subset of cell types (termed multipotency). They are responsible for the
repair or regeneration of tissues throughout the life of adult organism. To achieve this,
they are able to resist DNA damage by expressing high levels of DNA repair machinery.
Adult stem cells can resist the effects of toxins, primarily by expressing high levels of
cell surface transport proteins with which they can expel harmful compounds. Finally,
they have several mechanisms by which to avoid apoptosis (Hemmings).
144
Typically, adult stem cells will stay quiescent until they are needed to generate a
differentiated daughter cell by injury or the turnover of old cells as they become
senescent. This is achieved by the adult stem cell undergoing asymmetric division,
producing one identical daughter stem cell (thus perpetually renewing this population)
and an intermediate precursor, or transit amplifying (TA), daughter cell. Transit
amplifying cells are often also multipotent, but have limited ability to self-renew. They
will undergo repeated (but limited) rounds of asymmetric cell division, each producing
another transit amplifying cell and a terminally differentiated somatic cell. Stem cells are
thought to reside in a “niche” which provides the signals necessary to maintain the stem
like phenotype. When stem cells undergo asymmetric division, the daughter stem cell
stays in the niche, whilst the more differentiated daughter cell is pushed out of it. This
physically removes it from the signals required for maintaining the stem cell phenotype,
so they adopt a more differentiated phenotype. As these cells undergo subsequent rounds
of asymmetric division, their daughters are progressively pushed further out of the niche
providing the pro-stem signals. Thus, daughter cells become progressively more
differentiated with each asymmetric division, and are decreasingly able to self-renew and
undergo asymmetric divisions themselves. Each cell division requires the replication of
DNA, which is intrinsically imperfect, i.e. can introduce mutations. Thus, by allowing
expendable TA cells to do the majority of the necessary cell divisions required to
regenerate tissues and organs, the stem cells maintain the integrity of their genome and
allow the organism a longer healthy lifespan.
145
Cancer Stem Cells
Traditionally, it has been hypothesized that cancer occurs when a somatic cell acquires
mutations which confer upon it immortality and the ability to undergo uncontrolled cell
division. As a result of the uncontrolled clonal replication of this cell, a tumor forms.
With rapid division comes an increased likelihood of acquiring yet more mutations,
which may confer abilities upon the tumor such as resistance to chemotherapeutic agents,
resistance to apoptosis and the capacity for metastatic spread. Thus, while conventional
anticancer therapies have a great deal of initial success in debulking the tumor, a small
minority of its cells will have the ability to resist those therapies (Wang). Such cells are
responsible for the common recurrence of tumors after effective primary cancer treatment
that is so dreaded by patients and physicians.
Recently, this stochastic model of tumorigenesis has been challenged by the cancer stem
cell hypothesis (Hemmings). It posits that the cell of origin for cancer is actually a
somatic stem cell which acquires mutations causing it to adopt a malignant phenotype
whilst maintaining the hallmarks of “stemness”, i.e. the ability to self-renew and produce
differentiated daughter cells. Like normal stem cells, these cancer stem cells are relatively
rare compared to the population of terminally differentiated tumor cells. Cancer stem
cells share several key properties with their normal counterparts that can help explain the
regeneration of tumors after initial treatment. For example, their quiescence and highly
active DNA repair mechanisms protect them from radiotherapy. They are able to resist
many chemotherapeutic agents by virtue of their ability to efficiently expel chemicals,
146
and as a result of the fact that many such drugs (such as those that interfere with DNA
replication) target rapidly proliferating cells. Most conventional cancer therapies are
characterized by severe side effects, and cannot be tolerated in the long term by patients.
Treatment is terminated when a detectable tumor is no longer present. As a result of their
intrinsic properties of self-preservation, cancer stem-like cells are far more likely than
their differentiated daughter cells to survive the entire duration of a course of cancer
therapy. If this is the case, then hypothetically even a single surviving cancer stem-like
cell could begin dividing again after treatment has been stopped and regenerate the entire
tumor. Complete and apparently permanent tumor regression does occur in response to
radiotherapy and/or chemotherapy. In such cases, the treatment administered must have
been applied long enough to terminally damage all of the cancer stem cells. This may
explain why the patients who can tolerate the longest courses of treatment tend to have
better outcomes; there is simply more time for rare divisions of cancer stem cells to
occur, thereby allowing them to be killed by the therapeutic agent in use. However, these
cells may not divide for years at a time. Simply put, the majority of patients is not
capable of surviving the duration of conventional anticancer treatment that would be
required to guarantee that all cancer stem cells are eradicated. If the ultimate goal in
oncology is to be reached – total annihilation of the tumor – then cancer stem cells must
be characterized and specifically targeted.
147
Identification of Cancer Stem Cells
Currently, the only way to unambiguously identify a cancer stem cell is by its functional
properties. The first such property is the ability of a cancer stem cell to initiate tumor
formation. This is typically done via implantation of the putative cancer stem cell into
immune incompetent mice. Hypothetically, a tumor should be able to develop from the
successful implantation of a single true cancer stem cell. The second such property is the
ability of the putative cancer stem cell to self-renew. This is achieved by repeatedly
dissociating the tumor and reimplanting hypothetical cancer stem cells, which should be
phenotypically identical to the parental cell if they are truly self-renewing, and thus they
should be equally tumorigenic.
Many cancer cell lines are used in implantation models are used to model human disease.
In most cases, the implantation of at least a few thousand cells is required in order to
generate a tumor. Cell lines are characterized by varying tumorgenicity, with the “most
tumorigenic” cell lines requiring challenge of mice with fewer cells such that growth of
consistently-sized tumors in a reasonable timeframe for experiments is assured. The
tumorigenicity of a given line is a reflection of how common cancer stem cells are within
that cell line. For example, if 0.1% of cells in a cancer cell line are cancer stem cells, then
stochastically 1000 cells would need to be injected into a mouse, assuming that every
tumor initiating cell that is injected will find the correct microenvironment to grow and
form a tumor. The tumorigenicity of cancer cell lines can often be increased if they are
selected for cell surface markers that are thought to identify cells with stem-like
148
characteristics. For example, selecting prostate cancer cells based the CD49f
hi
Sca-
1
+
CD117
+
profile of cell surface markers dramatically reduces the number of cells that
are required to form a tumor in a challenged mouse (Mulholland et al., 2009) However,
not every such cell is tumorigenic, indicating that only a subset of this cell population is
the true stem cell fraction.
The identification of cell surface markers that enrich for the tumor initiating cell
phenotype has resulted in the identification of putative cancer stem cell populations in
several different cancers. These include cancers of the prostate, breast, colon, brain,
pancreas, head and neck, lung, skin and haematopoeitic system. Many of these cancer
stem cell enrichment markers are common to normal stem cells and to several different
cancers. For example, CD133 is a very common stem cell marker that has been identified
as being expressed in populations of breast and glial cancer stem-like cells. However, this
molecule was recently demonstrated to be of very limited use as a prostate cancer stem
cell marker (Garraway et al.).
Prostate Cancer Stem Cells
Prostate tumors are largely androgen dependent, and regress upon castration and/or
pharmacological androgen ablation. However, both the prostate and prostate tumors will
regenerate if androgen is made available again. In addition, hormone refractory prostate
cancer – in which tumors adapt to the low androgen milieu in castrate conditions –
inevitably occurs in both humans and mouse models. Thus, there must exist an androgen-
149
independent cell type with stem-like properties which can regenerate the prostate/prostate
tumor under the correct conditions. Great advances in understanding the biology of
prostate and prostate cancer stem cells have been made in recent years. Markers
identifying populations of prostate/prostate cancer cells at various stages of
differentiation have been identified (Figure 4.1). Both prostate and prostate cancer stem
cells share many features with the cells of the prostate basal layer, including expression
of cytokeratins 5 and 14, CD44, p63 and Notch1 (Tran et al., 2002). It was demonstrated
very recently that the cell of origin in human prostate cancer is in the basal layer
(Goldstein et al.). As described above, cells with the cell surface marker expression
profile CD49f
hi
Sca-1
+
CD117
+
are strongly enriched for cells with prostate/prostate
cancer stem-like properties. Single cells with this phenotype are capable of generating
functional prostates when implanted in vivo in mice (Leong et al., 2008).
150
Figure 4.1
Figure 4.1: Cell surface markers identifying prostate/prostate cancer cells as they
proceed down their differentiation pathway. Adapted from (Tran et al., 2002).
151
Notch Signaling
Notch signaling is an extremely common means by which cells communicate and alter
their gene expression profiles in response to environmental stimuli. In mammals, there
are four Notch cell surface receptors (Notch1-4), all of which share a similar molecular
structure and are all type I transmembrane proteins. Mammalian cells can express five
different Notch ligands (Jagged1-2 and Delta-like1-3), also as type I transmembrane
proteins. Autocrine and juxtacrine signaling can be mediated via interactions between
Notch receptors and their ligands. In all cases, these interactions result in a
conformational change in the Notch receptor, allowing sequential cleavage by the TNFα-
converting enzyme (TACE) and the γ-secretase complex. This results in the release of the
Notch intracellular domain (NICD) into the cytoplasm, from whence it can translocate to
the nucleus and act as a transcription factor. Pharmacological γ-secretase inhibitors are
powerful suppressors of Notch signaling, as inhibition of this enzyme prevents the release
of the NICD.
152
Hypotheses and Aims
Notch signaling is thought to play a critical role in the maintenance of both healthy and
cancerous stem cells of the haematopoeitic system (Armstrong et al., 2009) and of the
breast (Farnie et al., 2007). Notch receptors and the Notch receptor ligand Jagged1 are
expressed in cells of the basal layer of the prostate, where the prostate cancer cell of
origin was recently shown to reside (Goldstein et al.). Furthermore, Notch is known to be
expressed in every human prostate cancer cell line tested thus far (Leong and Gao, 2008).
Thus, it was hypothesized that Notch signaling plays an important role in the
maintenance of the prostate cancer stem cell phenotype. Further, it was hypothesized that
treatment of prostate cancer stem cells with γ-secretase inhibitors would disrupt the
maintenance of this phenotype, forcing prostate cancer stem cells to differentiate and thus
eliminating the pool of cells capable of regenerating the prostate tumor. Finally, it is
known that, despite the name, prostate and prostate cancer stem cells do not express
prostate stem cell antigen (PSCA), a well known prostate tumor-associated antigen
against which the excellent therapeutic prostate cancer vaccine described in Chapter Two.
However, it is also known that cells further down the prostate cancer differentiation
pathway – namely late intermediate precursors – do express PSCA (Tran et al., 2002).
Therefore it was hypothesized that forced differentiation of prostate cancer stem cells by
γ-secretase treatment may act in synergy with therapeutic cancer vaccination against
mPSCA.
153
Specific Aims:
1.) Determine whether Notch signaling can be interrupted using γ-secretase inhibitors
in TRAMP-C2 cells
2.) Determine whether successful disruption of Notch signaling in TRAMP-C2 cells
can abrogate prostate cancer stem cell maintenance and force them to differentiate
3.) Determine whether disruption of Notch signaling in TRAMP-C2 cells can act
synergistically with therapeutic vaccination directed against mPSCA
154
MATERIALS AND METHODS
Mice and cell lines
C57BL/6 mice were obtained from Taconic farms (Germantown, NY). TRAMP mice
(Greenberg et al., 1995) on the C57BL/6 background were bred at USC. Research was
conducted in compliance with the Institutional Animal Care and Use Committee
guidelines. TRAMP-C2 prostate cancer cells were cultured in IMDM medium
supplemented with 5% heat inactivated fetal calf serum (FCS) (JRH Biosciences, Lenexa,
KS), 5% NuSerum (Collaborative
Biomedical Products, Bedford, MA), 2 mM L-
glutamine, 100 μg/ml
kanamycin, 0.01 nM dihydrotestosterone (Sigma Chemical
Co. St
Louis, MO) and 5 μg/ml insulin (Sigma
Chemical Co.).
Prostasphere culture
Prostaspheres were cultured in IMDM medium supplemented with 1x B-27 (Invitrogen),
20 ng/ml EGF (Invitrogen), 20 ng/ml bFGF (RD Systems), 4 μg/ml heparin (Sigma), 2
mM glutamine (Invitrogen), 100 IU/ml penicillin/streptomycin (Invitrogen), 5 μg/ml
insulin and 0.5 μg/ml hydrocortisone. This medium is termed prostasphere medium
(PSM). In each well of a six well sterile tissue culture plate, 25,000 TRAMP-C2 cells
were plated in 4 ml of PSM. Visible prostaspheres were assayed under 10x light
microscopy after 10 days. True prostaspheres have a spherical morphology that is easily
distinguished from cell clumps by visual inspection.
155
In vitro γ-secretase inhibition
For all assays involving Notch inhibition, γ-secretase I (GSI I) was used (Calbiochem, La
Jolla, CA). For in vitro assays, 1 mg GSI I was diluted in 2.1 ml DMSO to yield a 1 mM
stock solution. This was stored in 50 μl aliquots at -80
o
C until required. GSI I stock
solution was diluted to the required assay concentration in PSM (for prostasphere assays)
or TRAMP-C2 medium (for assays involving TRAMP-C2 cells in standard culture),
taking care to include an equal amount of DMSO vector at all final concentrations of GSI
I.
Tumor challenge, vaccination and in vivo γ-secretase treatment
Male C57BL/6 mice (8-10 weeks old) were challenged subcutaneously with 5x10
5
TRAMP-C2 cells resuspended in 100 μl HBSS (Sigma). Tumor growth was monitored
twice weekly using engineer calipers. When tumors developed to approximately 20 mm
3
in volume, mice were randomized into four groups (vaccinated and control vaccinated,
each either receiving GSI I or not) such that the average tumor volume per group was 20
mm
3
. Mice were anesthetized ip with 2.4 mg ketamine (Phoenix Pharmaceutical Inc, St
Joseph, MO) and 480μg xylazine (Phoenix). DNA-gold particles were delivered to a
shaved area on the abdomen using a helium-driven gene gun (BioRad) with a discharge
pressure of 400 psi. Each mouse received 2 μg of murine PSCA cDNA vaccine. Two
days after vaccination, mice received intraperitoneal injection of either 125 μM GSI I in
100 μl 30% DMSO/PBS, or 100 μl 30% DMSO/PBS without GSI I. This was repeated
two days later. Two days after the second GSI I treatment, mice were subcutaneously
156
boosted 1 cm from the tail base with 10
6
infectious units (IU) mPSCA-VRP. As control
groups, C57BL/6 or TRAMP mice were vaccinated with pcDNA3 and boosted with 10
6
IU GFP-VRP. Survival was followed until tumors reached volumes greater than 1000
mm
3
, in accordance with our IACUC guidelines.
