University of Southern California Dissertations and Theses

Studies of murine prostate cancer stem / progenitor cells

(USC Thesis Other)

Studies of murine prostate cancer stem / progenitor cells

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content STUDIES OF MURINE PROSTATE CANCER STEM /
PROGENITOR CELLS

by

Chun-Peng Liao

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

August 2008

Copyright 2008 Chun-Peng Liao

ii
DEDICATION
This work is dedicated to my parents, Mei-Fong Tsai and Ming-Chih Liao
and to my love, Man-Chun Ting.

iii
ACKNOWLEDGEMENTS

It is a pleasure to thank the many people who made this thesis possible.
I would like to express my deep and sincere gratitude to my supervisor,
Dr. Pradip Roy-Burman. His wide knowledge and his logical way of thinking have
been of great value for me. His understanding, encouraging and personal guidance
have provided a good basis for the present thesis
My warm thanks are due to Dr. Baruch Frenkel and Dr. Louis Dubeau for serving
as members of my dissertation committee and for their inspiring advice. I also wish
to thank Dr. Alexander Nikitin and Dr. Zongxiang Zhou for their help in our research
collaboration.
I am indebted to my many colleagues who have assisted me during my thesis
work. Dr. Chen Zhong and Gohar Saribekyan collaborated with me in the
development of the mouse models. Dr. Shangxin Yang and Helty Adisetiyo helped
me to characterize murine carcinoma-associate fibroblasts and prostate stem cells.
Kumkum Mittra gave me a lot of advice when I met difficulties in the laboratory. I
want to thank the 2002-2008 members of the Roy-Burman lab for all the emotional
support, comraderie, entertainment, and caring they provided.
Lastly, I owe my loving thanks to Man-Chun Ting. Without her encouragement
and understanding it would have been impossible for me to finish this work.

iv
TABLE OF CONTENTS
Dedication……………………………………………………………...ii
Acknowledgements…………………………………………..…….….iii
List of Figures…………………………………………………………vi
Abstract………………………………………………………………viii
Chapter 1: Introduction………………………………………………...1
Prostate Cancer…………………….……………….1
Cancer Stem Cells………………………………......2
Prostate Cancer Stem Cells……………….……..….3
Animal Models of Prostate Cancer…………………6
Stem Cell Niche……………………………………10
Prostate Cancer Microenvironment...………………12
Hypothesis and Rationale…….……..……………...13

Chapter 2: Mouse Models of Prostate Adenocarcinoma with Capacity for
Monitoring Spontaneous Carcinogenesis by Bioluminescence
or Fluorescence………………………………….....……...15
Abstract……………………………………………15
Introduction………………………………………..16
Materials and Methods…………………………….18
Results……………………………………………..24
Discussion…………………………………………42

Chapter 3: Isolation and Characterization of Murine Prostate Cancer
Stem / Progenitor Cells………….…………………………46
Abstract…………………………………………….46
Introduction………………………...……………...48
Materials and Methods…………………..………....51
Results……….………...…………………………...56
Discussion…..……………………………………...72

Chapter 4: Conclusions and Future Directions………………….……..77
Conclusions…………………………………………….77

v
Future Directions……………………………………….79

References……………………………………………………………...83

vi
LIST OF FIGURES

Figure 1. In vivo and ex vivo bioluminescence images of cPten
-/-
L mice..……....….25

Figure 2. Fluorescence analysis of tissues from cPten
-/-
G males….......................…30

Figure 3. Longitudinal BLI monitoring of living cPten
-/-
L animals………………...32

Figure 4. Other characteristics of the models…………………….………...……….37

Figure 5. Comparative immunohistochemical analysis of epithelial cells from the
normal prostate and primary and recurrent tumors…...……………….....39

Figure 6. FACS analyses of the Lin
-
population isolated from murine prostate
tissues.…..…………...…………………………………………………...57

Figure 7. Characterization of stem / Progenitor cells with respect to intracellular
protein expression……………………………………………...………...61

Figure 8. Analysis of spheroid forming ability of the Lin
-
Sca-1
+
cell
subpopulation.............................................................................................63

Figure 9. Comparative Morpholoical and histological analyses of spheroids formed
from Lin
-
Sca-1
+
cells …………………...………………………………..66

Figure 10. Characterization of LSC cell subpopulation isolated from AD-Ca……..70

vii
ABSTRACT

The application of our prostate epithelium-specific Cre/loxP system to inactivate
tumor suppressor genes had resulted in successful development of mouse models of
prostate cancer. We further increased the efficiency of the conditional Pten deletion
model of prostate adenocarcinoma by combining it with either a conditional
luciferase or EGFP reporter line. The growth kinetics of the androgen dependent
cancer (AD-Ca) and androgen-depletion independent recurrent cancer (ADI-Ca)
could be followed non-invasively in live animals by bioluminescence imaging. We
expect that such an investigation will be facilitated by timing the collection of tumors
at specific growth or re-growth points, an advantage that is provided by the model.
The EGFP model can provide an opportunity to locate tumors or to isolate enriched
populations of cancer cells from tumor tissues via fluorescence-based technologies.
Previous studies have shown a small cell subpopulation with Lin
-
Sca-1
+
CD49f
+

(LSC) cell surface marker phenotypes in the normal murine prostate to have the
capacities of stemness. We examined the presence of such cells in our mouse models
of prostate cancer, and found a much higher percentage of the LSC cell
subpopulation among the Lin
-
cells in AD-Ca and ADI-Ca compared to that in the
proximal region of the normal counterparts. To characterize the stemness abilities of
cancer LSC cells, we conducted spheroid-forming analyses by the matrigel-based co-
culture system. Spheroids formed from the cancer LSC cells were larger than those
form normal LSC cells. The spheroids could be passaged serially. In the presence of

viii
carcinoma associated fibroblasts, they showed a prostate cancer glandular-like
structure. We also observed that only cancer LSC cells, not non-LSC, cells formed
spheroids. The differentiation to various cell types in spheroids was identified by
dual immunofluorescence staining. In the spheroids generated from normal LSC
cells, CK8
+
luminal cells were located in the intra-lumen layers, and p63
+
basal cells
in the outer layers. However, in the spheroids formed from cancer LSC cells, most of
the cells in the inner layers are p63
+
CK8
+
dual-positive indicating an intermediate
phenotype. Taken together, these results describe the opportunity present in the
model system to explore the role of stem cells in prostate cancer.

1
CHAPTER 1
Introduction
Prostate Cancer
Prostate cancer is a molecularly, phenotypically, and clinically heterogeneous
disease, which is the most common diagnosed malignancy in North America. This
cancer contains extensive phenotypic heterogeneity, in both morphology and
molecular genetics (DeMarzo et al., 2003; Roy-Burman et al., 1997). Because the
natural history of this disease has been difficult to ascertain, once a man is diagnosed
with this disease, it is hard to predict if the cancer will stay in a quiescent stage
throughout the patient’s life or become aggressive. Despite these difficulties, there
has been some progress in understanding the clinical course and the molecular
parameters of this disease (Abbas and Scardino, 1997; Gittes, 1991; Jemal et al.,
2003). The development of this disease proceeds through a series of defined states,
apparently initiating from prostatic intraepithelial neoplasia (PIN) to prostate cancer
in situ, invasive cancer and then to metastatic cancer (Isaacs et al., 2002; McNeal et
al., 1991). The prostate is an androgen-dependent organ that undergoes involution
after androgen withdrawal. So, the standard treatment for prostate cancer is androgen
deprivation therapy (ADT) that initially causes tumor regression. However, tumor
cells will eventually relapse and develop into androgen-depletion independent cancer
(ADI-Ca) (Denis and Murphy, 1993; Isaacs, 1999; Roy-Burman et al., 2005; Zhou et
al., 2007)

2
Cancer Stem Cells
Human cancers have been recognized as a morphologically heterogeneous
population of cells for more then a century. What has become clear in the end of the
last century is that these cells are also functionally heterogeneous. The hypothesis
that cancers contained stem cells had been proposed over twenty years ago by
Mackillop et al (Mackillop et al., 1983). Properties shared by neoplastic and stem
cells indicate a possibility that normal somatic stem cells or transit-amplifying (TA)
cells that have reacquired stem cell properties, particularly the abilities for self-
renewal and differentiation, may represent favorable targets for malignant
transformation. The cancer stem cell subpopulations in tumors, like the normal stem
cells in normal adult tissues, may self-renew and also give rise to a large population
of differentiated progeny that make up the bulk of the tumor but lack tumorigenetic
potential (Al-Hajj et al., 2004; Knudson et al., 1973; Morrison et al., 2002).
Cancer stem cells were first identified in the haemopoietic system by Bonnet and
Dick in 1997 (Bonnet and Dick, 1997). They were able to separate tumor-initiating
cells, which share the same cell surface marker (CD34
+
CD38
-
) phenotype with
normal haematopoietic stem cells, from acute myeloid leukemia patient samples.
Although the number of this subpopulation is small, these are the only cells capable
of transfering acute myeloid leukemia from patients to NOD/SCID mice. It is now
being identified that solid tumors also contain cancer stem cell populations,
including the mammary gland, brain, lung, kidney, pancreas and prostate (Al-Hajj et

3
al., 2003; Collins et al., 2005; Li et al., 2007; O'Brien et al., 2007; Ricci-Vitiani et
al., 2007).
The origin of cancer stem cells is still unclear. Since cancer cells must overcome
the tight genetic pressure on both self-renewal and proliferation to maintain the
disease (Morrison et al., 2002), cancer stem cells may derive from their normal stem
cell counterparts, progenitor cells, or matured cells. The fact that multiple mutations
are necessary for a cell to become cancerous has implications for the cellular origin
of cancer cells (Knudson et al., 1973).