Real-time quantitative PCR
Single cell suspensions of cultured TRAMP-C2 cells or prostaspheres were generated by
1x trypsin digestion at 37
o
C. Total RNA was isolated using an RNeasy kit (Qiagen).
DNase-treated RNA was reverse transcribed with random hexamer primers and
SuperScript III (Invitrogen). Quantitative PCR was performed using SensiMix SYBR
QPCR Master Mix, following the manufacturer’s protocol (Bioline, Tauton, MA). Primer
sequences for GAPDH (Forward TCAATGAAGGGGTCGTTGAT, Reverse
CGTCCCGTAGACAAAATGGT), CD49f (Forward AGTGCTTCTGCCCGAGGT,
Reverse GGAGCCTCTTCGGCTTCTC), Sca-1 (Forward
GGCAGATGGGTAAGCAAAGA, Reverse CAATTACCTGCCCCTACCCT) and
PSCA (Forward TCATCTGTGCTGTGC ATGAAT, Reverse
GCTCACTGCAACCATGAAGA) were obtained from qPrimerDepot
(primerdepot.nci.nih.gov). Quantitative PCRs were performed using a CFX system (BIO-
RAD, Carlsbad, CA). The relative level of mRNA expression for each gene in TRAMP-
C2 cell/prostasphere treatment group was first normalized to the expression of GAPDH
RNA in that tumor and then normalized to the level of mRNA expression in
cells/prostaspheres treated with DMSO vector control only.
157
Western Blot
TRAMP-C2 cells were harvested by trypsin digestion and washed twice in PBS. Cellular
extracts were prepared using the Mammalian Protein Extraction Reagent (Pierce) in the
presence of 1x protease inhibitors (Pierce). Normalized aliquots of cell lysates were
electrophoresed on 7% NuPage Novex Bis-Tris gels (Invitrogen) and transferred to
nitrocellulose membranes. Western blot was performed using rabbit anti-mouse activated
Notch1 (*Notch1) primary antibody (Abcam) diluted 1:800 in StartingBlock (Pierce) and
IRDye 800-conjugated goat anti-rabbit secondary antibody (LI-COR Bioscience) diluted
1:10,000 in StartingBlock. As a housekeeping control, the blot was simultaneously
probed with mouse anti-GAPDH (Abcam) diluted 1:1,000 in StartingBlock and IRDye
680-conjugated goat anti-mouse secondary antibody (LI-COR Bioscience) diluted
1:10,000 in StartingBlock. Bands were visualized and their mean fluorescence intensity
calculated using an Odyssey system (LI-COR Bioscience).
Statistical analyses
Real-time quantitative PCR results and prostasphere numbers were analysed by one-way
ANOVA. Tumor growth was analyzed by independent t test.
158
RESULTS
Notch1 is expressed in every human prostate cancer cell line that has been tested to date
(Leong and Gao, 2008). It is known that mRNA transcripts of Notch1 can be detected in
the mouse prostate cancer cell line TRAMP-C2 (Leong and Gao, 2008). It was thus
hypothesized that Notch1 receptor expression would be detectable in TRAMP-C2 cells.
Furthermore, it was hypothesized that active Notch signaling could be abrogated in these
cells by pharmacological γ-secretase inhibition. To test these hypotheses, protein was
isolated from TRAMP-C2 cells cultured for 48 hours in the presence of 0 μM, 0.3 μM
and 0.6 μM GSI I (Calbiochem) and analyzed by fluorescent Western blot (Odyssey
system, LICOR) using an antibody specific for the activated Notch1 receptor. GSI I is
dissolved in 100% DMSO as a vector. To control for toxic effects due to the presence of
DMSO, culture media contained the same final concentration of the vector regardless of
the final concentration of GSI I. It was determined that the Notch1 receptor is expressed
by TRAMP-C2 cells (Figure 4.2). GSI I treatment made no difference to the expression
of a housekeeping control protein, GAPDH. However, a marked reduction in activated
Notch1 was detected in TRAMP-C2 cells cultured in both 0.3 μM and 0.6 μM GSI I
compared to the negative control (18.8% and 47.1%, respectively) (Figure 4.2).
159
Figure 4.2: TRAMP-C2 cells express Notch1, the activation of which can be
inhibited by a γ-secretase inhibitor. TRAMP-C2 cells were cultured for 48 hours in
media supplemented with 0 μM, 0.3 μM and 0.6 μM GSI I. Cells were harvested and
washed, then protein isolated in the presence of protease inhibitors. Western blot was
performed using rabbit anti-mouse activated Notch1 (*Notch1) primary antibody diluted
1:800 in StartingBlock (Pierce) and IRDye 800-conjugated goat anti-rabbit secondary
antibody diluted 1:10,000 in StartingBlock. As a housekeeping control, the blot was
simultaneously probed with mouse anti-GAPDH diluted 1:1,000 in StartingBlock and
IRDye 680-conjugated goat anti-mouse secondary antibody diluted 1:10,000 in
StartingBlock. Bands were visualized and their mean fluorescence intensity calculated
using the LI-COR Odyssey system. Bars represent mean fluorescence intensity of all
pixels comprising each band.
160
Figure 4.2 continued
161
Next it was determined whether the reduction in Notch1 activation mediated by GSI I
treatment of TRAMP-C2 cells could cause changes in the expression of downstream
target genes that are under the control of Notch signaling. The hairy/enhancer-of-split-1
(Hes1) gene is one such target, its expression being activated as a result of the NICD/CSL
complex binding to its promoter upon Notch signaling (Leong and Gao, 2008). Real-time
quantitative PCR analysis demonstrated that transcription of Hes1 was reduced with
increasing GSI in TRAMP-C2 cells (Figure 4.3), indicating that GSI I treatment
interfered with the Notch signaling pathway in these cells.
162
Figure 4.3
0 0.3 0.6 1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
[GSI] (uM)
Relative Hes1 mRNA
Expression
Figure 4.3: Treatment of TRAMP-C2 cells with γ-secretase inhibitors inhibits
transcription of Hes1, a critical downstream mediator of Notch signaling. TRAMP-
C2 cells were cultured for 48 hours in media supplemented with 0 μM, 0.3 μM, 0.6 μM
and 1 μM GSI I. Cells were harvested and washed, and then total mRNA isolated
(RNeasy kit, QIAGEN). Isolated RNA was quantitated (NanoDrop), and used as a
template to make cDNA (First Strand cDNA Synthesis Kit, Invitrogen) using the
supplied random hexamer primers. The cDNA was used as a template in real-time
quantitative PCR. Hes1 expression was calculated relative to GAPDH expression in the
same sample, with the relative Hes1 expression of the 0 μM GSI I treated sample being
assigned a relative expression value of 1. Bars represent mean Hes1 expression ± SEM as
determined in two independent experiments.
163
Notch signaling is required for the maintenance of a population of breast cancer stem
cells (Farnie et al., 2007). Therefore, it was hypothesized that Notch1 signaling may also
be required to maintain prostate cancer stem cells. In order to test this hypothesis, it was
first determined whether a subpopulation of TRAMP-C2 cells existed that might include
a putative prostate cancer stem cell population. It has been demonstrated that
CD49f
hi
Sca-1
+
prostate cells have a primitive phenotype and are enriched with cells with
stem-like phenotypes that are capable of tumor initiation in vivo (Goldstein et al.). In
contrast, PSCA is a marker that is highly expressed by the vast majority of TRAMP-C2
prostate tumor cells (Garcia-Hernandez Mde et al., 2008), suggesting that it is a marker
of terminal differentiation in this cell line. It was hypothesized that if Notch signaling
was required to maintain the primitive stem-like population within the overall population
of TRAMP-C2 cells, then expression of CD49f and Sca-1 should decrease whilst PSCA
expression should increase when GSI I in increasing concentrations to culture media.
TRAMP-C2 cells were cultured for 48 hours in the presence of GSI I at concentrations
ranging from 0 μM to 1 μM and analyzed by real-time quantitative PCR and flow
cytometry for expression of these markers of prostate cancer differentiation status (Figure
4.4). Expression of both CD49f (Figure 4.4a) and Sca-1 (Figure 4.4b) were both reduced
with increasing GSI I concentration, though this was not statistically significant.
However, expression of PSCA was highly upregulated in response to GSI I treatment
(Figure 4.4c, ANOVA, p = 0.0135). Specifically, the 13.1-fold increase in mean relative
PSCA expression in TRAMP-C2 cells treated with 1 μM GSI was statistically
significantly increased compared to the mean relative PSCA expression in TRAMP-C2
164
cells treated with GSI at 0 μM (1 ± 0 relative expression), 0.3 μM (1.9 ± 0.6 relative
expression) and 0.6 μM (4.1 ± 1.0 relative expression) (Figure 4.4c, Tukey’s multiple
comparison test, p < 0.05 in all cases. These data indicate that inhibition of Notch
signaling results in TRAMP-C2 cells adopting a more differentiated phenotype.
165
Figure 4.4: Treatment of TRAMP-C2 cells with a γ-secretase inhibitor causes them
to express a marker profile associated with a more differentiated state. TRAMP-C2
cells were cultured for 48 hours in media supplemented with 0 μM, 0.3 μM, 0.6 μM and 1
μM GSI I. Cells were harvested and washed, and then total mRNA isolated (RNeasy kit,
QIAGEN). Isolated RNA was quantitated (NanoDrop), and used as a template to make
cDNA (First Strand cDNA Synthesis Kit, Invitrogen) using the supplied random hexamer
primers. The cDNA was used as a template in real-time quantitative PCR. CD49f (A),
Sca-1 (B) and PSCA (C) expression was calculated relative to GAPDH expression in the
same sample, with the relative expression of the 0 μM GSI I treated sample being
assigned a relative expression value of 1 in each case. Bars represent mean target
expression ± SEM as determined in two independent experiments. The * symbol
indicates p < 0.05
166
Figure 4.4 continued
0 0.3 0.6 1
0.00
0.25
0.50
0.75
1.00
[GSI] (uM)
Relative CD49f mRNA
Expression
A
0 0.3 0.6 1
0.00
0.25
0.50
0.75
1.00
1.25
B
[GSI] (uM)
Relative Sca-1 mRNA
Expression
0 0.3 0.6 1
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
C
[GSI] uM
Relative PSCA mRNA
Expression
*
*
*
167
In order to investigate the effects of Notch inhibition on the maintenance of the stem-like
population, a functional assay of the stem cell properties was required. A contact-
independent prostate spheroid colony formation (“prostasphere”) assay was performed. In
this assay, prostate cancer cells are cultured in a specialized medium (prostasphere
medium, PSM) that maintains their undifferentiated state. Under such conditions, an
individual prostate cancer stem cell can undergo asymmetric division to generate one or
more transit amplifying cells, which in turn undergo many divisions to produce a single
prostasphere. This cannot be achieved by more differentiated cells, thus each spheroid
colony that forms represents a single prostate cancer stem cell that was seeded into the
culture medium. TRAMP-C2 cells were seeded at 2.5x10
4
cells per well in 6-well culture
plates in PSM containing GSI I at concentrations ranging from 0 μM to 1 μM, and
prostasphere colony formation followed for ten days (Figure 4.5). Statistically
significantly fewer prostaspheres formed in the presence of increasing GSI I (Figure 4.5a,
ANOVA, p = 0.0353). In addition, the prostaspheres that did form were smaller,
containing fewer cells per spheroid with increasing GSI I concentration (Figure 4.5b).
168
Figure 4.5: Treatment of TRAMP-C2 cells with a γ-secretase inhibitor results in
formation of fewer and smaller prostaspheres. TRAMP-C2 cells were cultured for 10
days in prostasphere medium supplemented with 0 μM, 0.3 μM, 0.6 μM and 1 μM GSI I.
A total of 25,000 cells were seeded into each well of a six well culture plate. Three wells
per concentration of GSI I were used. (A) The number of prostaspheres was counted at
day 10. Bars represent mean number of prostaspheres ± SEM. (B) Prostaspheres from all
wells for a given concentration were harvested and pooled together, and then were
dissociated into a single cell suspension by trypsin digestion. Cells were then counted,
and the total per prostasphere calculated. Bars represent mean number of cells per
prostasphere ± SEM. Figure is representative of three independent experiments with
similar results.
169
Figure 4.5 continued
0 0.3 0.6 1
0
5
10
15
20
A
[GSI] (uM)
Number Prostapheres
0 0.3 0.6 1
0
1000
2000
3000
4000
B
[GSI] (uM)
Cells per Prostasphere
170
Given that γ-secretase is involved in several cell signaling pathways, it was important to
test whether the effect of GSI I treatment observed was specific to the Notch signaling
pathway. To this end, TRAMP-C2 cells were transiently transfected with small
interfering RNAs (siRNAs) designed to target murine Notch1. After 48 hours, Notch1
mRNA expression was measured by real-time quantitative PCR. All three of the Notch1
siRNAs tested knocked down Notch mRNA expression in TRAMP-C2 cells (Figure 4.6a,
siRNA A, siRNA B and siRNA C yielding 79%, 56% and 44% knockdown,
respectively). When these cells were cultured for ten days in PSM, fewer prostaspheres
formed, though this was not statistically significant (Figure 4.6b, ANOVA, p = 0.3709).
Taken together, these data suggest that Notch signaling plays at least some role in the
proper control of prostasphere formation, indicating a role in the control of prostate
cancer cell differentiation.
171
Figure 4.6: Transient siRNA-mediated knockdown of Notch partially recapitulates
the γ-secretase-mediated effect on prostasphere formation. TRAMP-C2 cells were
transiently transfected with one of three different siRNAs directed against Notch1
(Ambion). As negative controls, cells were transiently transfected in the same manner
using a scrambled siRNA, or left untreated. After 48 hours, cells were harvested and
washed, and then total mRNA isolated (RNeasy kit, QIAGEN). Isolated RNA was
quantitated (NanoDrop), and used as a template to make cDNA (First Strand cDNA
Synthesis Kit, Invitrogen) using the supplied random hexamer primers. The cDNA was
used as a template in real-time quantitative PCR. (A) Notch1 expression was calculated
relative to GAPDH expression in the same sample, with the relative expression of the
untreated cells being assigned a relative expression value of 1. Bars represent mean
Notch1 expression ± SEM. (B) Transfected cells were cultured for 10 days in
prostasphere medium. A total of 25,000 cells were seeded into each well of a six well
culture plate. Three wells per treatment group were used. The number of prostaspheres
was counted at day 10. Bars represent mean number of prostaspheres ± SEM.
172
Figure 4.6 continued
No Tx
Notch1 siRNA A
Notch1 siRNA B
Notch1 siRNA C
Scrambled siRNA
0.00
0.25
0.50
0.75
1.00
1.25
1.50
A
Relative Notch1
Expression
No Tx
Notch1 siRNA A
Notch1 siRNA B
Notch1 siRNA C
Scrambled siRNA
0
5
10
15
20
B
Number Prostaspheres
173
It was a possibility that Notch signaling only played a role in prostasphere formation in
the TRAMP-C2 cell line, rather than being a feature common to all TRAMP prostate
tumors. To address this, single cell suspensions were made from spontaneously arising
prostate tumors isolated from four TRAMP mice and cultured in PSM containing 0 μM to
1 μM GSI. In all cases, prostaspheres were formed by cancer cells from TRAMP mice
with similar efficiency as observed with cells of the TRAMP-C2 line (Figure 4.7a). In
addition, prostasphere size was reduced by GSI I treatment in a similar manner to that
observed in the TRAMP-C2 cell line (Figure 4.7b, ANOVA, p < 0.0001).
174
Figure 4.7: Treatment of primary prostate cancer cells from TRAMP mice with γ-
secretase inhibitor results in formation of fewer and smaller prostaspheres. Primary
prostate tumors were isolated from four TRAMP mice. Single cell suspensions of
prostate tumor cells were made via manual dissociation and enzymatic dissociation with
0.1% dispase. Cells were counted, and were cultured for 10 days in prostasphere medium
supplemented with 0 μM, 0.3 μM, 0.6 μM and μM GSI I. A total of 25,000 cells were
seeded into each well of a six well culture plate. Eight wells per concentration of GSI I
were used. (A) The number of prostaspheres was counted at day 10. Bars represent mean
number of prostaspheres ± SEM. (B) Representative images of prostaspheres formed
from primary prostate cancer cells grown in prostasphere media supplemented with 0
μM, 0.3 μM and 0.6 μM GSI I. The * and *** symbols represent p < 0.05 and p < 0.001,
respectively.
175
Figure 4.7 continued
0 0.3 0.6 1
0
5
10
15
20
A
[GSI] (uM)
Number Prostaspheres
***
***
***
*
B
[GSI] (uM)
0 0.3 0.6
B
[GSI] (uM)
0 0.3 0.6
176
To determine whether Notch inhibition caused the cells comprising prostaspheres to be
more differentiated in a similar manner to what was observed in TRAMP-C2 cells in
normal tissue culture, prostospheres cultured for ten days in the presence of GSI I at
concentrations ranging from 0 μM to 1 μM. Expression of Sca-1 was substantially
reduced in prostaspheres with increasing GSI I concentration (Figure 4.8a, ANOVA, p =
0.0124). Specifically, mean Sca-1 expression was statistically significantly reduced in
prostaspheres treated with 0.3 μM and 0.6 μM GSI compared to mean Sca-1 expression
in untreated prostaspheres (Figure 4.8a, Tukey’s multiple comparison test, p < 0.05 in
both cases). Interestingly, expression of PSCA was also reduced in prostaspheres with
increasing GSI I concentration (Figure 4.8b, ANOVA, p = 0.0019). Specifically, mean
PSCA expression was statistically significantly reduced in prostaspheres treated with 0.3
μM and 0.6 μM GSI compared to mean PSCA expression in untreated prostaspheres
(Figure 4.8a, Tukey’s multiple comparison test, p < 0.05 and p < 0.01, respectively).
Unlike normal TRAMP-C2 culture medium, no dihydrotestosterone is added to PSM.
With this in mind, these data suggest that the large increase in PSCA expression observed
in TRAMP-C2 cells cultured in the presence of GSI must occur in androgen-dependent
cells.
177
Figure 4.8: Growth of prostaspheres in the presence of a γ-secretase inhibitor causes
them to express a marker profile associated with a more differentiated state.
TRAMP-C2 cultured for 10 days in prostasphere medium supplemented with 0 μM, 0.3
μM, 0.6 μM and 1 μM GSI I. A total of 25,000 cells were seeded into each well of a six
well culture plate. Three wells per concentration of GSI I were used. Cells were harvested
and washed, and then total mRNA isolated (RNeasy kit, QIAGEN). Isolated RNA was
quantitated (NanoDrop), and used as a template to make cDNA (First Strand cDNA
Synthesis Kit, Invitrogen) using the supplied random hexamer primers. Insufficient
material was recovered from the 1 μM group to proceed with this step. The cDNA was
used as a template in real-time quantitative PCR. Sca-1 (A) and PSCA (B) expression
was calculated relative to GAPDH expression in the same sample, with the relative
expression of the 0 μM GSI I treated sample being assigned a relative expression value of
1 in each case. Bars represent mean Sca-1/PSCA expression ± SEM as determined in two
independent experiments. The * and ** symbols represent p < 0.05 and p < 0.01,
respectively.
178
Figure 4.8 continued
0 0.3 0.6
0.00
0.25
0.50
0.75
1.00
1.25
A
[GSI] (uM)
Relative Sca-1
Expression
*
*
0 0.3 0.6
0.00
0.25
0.50
0.75
1.00
1.25
B
[GSI] (uM)
Relative PSCA
Expression
*
**
179
Taken together, the data presented above indicate that Notch signaling is required for the
maintenance of cells capable of forming spheroid colonies. In addition, the data indicate
that Notch inhibition tends to force TRAMP-C2 cells in both standard culture and in
prostaspheres to adopt a more differentiated phenotype. Given these considerations, it
was hypothesized that Notch signaling was required to maintain a population of stem-like
prostate cancer cells. Stem cell populations self-renew by undergoing an asymmetric
division that yields an identical stem cell and a more differentiated early intermediate
precursor (transit amplifying) cell. It was hypothesized that Notch signaling maintained
the prostate cancer stem cell phenotype by allowing one daughter cell to maintain the
stem-like phenotype, i.e. Notch signaling is directly responsible for self-renewal during
the asymmetric division. To test this, an experiment was carried out to determine whether
the self-renewal capacity of prostasphere forming cells was maintained after γ-secretase
inhibitor treatment. TRAMP-C2 cells were seeded at 2.5x10
4
cells per well in 6-well
culture plates in PSM containing GSI I at concentrations ranging from 0 μM to 1 μM, and
prostasphere grown for ten days. A known number of prostaspheres (“PS In”) from wells
of each concentration were harvested and pooled together, and then were dissociated into
a single cell suspension by trypsin digestion. All recovered cells from prostaspheres at
each GSI I treatment concentration were used to reseed six wells of a six well tissue
culture plate containing fresh prostasphere medium without GSI I. Cells were cultured for
an additional ten days, and the total number of prostaspheres recovered from reseeded
cells from prostaspheres treated with each GSI I treatment concentrations was calculated
(“PS Out”). Most prostaspheres are of clonal origin, i.e. each is formed from a single
180
prostate cancer stem cell (Garraway et al.). Thus, it was expected that each cell that
formed a prostasphere in the absence of GSI in the first generation would form a new
second generation sphere. This was indeed observed, with cells isolated from 53 primary
prostaspheres yielding 56 secondary prostaspheres (Figure 4.9). The fact that more
secondary spheroids formed indicates that either some rare primary spheroids were
amalgams formed from two or more spheroids derived from different stem cells, or that
some stem cells failed to form spheroids in the first generation but nevertheless were still
viable when transferred to the secondary culture. If as hypothesized Notch signaling was
required for prostate cancer stem cell self-renewal, then fewer spheroids should have
formed in the second generation when the first was treated with GSI. However, this was
not the case. At every concentration of GSI I treated, the number of secondary spheroids
formed was equal to or slightly greater than the number of primary prostaspheres
harvested (Figure 4.9). These data indicate that Notch signaling is not required for self-
renewal of TRAMP-C2 prostate cancer stem cells.
181
Figure 4.9
0 0.3 0.6 1
0
10
20
30
40
50
60
70
80
90
PS In
PS Out
[GSI] uM
Number Prostaspheres
Figure 4.9: Notch signaling is not required for the maintenance of the founding
prostate cancer stem cell in prostaspheres. TRAMP-C2 cells were cultured for 10 days
in prostasphere medium supplemented with 0 μM, 0.3 μM, 0.6 μM and 1 μM GSI I. A
total of 25,000 cells were seeded into each well of a six well culture plate. Three wells
per concentration of GSI I were used. A known number of prostaspheres (“PS In”) from
wells for a given concentration were harvested and pooled together, and then were
dissociated into a single cell suspension by trypsin digestion. All recovered cells from
prostaspheres at each GSI I treatment concentration were used to reseed six wells of a six
well tissue culture plate containing fresh prostasphere medium without GSI I. Cells were
cultured for an additional ten days, and the total number of prostaspheres recovered from
reseeded cells from prostaspheres treated with each GSI I treatment concentrations was
calculated (“PS Out”).
182
Given the data presented above, it was hypothesized that γ-secretase inhibitor treatment
may act synergistically with therapeutic vaccination against PSCA to inhibit TRAMP-C2
tumor growth in vivo, for two reasons. Firstly, the increased expression of PSCA by
TRAMP-C2 cells treated with GSI I may make them more immunogenic targets for
PSCA-specific CD8
+
cytotoxic T cells induced by vaccination against that TAA.
Secondly, the inhibition of the ability of the prostate cancer stem cell population to
generate prostaspheres suggests that GSI I treatment may attenuate the tumor’s ability to
replace cells killed by the antitumor immune response. To test this, four groups of 8-10
week old C57BL/6 mice were challenged with 5x10
5
TRAMP-C2 cells subcutaneously in
the flank. When tumors reached approximately 20 mm
3
in volume, mice were either
DNA vaccinated with 2 μg plasmid encoding mPSCA or were mock vaccinated with 2 μg
empty plasmid vector via helium-driven gene gun. Two days after vaccination, mice were
injected intraperitoneally with either GSI I sufficient to achieve a physiological
concentration of 5 μM in a 25 g mouse, or an equal volume of vector (125 μl 30% DMSO
in PBS). This was repeated once, each dose being administered two days apart. Two days
after the final dose of GSI I, mice were boosted by subcutaneous injection of either 10
6
VRP encoding mPSCA (for mice vaccinated with plasmid encoding mPSCA) or 10
6
VRP
encoding GFP (as negative controls). In control vaccinated mice, GSI I treatment alone
was not capable of statistically significantly reducing mean tumor volume (537 ± 48
mm
3
) compared to vector treated controls (497 ±160 mm
3
) at 42 days post challenge
(Figure 4.11a). Without GSI I treatment, mean TRAMP-C2 tumor volume was
substantially retarded in mPSCA vaccinated mice (248 ± 118 mm
3
) compared to control
183
vaccinated mice (497 ±160 mm
3
) by 42 days post challenge, though this was not
statistically significant (Figure 4.11b). Contrary to the expected findings, mean tumor
volume was not statistically significantly changed in GSI I treated mPSCA vaccinated
mice (634 ± 111 mm
3
) compared to either GSI I treated control vaccinated mice (497
±160 mm
3
) or vector treated control vaccinated mice (497 ± 160 mm
3
). Surprisingly,
these data indicate that the reduction in mean tumor volume observed in vector treated
mPSCA vaccinated mice (248 ± 118 mm
3
) is lost when mPSCA vaccination is combined
with GSI I treatment (634 ± 111 mm
3
) (Figure 4.11c, independent t test, p = 0.0499).
184
Figure 4.10: Treatment with γ-secretase inhibitor cannot attenuate TRAMP-C2
tumor growth in vivo and inhibits the antitumor immune response elicited by
vaccination against mPSCA. 8-10 week old C57BL/6 mice were challenged with 5x10
5
TRAMP-C2 cells subcutaneously in the flank. When tumors reached approximately 20
mm
3
in volume (day 27 post challenge), groups of mice were either DNA vaccinated with
2 μg plasmid encoding mPSCA or were mock vaccinated with 2 μg empty plasmid vector
via helium-driven gene gun. Two days after vaccination (day 29 post challenge), mice
were injected intraperitoneally with either GSI I sufficient to achieve a physiological
concentration of 5 μM in a 25 g mouse, or an equal volume of DMSO vector (125 μl 30%
DMSO in PBS). This was repeated on day 31 post challenge. Two days after the final
dose of GSI I (day 33 post challenge), mice were boosted by subcutaneous injection of
either 10
6
VRP encoding mPSCA (for mice vaccinated with plasmid encoding mPSCA)
or 10
6
VRP encoding GFP (as negative controls). (A) Comparison of TRAMP-C2 tumor
growth between control vaccinated mice treated with GSI (n = 3) and DMSO vector (n =
7). (B) Comparison of TRAMP-C2 tumor growth between control vaccinated mice
treated with DMSO vector (n = 7) and mPSCA vaccinated mice treated with DMSO
vector (n = 4). (C) The mean TRAMP-C2 tumor volume of mPSCA vaccinated mice
treated with GSI (n = 6) was statistically significantly increased compared to that of
mPSCA vaccinated mice treated with DMSO vector (n = 4) (Independent t test, p < 0.05).
The * symbol indicates p < 0.05).
185
Figure 4.10 continued
186
DISCUSSION
Cancer stem cells are emerging as a critical population confounding efforts to truly
eradicate tumors. Given their unique characteristics that protect them from conventional
anticancer therapies, and the fact that a single cancer stem cell can initiate the growth (or
regrowth) of a tumor, understanding their biology is an extremely high priority in
oncology.
The data presented here demonstrate a central role for Notch signaling in maintaining the
homeostasis of the differentiation pathway down which prostate cancer cells proceed. It
was demonstrated that Notch1 is expressed by TRAMP-C2 cells (Figure 4.2), and that γ-
secretase inhibition can abrogate signaling via Notch1 (Figures 4.2 and 4.3). In standard
TRAMP-C2 tissue culture γ-secretase inhibition led to a loss of expression of markers
(Sca-1 and CD49f) associated with a primitive fraction of prostate cancer cells within
which the prostate cancer stem cell population is thought to reside (Goldstein et al.). In
addition, γ-secretase inhibition dramatically upregulated expression of PSCA, a marker
known to be expressed by the vast majority of cells in TRAMP mice (Garcia-Hernandez
Mde et al., 2008). These findings led to the investigation of the role that Notch signaling
was playing in prostate cancer stem cell maintenance and differentiation.
In order to be considered a stem cell, a cell must demonstrate the ability to self-renew and
must have the capacity to generate more differentiated daughter cells. In vivo assays to
demonstrate these properties are time consuming and expensive. An alternative has been
187
developed in which stem cells cultured under certain conditions will maintain their
undifferentiated status and form anchorage-independent spheroid colonies. This
methodology has been used to identify and study stem cells (both healthy and cancerous)
in breast (Dontu et al., 2003), brain (Reynolds and Weiss, 1996) and most recently
prostate (Wang, 2009). The colonies that form in these assays from the stem cells/cancer
stem cells derived from each of these tissue types are termed mammospheres,
neurospheres and prostaspheres, respectively. It has been demonstrated that human
prostaspheres derived from a single cell contain a heterogeneous population of
proliferating and differentiating prostate cancer cells (Bisson and Prowse, 2009). Notch
signaling was shown to have a role in the maintenance of a breast cancer stem cell
population (Farnie et al., 2007). Given that expression of differentiation markers seemed
to be affected by blockage of Notch signaling in cultured TRAMP-C2 cells, it was
investigated whether Notch had a similar role in these cells. Treatment with a γ-secretase
inhibitor did inhibit the formation of prostaspheres in anchorage-independent assays. Not
only did fewer prostaspheres form in the presence of γ-secretase inhibition, those that did
form were smaller than those grown in the absence of Notch inhibition (Figure 4.5). A
similar phenotype was observed when Notch signaling was interrupted by siRNA-
mediated knockdown of Notch1 mRNA (Figure 4.6). However, this effect was not as
pronounced as that observed with γ-secretase inhibition. There are a number of possible
explanations for this. The first is that γ-secretase is acting on another cell signaling
pathway in addition to Notch signaling that is also involved in prostasphere formation.