Prostate Cancer Stem Cells
Prostate cancer stem cells are a fraction of tumor cells that give rise to malignant
cells in prostates. Goldie and Coldman provided a hypothesis in 1979 suggesting that
a small percentage of cells in a tumor obtained fundamental characteristics that made
them resistant to treatment (Goldie and Coldman, 1979). Their hypothesis fits the
cancer stem cell hypothesis and can be used to explain why some prostate cancer
patients who accept ADT treatments may develop recurrent cancers several months
after starting therapies. Since prostate cancer stem cells do not express androgen
receptor (AR) (Collins et al., 2005), they should not directly respond to ADT
therapy. Thus, the survival of a small group of cancer cells with unique
characteristics may obtain the ability to give rise to a new population of cells with a
resistant phenotype to androgen ablation, and then initiate ADI-Ca. Thus, prostate

4
cancer stem cells may undergo molecular changes such as amplification of the
androgen receptor gene resulting in gain-of-function mutations in AR, or other
epigenetic changes, implying an acquired resistance to androgen deprivation.
(Collins and Maitland, 2006; Dehm and Tindall, 2006; Feinberg et al., 2006; Nikitin
et al., 2007; Nikitin et al., (In Press); Sharifi et al., 2006).
Evidence from in vivo works have shown that there is a cell population containing
stem cell properties within the basal cell layer with the phenotype of cytokeratin 5
+
,
cytokeratin 14
+
, CD133
+
, CD44
+
and high expression of β1 integrins (Collins et al.,
2005; Collins et al., 2001; Richardson et al., 2004). This prostate stem cell
subpopulation is relatively rare and comprises only 1% of basal epithelial cells.
Collins et al. used three surface markers (CD44, α
2
β
1
hi
, CD133) , which are also used
for the normal prostate stem cells, to isolate a putative prostate cancer stem cell from
metastatic and primary human prostate tumors (Collins et al., 2005). This cellular
subpopulation demonstrated increased self-renewal, long-term proliferation,
increased anchorage-independent survival, and a more invasive ability through
matrigel as compared to their CD44
-
α
2
β
1
low
counterparts. These cells do not express
AR, but when exposed to dihydrotestosterone and serum, they demonstrated
decreased CD133 expression and began to exhibit more of a differentiated secretory
luminal cell phenotype with expressions of AR, cytokeratin 18, and prostatic acid
phosphatase. Although in vivo transplant data are needed to confirm that this cellular
subpopulation contains prostate cancer stem cells, this study provided compelling in

5
vitro data supporting the view that CD44
+
α
2
β
1
hi
CD133
+
cells demonstrate properties
of human prostate cancer stem cells.
Patrawala et al. separated cells with CD44 expression from prostate cancer cell
lines and characterized the tumorigenic properties of this cellular population
(Patrawala et al., 2006). The results revealed that CD44
+
cells contain much higher
inherited tumorigenic and metastatic properties in NOD/SCID mice as compared to
the CD44
-
counterparts isolated from the same cell line. CD44
+
cells were also found
to have increased mRNA levels of genes associated with stem cells, such as Oct-3/4,
BMI1, SMO and β-catenin. Additionally, a very small percentage of CD44
+
cells
appears to undergo asymmetric cell division in colony forming analyses. It is
postulated that the CD44
+
population is enriched in cells with stem cell properties
and tumorigenic potential. Importantly and consistent with the results of Collins et
al, they found that the putative stem cells do not express AR and are capable of
differentiation into AR
+
cells.
Interestingly, they observed an isolated population of CD44
-
cells would slowly
generate a small amount of CD44
+
cells in in vivo and in vitro experiments. Although
it was postulated in this experiment that the CD44
+
cells may derive from more
undifferentiated CD44
-
cells, this may suggest a possibility of de-differentiation into
cancer stem cells. It would be necessary to determine if these CD44
+
cells arising
from CD44
-
cells obtain the same stemness and tumorigenic potential as the CD44
+

cells isolated from the original prostate cancer cell lines.

6
Animal models for prostate cancer stem cell studies
Mouse models have been widely utilized in the studies of stem cell biology
(Guasch and Fuchs, 2005). Currently, most laboratories are using human cell lines
and/or xenografts or genetically engineered models (GEM) to study prostate stem
cells. GEM models are used in studies of the molecular events of prostate
tumorigensis. These models can be separated into two areas: One is generated by
overexpression of an oncogene with a prostate specific promoter; the other one is
with targeted deletion of specific genes.
The transgenic mouse model of prostate (TRAMP) has been used by a variety of
investigators to study molecular events in carcinogenesis of the prostate and for
preclinical testing of new therapies (Gingrich et al., 1999; Gingrich et al., 1996).
This model utilized the SV40 T antigen (Tag) transgene specifically overexpressed
by probasin promoter in the epithelial cells of the prostate which results in the
development of prostate cancers from primary malignancies to metastatic tumors in
distant organs, such as lymph nodes and lung. Since the development of TRAMP,
several other transgenic models have become available. Recently, Reiner, T. et al.
develop the FG/TG transgenic mouse model using the human fetal globin promoter
linked to Tag. This model develops a similar progression of prostate cancer seen in
humans from high-grad prostate intraepithelial neoplasia (PIN) to adenocarcinoma.
Their observation showed that Tag is targeted to a subset of p63
+
basal epithelial
cells before the production of PIN. However, p63 is lost in the prostate once the PIN

7
develops (Reiner et al., 2007). The p63
+
basal epithelial cell is required for the
development of basal and luminal cells during normal differentiation of the prostate
and functions as adult prostate stem cells (Reiner et al., 2007; Signoretti et al., 2005).
This suggests that p63 may be one of the marker expressed in prostate cancer stem
cells.
Several murine models generated by disruption or overexpression of genes in the
prostate develop premalignant and malignant lesions. The best characterized of these
models is the loss of the tumor suppressor gene Pten. The lipid phosphatase PTEN,
acts as a tumor suppressor gene by acting as a negative regulator of the
phosphatidylinositol 3-kinase/AKT pathway. Approximately 70% of primary
prostate cancers exhibit a loss of at least one PTEN allele and loss of both alleles is
associated with advanced disease (Carver and Pandolfi, 2006; Shen and Abate-Shen,
2007). In mice, loss of one allele of Pten is associated with the development of high-
grade PIN (Podsypanina et al., 1999) and conditional loss of both Pten alleles in
prostate results in developing adenocarcinoma in situ at the age of 9 weeks and
invasive prostate cancer that metastasizes to lymph nodes and lung at the age of 12
weeks in animals (Wang et al., 2003). This conditional Pten deletion mouse model
(cPen
-/-
) is a combination of PB-Cre4 (Wu et al., 2001) and Pten
floxP
(Wang et al.,
2003) mouse lines and, therefore, the loss of Pten alleles in prostate is mediated by
the Cre/loxP system. In prostate epithelial cells in this model, an androgen-regulated
and prostate epithelium-specific promoter, ARR
2
PB, actives Cre expression in

8
postnatal mice. Cre then causes a DNA recombinant deletion in Pten exon 5, which
is floxed by two loxP sites to make PTEN functionless. Because of the Cre/loxP
system, the conditional Pten model was also able to recapitulate the progression of
the recurrent ADI-cancer observed in humans. Global assessment of molecular
changes caused by a homozygous Pten deletion identified key genes known to be
relevant to human prostate cancer such as up-regulation of cyclin A, clasterin, PSCA,
S100 calcium binding protein P, early growth response 1, and osteopontin, as well as
down-regulation of NKX3.1 and myosin, heavy polypeptide 11, smooth muse(Wang
et al., 2003; Wu et al., 2001).
Stem cell antigen-1 (Sca-1) is a marker that has been demonstrated to enrich for
stem cells in hematopietic, skin, cardiac and testicular tissues (Falciatori et al., 2004;
Matsuura et al., 2004; Montanaro et al., 2003; Spangrude et al., 1988) in mouse,
though the function of Sca-1 is not well understood. It is an 18-kDa mouse glycosyl
phosphatidylinositol-anchored

cell surface protein (GPI-AP) of the Ly6 gene family
and does not have a human ortholog (Holmes and Stanford, 2007). The murine Ly6
gene family encodes at least 18

highly homologous, cross-hybridizing genes closely
linked on

mouse chromosome 15, many of which demonstrate greater than

80%
sequence similarity with Sca-1(Kamiura et al., 1992; Patterson et al., 2000; van de
Rijn et al., 1989). Burger and colleagues found that normal murine prostate cells
with high Sca-1 expression (Sca-1
+high
) had considerably more growth potential and
proliferative capabilities than cells expressing low or no Sca-1 antigen (Burger et al.,

9
2005). Sca-1
+
cells were also concentrated in the proximal region in prostate, in a
pattern similar to BrdU label-retaining cells (Xin et al., 2003; Xin et al., 2005).
Interestingly, Sca-1
+
cell assortments contained both more quiescent cells and more
cycling cells than Sca-1 negative cells. Additionally, androgen ablation enriches for
Sca-1
+
cells (Xin et al., 2005).
To determine the cell of origin of the prostate cancer, Xin et al. genetically
perturbed the PTEN/AKT signaling pathway in both Sca-1
+
and Sca-1
-
cellular
subpopulations from murine prostates and used the renal capsule reconstitution assay
to demonstrate that only the Sca-1
+
fractions are able to contribute to tumorigenic
potential in SCID mice (Xin et al., 2005). Prostate cancer initiation in this
experiment was with an increased Sca-1
+
subpopulation. This suggests that prostate-
regenerating cells have the oncogenic potential and only a limited number of normal
prostate cells are capable to transform, and prostate cancers are likely to be initiated
and maintained by prostate cancer stem cells. In summary, Sca-1
+
cell fractions
contain a higher proportion of cells demonstrating progenitor cell characteristics,
which include replication quiescence, androgen independence, and multilineage
differentiation potentials, indicating that Sca-1 is a prostate stem cell marker.
Recent studies have shown that the expansion of the Sca-1-positive subpopulation
in prostate is related to Pten deletion. Wang S. and his colleges indicated that altered
stem cell proliferation is directly associated with prostate tumor initiation and
progression in the conditional Pten deletion murine model (Wang et al., 2006).

10
Because PTEN negatively regulates basal cell proliferation, Pten deletion in the p63-
basal cells leads to expansion of Sca-1
+
and BCL-2
+
cell subpopulations with
concomitant differentiation. Their works illustrate that Sca-1
+
cell proliferation is an
important event for prostate cancer initiation and early progression.
Except the PTEN signaling, other signaling pathways may also participate in the
regulations of prostate cancer stem cells. Prostate epithelium-specific deficiency for
p53 and Rb tumor suppressors leads to highly aggressive, poorly differentiated, and
metastatic carcinomas in a novel mouse model using the same Cre/loxP mediating
system as the conditional Pten deletion model (Wu et al., 2001; Zhou et al., 2006).
Interestingly, the cells of the earliest neoplastic lesions express Sca-1 and are not
sensitive to androgen withdrawal. The early mutation cells isolated from the
proximal region of the prostatic ducts are capable of forming neoplasms within SCID
mice (Zhou et al., 2007). Thus, this model has shown that p53 and Rb are also
important for the regulation of the prosatic stem cell compartment.

Stem Cell Niche
Maintaining homeostasis through a fine balance between stem cell self-renewal
and loss of cells through differentiation or apoptosis is important in both normal and
tumor tissues. To keep this balance stem cells most interact with their
microenvironment, or “niche”. The factors that niches provide include soluble
growth factors, the extracellular matrix, or neighboring cells. The niche maintains

11
the quiescent and undifferentiated state of stem cells and also these cells’
proliferation and differentiation potentials (Fuchs et al., 2004). Signals that control
the self-renewal property of stem cells may include the Wnt pathway (Verras et al.,
2004; Yardy and Brewster, 2005) Delta/Notch1 pathway (Lowell et al., 2000; Shou
et al., 2001; Wang et al., 2004) , hedgehog (Berman et al., 2004; Karhadkar et al.,
2004; Stecca et al., 2005), and transforming growth factor β (TGF-β) (Bruckheimer
and Kyprianou, 2002; Nemeth et al., 1997; Tsujimura et al., 2002). The molecules
that may be involved in the regulation within stem cells may be p27
kip1
(De Marzo et
al., 1998)

and Polycomb group proteins (Di Cristofano et al., 2001).
Just as with normal stem cells, the niche in tumors may be important for
maintaining asymmetric division of cancer stem cells and for attracting cancer stem
cells close to signals that maintain stem-like properties (Sneddon et al., 2006). In the
head and neck squamous cell carcinoma, the cancer stem cells lie closely to the
stroma, suggesting that there may be crucial interactions between cancer stem cells
and stroma in carcinoma (Prince et al., 2007). An understanding of the cancer stem
cell niche in addition to the cancer stem cells themselves should lead to a better
knowledge of the signals that are important for the self-renewal and/or differentiation
abilities of cancer stem cells in cancers.