However, the transient nature of the transfection protocol followed, the fact that TRAMP-
188
C2 cells also express Notch2 in addition to Notch1 (data not shown) and the partial
knockdown of Notch1 mRNA means that Notch1 signaling might not have been as
heavily abrogated by siRNA as it is with γ-secretase inhibition, perhaps explaining the
partial response observed. Though this is a concern, the data presented here do indicate
that at least part of the effect of the γ-secretase inhibitor on prostasphere formation was
due to the compound’s effect on Notch signaling.
Given that immortalized cancer cell lines can develop mutations in vitro that confer upon
them characteristics not normally seen in similar primary tumors, the effect of γ-secretase
inhibition on the ability of cancer cells isolated from primary mouse prostate tumors to
form prostaspheres in culture was investigated. In all four mice tested, single cells
isolated from their prostate tumors were capable of forming prostaspheres at a frequency
comparable to the TRAMP-C2 cell line (Figure 4.7). Furthermore, prostasphere
formation was inhibited by γ-secretase treatment in all four mice in a manner that
recapitulated the observations made in the TRAMP-C2 cell line (Figure 4.7). These data
indicate that the phenomenon observed is not merely an artifact of the TRAMP-C2 cell
line, but is a fundamental property of TRAMP mouse prostate tumors.
The initial hypothesis being tested was that Notch is required for the maintenance of the
prostate cancer stem cell population by allowing one daughter cell in the first asymmetric
division to maintain the stem cell phenotype. In order to test the hypothesis, an attempt
was made to grow secondary prostaspheres from single cells isolated from prostaspheres
189
that had been grown in the presence or absence of γ-secretase inhibition. Without any
intervention, by definition the prostate cancer stem cell responsible for the formation of
the primary prostasphere must have self-renewed during the growth of that spheroid, and
should maintain its “stemness”. Thus, when recultured in fresh PSM without GSI-I, that
prostasphere-initiating cell should have an identical capacity to make a secondary
spheroid colony. If Notch signaling is required for one cell to keep the stem cell
phenotype, then this cell type would be lost upon γ-secretase treatment and no (or fewer)
secondary prostaspheres would form. This was not the case; at least one secondary
prostasphere formed per primary prostasphere treated with GSI I.
A second hypothesis is thus proposed. Notch signaling is required in the first asymmetric
division to allow one daughter cell to adopt a different (i.e. more differentiated)
phenotype than that of the parental stem cell. This is much more in keeping with the data
obtained. The limited growth of prostaspheres in the presence of γ-secretase is due to a
lack of formation of early intermediate precursors and therefore late intermediate
precursors (which form the the bulk of prostasphere’s mass). Interestingly, late
intermediate precursors (produced as the result of asymmetric divisions of early
intermediate precursors) express PSCA. Thus, the reduction of PSCA observed in GSI I-
treated prostaspheres (Figure 4.8b) is due to a lack of early intermediate precursor cells to
drive the production of PSCA-expressing late intermediate precursors. This hypothesis
also helps to explain the dramatic relative increase in PSCA expression in GSI I-treated
TRAMP-C2 cells (Figure 4.4c). The vast majority of TRAMP-C2 cells in standard
190
culture express both PSCA and the AR. They are androgen dependent, and to facilitate
their growth dihydrotestosterone is added to TRAMP-C2 medium. These terminally
differentiated prostate tumor cells do not express Notch and will not be affected by GSI I
treatment. Under such conditions, they will proliferate readily. In contrast, GSI I
treatment will inhibit the proliferation and generation of more primitive intermediate
precursor cells. Thus, proportionately more PSCA-expressing cells are present in culture
under conditions inhibiting Notch function. A schematic of this concept is shown in
Figure 4.11.
Interestingly, this model that Notch signaling is required for the differentiation of
primitive prostate cancer cells reconciles the data presented above with findings from
other studies, which at first glance seem completely contrary to the observations made
here. The first was a study in which the constitutive expression of the Notch intracellular
domain (NICD) in several different prostate cancer cell lines (DU145, LNCaP and PC3)
caused growth arrest (Shou et al., 2001). The second was a study in which the
constitutive expression of the Notch intracellular domain (NICD) in prostate stem cells
resulted in the inhibition of prostasphere formation, and disrupted the formation of
prostatic acini in in vivo prostate reconstitution assays (Shahi et al.). Under normal
circumstances, the signals given to cells that cause them to undergo asymmetric division
are spatially regulated in the stem cell niche. The daughter stem cell within the niche is
not exposed to Notch ligands, and therefore maintains its stemness. On the contrary, the
daughter cell that is pushed out of the niche during division is forced into a new
191
microenvironment, in which it is exposed to Notch ligands (possibly Jagged1, known to
be expressed in the prostate basal layer (Tran et al., 2002)). Notch signaling in this
daughter cell is thus activated, and it becomes more differentiated. If this hypothesis is
correct, then constitutive Notch signaling will bypass the delicate spatial regulation of
Notch signaling. In this case, both daughter cells have their Notch pathways activated,
thus both differentiate. As a result, there are no prostate cancer stem cells to drive the
growth of the cell line in vitro (Shou et al., 2001). In addition, without a prostate stem
cell population, prostate regeneration cannot occur and nor can prostaspheres form (Shahi
et al.). The apparent role of Notch signaling in the differentiation of primitive prostate
cells can therefore explain why both inhibition of Notch signaling and its constitutive
activation can have similar phenotypic effects.
Figure 4.11
= Terminal tumor cell
Notch- PSCA+ AR+
Asymmetrical PCSC and
intermediate precursor division
allows unlimited tumor growth in
presence of androgen.
All cell types proliferate
Unlimited
self-
renewal
Limited
self-
renewal
Very
limited
self-
renewal
GSI limits asymmetrical PCSC
and intermediate precursor
division, prevents unlimited tumor
growth. Few precursors to drive
proliferation of terminal AR
+
cells
. Only late intermediate
precursors and terminal tumor
cells proliferate
Unlimited
self-
renewal
Limited
self-
renewal
Very
limited
self-
renewal
GSI
GSI
Asymmetrical PCSC and
intermediate precursor division
allows growth of prostasphere.
Growth limited by requirement of
terminal cells of androgen.
Only AR negative precursor
cells proliferate
Unlimited
self-
renewal
Limited
self-
renewal
Very
limited
self-
renewal
GSI limits asymmetrical PCSC and
intermediate precursor division
allows prevents unlimited tumor
growth. Limited precursors to drive
proliferation of terminal AR
+
cells.
Virtually no cells proliferate
Unlimited
self-
renewal
Limited
self-
renewal
GSI
GSI
TRAMP-C2 Media
Without GSI
TRAMP-C2 Media
With GSI
Prostasphere Media
Without GSI
Prostasphere Media
With GSI
= PCSC
= Early intermediate
precursor
= Late intermediate
precursor
Notch+ PSCA- AR- Notch- PSCA+ AR- Notch+ PSCA- AR-
Key:
= Terminal tumor cell
Notch- PSCA+ AR+
Asymmetrical PCSC and
intermediate precursor division
allows unlimited tumor growth in
presence of androgen.
All cell types proliferate
Unlimited
self-
renewal
Limited
self-
renewal
Very
limited
self-
renewal
GSI limits asymmetrical PCSC
and intermediate precursor
division, prevents unlimited tumor
growth. Few precursors to drive
proliferation of terminal AR
+
cells
. Only late intermediate
precursors and terminal tumor
cells proliferate
Unlimited
self-
renewal
Limited
self-
renewal
Very
limited
self-
renewal
GSI
GSI
Asymmetrical PCSC and
intermediate precursor division
allows growth of prostasphere.
Growth limited by requirement of
terminal cells of androgen.
Only AR negative precursor
cells proliferate
Unlimited
self-
renewal
Limited
self-
renewal
Very
limited
self-
renewal
GSI limits asymmetrical PCSC and
intermediate precursor division
allows prevents unlimited tumor
growth. Limited precursors to drive
proliferation of terminal AR
+
cells.
Virtually no cells proliferate
Unlimited
self-
renewal
Limited
self-
renewal
GSI
GSI
TRAMP-C2 Media
Without GSI
TRAMP-C2 Media
With GSI
Prostasphere Media
Without GSI
Prostasphere Media
With GSI
= PCSC
= Early intermediate
precursor
= Late intermediate
precursor
Notch+ PSCA- AR- Notch- PSCA+ AR- Notch+ PSCA- AR-
Key:
Figure 4.11: Proposed scheme of the role of Notch signaling in prostate cancer stem cell differentiation and
proliferation.
192
193
In the presence of androgen – such as in non-castrated male mice – γ-secretase inhibition
promotes the expansion of PSCA expressing cells and inhibits the proliferation of the
intermediate precursor cells that ultimately drive tumor growth. Thus, it was
hypothesized that treatment of TRAMP-C2 tumor-bearing mice with γ-secretase inhibitor
would be synergistic with therapeutic vaccination against mPSCA, as this would result in
more immunogenic tumors which would grow more slowly. C57BL/6 mice were
challenged with TRAMP-C2 tumors, and either DNA vaccinated against mPSCA or were
given an empty vector mock DNA vaccine. Between DNA priming and VRP boosting
eight days later, mice were either treated with GSI I (at a dose equivalent of five times the
maximum used in the in vitro studies presented here) or were injected with a vector
control. Treatment with γ-secretase alone did not change the rate of tumor growth (Figure
4.10), indicating that either the dosage of GSI I reaching the tumor cells in vivo is not as
high as that reaching them in the in vitro studies presented here, or that its in vivo half life
is not as long as that observed in vitro. Though mPSCA vaccination in the absence of
GSI I treatment did inhibit TRAMP-C2 tumor growth as expected, this effect was
completely lost when mPSCA vaccination was combined with γ-secretase treatment.
Given the rapid growth of TRAMP-C2 tumors in vivo it was necessary to administer GSI
I in the middle of the immunization protocol. Unfortunately, this meant that the well-
documented negative effects of γ-secretase inhibition on T cell activation and
proliferation far outweighed its potential benefits on changing the phenotype of the tumor
itself. If, as originally hypothesized, γ-secretase inhibition had permanent deleterious
effects on the prostate cancer stem cell population, then it could still be conceivable that
194
GSI I could be successfully combined with therapeutic cancer vaccination. There would
need to be a significant delay in the administration of the γ-secretase inhibitor and the
onset of vaccination, requiring the use of a relatively slow-growing prostate tumor system
such as the autochthonous TRAMP model of prostate cancer. However, given the data
presented here that γ-secretase inhibition does not permanently remove the prostate
cancer stem cell population – and that they continue to promote the outgrowth of prostate
tumor structures as soon as released from Notch inhibition – this is unlikely to be of
significant clinical value. The results presented here suggest that forced Notch signaling
would eliminate the prostate cancer stem cell population. It would be difficult to use this
finding clinically, but it is highly significant nonetheless.
Conclusion
This study demonstrates the critical role of Notch signaling in the homeostasis of prostate
cancer development and differentiation. Inhibition of Notch signaling can slow the
growth of tumor cells and temporarily limit the growth of prostaspheres in culture.
Intriguingly, Notch is not required for the maintenance of the prostate cancer stem cell
population as was initially hypothesized. These cells are fully capable of self-renewal and
of forming complex prostaspheres consisting of differentiated daughter cells even after
exposure to high levels of Notch inhibition. Thus, γ-secretase inhibitors alone are likely
to be of very limited benefit in the treatment of prostate cancer. Given the failure to
remove the prostate cancer stem cell population that do not express common prostate
cancer tumor-associated antigens, and the extremely deleterious effects of Notch
195
inhibition on T cell function, γ-secretase inhibition is not a good candidate for
combination with therapeutic vaccination against prostate tumor associated antigens
either. The data presented here indicate that Notch signaling is in fact required for
differentiation of prostate/prostate cancer transit amplifying cells. This is a highly
significant and unexpected finding, and should guide the field in developing methods –
for example, forced expression of Notch receptor ligands within the prostate cancer stem
cell niche – which exploit this role of Notch signaling in order to abrogate the function of
prostate cancer stem cells.
196
RESEARCH SUMMARY
The projects comprising this doctoral thesis focused on the mechanisms by which
therapeutic cancer vaccination can fail, and means by which to target these mechanisms
in order to improve vaccine efficacy.
It was demonstrated that vaccination against the prostate tumor associated antigen PSCA
using a unique heterologous DNA prime/VEE virus replicon particle boost at the earliest
stage of carcinogenesis yields vastly superior protection against prostate cancer than
immunization carried out once invasive cancer has already developed. The inferior
protection mediated by late vaccination is likely due to the accumulation of
immunosuppressive cells and molecules within the prostate tumor as it advances. This is
significant because most cancer immunotherapy clinical trials to date have involved
terminally ill human patients and the results have almost universally been disappointing.
These patients have advanced cancer and are frequently immune compromised. It has
become apparent that while the vaccines themselves have the potential to elicit potent
anti-tumor immune responses, they must be used in patients that are capable of mounting
a robust immune response if they are to mediate inhibition of tumor growth. The
compromised immunity in patients with advanced cancer is largely unavoidable and,
currently, untreatable. This is broadly true even in instances where conventional
anticancer treatments have been used to generate a scenario of minimal residual disease
in which to administer immunotherapy. Tumor- and treatment-mediated immune
suppression in cancer patients can be avoided by vaccinating them early in disease,
197
before other treatments are dispensed and prior to the establishment of systemic immune
failure by the tumor.
One of the most significant cell types that mediate immune suppression is the regulatory
T cell. It was shown here that these cells accumulate in the prostate tumors as they
advance. A novel murine model – the TRAMP DEREG mouse – was developed in order
to test whether these cells are actually responsible for limiting the immune responses to
therapeutic vaccinations. The TRAMP DEREG mouse spontaneously develops prostate
cancer, is immunologically intact, and can be depleted of FOXP3-expressing Tregs at the
discretion of the researcher. As such, they represent an excellent tool with which to
investigate the role of regulatory T cells in responses to therapeutic prostate cancer
vaccines. Even when TRAMP DEREG mice were vaccinated at eight weeks – prior to
the establishment of a strong tumor-mediated immunosuppressive network – the response
to vaccination was modestly improved. Further work is being carried out to establish
whether this improvement will be even more significant if Tregs are depleted from mice
with highly suppressive prostate tumors.
Tumor growth and regeneration depends upon a small subset of cancer stem cells. These
cells are resistant to most anticancer therapies, including immunotherapy. It was known
that Notch signaling was important in maintaining the stem cell population in other
cancers. Thus, it was investigated whether there was a similar role for Notch in
maintaining prostate cancer stem cells, and if so whether inhibition of Notch signaling
198
could be exploited in order to render tumors more vulnerable to immunotherapy. It was
determined that Notch signaling is actually required for differentiation of primitive
prostate cancer cells. Thus, inhibition of Notch signaling cannot eradicate the true
prostate cancer stem cell population and thus will likely not be of clinical benefit. In
addition, the severe deleterious effects of Notch inhibition on the immune response
elicited by therapeutic vaccination render it a poor candidate for combination with
immunotherapy. However, these results have shed light on the critical role of Notch
signaling in prostate cancer cell differentiation, and thus on prostate tumor growth and
recurrence. Thus, these studies will guide the field in targeting Notch signaling in
prostate cancer in a manner that is capable of disrupting tumor outgrowth and
regeneration.
Taken together, the findings presented here indicate that the best possible time to apply
immunotherapies is at the very onset of carcinogenesis. This is the polar opposite of the
paradigm that is currently followed. Therefore, it is proposed here that clinical trials of
cancer vaccines should be carried out in the preventive setting, when cancer patients are
first diagnosed with disease, in order to stand the best chance of being successful.
199
RELATED PUBLICATIONS BY THE AUTHOR
1. Garcia-Hernandez Mde, L., A. Gray, B. Hubby, and W. M. Kast. 2007. In vivo
effects of vaccination with six-transmembrane epithelial antigen of the prostate: a
candidate antigen for treating prostate cancer. Cancer Res 67:1344-1351
2. Garcia-Hernandez Mde, L., A. Gray, B. Hubby, O. J. Klinger, and W. M. Kast.
2008. Prostate stem cell antigen vaccination induces a long-term protective
immune response against prostate cancer in the absence of autoimmunity. Cancer
research 68:861-869.
3. Gray, A., A. B. Raff, M. Chiriva-Internati, S. Y. Chen, and W. M. Kast. 2008. A
paradigm shift in therapeutic vaccination of cancer patients: the need to apply
therapeutic vaccination strategies in the preventive setting. Immunol Rev 222:316-
327.
4. Raff, A. B., A. Gray, and W. M. Kast. Prostate stem cell antigen: a prospective
herapeutic and diagnostic target. Cancer letters 277:126-32.
5. Koh, Y. T., A. Gray, S. A. Higgins, B. Hubby, and W. M. Kast. 2009. Androgen
ablation augments prostate cancer vaccine immunogenicity only when applied
after immunization. Prostate 69:571-584.
6. Gray, A., M. de la Luz Garcia-Hernandez, M. van West, S. Kanodia, B. Hubby,
and W. M. Kast. 2009. Prostate cancer immunotherapy yields superior long-term
survival in TRAMP mice when administered at an early stage of carcinogenesis
prior to the establishment of tumor-associated immunosuppression at later stages.
Vaccine 27 Suppl 6:G52-59.
7. Gray, A., L. Yan, and W. M. Kast. Prevention is better than cure: the case for
clinical trials of therapeutic cancer vaccines in the prophylactic setting. Molecular
interventions 10:197-203.
200
REFERENCES
Antonia, S.J., Mirza, N., Fricke, I., Chiappori, A., Thompson, P., Williams, N., Bepler,
G., Simon, G., Janssen, W., Lee, J.H., et al. (2006). Combination of p53 cancer vaccine
with chemotherapy in patients with extensive stage small cell lung cancer. Clin Cancer
Res 12, 878-887.
Apostolou, I., Sarukhan, A., Klein, L., and von Boehmer, H. (2002). Origin of regulatory
T cells with known specificity for antigen. Nat Immunol 3, 756-763.
Arlen, P.M., Gulley, J.L., Parker, C., Skarupa, L., Pazdur, M., Panicali, D., Beetham, P.,
Tsang, K.Y., Grosenbach, D.W., Feldman, J., et al. (2006). A randomized phase II study
of concurrent docetaxel plus vaccine versus vaccine alone in metastatic androgen-
independent prostate cancer. Clin Cancer Res 12, 1260-1269.
Arlen, P.M., Gulley, J.L., Todd, N., Lieberman, R., Steinberg, S.M., Morin, S., Bastian,
A., Marte, J., Tsang, K.Y., Beetham, P., et al. (2005). Antiandrogen, vaccine and
combination therapy in patients with nonmetastatic hormone refractory prostate cancer. J
Urol 174, 539-546.
Armstrong, F., Brunet de la Grange, P., Gerby, B., Rouyez, M.C., Calvo, J., Fontenay,
M., Boissel, N., Dombret, H., Baruchel, A., Landman-Parker, J., et al. (2009). NOTCH is
a key regulator of human T-cell acute leukemia initiating cell activity. Blood 113, 1730-
1740.
Arnoldussen, Y.J., Wang, L., and Saatcioglu, F. Regulation of apoptosis by androgens in
prostate cancer cells. Methods in molecular biology (Clifton, NJ 776, 349-360.
Bahrenberg, G., Brauers, A., Joost, H.G., and Jakse, G. (2000). Reduced expression of
PSCA, a member of the LY-6 family of cell surface antigens, in bladder, esophagus, and
stomach tumors. Biochem Biophys Res Commun 275, 783-788.
Bennett, C.L., Christie, J., Ramsdell, F., Brunkow, M.E., Ferguson, P.J., Whitesell, L.,
Kelly, T.E., Saulsbury, F.T., Chance, P.F., and Ochs, H.D. (2001). The immune
dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by
mutations of FOXP3. Nat Genet 27, 20-21.
201
Berd, D., Sato, T., Cohn, H., Maguire, H.C., Jr., and Mastrangelo, M.J. (2001). Treatment
of metastatic melanoma with autologous, hapten-modified melanoma vaccine: regression
of pulmonary metastases. Int J Cancer 94, 531-539.
Berd, D., Sato, T., Maguire, H.C., Jr., Kairys, J., and Mastrangelo, M.J. (2004).
Immunopharmacologic analysis of an autologous, hapten-modified human melanoma
vaccine. J Clin Oncol 22, 403-415.
Berek, J.S., Taylor, P.T., Gordon, A., Cunningham, M.J., Finkler, N., Orr, J., Jr., Rivkin,
S., Schultes, B.C., Whiteside, T.L., and Nicodemus, C.F. (2004). Randomized, placebo-
controlled study of oregovomab for consolidation of clinical remission in patients with
advanced ovarian cancer. J Clin Oncol 22, 3507-3516.
Berntsen, A., Geertsen, P.F., and Svane, I.M. (2006). Therapeutic dendritic cell
vaccination of patients with renal cell carcinoma. Eur Urol 50, 34-43.
Bisson, I., and Prowse, D.M. (2009). WNT signaling regulates self-renewal and
differentiation of prostate cancer cells with stem cell characteristics. Cell research 19,
683-697.
Brinkman, J.A., Hughes, S.H., Stone, P., Caffrey, A.S., Muderspach, L.I., Roman, L.D.,
Weber, J.S., and Kast, W.M. (2007). Therapeutic vaccination for HPV induced cervical
cancers. Dis Markers 23, 337-352.
Bronte, V., and Zanovello, P. (2005). Regulation of immune responses by L-arginine
metabolism. Nature reviews 5, 641-654.
Brunkow, M.E., Jeffery, E.W., Hjerrild, K.A., Paeper, B., Clark, L.B., Yasayko, S.A.,
Wilkinson, J.E., Galas, D., Ziegler, S.F., and Ramsdell, F. (2001). Disruption of a new
forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of
the scurfy mouse. Nat Genet 27, 68-73.
Burch, P.A., Croghan, G.A., Gastineau, D.A., Jones, L.A., Kaur, J.S., Kylstra, J.W.,
Richardson, R.L., Valone, F.H., and Vuk-Pavlovic, S. (2004). Immunotherapy
(APC8015, Provenge) targeting prostatic acid phosphatase can induce durable remission
of metastatic androgen-independent prostate cancer: a Phase 2 trial. Prostate 60, 197-204.
202
Campbell, D.J., and Koch, M.A. Phenotypical and functional specialization of FOXP3+
regulatory T cells. Nature reviews 11, 119-130.
Casati, A., Zimmermann, V.S., Benigni, F., Bertilaccio, M.T., Bellone, M., and Mondino,
A. (2005). The immunogenicity of dendritic cell-based vaccines is not hampered by
doxorubicin and melphalan administration. J Immunol 174, 3317-3325.
Cassetti, M.C., McElhiney, S.P., Shahabi, V., Pullen, J.K., Le Poole, I.C., Eiben, G.L.,
Smith, L.R., and Kast, W.M. (2004). Antitumor efficacy of Venezuelan equine
encephalitis virus replicon particles encoding mutated HPV16 E6 and E7 genes. Vaccine
22, 520-527.
Celis, E. (2007). Overlapping human leukocyte antigen class I/II binding peptide vaccine
for the treatment of patients with stage IV melanoma: evidence of systemic immune
dysfunction. Cancer 110, 203-214.
Chen, W., Liang, X., Peterson, A.J., Munn, D.H., and Blazar, B.R. (2008). The
indoleamine 2,3-dioxygenase pathway is essential for human plasmacytoid dendritic cell-
induced adaptive T regulatory cell generation. J Immunol 181, 5396-5404.
Chung, D.J., Rossi, M., Romano, E., Ghith, J., Yuan, J., Munn, D.H., and Young, J.W.
(2009). Indoleamine 2,3-dioxygenase-expressing mature human monocyte-derived
dendritic cells expand potent autologous regulatory T cells. Blood.
Chung, M.H., Gupta, R.K., Hsueh, E., Essner, R., Ye, W., Yee, R., and Morton, D.L.
(2003). Humoral immune response to a therapeutic polyvalent cancer vaccine after
complete resection of thick primary melanoma and sentinel lymphadenectomy. J Clin
Oncol 21, 313-319.
Comes, A., Rosso, O., Orengo, A.M., Di Carlo, E., Sorrentino, C., Meazza, R., Piazza, T.,
Valzasina, B., Nanni, P., Colombo, M.P., et al. (2006). CD25+ regulatory T cell
depletion augments immunotherapy of micrometastases by an IL-21-secreting cellular
vaccine. J Immunol 176, 1750-1758.
203
Corona Gutierrez, C.M., Tinoco, A., Navarro, T., Contreras, M.L., Cortes, R.R., Calzado,
P., Reyes, L., Posternak, R., Morosoli, G., Verde, M.L., et al. (2004). Therapeutic
vaccination with MVA E2 can eliminate precancerous lesions (CIN 1, CIN 2, and CIN 3)
associated with infection by oncogenic human papillomavirus. Hum Gene Ther 15, 421-
431.
Curcio, C., Di Carlo, E., Clynes, R., Smyth, M.J., Boggio, K., Quaglino, E., Spadaro, M.,
Colombo, M.P., Amici, A., Lollini, P.L., et al. (2003). Nonredundant roles of antibody,
cytokines, and perforin in the eradication of established Her-2/neu carcinomas. The
Journal of clinical investigation 111, 1161-1170.
Curiel, T.J. (2007). Tregs and rethinking cancer immunotherapy. J Clin Invest 117, 1167-
1174.
Curiel, T.J. (2008). Regulatory T cells and treatment of cancer. Current opinion in
immunology 20, 241-246.
Curiel, T.J., Coukos, G., Zou, L., Alvarez, X., Cheng, P., Mottram, P., Evdemon-Hogan,
M., Conejo-Garcia, J.R., Zhang, L., Burow, M., et al. (2004). Specific recruitment of
regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced
survival. Nature medicine 10, 942-949.
D'Amaro, J., Houbiers, J.G., Drijfhout, J.W., Brandt, R.M., Schipper, R., Bavinck, J.N.,
Melief, C.J., and Kast, W.M. (1995). A computer program for predicting possible
cytotoxic T lymphocyte epitopes based on HLA class I peptide-binding motifs. Hum
Immunol 43, 13-18.
Danna, E.A., Sinha, P., Gilbert, M., Clements, V.K., Pulaski, B.A., and Ostrand-
Rosenberg, S. (2004). Surgical removal of primary tumor reverses tumor-induced
immunosuppression despite the presence of metastatic disease. Cancer Res 64, 2205-
2211.
Dannull, J., Su, Z., Rizzieri, D., Yang, B.K., Coleman, D., Yancey, D., Zhang, A., Dahm,
P., Chao, N., Gilboa, E., et al. (2005). Enhancement of vaccine-mediated antitumor
immunity in cancer patients after depletion of regulatory T cells. J Clin Invest 115, 3623-
3633.
204
Davis, N.L., Caley, I.J., Brown, K.W., Betts, M.R., Irlbeck, D.M., McGrath, K.M.,
Connell, M.J., Montefiori, D.C., Frelinger, J.A., Swanstrom, R., et al. (2000).
Vaccination of macaques against pathogenic simian immunodeficiency virus with
Venezuelan equine encephalitis virus replicon particles. J Virol 74, 371-378.
Degl'Innocenti, E., Grioni, M., Capuano, G., Jachetti, E., Freschi, M., Bertilaccio, M.T.,
Hess-Michelini, R., Doglioni, C., and Bellone, M. (2008). Peripheral T-cell tolerance
associated with prostate cancer is independent from CD4+CD25+ regulatory T cells.
Cancer research 68, 292-300.
DiPaola, R.S., Plante, M., Kaufman, H., Petrylak, D.P., Israeli, R., Lattime, E., Manson,
K., and Schuetz, T. (2006). A phase I trial of pox PSA vaccines (PROSTVAC-VF) with
B7-1, ICAM-1, and LFA-3 co-stimulatory molecules (TRICOM) in patients with prostate
cancer. J Transl Med 4, 1.
Dontu, G., Abdallah, W.M., Foley, J.M., Jackson, K.W., Clarke, M.F., Kawamura, M.J.,
and Wicha, M.S. (2003). In vitro propagation and transcriptional profiling of human
mammary stem/progenitor cells. Genes & development 17, 1253-1270.
Eder, J.P., Kantoff, P.W., Roper, K., Xu, G.X., Bubley, G.J., Boyden, J., Gritz, L.,
Mazzara, G., Oh, W.K., Arlen, P., et al. (2000). A phase I trial of a recombinant vaccinia
virus expressing prostate-specific antigen in advanced prostate cancer. Clin Cancer Res 6,
1632-1638.
Einstein, M.H., Kadish, A.S., Burk, R.D., Kim, M.Y., Wadler, S., Streicher, H.,
Goldberg, G.L., and Runowicz, C.D. (2007). Heat shock fusion protein-based
immunotherapy for treatment of cervical intraepithelial neoplasia III. Gynecol Oncol 106,
453-460.
El Andaloussi, A., Han, Y., and Lesniak, M.S. (2006). Prolongation of survival following
depletion of CD4+CD25+ regulatory T cells in mice with experimental brain tumors. J
Neurosurg 105, 430-437.
Emens, L.A., and Jaffee, E.M. (2005). Leveraging the activity of tumor vaccines with
cytotoxic chemotherapy. Cancer Res 65, 8059-8064.
205
Ercolini, A.M., Ladle, B.H., Manning, E.A., Pfannenstiel, L.W., Armstrong, T.D.,
Machiels, J.P., Bieler, J.G., Emens, L.A., Reilly, R.T., and Jaffee, E.M. (2005).
Recruitment of latent pools of high-avidity CD8(+) T cells to the antitumor immune
response. J Exp Med 201, 1591-1602.