12
Prostate Cancer Microenrivonment
Prostatic tumorigenesis is coupled with changes in the interactions between
epithelial cells and the stromal microenvironmental signals that in turn promote
incipient tumor cells to full malignancy. The interactions between epithelium and
mesenchyme are believed to be mediated by paracrine signals (Cunha et al., 1980).
In response to tumorigenesis in adjacent epithelial cells, fibroblasts, a major stromal
component, also undergo genetic and epigenetic changes that may alter the normal
epithelial-mesenchymal interactions (Bergers and Coussens, 2000; Olumi et al.,
1999). For example, Hill et al. demonstrated that tumor cells can induce
overexpression of p53 in stromal fibroblasts in the mouse model of prostate cancer.
This process creates a selective pressure that induces the expansion of a
subpopulation of p53 null fibroblasts, and these stromal cells then contribute to
tumor progression. (Hill et al., 2005). Yang et al. isolated carcinoma-associated
fibroblasts (CAF) from tumored mice and normal prostate fibroblasts from littermate
controls to study the role of stromal cells in tumorigenesis. They found that BMPs
can strikingly stimulate RNA expression and protein secretion of SDF-1 in CAF,
which, in turn, can significantly induce microvascular tube formation (Yang et al.,
2008). Therefore, this suggested that CAF can enhance the tumorigenic properties of
the epithelial compartment; however, whether and how the cancer stem cell niche
controls and regulates the self-renewal and differentiation properties in cancer stem
cells in prostate cancers is still not clear.

13
Hypothesis and Rationale
Most human prostate cancers are adenocarcinomas and associated with the
expression of luminal epithelial cell markers. Many nonsurgical prostate cancer
therapies are directed against the androgen-receptor axis, which is active in most
tumor cells. However, any residual cells which do not express the androgen receptor
may survive to form the basis for developing the new androgen-depletion
independent cancer (ADI-Ca) (Roy-Burman et al., 2004). Experiments have
demonstrated that the proximal region of prostatic ducts is enriched in a
subpopulation of epithelial cells that are cycling slowly (Tsujimura et al., 2002).
These cells lack expression of androgen receptors, can survive androgen ablation and
regenerate prostatic tissue fully once androgens are administered (Goto et al., 2006).
Both of these attributes would be expected in a stem cell population from these areas.
Because prostatic carcinoma eventually progresses to an androgen-depletion
independent tumor (ADI-Ca), this may reflect a stem cell-like phenotype (Reya et
al., 2001). Since prostate cancer arises as a result of unbalanced cell proliferation,
differentiation, and death, the altered stem cell may play an important role in prostate
cancer progression and recurrence. Therefore, the isolation and characterization of
these stem cells from mouse prostate cancer at different stages may lead to new
therapeutic approaches.
In the following chapter, I will describe the development and characterization of
two novel mouse lines based on our conditional Pten mouse model (cPten
-/-
). This

14
cPten
-/-
model has been proven to efficiently generate murine prostatic AD-Ca in
early age and ADI-Ca after castration surgery; however, the growth and progression
of cancer are different in individual mice. To facilitate the studies of cancer stem
cells in collecting murine prostate tumors at different stages of progression of
tumorigenesis, we combined this mouse line with Cre mediated reporter genes,
luciferase or green fluorescence protein (EGFP), to improve its applications.
Chapter three will address isolation and characterization of the putative prostate
cancer stem / progenitor cells derived from murine prostate tissues. The Lin
-
Sca-
1
+
CD49f
+
(LSC) cell subpopulation, isolated from the normal murine prostate, have
earlier been shown to have stem cell-like properties (7). We proceeded to examine
the presence of such cells in the adenocarcinoma of our conditional Pten deletion
mouse model of prostate adenocarcinoma. Our findings suggest that cancer LSC
cells maintain stemness properties and can develop in vitro into structures similar to
cancerous prostate glands.
In the near future, we plan to use these putative murine prostate stem cells to
study the interactions between the cancer stem cell niche and cancer stem cells and
the initiation of ADI-cancer. The details are illustrated in chapter four.

15
CHAPTER 2
Mouse Models of Prostate Adenocarcinoma with Capacity for
Monitoring Spontaneous Carcinogenesis by Bioluminescence or
Fluorescence

Abstract
The application of Cre/loxP technology has resulted in a new generation of
conditional mouse models of prostate cancer. Here, we describe improvement of the
conditional Pten deletion model of prostate adenocarcinoma by combining it with
either a conditional luciferase or EGFP reporter line. In these models, the
recombination mechanism that inactivates the Pten alleles also activates the reporter
gene. In the luciferase reporter model, the growth of the primary cancer can be
followed non-invasively by bioluminescence imaging (BLI). Surgical castration of
tumor-bearing animals leads to a reduced bioluminescence signal corresponding to
tumor regression that is verified at necropsy. When castrated animals are maintained,
the emergence of androgen depletion-independent (ADI) cancer is detected by BLI at
times varying from 7 to 28 weeks post-castration. The ability to monitor growth,
regression or relapse of the tumor by BLI led to collection of tumors at different
stages of development. By comparing the distribution of phenotypically distinct
populations of epithelial cells in cancer tissues, we noted that the degree of
hyperplasia of cells with neuroendocrine differentiation significantly increases in the
recurrent cancer relative to the primary cancer, a characteristic which may parallel

16
appearance of neuroendocrine phenotype in human ADI cancer. The EGFP model, at
necropsy, can provide an opportunity to locate or assess tumor volume or to isolate
enriched populations of cancer cells from tumor tissues via fluorescence-based
technologies. These refined models should be useful in the elucidation of
mechanisms of prostate cancer progression, and for the development of approaches
to preclinical intervention.

Introduction
There are two primary objectives in the modeling human prostate cancer in mice.
The first is to recapitulate the pathophysiologic characteristics of the human disease
in a “natural” manner in immuno-competent mice to facilitate the understanding of
the complex molecular mechanisms underlying prostate cancer. The second is to use
the models to develop or test new targeted therapies. Because the growth and
progression of prostate cancer varies widely from animal to animal, postmortem
tissue analysis of large cohorts of animals is often required to derive statistically
meaningful data. The recent development of miniaturized non-invasive imaging
techniques has made it possible to follow tumor progression in individual mice and
other small animals (Massoud and Gambhir, 2003). Among the different noninvasive
imaging techniques with potential to provide tumor-specific information in
individual living mice, bioluminescence imaging (BLI) has drawn much attention

17
(Adams et al., 2002; Lyons, 2005). Recently, transgenic mouse models were
developed to express firefly luciferase specifically in the prostate in an androgen-
dependent fashion (Ellwood-Yen et al., 2006; Hsieh et al., 2005; Lyons et al., 2006).
In one case, such a system was combined with a SV40 T-antigen induced prostate
tumor model for the purpose of following tumor development by BLI (Lyons et al.,
2006).
Several prostate epithelium-specific Cre/loxP systems have been developed
(Abdulkadir et al., 2002; Ma et al., 2005; Maddison et al., 2000; Wu et al., 2001),
and shown to be useful in generating prostate preneoplastic or neoplastic mouse
models (Abdulkadir et al., 2002; Chen et al., 2005; Huang et al., 2002; Ma et al.,
2005; Maddison et al., 2000; Trotman et al., 2003; Wang et al., 2003; Zhou et al.,
2006). However, there are no reports of incorporation of Cre/loxP-mediated
luciferase activation in any of these systems. The Cre line, PB-Cre4 that we
developed (Wu et al., 2001) has been proven to be robust in not only causing
recombination of both alleles of a target gene (Huang et al., 2002; Trotman et al.,
2003; Wang et al., 2003; Zhong et al., 2006) but also in the efficient switching of up
to four alleles in a single cell (Chen et al., 2005; Zhou et al., 2006). Encouraged by
this powerful expression of Cre in the prostate, we proceeded to increase the
efficiency and utility of the conditional Pten deletion (cPten
-/-
) model (Khodavirdi et
al., 2006; Wang et al., 2003; Zhong et al., 2006) by combining it with a conditional
reporter allele based on the idea that the cells in which Pten alleles are deleted would

18
also be subject to activation of the reporter allele by the same Cre-mediated
recombination. We used the luciferase reporter line (Lyons et al., 2003) that was
demonstrated to be useful in the visualization of spontaneous tumorigenesis in other
tissue-specific conditional mouse models. Similarly, the ROSA26-EGFP
loxP
line
(Mao et al., 2001) offered an opportunity to implement fluorescence imaging of the
cPten
-/-
model.
Here we present evidence that each of the conditional reporter alleles, luciferase
and EGFP can be used to assess the primary tumor burden in the prostate.
Furthermore, we show that longitudinal BLI in the combinatorial luciferase model
can register the growth of the primary tumor, its regression through androgen
depletion, and most importantly, in the recurrence of androgen depletion-
independent (ADI) cancer (Roy-Burman et al., 2005), all in individual living mice.

Materials and Methods
Generation of compound transgenic mice
The luciferase reporter construct that was used to generate the luciferase
transgenic line contained β-actin promoter / loxp / GFP / polyadenylation sequence /
loxp / luciferase / polyadenylation sequence in 5’ to 3’ orientation (18). While the
transgene expression driven by the β-actin promoter is ubiquitous in this mouse line,
the transcription is terminated at the floxed polyadenylation site before the luciferase
gene. However, there is luciferase gene expression when the floxed polyadenylation

19
sequence is deleted by Cre-mediated recombination. In EGFP reporter mice, a floxed
DNA fragment that includes PGK-cytosine deaminase / PGK-Puro / “Stop”
sequences is inserted between ROSA26 promoter and EGFP coding region, so that
EGFP is only expressed upon Cre-mediated excision of this floxed DNA segment
(19). To generate mice with conditional inactivation of Pten alleles and activation of
luciferase or EGFP reporter, male mice of ARR2PB promoter-driven (Zhang et al.,
2000), prostate epithelium-specific Cre line PB-Cre4 on C57B/6xDBA2 background
(Wu et al., 2001) were first crossed to homozygous floxed Pten mice on the
129/BALB/c background (Lesche et al., 2002). The male offspring carrying floxed
Pten alleles and PB-Cre4 transgene (cPten
-/-
) were then crossed to floxed luciferase
(L) or floxed EGFP (G) reporter female mice carrying homozygous floxed Pten
gene. The background of the L reporter line was FVB/N (Lyons et al., 2003), while
that of the G line was C57BL/6 (Mao et al., 2001). The resulting
cPten
-/-
L or cPten
-/-
G mice were consequently of mixed genetic background, a fact
that was readily apparent from variable skin color ranging from white to brown to
black. Only male compound mutant mice were used for the study. Non-recombinant
littermates, such as Pten
floxed/ floxed
L or Pten
floxed/floxed
G without the Cre gene, served as
controls. All mice were maintained under identical conditions and animal
experimentation was conducted using the standards for humane care in accordance
with the NIH Guide for the Care and Use of Laboratory Animals.