Farnie, G., Clarke, R.B., Spence, K., Pinnock, N., Brennan, K., Anderson, N.G., and
Bundred, N.J. (2007). Novel cell culture technique for primary ductal carcinoma in situ:
role of Notch and epidermal growth factor receptor signaling pathways. Journal of the
National Cancer Institute 99, 616-627.
Flavell, R.A., Sanjabi, S., Wrzesinski, S.H., and Licona-Limon, P. The polarization of
immune cells in the tumour environment by TGFbeta. Nature reviews 10, 554-567.
Fong, L., Brockstedt, D., Benike, C., Breen, J.K., Strang, G., Ruegg, C.L., and Engleman,
E.G. (2001). Dendritic cell-based xenoantigen vaccination for prostate cancer
immunotherapy. J Immunol 167, 7150-7156.
Fontenot, J.D., Gavin, M.A., and Rudensky, A.Y. (2003). Foxp3 programs the
development and function of CD4+CD25+ regulatory T cells. Nat Immunol 4, 330-336.
Fuessel, S., Meye, A., Schmitz, M., Zastrow, S., Linne, C., Richter, K., Lobel, B.,
Hakenberg, O.W., Hoelig, K., Rieber, E.P., et al. (2006). Vaccination of hormone-
refractory prostate cancer patients with peptide cocktail-loaded dendritic cells: results of
a phase I clinical trial. Prostate 66, 811-821.
Gabrilovich, D.I., and Nagaraj, S. (2009). Myeloid-derived suppressor cells as regulators
of the immune system. Nature reviews 9, 162-174.
Garcia-Hernandez, E., Gonzalez-Sanchez, J.L., Andrade-Manzano, A., Contreras, M.L.,
Padilla, S., Guzman, C.C., Jimenez, R., Reyes, L., Morosoli, G., Verde, M.L., et al.
(2006). Regression of papilloma high-grade lesions (CIN 2 and CIN 3) is stimulated by
therapeutic vaccination with MVA E2 recombinant vaccine. Cancer Gene Ther 13, 592-
597.
Garcia-Hernandez Mde, L., Gray, A., Hubby, B., and Kast, W.M. (2007). In vivo effects
of vaccination with six-transmembrane epithelial antigen of the prostate: a candidate
antigen for treating prostate cancer. Cancer Res 67, 1344-1351.
206
Garcia-Hernandez Mde, L., Gray, A., Hubby, B., Klinger, O.J., and Kast, W.M. (2008).
Prostate stem cell antigen vaccination induces a long-term protective immune response
against prostate cancer in the absence of autoimmunity. Cancer research 68, 861-869.
Garraway, I.P., Sun, W., Tran, C.P., Perner, S., Zhang, B., Goldstein, A.S., Hahm, S.A.,
Haider, M., Head, C.S., Reiter, R.E., et al. Human prostate sphere-forming cells represent
a subset of basal epithelial cells capable of glandular regeneration in vivo. The Prostate
70, 491-501.
Giaccone, G., Debruyne, C., Felip, E., Chapman, P.B., Grant, S.C., Millward, M.,
Thiberville, L., D'Addario, G., Coens, C., Rome, L.S., et al. (2005). Phase III study of
adjuvant vaccination with Bec2/bacille Calmette-Guerin in responding patients with
limited-disease small-cell lung cancer (European Organisation for Research and
Treatment of Cancer 08971-08971B; Silva Study). J Clin Oncol 23, 6854-6864.
Gingrich, J.R., Barrios, R.J., Kattan, M.W., Nahm, H.S., Finegold, M.J., and Greenberg,
N.M. (1997). Androgen-independent prostate cancer progression in the TRAMP model.
Cancer Res 57, 4687-4691.
Gjertsen, M.K., Buanes, T., Rosseland, A.R., Bakka, A., Gladhaug, I., Soreide, O.,
Eriksen, J.A., Moller, M., Baksaas, I., Lothe, R.A., et al. (2001). Intradermal ras peptide
vaccination with granulocyte-macrophage colony-stimulating factor as adjuvant: Clinical
and immunological responses in patients with pancreatic adenocarcinoma. Int J Cancer
92, 441-450.
Goldberg, S.M., Bartido, S.M., Gardner, J.P., Guevara-Patino, J.A., Montgomery, S.C.,
Perales, M.A., Maughan, M.F., Dempsey, J., Donovan, G.P., Olson, W.C., et al. (2005).
Comparison of two cancer vaccines targeting tyrosinase: plasmid DNA and recombinant
alphavirus replicon particles. Clin Cancer Res 11, 8114-8121.
Goldstein, A.S., Huang, J., Guo, C., Garraway, I.P., and Witte, O.N. Identification of a
cell of origin for human prostate cancer. Science (New York, NY 329, 568-571.
Goldstein, A.S., Stoyanova, T., and Witte, O.N. Primitive origins of prostate cancer: in
vivo evidence for prostate-regenerating cells and prostate cancer-initiating cells.
Molecular oncology 4, 385-396.
207
Gray, A., de la Luz Garcia-Hernandez, M., van West, M., Kanodia, S., Hubby, B., and
Kast, W.M. (2009). Prostate cancer immunotherapy yields superior long-term survival in
TRAMP mice when administered at an early stage of carcinogenesis prior to the
establishment of tumor-associated immunosuppression at later stages. Vaccine 27 Suppl
6, G52-59.
Gray, A., Raff, A.B., Chiriva-Internati, M., Chen, S.Y., and Kast, W.M. (2008). A
paradigm shift in therapeutic vaccination of cancer patients: the need to apply therapeutic
vaccination strategies in the preventive setting. Immunol Rev 222, 316-327.
Gray, A., Yan, L., and Kast, W.M. Prevention is better than cure: the case for clinical
trials of therapeutic cancer vaccines in the prophylactic setting. Molecular interventions
10, 197-203.
Greenberg, N.M., DeMayo, F., Finegold, M.J., Medina, D., Tilley, W.D., Aspinall, J.O.,
Cunha, G.R., Donjacour, A.A., Matusik, R.J., and Rosen, J.M. (1995). Prostate cancer in
a transgenic mouse. Proc Natl Acad Sci U S A 92, 3439-3443.
Gu, Z., Thomas, G., Yamashiro, J., Shintaku, I.P., Dorey, F., Raitano, A., Witte, O.N.,
Said, J.W., Loda, M., and Reiter, R.E. (2000). Prostate stem cell antigen (PSCA)
expression increases with high gleason score, advanced stage and bone metastasis in
prostate cancer. Oncogene 19, 1288-1296.
Gulley, J.L., Arlen, P.M., Bastian, A., Morin, S., Marte, J., Beetham, P., Tsang, K.Y.,
Yokokawa, J., Hodge, J.W., Menard, C., et al. (2005). Combining a recombinant cancer
vaccine with standard definitive radiotherapy in patients with localized prostate cancer.
Clin Cancer Res 11, 3353-3362.
Guy, C.T., Webster, M.A., Schaller, M., Parsons, T.J., Cardiff, R.D., and Muller, W.J.
(1992). Expression of the neu protooncogene in the mammary epithelium of transgenic
mice induces metastatic disease. Proceedings of the National Academy of Sciences of the
United States of America 89, 10578-10582.
Hasegawa, K., Martin, F., Huang, G., Tumas, D., Diehl, L., and Chan, A.C. (2004). PEST
domain-enriched tyrosine phosphatase (PEP) regulation of effector/memory T cells.
Science 303, 685-689.
208
Heiser, A., Coleman, D., Dannull, J., Yancey, D., Maurice, M.A., Lallas, C.D., Dahm, P.,
Niedzwiecki, D., Gilboa, E., and Vieweg, J. (2002). Autologous dendritic cells
transfected with prostate-specific antigen RNA stimulate CTL responses against
metastatic prostate tumors. J Clin Invest 109, 409-417.
Hemmings, C. The elaboration of a critical framework for understanding cancer: the
cancer stem cell hypothesis. Pathology 42, 105-112.
Higano, C.S., Schellhammer, P.F., Small, E.J., Burch, P.A., Nemunaitis, J., Yuh, L.,
Provost, N., and Frohlich, M.W. (2009). Integrated data from 2 randomized, double-
blind, placebo-controlled, phase 3 trials of active cellular immunotherapy with
sipuleucel-T in advanced prostate cancer. Cancer 115, 3670-3679.
Hoedemaeker, R.F., Vis, A.N., and Van Der Kwast, T.H. (2000). Staging prostate cancer.
Microscopy research and technique 51, 423-429.
Howell, A., Cuzick, J., Baum, M., Buzdar, A., Dowsett, M., Forbes, J.F., Hoctin-Boes,
G., Houghton, J., Locker, G.Y., and Tobias, J.S. (2005). Results of the ATAC (Arimidex,
Tamoxifen, Alone or in Combination) trial after completion of 5 years' adjuvant
treatment for breast cancer. Lancet 365, 60-62.
Hsu, F.J., Caspar, C.B., Czerwinski, D., Kwak, L.W., Liles, T.M., Syrengelas, A., Taidi-
Laskowski, B., and Levy, R. (1997). Tumor-specific idiotype vaccines in the treatment of
patients with B-cell lymphoma--long-term results of a clinical trial. Blood 89, 3129-3135.
Hsueh, E.C., Essner, R., Foshag, L.J., Ollila, D.W., Gammon, G., O'Day, S.J., Boasberg,
P.D., Stern, S.L., Ye, X., and Morton, D.L. (2002). Prolonged survival after complete
resection of disseminated melanoma and active immunotherapy with a therapeutic cancer
vaccine. J Clin Oncol 20, 4549-4554.
Hsueh, E.C., Famatiga, E., Shu, S., Ye, X., and Morton, D.L. (2004). Peripheral blood
CD4+ T-cell response before postoperative active immunotherapy correlates with clinical
outcome in metastatic melanoma. Ann Surg Oncol 11, 892-899.
Humphrey, P.A. (2004). Gleason grading and prognostic factors in carcinoma of the
prostate. Mod Pathol 17, 292-306.
209
Jaffee, E.M., Hruban, R.H., Biedrzycki, B., Laheru, D., Schepers, K., Sauter, P.R.,
Goemann, M., Coleman, J., Grochow, L., Donehower, R.C., et al. (2001). Novel
allogeneic granulocyte-macrophage colony-stimulating factor-secreting tumor vaccine for
pancreatic cancer: a phase I trial of safety and immune activation. J Clin Oncol 19, 145-
156.
Jaini, R., Kesaraju, P., Johnson, J.M., Altuntas, C.Z., Jane-Wit, D., and Tuohy, V.K. An
autoimmune-mediated strategy for prophylactic breast cancer vaccination. Nature
medicine.
Jaini, R., Kesaraju, P., Johnson, J.M., Altuntas, C.Z., Jane-Wit, D., and Tuohy, V.K. An
autoimmune-mediated strategy for prophylactic breast cancer vaccination. Nature
medicine 16, 799-803.
Jemal, A., Siegel, R., Xu, J., and Ward, E. Cancer statistics, 2010. CA: a cancer journal
for clinicians 60, 277-300.
Kanodia, S., Fahey, L.M., and Kast, W.M. (2007). Mechanisms used by human
papillomaviruses to escape the host immune response. Curr Cancer Drug Targets 7, 79-
89.
Kantoff, P.W., Schuetz, T.J., Blumenstein, B.A., Glode, L.M., Bilhartz, D.L., Wyand, M.,
Manson, K., Panicali, D.L., Laus, R., Schlom, J., et al. Overall survival analysis of a
phase II randomized controlled trial of a Poxviral-based PSA-targeted immunotherapy in
metastatic castration-resistant prostate cancer. J Clin Oncol 28, 1099-1105.
Kaplan-Lefko, P.J., Chen, T.M., Ittmann, M.M., Barrios, R.J., Ayala, G.E., Huss, W.J.,
Maddison, L.A., Foster, B.A., and Greenberg, N.M. (2003). Pathobiology of
autochthonous prostate cancer in a pre-clinical transgenic mouse model. The Prostate 55,
219-237.
Katz, J.B., Muller, A.J., and Prendergast, G.C. (2008). Indoleamine 2,3-dioxygenase in
T-cell tolerance and tumoral immune escape. Immunological reviews 222, 206-221.
Kaufman, H.L., Wang, W., Manola, J., DiPaola, R.S., Ko, Y.J., Sweeney, C., Whiteside,
T.L., Schlom, J., Wilding, G., and Weiner, L.M. (2004). Phase II randomized study of
vaccine treatment of advanced prostate cancer (E7897): a trial of the Eastern Cooperative
Oncology Group. J Clin Oncol 22, 2122-2132.
210
Kawata, H., Ishikura, N., Watanabe, M., Nishimoto, A., Tsunenari, T., and Aoki, Y.
Prolonged treatment with bicalutamide induces androgen receptor overexpression and
androgen hypersensitivity. The Prostate 70, 745-754.
Kenter, G.G., Welters, M.J., Valentijn, A.R., Lowik, M.J., Berends-van der Meer, D.M.,
Vloon, A.P., Essahsah, F., Fathers, L.M., Offringa, R., Drijfhout, J.W., et al. (2009).
Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia. The New
England journal of medicine 361, 1838-1847.
Kimura, H., and Yamaguchi, Y. (1997). A phase III randomized study of interleukin-2
lymphokine-activated killer cell immunotherapy combined with chemotherapy or
radiotherapy after curative or noncurative resection of primary lung carcinoma. Cancer
80, 42-49.
Kirkwood, J.M., Ibrahim, J.G., Sosman, J.A., Sondak, V.K., Agarwala, S.S., Ernstoff,
M.S., and Rao, U. (2001). High-dose interferon alfa-2b significantly prolongs relapse-
free and overall survival compared with the GM2-KLH/QS-21 vaccine in patients with
resected stage IIB-III melanoma: results of intergroup trial E1694/S9512/C509801. J Clin
Oncol 19, 2370-2380.
Koh, Y.T., Gray, A., Higgins, S.A., Hubby, B., and Kast, W.M. (2009). Androgen
ablation augments prostate cancer vaccine immunogenicity only when applied after
immunization. Prostate 69, 571-584.
Kretschmer, K., Apostolou, I., Jaeckel, E., Khazaie, K., and von Boehmer, H. (2006).
Making regulatory T cells with defined antigen specificity: role in autoimmunity and
cancer. Immunological reviews 212, 163-169.
Lahl, K., Loddenkemper, C., Drouin, C., Freyer, J., Arnason, J., Eberl, G., Hamann, A.,
Wagner, H., Huehn, J., and Sparwasser, T. (2007). Selective depletion of Foxp3+
regulatory T cells induces a scurfy-like disease. J Exp Med 204, 57-63.
Lahl, K., and Sparwasser, T. In vivo depletion of FoxP3+ Tregs using the DEREG mouse
model. Methods in molecular biology (Clifton, NJ 707, 157-172.
211
Lambeck, A.J., Crijns, A.P., Leffers, N., Sluiter, W.J., ten Hoor, K.A., Braid, M., van der
Zee, A.G., Daemen, T., Nijman, H.W., and Kast, W.M. (2007). Serum cytokine profiling
as a diagnostic and prognostic tool in ovarian cancer: a potential role for interleukin 7.