20
Bioluminescence Imaging
Mice were given a single intraperitoneal (ip) injection of Ketamin (50 mg/kg) and
Xylazine (10 mg/kg) followed by intravenous (iv) injection of luciferin (50 mg/kg).
After waiting for 4.5 minutes to allow proper distribution of luciferin, the mice were
placed in the chamber of an IVIS 200 optical imaging system (Xenogen Corp.,
Alameda, CA). Photons were collected for a period of 1 min, and images were
analyzed using LIVING IMAGE software v. 2.50 (Xenogen). Bioluminescence
imaging of excised tissues was performed after euthanasia of the animals previously
injected iv with luciferin. Signal intensity was quantified for defined regions of
interest as photon count rate per unit body area per unit solid angle subtended by the
detector (units of photons/s/cm
2
/steradian). Images simulating the three-dimensional
location of bioluminescent sources within mice were generated using the single-view
diffuse tomography capability of the IVIS 200 and LIVING IMAGE 3D v. 2.50.
Immunohistochemistry and immunofluorescence
Immunohistochemical analysis of

parallel paraffin sections of paraformaldehyde-
fixed tissue was done

by a modified avidin-biotin-peroxidase (ABC) technique as
described previously (Zhou et al. 2006). Briefly,

antigen retrieval was done by
boiling the slides in 10 mmol/L

citric buffer (pH 6.0) for 15 minutes. Antibodies to
Ki67

(Novocastra Laboratories, Newcastle upon Tyne, United Kingdom,

#NCL-
Ki67p; 1:1,000 dilution), cytokeratin 8 (CK8; TROMA-1 antibody, Developmental

Studies Hybridoma Bank, University of Iowa; 1:50), cytokeratin

5 (CK5; Covance,

21
Berkeley, CA, #PRB-160P; 1:1,000), androgen receptor

(AR; Upstate,
Charlottesville, VA, #06-680 1:60), and synaptophysin [SYN; Dako, Carpinteria,
CA, #A0010 (1:100)

or #M0776 (1:20)] or Chromagranin A (Zymed Laboratories,
San Francisco, CA. #18-0094, 1:100) were incubated with de-paraffinized sections

for overnight at 4 C. Sections were subsequently incubated with biotinylated
secondary

antibody for 30 minutes at room temperature and then detected with the
ABC Elite kit (Vector

Laboratories, Burlingame, CA) and 3,3'-diaminobenzidine
(Sigma,

St. Louis, MO) as substrate. All transverse sections of the whole prostate
were scanned by ScanScope (Aperio Technologies, San Diego, CA) with 40X
objective followed by lossless compression and assessment of all immunostainings
in identical anatomical regions. Double immunofluorescence staining

was done with
rat monoclonal antibody CK8 (1:5) and rabbit polyclonal

antibody SYN (1:10), or
mouse monoclonal antibody

SYN (1:2) and rabbit polyclonal antibody AR (1:10),
followed by fluorescein (FITC)-conjugated anti-rat

or fluorescein (FITC)-conjugated
anti-mouse and rhodamine (TRITC)-conjugated

anti-rabbit secondary antibodies
(Jackson ImmunoResearch Laboratories,

West Grove, PA). To stain cell nuclei,
sections were incubated

with a 10 µg/mL solution of 4,6-diamidino-2-phenylindole

(DAPI) for 4 minutes.
Detection of EGFP fluorescence in tissues and tissue sections
EGFP expression in tissues was initially examined under a Leica Z16 APO (Leica
Microsystems Inc., Bannockburn, IL) dissecting microscope with a GFP filter. The

22
freshly collected tissues from euthanized mice were placed under the microscope and
images recorded with Leica DFC300FX Digital Color Camera (Leica Microsystems
Inc., Bannockburn, IL) and ImagePro MC5.1 (Media Cybernetics Inc., Silver Spring,
MD). We used confocal microscopy following a modification of a previously
described procedure in order to detect EGFP fluorescence in tissue sections
(Shariatmadari et al., 2001). Briefly, tissues were removed from euthanized mice and
then fixed for two hours in 4% paraformaldehyde at room temperature. Fixed tissues
were washed once in PBS and embedded in Tissue-Tek at room temperature. The
embedded tissues were kept in the dark at 4 C overnight and then slowly frozen at -
80 C in a container wrapped with an insulating material. Tissues were placed at -
20 C for 30 minutes before sectioning. The frozen sections (10 μm) were covered
with mounting medium containing DAPI. The slides were kept overnight at -20 C,
then examined by Zeiss LSM-510 laser scanning confocal microscope (Carl Zeiss,
Thornwood, NY). Images were recorded using the LSM 510 software version 3.2
SP2.
RT-PCR
Total RNA was extracted by TRIzol Reagent (Invitrogen) following the protocol
recommended by the manufacturer. RNA (2 μg) was reverse-transcribed by random
hexamers and SuperScript III reverse transcriptase (Invitrogen) in a volume of 21 μl.
The RT reaction (2 μl) was used as a template in a 20 μl PCR reaction mixture using
a primer set specific for the firefly luciferase reporter gene (Vooijs et al., 2001). An

23
equivalent amount of RNA without reverse transcription served as negative control.
As a positive control for amplification from the cDNA, we used the Gapdh primers,
5’-CAGCCTCGTCCCGTAGACAAAATGG-3’and 5’-TTCTGGGTGGCAGTGAT
GGCATGGA-3’ leading to a product of 520 bp.
Preparation of single-cell suspensions for FACS analyses
Freshly collected prostate tissues were minced with crossed scalpels (size 11
blades), transferred to a 5 ml tube and incubated in DMEM/F12 medium containing
10% fetal bovine serum, collagenase (1 mg/ml), hyaluronidase (1 mg/ml), and
DNase I (1 μg/ml) at 37
o
C overnight on a rotator. Collagenase, hyluronidase and
DNase I were purchased from Sigma,

St. Louis, MO. After low speed centrifugation
the single cells and cell clumps were collected and subjected to treatment with 0.05%
Trypsin-EDTA for 10 min. and then diluted with an equal amount of medium
containing 10% FBS. The preparation was passed through 100 μm and 40 μm cell
strainers sequentially for the isolation of single cell suspensions for FACS analyses.
GFP-expressing cells were detected using the FL1 channel (absorption spectra
530/30 nm) using a MoFlo Cell Sorter with Summit v.3.1 (Dako Cytomation, Fort
Collins, CO),

24
Results
Characterization of cPten
-/-
L and cPten
-/-
G mice. Considering that there could be
significant differences in the recombination efficiency of different alleles in vivo
(Vooijs et al., 2001), we first examined whether the reporter genes were expressed in
a tissue-specific manner. For cPten
-/-
L mice, we administered luciferin, the substrate
for luciferase by iv injection to male mice beginning at seven weeks of age, and
sequential images were recorded from the ventral side. Each experimental mouse
was paired with a corresponding littermate control lacking the Cre transgene. A
representative BLI picture of such a pair of mice taken approximately 5 minutes after
luciferin injection is shown in Fig. 1A. The image shows a strong bioluminescent
signal, specifically from an area corresponding to the prostate while the control
mouse shows only a very weak background signal. We also used three-dimensional
imaging to further identify where the bioluminescence originated in living animals.
For this purpose, we imaged a sexually mature (9 week-old) Cre-positive Pten
floxed/
wild-type
L male (no.1492) that should have a non-cancerous prostate but with Cre-
mediated reporter activation. The ventral image (Fig. 1B) confirmed that the sources
of relatively strong signals (dark dots) were located in the lower abdomen, and the
lateral image showed the source to be concentrated anteriorly in the expected
location of the prostate gland. When a cPten
-/-
L mouse (no.2425) was imaged
similarly, the results also corresponded to the location of the prostate and, as
expected, displayed a much higher (about 100-fold) signal intensity (Fig. 1B).

25
D
B
C
A

26
Fig. 1. In vivo and ex vivo bioluminescence images of cPten
-/-
L mice.
A, comparison of in vivo BLI signals from the abdominal region in a 12 week-old
cPten
-/-
L male (no.1424), and its littermate control (no.1423) that lacked the Cre
transgene. B, 3D imaging of a 9 week-old cPten
floxed/wildtype
L male (no.1492) and a
24 week-old cPten
-/-
L male (no.2425) indicating the major source of BLI signal to
be inside the abdomen at a location corresponding to the prostate. The BLI intensity
from the non-cancerous recombined mouse (no.1492) appeared two logs lower than
that observed in recombined cancerous mouse no.2425. The reverse rainbow color
table represents photo density detected from the surface. The BlackRed color table
represents the source intensity from low to high using a color scale from black to red.
C, ex vivo bioluminescence images of the urogenital system of a 15 week- old cPten
-
/-
L (no.2160) shown next to its control (no.2159). The regions of prostate, seminal
vesicle, spermatic cord, and testes are denoted by P, SV, SC, and T, respectively. D,
BLI indicative of lymph node metastasis in an 18 week-old cPten
-/-
L (no.1309).
Besides the strong signal from the prostate area, some of the other “hot spots”
corresponded to regional lumbar (L) and caudal (C) lymph nodes.

27
To confirm the prostate tissue-specificity of luciferase expression, we examined
several other cPten
-/-
L and Pten
floxed/floxed
L pairs, ranging in age from 10 to 15 weeks
by ex vivo BLI imaging. Mice were euthanized immediately following luciferin
administration and the urogenital system (UGS) was isolated and imaged. An
example of the images obtained at 18 minutes after luciferin administration is
displayed in Fig. 1C. It is clear that the UGS from the cPten
-/-
L mouse presented a
high level of BLI signal from prostate lobes, although weak but still detectable
signals were also found in regions of the seminal vesicles, spermatic cords, and
testes. The signal intensity was either very low or not detected in the reproductive
organs of the littermate controls when examined under the same conditions of
isolation and imaging. Taken together, these results supported the validity of BLI to
study prostate tumorigenesis in vivo in the cPten
-/-
L mice. However, it should be
noted that we encountered several factors, some related to the mixed genetic
background of the animals that influenced the intensity of the BLI signal. We found
that iv injection of the substrate was superior to ip injection in reducing the
bioluminescence background. The strength of the light was also significantly
affected by the fluid content in the bladder. Emitted light is absorbed by the skin or
fur, and a full bladder significantly increases the depth and hence the attenuation of
the signal (Rice et al., 2001). For these reasons, we routinely massaged all animals in
an attempt to release urine, and also shaved mice with dark fur to reduce attenuation
of the light. These limitations hindered comparisons of signal intensity between

28
animals and between different time points within the same animal. Besides
indicating the primary cancer, BLI was also effective in revealing lymph node
metastases in a few instances. This is illustrated in the case of an 18 week-old cPten
-
/-
L mouse shown in Fig. 1D. The dorsal image of this mouse revealed three “hot
spots” superior to the prostate region. When the image of the spots was enlarged and
flipped left-right, it matched very well with the ventral location of the caudal and
lumbar lymph nodes (URL:
http://www.eulep.org/Necropsy_of_the_Mouse/printable.php). These lymph nodes,
located in proximity to the bifurcation of the aorta in the abdominal cavity and close
to the backbone, were previously identified as preferential sites for metastasis in
orthotopic mouse models of prostate cancer (El Hilali et al., 2002; Rubio et al.,
2000). The frequency of detection of lymph node metastases in living animals by the
BLI approach was, however, quite low since we could identify only three such cases
from approximately 120 cPten
-/-
L live mice that were imaged. Since the
lymphovascular metastatic lesions in the cPten
-/-
model are mostly microscopic in
size, and for that matter many could even be missed during histology sectioning (12),
it is not surprising to find the sensitivity of BLI to detect metastases in this model to
be much lower than that of histology sectioning. In addition, it remains to be
determined whether changes in the mixed genetic background in cPten
-/-
L relative to
cPten
-/-
mice might have altered the incidence of micrometastases.

29
The prostate tissues of different cPten
-/-
G male mice ranging in age from 16 to 52
weeks were evaluated with UV epiflourescence microscopy. A view of the
fluorescence images derived from UGS of a cPten
-/-
G mouse relative to that of
controls is presented in Fig. 2. The autoflourescence in the prostate tissue is minimal
in the Pten
floxed/floxed
G mouse lacking the Cre gene (Panel A). The only tissue
exhibiting significant autofluorescence appeared to be the bladder. In comparison to
this background, a Cre-containing non-cancerous prostate of a Pten
floxed/wild-type
G
mouse showed fluorescence, although with variable distribution of intensities within
the lobes (Panel B). While the DLP and VP had uneven but still notable fluorescence
throughout, the AP exhibited the least and only focal fluorescence. This apparent
inefficiency in recombination or marker gene expression in AP appeared similar to
the pattern that we described earlier in PB-Cre4/R26R double transgenic mice (8).
However, the overall efficiency in EGFP expression in the mixed genetic
background of the current EGFP mice was relatively weak. To what extent this
property might be a consequence of the efficiency of recombination of the floxed
EGFP allele or epigenetic modification of marker gene expression is unclear.
It is noteworthy, however, that tumor areas in the prostate, irrespective of the lobe
of origin, had significantly higher levels of fluorescence. This is shown in Fig. 2C in
a 24 week-old cPten
-/-
G mouse for which the littermate control is presented in Panel
A. Of the fifteen cPten
-/-
G mice sacrificed to date, fluorescence from EGFP in the
prostate tumors was detected as early as four months of age. Confocal microscopic

30
Fig. 2. Fluorescence analysis of tissues from cPten
-/-
G males.
A, dorsal and ventral views of UGS of a 24 week-old Pten
floxed/wildtype
G male mouse
lacking the Cre gene(no.465) shown under bright field or UV. B, similar views of a
48 week-old cPten
floxed/wild-type
G mouse (no.343) carrying the Cre gene. C, views of
the prostate of cPten
-/-
G mouse (no.464) for which the control was no.465 (panel A).
On the photomicrographs obtained under bright field, the regions of dorsolateral
prostate (DLP), anterior prostate (AP), ventral prostate (VP), seminal vesicle (SV),
and bladder (B) are indicated. The arrow in the lower right corner of panel C
highlights the glandular structure within the tumor in VP. D, confocal
photomicrographs after nuclear DAPI staining, EGFP fluorescence, and their overlay
are illustrated using the prostate of a 20 week-old cPten
-/-
G male (no.111) relative to
its control (no.112).

31
analysis of another set of animals, aged 20 weeks identified prostatic epithelium as
the primary source of GFP fluorescence in the cPten
-/-
G mouse (Fig. 2D), with the
stromal cells showing only scattered spots of autofluorescence detectable in both the
recombinant and control tissues. Similar to the cPten
-/-
L system, lymph nodes with
prominent fluorescence were rarely identified in the cPten
-/-
G mouse except for a
single caudal node that exhibited focally intense fluorescence (data not shown).
Although auto-fluorescence of tissue materials could generally complicate analysis
of EGFP fluorescence, we did not encounter, as described above, a high level of
auto-fluorescence in the tissue of primary interest, the prostate. The bladder had
detectable autofluorescence as did tissues of the GI system which contain bacterial
flora. There was some variable and patchy fluorescence in testes and spermatic cords
irrespective of the presence or absence of the Cre gene, consistent with
autofluorescence.

Longitudinal monitoring of prostate tumor growth, regression and recurrence
in cPten
-/-
L mice. Beginning at 6 to 8 weeks of age, a cohort of mice was
monitored by BLI at intervals of > 4 weeks up to about 52 weeks. Images obtained
from one such mouse are presented in Fig. 3A, and the measured BLI intensity
originating from the region of interest for this and some other mice, including two
Cre-negative littermate controls is graphically presented in Fig. 3B. In general, the
signal increased with mouse age, although occasional decreases in BLI intensity

32

33
Fig. 3. Longitudinal BLI monitoring of living cPten
-/-
L animals.
A, serial images of an individual male (no.1470) over a period exceeding a year. B,
graphical presentation of such BLI signal measurements for a cohort of four cPten
-/-
L
(solid lines) and two control (dotted lines) animals. C, BLI images of a cPten
-/-
L
mouse (no.1588) at times before and after castration that was carried out at about 5
months of age. D, BLI monitoring of a cohort of several cPten
-/-
L animals (solid
lines) and four controls (dotted lines) castrated at a similar age. The vertical dotted
line denotes the castration time point. Signal intensity was measured for regions of
interest drawn around the lower abdomen.

34
followed by a rise were also apparent. For the cPten
-/-
L mouse no.1470, the BLI
increased at least five-fold over the course of 43 weeks, while that for no.1425
increased 50-fold during the 30 weeks of observation. There was, however, a two-
fold difference in the maximal signal strength attained between these animals. In
other mice, the signal intensity was lower, although an increase with age was still
quite evident. Considering that the spatial resolution of BLI is limited, the fact that
tumor sizes varied from animal to animal, and that several other factors could
influence BLI intensity, the consistent finding of a temporal increase in BLI in
individual cPten
-/-
L mice was very encouraging, especially when littermate controls,
monitored under analogous conditions virtually lacked detectable BLI throughout the
course of the observation. It is clear that this technology will allow evaluation of
tumor growth in individual animals, each one serving as its own control. Prostate
tissues of animals with BLI that were sacrificed between 12 and 52 weeks of age
were confirmed histopathologically to harbor adenocarcinomas, consistent with
previous reports (Khodavirdi et al., 2006; Wang et al., 2003; Zhong et al., 2006).
To investigate the effect of androgen depletion on the growth of prostate cancer in
the cPten
-/-
L

model, a cohort of recombined male mice was castrated at
approximately five months of age after the BLI signal had been recorded from the
prostate region. Imaging was next performed at two to three weeks post-castration,
and, in all cases, a sharp decline in BLI was evident. This is illustrated by a set of
serial images from a mouse in Fig. 3C, and by intensity curves generated from this

35
mouse and others, including four different littermate controls in Fig 3D. Following a
period of latency varying from 7 to 28 weeks post-castration, the signal from the
prostate began to increase again indicating emergence of ADI proliferating lesions.
This was very encouraging since it showed that growth of a recurrent tumor, like that
of the primary tumor, could be followed by BLI in living animals.
In some older cPten
-/-
L mice we also noted a variable signal in the thoracic area.
These mice were mostly 52 weeks of age or more, although a few were as young as
28 weeks. The BLI in the upper body region, seen in both intact and castrated older
mice (Fig. 3A, D), was not detected in the littermate controls lacking the Cre gene.
When we imaged various isolated tissues from some of these animals by ex vivo BLI,
we detected intensities higher than background in the heart and the rib cage. The
observation in the rib cage led us to search for potential skeletal metastases by
histopathologic analyses of bone marrow and bone tissues.
However, we did not detect cancer cells in bone structures in question (data not
shown). The possibility that Cre-mediated recombination in inflammatory cells or
other cell types could have occurred in older animals remained to be examined.
Although BLI from non-recombined tissues in the luciferase reporter line was
described to be low (Lyons et al., 2003), in consideration of the mixed genetic
background of the cPten
-/-
L mice we tested whether leaky expression of the reporter
could be a confounding factor in our analysis. When tissues (lung, liver, spleen, body
muscle, kidney, brain and testes) from Pten
floxed/ floxed
L or Pten
floxed/ wild-type
L allelic

36
mice without the Cre gene were examined for luciferase transcripts by RT-PCR,
readily detectable expression was found in muscle. This is illustrated in Fig. 4A.
Thus, it appears that the β-actin promoter-driven luciferase reporter that has a floxed
polyadenylation sequence before the luciferase open reading frame could undergo
detectable read-through transcription in some tissues. The reason for this effect
remains unclear but could be related to rearrangements that occur in the
cancatemeric transgene or to different insertion sites during segregation (17).
However, this was most likely not a significant problem in altering BLI specificity,
since signals from the Pten
floxed/ floxed
L mice were used to establish the base line.

Identification and phenotypic characterization of cancer cells in the models.
The regional lymph nodes from the cPten
-/-
L mice that were found to be positive by
BLI were examined by histopathology. Metastasis of prostate cancer cells into these
lymph nodes was evident when tissue sections were examined by
immunohistochemistry. This is illustrated by the detection of metastatic cells within
a lumbar lymph node that stained positive for AR and CK8 (Fig. 4B and D). In
regard to the cPten
-/-
G model, we attempted to sort cells from primary tumors on the
basis of EGFP fluorescence. These results relative to the corresponding control
materials are depicted in Fig. 4C. While there was no evidence of EGFP expressing
cells in the control prostate, the neoplastic prostate indicated 5% of the total cell
population to be EGFP-expressing. Further analyses of four other such pairs

37
Fig. 4. Other characteristics of the models.
A, tissues from two non-recombined males, one Pten
floxed/ wild-type
L (no.1382), and the
other Pten
floxed/ floxed)
L (no.1410), both lacking the Cre gene, were analyzed by RT-
PCR for potential leakiness in luciferase gene transcription. Of the various tissues
examined muscle displayed a readily detectable PCR band (lanes 2 and 4) that was
not present when the reverse transcription step was omitted (lanes 1 and 3). B, D,
detection of metastatic cells in the sub-capsular sinus of a lumbar lymph node from a
cPten
-/-
L mouse. Cells with nuclear staining of AR (B) and cytoplasmic staining of
CK8 (D) are detected. ABC Elite method was used with hematoxylin
counterstaining. Calibration bar,10 μm. D, illustration of fluorescence-based sorting
of cells from the neoplastic prostate of a cPten
-/-
G mouse (no.118) relative to the
prostate of a littermate control (no.112).