Clin Cancer Res 13, 2385-2391.
Leitner, W.W., Hwang, L.N., deVeer, M.J., Zhou, A., Silverman, R.H., Williams, B.R.,
Dubensky, T.W., Ying, H., and Restifo, N.P. (2003). Alphavirus-based DNA vaccine
breaks immunological tolerance by activating innate antiviral pathways. Nat Med 9, 33-
39.
Leong, K.G., and Gao, W.Q. (2008). The Notch pathway in prostate development and
cancer. Differentiation; research in biological diversity 76, 699-716.
Leong, K.G., Wang, B.E., Johnson, L., and Gao, W.Q. (2008). Generation of a prostate
from a single adult stem cell. Nature 456, 804-808.
Litzinger, M.T., Fernando, R., Curiel, T.J., Grosenbach, D.W., Schlom, J., and Palena, C.
(2007). The IL-2 immunotoxin denileukin diftitox reduces regulatory T cells and
enhances vaccine-mediated T-cell immunity. Blood.
Livingston, P.O., Wong, G.Y., Adluri, S., Tao, Y., Padavan, M., Parente, R., Hanlon, C.,
Calves, M.J., Helling, F., Ritter, G., et al. (1994). Improved survival in stage III
melanoma patients with GM2 antibodies: a randomized trial of adjuvant vaccination with
GM2 ganglioside. J Clin Oncol 12, 1036-1044.
Lizee, G., Radvanyi, L.G., Overwijk, W.W., and Hwu, P. (2006). Improving antitumor
immune responses by circumventing immunoregulatory cells and mechanisms. Clin
Cancer Res 12, 4794-4803.
Lohrisch, C., and Piccart, M. (2001). HER2/neu as a predictive factor in breast cancer.
Clinical breast cancer 2, 129-135; discussion 136-127.
MacDonald, G.H., and Johnston, R.E. (2000). Role of dendritic cell targeting in
Venezuelan equine encephalitis virus pathogenesis. J Virol 74, 914-922.
212
Markovic, S.N., Suman, V.J., Ingle, J.N., Kaur, J.S., Pitot, H.C., Loprinzi, C.L., Rao,
R.D., Creagan, E.T., Pittelkow, M.R., Allred, J.B., et al. (2006). Peptide vaccination of
patients with metastatic melanoma: improved clinical outcome in patients demonstrating
effective immunization. Am J Clin Oncol 29, 352-360.
Matsukawa, A., Lukacs, N.W., Standiford, T.J., Chensue, S.W., and Kunkel, S.L. (2000).
Adenoviral-mediated overexpression of monocyte chemoattractant protein-1
differentially alters the development of Th1 and Th2 type responses in vivo. J Immunol
164, 1699-1704.
Merlo, A., Casalini, P., Carcangiu, M.L., Malventano, C., Triulzi, T., Menard, S.,
Tagliabue, E., and Balsari, A. (2009). FOXP3 expression and overall survival in breast
cancer. J Clin Oncol 27, 1746-1752.
Miller, A.M., Lundberg, K., Ozenci, V., Banham, A.H., Hellstrom, M., Egevad, L., and
Pisa, P. (2006). CD4+CD25high T cells are enriched in the tumor and peripheral blood of
prostate cancer patients. J Immunol 177, 7398-7405.
Miller, A.M., and Pisa, P. (2007). Tumor escape mechanisms in prostate cancer. Cancer
Immunol Immunother 56, 81-87.
Mitchell, M.S. (2003). Combinations of anticancer drugs and immunotherapy. Cancer
Immunol Immunother 52, 686-692.
Mocellin, S., Panelli, M.C., Wang, E., Nagorsen, D., and Marincola, F.M. (2003). The
dual role of IL-10. Trends in immunology 24, 36-43.
Muderspach, L., Wilczynski, S., Roman, L., Bade, L., Felix, J., Small, L.A., Kast, W.M.,
Fascio, G., Marty, V., and Weber, J. (2000). A phase I trial of a human papillomavirus
(HPV) peptide vaccine for women with high-grade cervical and vulvar intraepithelial
neoplasia who are HPV 16 positive. Clin Cancer Res 6, 3406-3416.
Mulholland, D.J., Xin, L., Morim, A., Lawson, D., Witte, O., and Wu, H. (2009). Lin-
Sca-1+CD49fhigh stem/progenitors are tumor-initiating cells in the Pten-null prostate
cancer model. Cancer research 69, 8555-8562.
213
Murphy, G.P., Tjoa, B.A., Simmons, S.J., Ragde, H., Rogers, M., Elgamal, A., Kenny,
G.M., Troychak, M.J., Salgaller, M.L., and Boynton, A.L. (1999). Phase II prostate
cancer vaccine trial: report of a study involving 37 patients with disease recurrence
following primary treatment. Prostate 39, 54-59.
Mwau, M., Cebere, I., Sutton, J., Chikoti, P., Winstone, N., Wee, E.G., Beattie, T., Chen,
Y.H., Dorrell, L., McShane, H., et al. (2004). A human immunodeficiency virus 1 (HIV-
1) clade A vaccine in clinical trials: stimulation of HIV-specific T-cell responses by DNA
and recombinant modified vaccinia virus Ankara (MVA) vaccines in humans. J Gen
Virol 85, 911-919.
Nagai, H., Horikawa, T., Hara, I., Fukunaga, A., Oniki, S., Oka, M., Nishigori, C., and
Ichihashi, M. (2004). In vivo elimination of CD25+ regulatory T cells leads to tumor
rejection of B16F10 melanoma, when combined with interleukin-12 gene transfer. Exp
Dermatol 13, 613-620.
Nath, A., Chattopadhya, S., Chattopadhyay, U., and Sharma, N.K. (2006). Macrophage
inflammatory protein (MIP)1alpha and MIP1beta differentially regulate release of
inflammatory cytokines and generation of tumoricidal monocytes in malignancy. Cancer
Immunol Immunother 55, 1534-1541.
Nava-Parada, P., Forni, G., Knutson, K.L., Pease, L.R., and Celis, E. (2007). Peptide
vaccine given with a Toll-like receptor agonist is effective for the treatment and
prevention of spontaneous breast tumors. Cancer Res 67, 1326-1334.
Noguchi, M., Kobayashi, K., Suetsugu, N., Tomiyasu, K., Suekane, S., Yamada, A., Itoh,
K., and Noda, S. (2003). Induction of cellular and humoral immune responses to tumor
cells and peptides in HLA-A24 positive hormone-refractory prostate cancer patients by
peptide vaccination. Prostate 57, 80-92.
Noguchi, M., Yao, A., Harada, M., Nakashima, O., Komohara, Y., Yamada, S., Itoh, K.,
and Matsuoka, K. (2007). Immunological evaluation of neoadjuvant peptide vaccination
before radical prostatectomy for patients with localized prostate cancer. Prostate 67, 933-
942.
Parker, K.C., Bednarek, M.A., and Coligan, J.E. (1994). Scheme for ranking potential
HLA-A2 binding peptides based on independent binding of individual peptide side-
chains. J Immunol 152, 163-175.
214
Pound, C.R., Partin, A.W., Eisenberger, M.A., Chan, D.W., Pearson, J.D., and Walsh,
P.C. (1999). Natural history of progression after PSA elevation following radical
prostatectomy. Jama 281, 1591-1597.
Prasad, S.J., Farrand, K.J., Matthews, S.A., Chang, J.H., McHugh, R.S., and Ronchese, F.
(2005). Dendritic cells loaded with stressed tumor cells elicit long-lasting protective
tumor immunity in mice depleted of CD4+CD25+ regulatory T cells. J Immunol 174, 90-
98.
Pushko, P., Parker, M., Ludwig, G.V., Davis, N.L., Johnston, R.E., and Smith, J.F.
(1997). Replicon-helper systems from attenuated Venezuelan equine encephalitis virus:
expression of heterologous genes in vitro and immunization against heterologous
pathogens in vivo. Virology 239, 389-401.
Reinartz, S., Kohler, S., Schlebusch, H., Krista, K., Giffels, P., Renke, K., Huober, J.,
Mobus, V., Kreienberg, R., DuBois, A., et al. (2004). Vaccination of patients with
advanced ovarian carcinoma with the anti-idiotype ACA125: immunological response
and survival (phase Ib/II). Clin Cancer Res 10, 1580-1587.
Reiter, R.E., Gu, Z., Watabe, T., Thomas, G., Szigeti, K., Davis, E., Wahl, M., Nisitani,
S., Yamashiro, J., Le Beau, M.M., et al. (1998). Prostate stem cell antigen: a cell surface
marker overexpressed in prostate cancer. Proc Natl Acad Sci U S A 95, 1735-1740.
Ressing, M.E., van Driel, W.J., Celis, E., Sette, A., Brandt, M.P., Hartman, M., Anholts,
J.D., Schreuder, G.M., ter Harmsel, W.B., Fleuren, G.J., et al. (1996). Occasional
memory cytotoxic T-cell responses of patients with human papillomavirus type 16-
positive cervical lesions against a human leukocyte antigen-A *0201-restricted E7-
encoded epitope. Cancer Res 56, 582-588.
Reynolds, B.A., and Weiss, S. (1996). Clonal and population analyses demonstrate that
an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Developmental
biology 175, 1-13.
Roberts, W.B., and Han, M. (2009). Clinical significance and treatment of biochemical
recurrence after definitive therapy for localized prostate cancer. Surgical oncology 18,
268-274.
215
Roman, L.D., Wilczynski, S., Muderspach, L.I., Burnett, A.F., O'Meara, A., Brinkman,
J.A., Kast, W.M., Facio, G., Felix, J.C., Aldana, M., et al. (2007). A phase II study of
Hsp-7 (SGN-00101) in women with high-grade cervical intraepithelial neoplasia.
Gynecol Oncol 106, 558-566.
Ross, S., Spencer, S.D., Holcomb, I., Tan, C., Hongo, J., Devaux, B., Rangell, L., Keller,
G.A., Schow, P., Steeves, R.M., et al. (2002). Prostate stem cell antigen as therapy target:
tissue expression and in vivo efficacy of an immunoconjugate. Cancer Res 62, 2546-
2553.
Salgaller, M.L., Lodge, P.A., McLean, J.G., Tjoa, B.A., Loftus, D.J., Ragde, H., Kenny,
G.M., Rogers, M., Boynton, A.L., and Murphy, G.P. (1998). Report of immune
monitoring of prostate cancer patients undergoing T-cell therapy using dendritic cells
pulsed with HLA-A2-specific peptides from prostate-specific membrane antigen
(PSMA). Prostate 35, 144-151.
Sanderson, K., Scotland, R., Lee, P., Liu, D., Groshen, S., Snively, J., Sian, S., Nichol,
G., Davis, T., Keler, T., et al. (2005). Autoimmunity in a phase I trial of a fully human
anti-cytotoxic T-lymphocyte antigen-4 monoclonal antibody with multiple melanoma
peptides and Montanide ISA 51 for patients with resected stages III and IV melanoma. J
Clin Oncol 23, 741-750.
Saslow, D., Boetes, C., Burke, W., Harms, S., Leach, M.O., Lehman, C.D., Morris, E.,
Pisano, E., Schnall, M., Sener, S., et al. (2007). American Cancer Society guidelines for
breast screening with MRI as an adjunct to mammography. CA: a cancer journal for
clinicians 57, 75-89.
Schlom, J., Arlen, P.M., and Gulley, J.L. (2007). Cancer vaccines: moving beyond
current paradigms. Clin Cancer Res 13, 3776-3782.
Shahi, P., Seethammagari, M.R., Valdez, J.M., Xin, L., and Spencer, D.M. Wnt and
Notch pathways have interrelated opposing roles on prostate progenitor cell proliferation
and differentiation. Stem cells (Dayton, Ohio) 29, 678-688.
Sharma, M.D., Hou, D.Y., Liu, Y., Koni, P.A., Metz, R., Chandler, P., Mellor, A.L., He,
Y., and Munn, D.H. (2009). Indoleamine 2,3-dioxygenase controls conversion of Foxp3+
Tregs to TH17-like cells in tumor-draining lymph nodes. Blood 113, 6102-6111.
216
Shou, J., Ross, S., Koeppen, H., de Sauvage, F.J., and Gao, W.Q. (2001). Dynamics of
notch expression during murine prostate development and tumorigenesis. Cancer Res 61,
7291-7297.
Shu, S., Cochran, A.J., Huang, R.R., Morton, D.L., and Maecker, H.T. (2006). Immune
responses in the draining lymph nodes against cancer: implications for immunotherapy.
Cancer Metastasis Rev 25, 233-242.
Small, E.J., Fratesi, P., Reese, D.M., Strang, G., Laus, R., Peshwa, M.V., and Valone,
F.H. (2000). Immunotherapy of hormone-refractory prostate cancer with antigen-loaded
dendritic cells. J Clin Oncol 18, 3894-3903.
Small, E.J., Schellhammer, P.F., Higano, C.S., Redfern, C.H., Nemunaitis, J.J., Valone,
F.H., Verjee, S.S., Jones, L.A., and Hershberg, R.M. (2006). Placebo-controlled phase III
trial of immunologic therapy with sipuleucel-T (APC8015) in patients with metastatic,
asymptomatic hormone refractory prostate cancer. J Clin Oncol 24, 3089-3094.
Small, L.A., Da Silva, D.M., de Visser, K.E., Velders, M.P., Fisher, S.G., Potkul, R.K.,
and Kast, W.M. (2001). A murine model for the effects of pelvic radiation and cisplatin
chemotherapy on human papillomavirus vaccine efficacy. Clin Cancer Res 7, 876s-881s.
Sosman, J.A., Unger, J.M., Liu, P.Y., Flaherty, L.E., Park, M.S., Kempf, R.A.,
Thompson, J.A., Terasaki, P.I., and Sondak, V.K. (2002). Adjuvant immunotherapy of
resected, intermediate-thickness, node-negative melanoma with an allogeneic tumor
vaccine: impact of HLA class I antigen expression on outcome. J Clin Oncol 20, 2067-
2075.
Sportes, C., McCarthy, N.J., Hakim, F., Steinberg, S.M., Liewehr, D.J., Weng, D.,
Kummar, S., Gea-Banacloche, J., Chow, C.K., Dean, R.M., et al. (2005). Establishing a
platform for immunotherapy: clinical outcome and study of immune reconstitution after
high-dose chemotherapy with progenitor cell support in breast cancer patients. Biol
Blood Marrow Transplant 11, 472-483.
Su, Z., Dannull, J., Yang, B.K., Dahm, P., Coleman, D., Yancey, D., Sichi, S.,
Niedzwiecki, D., Boczkowski, D., Gilboa, E., et al. (2005). Telomerase mRNA-
transfected dendritic cells stimulate antigen-specific CD8+ and CD4+ T cell responses in
patients with metastatic prostate cancer. J Immunol 174, 3798-3807.
217
Tagawa, S.T., Cheung, E., Banta, W., Gee, C., and Weber, J.S. (2006). Survival analysis
after resection of metastatic disease followed by peptide vaccines in patients with Stage
IV melanoma. Cancer 106, 1353-1357.