38
indicated a similar ability to sort approximately 4 to 6% EGFP-positive cells. The
extent of homogeneity and the phenotypic characteristics of the sorted cells,
however, remained to be determined.
Using the cPten
-/-
L model, we wished to determine if the distribution of
phenotypically distinct populations of prostate epithelial cells might be altered in the
recurrent cancer relative to the primary cancer. We used CK8 and AR as markers for
the secretory luminal epithelial cells, CK5 for basal cells, SYN for neuroendocrine
(NE) cells, and Ki67 as a marker for proliferation. The results of such analyses with
three different pairs of tumors were similar. Representative photomicrographs are
shown in Fig. 5. Epithelial cells of the normal prostate and primary tumors had
positive nuclear staining for AR. In contrast, epithelial cells from recurrent tumor
displayed mostly a diffuse cytoplasmic AR staining (Fig. 5A). A majority of thr
atypical cells in the primary tumor and the recurrent tumor were CK8-positive, and
basal cells, marked by CK5 expression and separated from the stroma by the
basement membrane in the normal prostate, were commonly found inside both the
primary and recurrent tumors. While only a few cells in normal epithelium expressed
SYN, many cells in primary tumors and many more in recurrent tumors expressed
this NE marker. These cells were arranged both individually and in groups, in one
case substituted the entire thickness of the epithelium. Their NE differentiation was
confirmed by using an additional antibody against synaptophysin as well as by
detection of Chromagranin A. The characteristics of the SYN-expressing cells in the

39
A

40
B

41
Fig.5. Comparative immunohistochemical analysis of epithelial cells from the
normal prostate and primary and recurrent tumors.
A. Staining for hematoxylin and eosin (H&E), AR, CK8, CK5 and SYN on serial
sections of the normal (control) and neoplastic (primary tumor and recurrent tumor)
epithelium of the dorsolateral prostatic lobes. Arrows indicate areas enlarged in the
inserts. Note that prior to castration nuclear AR is detected in many of normal
prostatic cells and virtually all neoplastic cells. After castration expression of AR is
reduced and mainly cytoplasmic. The majority of neoplastic cells are CK8 positive in
both primary and recurrent tumors. The number of CK5 positive cells is increased
but not equally in all glands. Individual CK5 cells and their clusters are located
within both basal and luminal layers (arrow). SYN positive cells are very rare in the
distal part of the normal prostate and punctate SYN staining identifies nerve
terminals (arrow). Primary tumors have an increased number of SYN positive cells
(arrow) and their population further increases in castrated mice (arrow). Calibration
bar: low magnification, 100 μm: high magnification, 33 µm. B, characterization of
SYN positive cells by co-immunofluorescence. (Upper row) Co-localization of SYN
(red) and AR (green) in some (arrowhead) but not all (arrow) cells. (Middle row) Co-
localization of SYN (red) and Ki67 (green) in some (arrowhead) but not all (arrow)
cells. (Bottom row) Absence of co-localization of CK5 (green) and SYN (red,
arrows). DAPI counterstaining (blue) was used for multiple immunofluorescence.
Bar: 50 µm.

42
primary tumor were further examined by immunofluorescence analysis (Fig. 5B).
Interestingly, SYN and AR co-localized in some (arrowhead) but not all (arrow)
cells. Similarly, co-localization of SYN and Ki67 were detected in some (arrowhead)
but not all (arrow) cells and no co-localization of CK5 and SYN was observed.

Discussion
Bioluminescence imaging of prostate cancer in xenograft and spontaneous tumor
mouse models has been described (Adams et al., 2002; Jenkins et al., 2003; Lyons et
al., 2006; Scatena et al., 2004; Xie et al., 2004). In the spontaneous models reported
to date, the detection of BLI was based on prostate epithelium-specific and
androgen-responsive promoter driven expression of a luciferase reporter gene (Lyons
et al., 2006; Xie et al., 2004). Our approach has been different in the sense that the
prostate epithelial cells in which Pten alleles are inactivated by Cre/ loxP are also
targeted by the same mechanism for activation of the reporter gene, either luciferase
or EGFP. The initial recombination processes require Cre expression, which is
regulated by androgen. However, once the genes undergo recombination, the
affected cells or their progeny become independent of androgen since neither the
Pten allele nor the reporter transgene requires androgen for expression. Thus, the
models are ideal for monitoring growth, regression or relapse of the cancer
irrespective of hormonal manipulations, such as androgen deprivation. In fact, we

43
clearly demonstrate in the cPten
-/-
L model that BLI can register the growth kinetics
of the prostate cancer before and after castration, and most significantly, demarcate
the time points when the ADI recurrent disease emerges. The capacity to ascertain
the growth of the recurrent cancer non-invasively is an achievement that, to our
knowledge, has not been reported before. This has many implications. We have only
presented a snapshot of how the model could be exploited to delineate the
characteristics of recurrent cancer from those of the primary tumor. One of the
common features of recurrent prostate cancers is NE differentiation. Human prostate
adenocarcinomas exhibit at least focal positivity for NE markers in the range from 30
to 100% of cases (Abrahamsson, 1999; Abrahamsson and di Sant'Agnese, 1993; di
Sant'Agnese, 1992a, b; Young, 2000). NE differentiation has been reported to
increase in advanced tumors and ADI tumors (Bonkhoff et al., 1995; Bonkhoff et al.,
1991; di Sant'Agnese, 2001; Hoosein et al., 1993; Huang et al., 2006; Ito et al., 2001;
Jongsma et al., 2000; Vashchenko and Abrahamsson, 2005; Weinstein et al., 1996).
However, some other studies have led to findings that differed from such a
correlation. Thus, the role of focal NE differentiation in prostate cancer and its
connection to the development of ADI neoplasms remains controversial. Our results
indicate that in addition to well established hyperplasia of CK5-positive basal cell
compartment (Wang et al., 2006), Pten inactivation leads to the expansion of cells
with NE differentiation. The degree of hyperplasia of such differentiated cells
increases after castration, which may parallel the appearance of the NE phenotype in

44
human ADI tumors. Given that not all SYN+ express AR and none expresses CK5, it
is possible that those cells represent an expanding pool of luminal/NE progenitor
cells. With the availability of distinctive markers for prostate stem cells, the present
model will also make possible to a critical analysis of the hypothesis that recurrent
cancer has an origin in cancer stem cells. This type of investigation will be facilitated
by timing the collection of tumors at specific growth or re-growth points, an
advantage that is provided by the model.
We have also identified certain limitations of the cPten
-/-
L model. First, there is
detectable transcription from the luciferase reporter downstream of the poly-A
termination signal in non-recombined tissues, particularly muscle. Since the reporter
transgene is driven by the β-actin promoter (Lyons et al., 2003), it is not unexpected
that such read-through transcripts should turn out to be relatively more frequent in
muscle tissue where the promoter should be most active. Initially, when we
administered luciferin by ip injection, BLI signals were detected around the site of
injection in both cPten
-/-
L and Pten
floxed/ floxed
L controls. We speculate that such
activity is related, at least in part, to the above-noted leakiness. We were, however,
able to greatly minimize this problem by administering luciferin via the tail vein.
Another phenomenon that cannot be readily explained is the increasingly strong BLI
that became apparent with time in the thoracic area of some older experimental but
not the control animals. We were particularly interested in the signal that is emitted
from the rib cage. From extensive scrutiny of the associated skeletal structures, we

45
have determined that bone metastasis, if any, remains below the level of our
detection. In spite of these limitations, the model appears to serve efficiently when
BLI is used for detection, growth, atrophy or relapse of prostate cancer at the
primary organ site.
While the cPten
-/-
L mice are most useful for tumor studies in live animals, cPten
-/-
G mice, on the other hand, provide a means to highlight and localize primary tumors
at necropsy. Although it remains to be demonstrated, the anticipated spontaneous
EGFP expression in recurrent cancer should be similarly valuable. The ability to
isolate fluorescent cell populations from primary or recurrent cancers should provide
excellent resources for studies to derive clues that may distinguish cellular and
molecular properties of these cancer cells of varied origin. Additionally, isolation of
such cell types should facilitate the generation of cell lines for the study of signaling
aberrations. The refined preclinical models that we have developed should help to
uncover the underlying mechanisms of prostate cancer progression and serve as
convenient study systems to validate tests used for diagnosis, prevention or
treatment.

46
CHAPTER 3
Isolation and Characterization of Murine Prostate Cancer Stem /
Progenitor Cells

Abstract
Recent studies have shown that the Lin
-
Sca-1
+
CD49f
+
(LSC) subpopulation
isolated from the normal murine prostate can undergo self-renewal and form
spheroids in vitro for several generations, and can differentiate to produce prostatic
glandular structures in vivo. Considering this type of selection is likely to enrich a
putative epithelial stem cell-like cell population of the prostate, we applied this
approach to examine the presence of such cells in the adenocarcinoma of the
conditional Pten deletion mouse model of prostate cancer. While the LSC
subpopulation comprises 4% of the Lin
-
cells in the proximal region of normal
tissues, we found a higher percentage of LSC cells in androgen dependent primary
tumors and an even potentially higher percentage in androgen-depletion independent
recurrent tumors. To determine whether the stromal fibroblasts can influence the
differentiation of stem / progenitor cells, we conducted spheroid-forming analyses in
a matrigel-based co-culture system. Interestingly there is about a two-fold increase in
the number of spheroids in co-cultures of Lin
-
Sca-1
+
cells with carcinoma associated
fibroblasts (CAF) compared to the co-cultures of Lin
-
Sca-1
+
cells with embryonic
urogenital sinus mesenchyme or normal prostate fibroblasts (NPF). Spheroid

47
morphology also appeared to vary with the source of the Lin
-
Sca-1
+
cells. In the
presence of NPF the spheroids generated from cancer Lin
-
Sca-1
+
cells showed
cancer-like glandular structures while spheroids from normal Lin
-
Sca-1
+
cells
yielded mostly single or double layers of epithelial cells with only a few intra-lumen
cells. However, in the presence of CAF spheroids formed from the normal Lin
-
Sca-
1
+
cells were larger and with a denser lumen. In addition, we also noted that a
significant portion (8.94%) of the Lin
-
Sca-1
hi
cells from the androgen dependent
tumors might be lacking intracellular PTEN reactivity. Together, the results provide
a basis for the following contentions: (1) LSC cells enriched subpopulation isolated
from the mouse model of prostate adenocarcinoma does have the capacity to self-
renew and differentiate in vitro; (2) the increased percentage of LSC cells in the
cancer relative to the normal counterpart tissues implies that they may have an
important role in tumor growth and survival; (3) Sca-1 appears to be a prominent cell
surface marker of stem / progenitor cells both in normal and neoplastic context of the
mouse prostate; and (4) continued presence or even further enrichment of LSC cells
in the ADI cancer might serve as a clue in the strategies to better understand the
development of recurrent prostate cancer.

48
Introduction
Stem cell biology and tumorigenesis may be linked, and stem cells may have a
role in the etiology of cancer (Al-Hajj and Clarke, 2004; Al-Hajj et al., 2003; Reya et
al., 2001). Properties shared by neoplastic and stem cells indicate a possibility that
somatic stem cells or transit-amplifying (TA) cells that have maintained on
reacquired stem cell properties, particularly the ability for self-renewal, may
represent favorable targets for malignant transformation (Al-Hajj et al., 2003;
O'Brien et al., 2007; Ricci-Vitiani et al., 2007). These findings suggested the concept
of a cancer stem cell in which the tumor-initiating properties reside. These tumor-
initiating cells were identified by their unique and rare constellation of surface
markers that are different from those expressed on the majority of cells in the tumor.
A stem cell model for prostate organization proposes that a pluripotent stem cell is
the progenitor of differentiated basal, secretory luminal and neuroendocrine cells
(Isaacs and Coffey, 1989; Signoretti et al., 2005).
The hypothesis of the existence of prostate stem cells was initially supported by
the experiments done in animals. Androgen deprivation leads to rapid involution of
the gland with massive cell loss largely due to the loss of luminal cells, but when
androgens are restored, the gland regenerates completely (English et al., 1987; Evans
and Chandler, 1987; Isaacs et al., 1987). As this cycle of involution / restoration of
the gland can be repeated multiple times, this observation demonstrates the existence
of a population of stem cells that are able to survive in a low androgen environment

49
and that can reconstitute the organ when androgen levels are restored (Bonkhoff and
Remberger, 1996). Presence of stem cells in the adult human prostate is also
supported by the finding that a small number of cells within the prostate possess the
capacity to form glandular-like structures in reconstituted systems (Hudson et al.,
2000).
While the origin of “prostate cancer stem cell” remains unclear, these cells may
bear or carry the potential for a complete set of mutations responsible for the
carcinogenic properties (Reya et al., 2001), and may differ from their differentiated
progeny by epigenetic or other types of controls (Feinberg et al., 2006). According to
this concept, the tumor mainly consists of cells that underwent genetic and epigenetic
changes incompatible with the cancer stem or progenitor cell properties and do not
contribute to the maintenance of neoplasia. Such cells will be responsible for the
initial response to androgen deprivation therapy in prostate cancer, where most
differentiated cells are AR positive. However, since this therapy is not designed to
target cancer stem cells that do not express AR, the disease is likely to relapse later
as recurrent cancer (Collins and Maitland, 2006; Dehm and Tindall, 2006; Nikitin et
al., 2007; Sharifi et al., 2006).
Cell surface markers to isolate or separate prostate basal, luminal, neuroendocrine
and other prostatic cell types are yet to be identified. However, CD49f (integrin α6)
may be used as a surface marker for CK5-positive basal cells. Certain non-prostate
cell lineages that are present within the prostate may, however, be identified by

50
FACS using antibodies against CD45 (hematopoetic), Ter119 (red blood cell), and
CD31 (endothelial). These three non-prostate markers are collectively called “Lin”.
Recognizing that stem cells in other organs express a cell surface protein commonly
referred to as Sca-1, and that the proliferation of CK5-positive basal cells is
concomitant with the expression of Sca-1-positive cells in the mouse prostate, a
selection based on Lin
-
Sca-1
+
CD49f
+
(also named LSC) has resulted in enrichment
of prostate-regenerating cell populations (Lawson et al., 2007; Wang et al., 2006;
Xin et al., 2005). It should be pointed out that anatomically, each lobe of the mouse
prostate consists of a series of branching ducts each of which could be designated in
three parts: proximal region attached to the urethra, intermediate region, and a distal
region or acinus. While the differentiated cells and the TA cells occur predominantly
in the distal region, prostate stem cells are concentrated in the proximal region
(Burger et al., 2005; Lawson et al., 2007; Tsujimura et al., 2002; Xin et al., 2005;
Zhou et al., 2007). Sca-1 may also be expressed by both basal and luminal cells of
the proximal but not the distal region. The Sca-1-enriched subpopulation from the
proximal niche survives but does not proliferate in the absence of androgen.
However, they are capable of reconstituting the more differentiated distal region
when grafted admixed with urogenital sinus mesenchyme (UGSM) under the renal
capsule of SCID mice in the androgen environment (Burger et al., 2005; Xin et al.,
2005). There is evidence that Pten deletion leads to the expansion of the Sca-1
+

subpopulation (Wang et al., 2006), and that Sca-1
+
malignant epithelial cells from

51
the TRAMP mouse model, as well as those from normal prostate but with lentivirus-
mediated alteration of the PTEN/AKT signaling pathway, give rise to PIN lesions
and foci of cancer in vivo (Burger et al., 2005; Xin et al., 2005).
To determine if a cancer stem cell population could be identified in prostate
adenocarcinoma, we used Lin, Sca-1 and CD49f as the surface markers to isolated
LSC cells from prostate tumors collected from our mouse model of prostate
adenocarcinoma. We identified the Lin
-
Sca-1
+
CD49
+
subpopulation as putative
murine prostate cancer stem cells, which showed the stem cell properties of self-
renewal, the ability to produce differentiated progeny, and increased expression of
the developmental signaling molecule BMI1. Identification of prostate cancer stem
cells and further elucidation of the signaling pathways that regulate their growth and
survival may provide novel therapeutic approaches to treat prostate cancer.

Material and Methods
Animals
The cPten
-/-
L transgenic mouse strains were generated in our laboratory
previously (Liao et al., 2007). Mice were housed and bred under the regulation of the
NIH Guide for the Care and Use of Laboratory Animals. Mice were monitored
routinely by bioluminescence

imaging for the growth of tumors and then euthanized
at desired

time points to collect tumors. In this report, male mice were euthanized for
collection of prostate tissues at the age of 10 to 12 months.

52
Preparation of single-cell suspensions
Freshly collected prostate tissues were minced with crossed scalpels (size 11
blades), transferred to a 50 ml tube and incubated in 5 ml of DMEM/F12 medium
containing 10% fetal bovine serum, collagenase (1 mg/ml), hyaluronidase (1 mg/ml),
and DNase I (1 μg/ml) at 37
o
C overnight on a rotator. Collagenase, hyluronidase and
DNase I were purchased from Sigma,

St. Louis, MO. After low speed centrifugation
the single cells and cell clumps were collected and subjected to treatment with 0.05%
Trypsin-EDTA for 10 min at 37
o
C and then diluted with an equal amount of medium
containing 10% FBS. The preparation was passed through 100 μm and 40 μm cell
strainers sequentially for the isolation of single cell suspensions for FACS analyses.
Fluorescence-activated cell sorting (FACS)
Dissociated prostate cells were stained with biotinylated Lin antibodies (CD45,
CD31 and Ter119; final concentration: 0.1μg / 10
6
cells) for 10 minutes on ice,
washed with cell staining buffer (Biolegend), bound with streptavidin conjugated
iron beads, and separated using magnetic cell sorting system (Invitrogen). Lin
-
cells
were transferred to a new tube and stained with PE/Cy5-conjugated Sca-1 antibody
(Biolegend) and PE-conjugated CD49f antibody (Biolegend). The final
concentrations of Sca-1 and CD49f antibodies were 0.1 and 0.25μg / 10
6
cells.
Stained cells were kept on ice in the dark until sorting.
Fluorescence-activated cell sorting based on specific intracellular protein
expression

53
Lin
-
cells isolated from the magnetic cell sorting system (Invitrogen) were stained
by PE-conjugated Sca-1 antibody (Biolegend) with the final concentration of
0.1μg/10
6
cells if the purpose of the cells was for PTEN staining, or this PE-
conjugated Sca-1 antibody and Alex 647 conjugated CD49f (Biolegend) with the
final concentration of 0.25μg / 10
6
cells if the purpose of the cells was for BMI1
staining. Stained cells were fixed by PFA (final concentration: 0.65%) for 20
minutes, treated with Triton X-100 / PBS (final concentration: 0.1%) on ice for 10
minutes, centrifuged, and resuspended in cold 50% Methanol / PBS on ice for one
hour. Cells were resuspended in the incubation buffer (0.5% BSA in 1 x PBS) for 10
minutes for blocking and stained with rabbit monocolonal PTEN antibody (Cell
Signaling. 1:50) or rabbit polycolonal BMI1 (Santa Cruz. 1:100) antibody for 30
minutes. After washing with ice-cold incubation buffer, cells were stained with FITC
conjugated goat anti-rabbit secondary antibody (Sigma. 1:80) for 30 minutes. Cells
were washed again, resuspended in 0.5 ml cell staining buffer (Biolegend) and
analyzed by flow cytometer.
Prostate spheroid-forming assay
The conditions to culture and passage prostate spheres were adapted and modified
from previously published protocols (Lang et al., 2001; Xin et al., 2005; Xin et al.,
2007). Briefly, each sample of prostate

cells was counted and suspended in 1:1
Matrigel/PrEGM (BD Biosciences) in a total volume of 250 µl. The mixture was

plated in one well of a 24-well plate and allowed

to solidify for 15 min at 37°C

54
before 1 ml of PrEGM was added. Stromal cells were counted and seeded inside the
insert (BD Biosciences, pole size: 8.0 μm) above matrigel. This system was cultured
at 37°C, and one half volume of PrEGM was changed every 3 days. Spheres

were
counted 12 days after plating. For passaging of

spheres, media was aspirated and
Matrigel was digested by incubation

in 500 µl of dispase (Becton Dickinson)

for 30
min at 37°C. Digested cultures were pelleted and

incubated in 1 ml of F12/DMEM
containing collagenase (1 mg/ml), hyaluronidase (1 mg/ml), and DNase I (1
μg/ml)for 30

min at 37°C. Samples again were pelleted and incubated in

0.05%
Trypsin/EDTA for 10 min at room temperature, and passed over a 40-µm

filter. Cells
were counted and replated at

an appropriate density of cells per well after each
passage.
Isolation and culturing of primary stromal cells
Our method of culturing embryonic urogenital sinus mesenchyme (UGSM) was
modified from previous published reports (Xin et al., 2003; Xin et al., 2007) . E16.5
embryos were dissected and collected from euthanized parent female mice. UGS was
then isolated from these embryos in sterile PBS and dissected into two parts: UGSE
and UGSM. UGSM was mixed with 500 µl of the same F12/DMEM buffer
mentioned in the method of preparation of single-cell suspensions in a new tube.
After culturing in 37 °C for one to two hours, UGSM cells were centrifuged and
resuspended in Bfs medium for no longer than two weeks. The components of Bfs
were supplemented with

5% fetal bovine serum, 5% Nu serum (BD Biosciences), 0.5

55
µg/mL

R1881 (PerkinElmer), 5 µg/mL insulin (Sigma-Aldrich),

and 1%
penicillin/streptomycin.
For collecting the primary culture of carcinoma-associated fibroblasts (CAF), the
tumor tissues were first carefully

dissected, minced with crossed scalpels (size 11
blades), and then

cultured in Bfs medium. After 1 week of culturing, cells

that
migrated from the tissue clumps were trypsinized and transferred

to new culture
dishes. The primary cultures were maintained and used for experiments by 12
passages. The prostate lobes from littermate controls were similarly processed to
obtain the primary culture of normal prostate fibroblasts (NPF).
Histology and immunofluorescence
After twelve days of culturing in the in vitro spheroid-forming analyses, each
matrigel containing spheroids formed was covered with OCT and then stocked at -
80°C. Before the sectioning, matrigel with OCT was removed from the well,
embedded with OCT at -25 °C, and sectioned. The frozen sections (8 µm) were
stained by either H&E or immunofluorescence staining. The method of
immunofluorescence staining was based on the procedure previously mentioned in
chapter 2. Briefly, sections were fixed in 4% PFA for 20 minutes and washed with
1X PBS three times for a total of 30 minutes. Immunofluorescence staining was done
with rabbit polyclonal p63 antibody (Santa Cruz; 1:100) and rat monoclonal CK8
antibody (1:100), following by FITC-conjugated anti-rat (Sigma; 1:200) and

56
rhodamine (TRITC) -conjugaged anti-rabbit (Sigma; 1:200) antibodies. To stain cell
nuclei, sections were covered with mounting medium containing DAPI (Vector).