Takeuchi, H., Morton, D.L., Elashoff, D., and Hoon, D.S. (2005). Survivin expression by
metastatic melanoma predicts poor disease outcome in patients receiving adjuvant
polyvalent vaccine. Int J Cancer 117, 1032-1038.
Tanaka, H., Tanaka, J., Kjaergaard, J., and Shu, S. (2002). Depletion of CD4+ CD25+
regulatory cells augments the generation of specific immune T cells in tumor-draining
lymph nodes. J Immunother 25, 207-217.
Teng, M.W., Ngiow, S.F., von Scheidt, B., McLaughlin, N., Sparwasser, T., and Smyth,
M.J. Conditional regulatory T-cell depletion releases adaptive immunity preventing
carcinogenesis and suppressing established tumor growth. Cancer research 70, 7800-
7809.
Thomas-Kaskel, A.K., Zeiser, R., Jochim, R., Robbel, C., Schultze-Seemann, W., Waller,
C.F., and Veelken, H. (2006). Vaccination of advanced prostate cancer patients with
PSCA and PSA peptide-loaded dendritic cells induces DTH responses that correlate with
superior overall survival. Int J Cancer 119, 2428-2434.
Thompson, I.M., Goodman, P.J., Tangen, C.M., Lucia, M.S., Miller, G.J., Ford, L.G.,
Lieber, M.M., Cespedes, R.D., Atkins, J.N., Lippman, S.M., et al. (2003). The influence
of finasteride on the development of prostate cancer. The New England journal of
medicine 349, 215-224.
Thompson, I.M., Tangen, C.M., Goodman, P.J., Lucia, M.S., and Klein, E.A. (2009).
Chemoprevention of prostate cancer. The Journal of urology 182, 499-507; discussion
508.
Thompson, I.M., Tangen, C.M., Klein, E.A., and Lippman, S.M. (2005). Phase III
prostate cancer prevention trials: are the costs justified? J Clin Oncol 23, 8161-8164.
Tjoa, B.A., Simmons, S.J., Bowes, V.A., Ragde, H., Rogers, M., Elgamal, A., Kenny,
G.M., Cobb, O.E., Ireton, R.C., Troychak, M.J., et al. (1998). Evaluation of phase I/II
clinical trials in prostate cancer with dendritic cells and PSMA peptides. Prostate 36, 39-
44.
218
Tran, C.P., Lin, C., Yamashiro, J., and Reiter, R.E. (2002). Prostate stem cell antigen is a
marker of late intermediate prostate epithelial cells. Mol Cancer Res 1, 113-121.
Uemura, H., Fujimoto, K., Tanaka, M., Yoshikawa, M., Hirao, Y., Uejima, S.,
Yoshikawa, K., and Itoh, K. (2006). A phase I trial of vaccination of CA9-derived
peptides for HLA-A24-positive patients with cytokine-refractory metastatic renal cell
carcinoma. Clin Cancer Res 12, 1768-1775.
Vang, T., Congia, M., Macis, M.D., Musumeci, L., Orru, V., Zavattari, P., Nika, K.,
Tautz, L., Tasken, K., Cucca, F., et al. (2005). Autoimmune-associated lymphoid tyrosine
phosphatase is a gain-of-function variant. Nat Genet 37, 1317-1319.
Velders, M.P., McElhiney, S., Cassetti, M.C., Eiben, G.L., Higgins, T., Kovacs, G.R.,
Elmishad, A.G., Kast, W.M., and Smith, L.R. (2001). Eradication of established tumors
by vaccination with Venezuelan equine encephalitis virus replicon particles delivering
human papillomavirus 16 E7 RNA. Cancer research 61, 7861-7867.
Viehl, C.T., Moore, T.T., Liyanage, U.K., Frey, D.M., Ehlers, J.P., Eberlein, T.J.,
Goedegebuure, P.S., and Linehan, D.C. (2006a). Depletion of CD4(+)CD25 (+)
Regulatory T Cells Promotes a Tumor-Specific Immune Response in Pancreas Cancer-
Bearing Mice. Ann Surg Oncol 13, 1252-1258.
Viehl, C.T., Moore, T.T., Liyanage, U.K., Frey, D.M., Ehlers, J.P., Eberlein, T.J.,
Goedegebuure, P.S., and Linehan, D.C. (2006b). Depletion of CD4+CD25+ regulatory T
cells promotes a tumor-specific immune response in pancreas cancer-bearing mice. Ann
Surg Oncol 13, 1252-1258.
Vonderheide, R.H., Domchek, S.M., Schultze, J.L., George, D.J., Hoar, K.M., Chen,
D.Y., Stephans, K.F., Masutomi, K., Loda, M., Xia, Z., et al. (2004). Vaccination of
cancer patients against telomerase induces functional antitumor CD8+ T lymphocytes.
Clin Cancer Res 10, 828-839.
Waeckerle-Men, Y., Uetz-von Allmen, E., Fopp, M., von Moos, R., Bohme, C., Schmid,
H.P., Ackermann, D., Cerny, T., Ludewig, B., Groettrup, M., et al. (2006). Dendritic cell-
based multi-epitope immunotherapy of hormone-refractory prostate carcinoma. Cancer
Immunol Immunother 55, 1524-1533.
219
Wallack, M.K., Sivanandham, M., Ditaranto, K., Shaw, P., Balch, C.M., Urist, M.M.,
Bland, K.I., Murray, D., Robinson, W.A., Flaherty, L., et al. (1997). Increased survival of
patients treated with a vaccinia melanoma oncolysate vaccine: second interim analysis of
data from a phase III, multi-institutional trial. Ann Surg 226, 198-206.
Wang, D., Wang, H., Brown, J., Daikoku, T., Ning, W., Shi, Q., Richmond, A., Strieter,
R., Dey, S.K., and DuBois, R.N. (2006). CXCL1 induced by prostaglandin E2 promotes
angiogenesis in colorectal cancer. The Journal of experimental medicine 203, 941-951.
Wang, F., Bade, E., Kuniyoshi, C., Spears, L., Jeffery, G., Marty, V., Groshen, S., and
Weber, J. (1999). Phase I trial of a MART-1 peptide vaccine with incomplete Freund's
adjuvant for resected high-risk melanoma. Clin Cancer Res 5, 2756-2765.
Wang, J.C. Good cells gone bad: the cellular origins of cancer. Trends in molecular
medicine 16, 145-151.
Wang, S. (2009). Anchorage-independent growth of prostate cancer stem cells. Methods
in molecular biology (Clifton, NJ 568, 151-160.
Wang, X., Wang, J.P., Maughan, M.F., and Lachman, L.B. (2005a). Alphavirus replicon
particles containing the gene for HER2/neu inhibit breast cancer growth and
tumorigenesis. Breast Cancer Res 7, R145-155.
Wang, X., Wang, J.P., Rao, X.M., Price, J.E., Zhou, H.S., and Lachman, L.B. (2005b).
Prime-boost vaccination with plasmid and adenovirus gene vaccines control HER2/neu+
metastatic breast cancer in mice. Breast Cancer Res 7, R580-588.
Wang, Y., Harada, M., Yano, H., Ogasawara, S., Takedatsu, H., Arima, Y., Matsueda, S.,
Yamada, A., and Itoh, K. (2005c). Prostatic acid phosphatase as a target molecule in
specific immunotherapy for patients with nonprostate adenocarcinoma. J Immunother 28,
535-541.
Weber, J., Sondak, V.K., Scotland, R., Phillip, R., Wang, F., Rubio, V., Stuge, T.B.,
Groshen, S.G., Gee, C., Jeffery, G.G., et al. (2003). Granulocyte-macrophage-colony-
stimulating factor added to a multipeptide vaccine for resected Stage II melanoma.
Cancer 97, 186-200.
220
Weng, W.K., Czerwinski, D., Timmerman, J., Hsu, F.J., and Levy, R. (2004). Clinical
outcome of lymphoma patients after idiotype vaccination is correlated with humoral
immune response and immunoglobulin G Fc receptor genotype. J Clin Oncol 22, 4717-
4724.
Wrzesinski, S.H., Wan, Y.Y., and Flavell, R.A. (2007). Transforming growth factor-beta
and the immune response: implications for anticancer therapy. Clin Cancer Res 13, 5262-
5270.
Yang, D., Holt, G.E., Velders, M.P., Kwon, E.D., and Kast, W.M. (2001). Murine six-
transmembrane epithelial antigen of the prostate, prostate stem cell antigen, and prostate-
specific membrane antigen: prostate-specific cell-surface antigens highly expressed in
prostate cancer of transgenic adenocarcinoma mouse prostate mice. Cancer Res 61, 5857-
5860.
Yokokawa, J., Cereda, V., Remondo, C., Gulley, J.L., Arlen, P.M., Schlom, J., and
Tsang, K.Y. (2008). Enhanced functionality of CD4+CD25(high)FoxP3+ regulatory T
cells in the peripheral blood of patients with prostate cancer. Clin Cancer Res 14, 1032-
1040.
Zheng, S.G., Wang, J.H., Gray, J.D., Soucier, H., and Horwitz, D.A. (2004). Natural and
induced CD4+CD25+ cells educate CD4+CD25- cells to develop suppressive activity:
the role of IL-2, TGF-beta, and IL-10. J Immunol 172, 5213-5221.
Zhu, Z.Y., Zhong, C.P., Xu, W.F., Lin, G.M., Ye, G.Q., Ji, Y.Y., Sun, B., and Yeh, M.
(1999). PSMA mimotope isolated from phage displayed peptide library can induce
PSMA specific immune response. Cell Res 9, 271-280.
Zimmermann, V.S., Casati, A., Schiering, C., Caserta, S., Michelini, R.H., Basso, V., and
Mondino, A. (2007). Tumors Hamper the Immunogenic Competence of CD4+ T Cell-
Directed Dendritic Cell Vaccination. J Immunol 179, 2899-2909.
Abstract (if available)
Abstract
Immunotherapy has long been proposed as a novel method of specifically, safely and inexpensively treating cancer. Despite decades of research and hundreds of clinical trials, only one therapeutic cancer vaccine has been approved for human use. The vast majority of these clinical trials have been carried out in terminally ill patients with advanced cancer. These patients are severely immune compromised. The studies herein demonstrate that multiple immunosuppressive mechanisms develop within prostate tumors as they progress. These mechanisms include the accumulation of regulatory T cells in the prostate tumor, and increased expression of TGFβ and indoleamine 2,3 dioxygenase in the prostate tumor. Vaccination of mice with spontaneously arising prostate cancer against tumor-associated antigens is vastly superior to immunization at later stages of disease, when tumor microenvironments are more suppressive. A novel murine model of prostate cancer in which major mediators of immune suppression – FOXP3-expressing regulatory T cells – can be specifically depleted was developed. This model was termed the TRAMP DEREG mouse. Depletion of regulatory T cells improved the response to therapeutic cancer vaccinination in TRAMP DEREG mice. This was the case even though Treg depletion was carried out at a phase of carcinogenesis prior to the development of serious immune suppression, suggesting that even better results can be obtained if Tregs are inhibited when they are most prevalent in immunosuppressive prostate tumors. Prostate cancer stem cells are responsible for driving tumor growth and regeneration. Additionally, they do not express the tumor-associated antigens that are targeted in prostate cancer immunotherapy, thus rendering them resistant to immune-medidated eradication. An effort was made to eliminate this population by inhibition of Notch signaling, but it was found that Notch signaling is not involved in the maintenance of the prostate cancer stem cell phenotype. In addition, Notch inhibition severely limited the efficacy of concomitant therapeutic cancer vaccination. However, the novel finding that Notch signaling is critical to the differentiation of prostate cancer cells, and thus to tumor growth and regeneration, is extremely important. Taken together, these studies show that immune-mediated tumor eradication is extremely difficult to achieve in advanced cancer. Therefore it is proposed that clinical trials of cancer immunotherapies be carried out in the preventative setting as soon as possible.
Linked assets
University of Southern California Dissertations and Theses
Conceptually similar
PDF
LIGHT-ing up prostate cancer: remodeling the tumor microenvironment and enhancing therapeutic vaccine efficacy through forced LIGHT expression in prostate cancer
PDF
Exploration of the roles of cancer stem cells and survivin in the pathogenesis and progression of prostate cancer
PDF
Rationalized immunotherapy by immune signature characterization
PDF
Development of immunotherapy for small cell lung cancer using novel modified antigens
PDF
Suppressor cell therapy: targeting T regulatory cells in cancer
PDF
Engineering chimeric antigen receptor (CAR) -modified T cells for enhanced cancer immunotherapy
PDF
Study of the role of bone morphogenetic proteins in prostate cancer progression
PDF
Targeting molecular signals involved in the development of castration resistant prostate cancer
PDF
Oncolytic adenovirus based vaccine in cancer immunotherapy
PDF
Deregulation of CD36 expression in cancer presents a potential targeting therapeutic opportunity
PDF
Homologous cell systems for the study of progression of androgen-dependent prostate cancer to castration-resistant prostate cancer
PDF
Effects of androgen ablation and vaccine preparation on cancer vaccine efficacy
PDF
Development of immunotherapy for small cell lung cancer using iso-aspartylated antigen
PDF
Dendritic cell-specific vaccine utilizing antibody-mimetic ligand and lentivector system
PDF
Investigation into the use of repurposed influenza vaccines for immunotherapy of HPV-induced tumors
PDF
MAO a deficient mice exhibit an altered immune system in the brain and prostate
PDF
The role of glucose-regulated proteins in endometrial and pancreatic cancers
PDF
The cancer stem-like phenotype: therapeutics, phenotypic plasticity and mechanistic studies
PDF
Identification of novel epigenetic biomarkers and microRNAs for cancer therapeutics
PDF
Targeting glioma cancer stem cells for the treatment of glioblastoma multiforme
Asset Metadata
Creator
Gray, Andrew
(author)
Core Title
The effect of tumor-mediated immune suppression on prostate cancer immunotherapy
School
Keck School of Medicine
Degree
Doctor of Philosophy
Degree Program
Genetic, Molecular and Cellular Biology
Publication Date
11/22/2011
Defense Date
08/19/2011
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
cancer stem cells,immune suppression,immunotherapy,Notch signaling,OAI-PMH Harvest,prostate cancer,regulatory T cell,therapeutic vaccine,Tregs
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Chen, Si-Yi (
committee chair
), Hutchinson, Ian V. (
committee member
), Kast, W. Martin (
committee member
)
Creator Email
andrewgrayusc@gmail.com,graya@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c3-211636
Unique identifier
UC11292131
Identifier
usctheses-c3-211636 (legacy record id)
Legacy Identifier
etd-GrayAndrew-432-0.pdf
Dmrecord
211636
Document Type
Dissertation
Rights
Gray, Andrew
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Access Conditions
The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...
Repository Name
University of Southern California Digital Library
Repository Location
USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA
Tags
cancer stem cells
immune suppression
immunotherapy
Notch signaling
prostate cancer
regulatory T cell
therapeutic vaccine
Tregs