Result
LSC cells are increased in AD- and ADI-cancer. We have isolated and examined
the properties of the Lin
-
Sca-1
+
CD49f
+
from tumor tissues dissected and collected
from the cPten
-/-
L mice. Such LSC populations were also isolated from the proximal
ducts of the littermate controls under identical conditions. Fig. 6A illustrates the
FACS plot and gate of LSC cell subpopulation in an AD-Ca prostate tumor and a
normal prostate tissue. These tissues were collected from a cPten
-/-
L mouse (aged 12
months) and its littermate control. Interestingly, the percentage of LSC cells among
the Lin
-
cell population in this tumor (37.57%) was much higher than in the normal
counterpart (2.74%). Results collected from a greater number of normal and tumor
samples from mice of the ages between 10 to 12 months suggested that prostate
cancer tissue harbored a higher percentage of LSC among the Lin
-
cells than the
corresponding fraction from the normal proximal niche (4%, n=5), where this LSC
cell subpopulation should be more dense(Fig. 6B). There was an indication that LSC
may be further expanded in ADI tumor (43%; n=5) relative to androgen-dependent
(AD) tumor (33%; n=2). We also noticed that there was only about 20% of the LSC
cell subpopulation in the younger mice at the age of 4-5 months (data not show).
Therefore, these FACS results observed in this study indicated that the LSC

57
A
B
C

58
Fig 6. FACS analyses of the Lin
-
population isolated form murine prostate
tissues.
A, FACS plots show gates drawn for sorting LSC subpopulations from Lin
-
cells.
The prostate tissues were dissected from a ten-month old cPten
-/-
L mouse and its
normal littermate control. B, comparing the LSC fractions in Lin
-
population between
normal and tumor tissues of the prostate from 12 animals. C, comparison of either
the Lin
-
Sca-1
+
or the Lin
-
CD49f
+
subpopulation in the Lin
-
population between
normal and tumor tissues of the prostate.

59
subpopulation expands with mouse age. Because prostate tumors in our cPten
-/-
L
model are usually enlarged and become more aggressive with age, the cancer LSC
subpopulation in prostate should play an important role in maintaining and
promoting the progression of prostate tumors. The LSC cell subpopulation in ADI-
Ca should also be linked to the initiation and growth of recurrent cancers in the
prostate. Previous studies have shown that the LSC cell subpopulation isolated from
normal murine prostates contains prostate epithelial stem cells. Thus far, our
observations suggest a clue that the LSC cell subpopulation in tumors may be related
to cancer stem / progenitor cells.
To conclude which cell surface marker contributes more compared to other
markers to the increase of LSC cell subpopulation in AD- and ADI-Ca, we further
compared the cell population in regards to the expression of Sca-1 or CD49f in
tumors and in normal prostate tissues. The result is shown in Fig. 6C. There were
more than 60% Lin- cells expressing Sca-1 in either AD- (63.50%; n=3) or ADI-Ca
(69.60%; n=2) relative to the normal prostates (8.27%; n=4), meaning that there was
an eight-fold increase in Sca-1 in tumors. However, there was only a three-fold
increase when comparing the CD49f expressing cell populations in tumors to normal
tissues (AD-Ca and ADI-Ca: 55%; normal: 18.76%). Because Sca-1 is enriched in
normal prostatic epithelial stem cells (Burger et al., 2005), its expression in tumor
cells suggests the existence of the prostatic cancer stem cells in murine prostate

60
adenocarcinoma, and further indicating that Sca-1 has an important role in murine
prostate cancer stem / progenitor cells.
In the studies of normal prostate stem cells, the Sca-1 antigen is present on a
much larger population of cells than what can be considered to be true stem cells
(Lawson et al., 2005). Therefore, in this study, Sca-1 was probably most likely
expressed by more differentiated progenitor cells in prostate cancer as well.

Characterization of putative prostate stem / progenitor cells isolated from
cPten
-/-
L mice. We also attempted to determine if the Sca-1
+
fraction contained cells
that might have undergone recombination at the Pten alleles. In this initial study we
used the Lin
-
population that was permeabilized to facilitate detection of intracellular
proteins following published protocols (Askenasy and Koretsky, 2002; Chow et al.,
2005). The results illustrated in Fig. 7A indicated that there was a significant portion
(8.94%) of the Lin
-
cells with strong Sca-1 positivity from the AD prostate tumors
may potentially lack intracellular PTEN reactivity. We rarely detected cells with the
same type of expression pattern (0.18%) in the normal prostate tissue from the
littermate control mouse. This interesting issue remains to be investigated with the
LSC fraction and by using other more sensitive techniques.
To characterize LSC cells isolated from murine prostate cancers, we compared the
percentages of the Lin
-
Sca-1
+
CD49
+
Bmi1
+
cell subpopulation within Lin
-
cells from
different prostate tissues using fluorescence activated cell sorting. BMI1, one of the

61
A
B
Fig 7. Characterization of stem / Progenitor cells with respect to intracellular
protein expression.
A, a Lin
-
cell subpopulation with high Sca-1 and low or no PTEN expression was
identified in a mouse tumor but not in the age-matched normal control. B,
comparison of the percentage of BMI1
+
cells in the Lin- population. Samples were
collected form two pairs of normal and AD-Ca tissues of prostate. Another prostate
tissue was collected from ADI-Ca. (N: normal controls)

62
polycomb group genes and a common oncogene activated in lymphoma, has been
found to be required for the proliferation and self-renewal of normal and leukemia
stem cells (1). Results collected from two pairs of littermates are showed in Fig. 7B,
the percentage of the Lin
-
Sca-1
+
CD49
+
BMI1
+
cell subpopulation within the total Lin
-

cell population was increasing in AD-Ca by almost two folds when compared to the
normal sample. This percentage was even higher in ADI-Ca (~6 folds higher). Since
BMI1 expression is important in controlling self-renewal in stem cells, these results
may indicate that the LSC subpopulation contains the rare cancer stem cells in
murine prostate adenocarcinoma and that BMI1 related pathways participate in the
initiation and growth of recurrent prostate cancers.
In biological assays with the Lin
-
Sca-1
+
cells isolated from AD tumors, we found
that they were able to form spheroids more efficiently when co-cultured with
prostate fibroblasts derived from either normal or tumor tissues. Lin
-
Sca-1
+
cells
(2x10
4
) were mixed with matrigel and with the same number of fibroblast cells
seeded in an insert placed above the matrigel inside a well in a 24-well plate as
shown in Fig. 8A. The number of spheroids formed was counted at day 12. Initial
series of experiments show that fibroblast cells, in general, appear to help LSC cells
to form increased number of colonies compared to controls (LSC cells alone). While
there was not a significant difference between the number of spheroids obtained in
co-cultures with UGSM or NPF, there was about a further two-fold increase in the
number of spheroids in co-cultures with CAFs. The result is illustrated in Fig. 8B

63
A
B
C

64
Fig 8. Prostate spheroid-forming analyses of the Lin
-
Sca-1
+
cell subpopulation.
A, the plot of the co-culturing system used in prostate spheroid-forming ayalyses, in
which sorted Lin
-
Sca-1
+
cells were mixed with matrigel in the well. Above the
matrigel was an insert (pole size: 8.0 μ m) with stromal cells. B, comparing the
spheroid forming ability of the Lin
-
Sca-1
+
subpopulation in the co-culture with
different sources of stromal cells. Lin
-
Sca-1
+
cells (2x10
4
) were co-cultured with the
same amount of stromal cells for 12 days. C, comparison of the self-renewal
potential of normal and AD-Ca Lin-Sca-1+ cells. Cells (2x10
4
) isolated from
spheroids formed in the co-cultures with UGSM were passaged to a fresh co-culture
system with the same amount of UGSM cells and cultured for 12 days.

65
and C. Interestingly, with the same number of cells seeded at the initiation of
culturing, normal Lin
-
Sca-1
+
cells formed agreater number of spheroids than AD-Ca
Lin
-
Sca-1
+
cells.
We next examined the ability of self-renewal of AD-Ca Lin
-
Sca-1
+
cells by
passaging these cells isolated from spheroids formed into a fresh co-culture system.
A certain number of cells (2x10
4
) were counted and seeded into new matrigel with
inserts containing UGSM (2x10
4
). Twelve days later, spheroids formed were found
in both co-cultures (Fig. 8C). Interestingly, we found that cells isolated from tumor
Lin
-
Sca-1
+
spheroids formed generated a greater number of spheroids than those
separated from normal Lin
-
Sca-1
+
spheroids formed, implying that AD-Ca Lin
-
Sca-
1
+
cells have a stronger self-renewal property than normal Lin
-
Sca-1
+
cells.
Spheroid morphology also seemed to vary with respect to the source of the Lin
-
Sca-1
+
cells. The spheroids generated from cancer Lin
-
Sca-1
+
cells appeared to have
dense glandular structures while spheroids from normal Lin
-
Sca-1
+
cells presented a
smaller size with a clear lumen (Fig.9A). An exception is noted when the fibroblast
compartment was from the CAF cells. In this case the spheroid morphology appeared
to be different from those obtained with UGSM or NPF. In the H&E staining, the
spheroids generated from cancer Lin
-
Sca-1
+
cells appeared to have cancer-like
glandular structures while spheroids from normal Lin
-
Sca-1
+
cells presented one or
two epithelial cell layers with only a few intra-lumen cells. However, in the presence

66
A
B

67
C
Fig. 9. Comparative morpholoical and histological analyses of spheroids formed
form Lin
-
Sca-1
+
cells.
A and B, the morphology of spheroids formed. Typical phase contrast morphologies
(Bar, 100 μm) (A) and the H&E staining (Bar, 10μm) (B) of 8-um frozen sections of
spheroids formed. C, characterization of spheroids formed by coimmunofluorescence
staining. Sections of spheroids were stained with antibodies for the basal cell marker
p63 (red), the luminal cell marker CK8 (green) and DAPI (blue). Cells with the
expression of p63 and CK8 were found in the spheroids formed from the AD-Ca Lin
-
Sca-1
+
cell subpopulation (white arrows). Bar, 50 μm.

68
of CAF the lumen was denser and the size was also increased for the spheroids from
the normal Lin
-
Sca-1
+
cells (Fig.9B).
The cell type and morphology of the structure in spheroids formed were detected
by dual fluorescence staining with antibodies against p63 (basal cell marker in
nuclei) and CK8 (luminal cell marker in cytoplasm). As illustrated in Fig 9C, in the
co-cultures of NPF we found the spheroid formed from normal Lin
-
Sca-1
+
cells
contained p63 positive cells in the outer layers and CK8 positive cells in the intra-
lumen layers. However, as shown in Fig. 10C, the spheroid formed from cancer Lin
-
Sca-1
+
cells contained a number of cells simultaneously expressing p63 and CK8 in
the relatively thicker outer layer. We noticed that almost every CK8 positive luminal
cell in the intra-lumen layers expressed p63 as well. We further stained the spheroids
formed from the co-cultures of CAF. On average these spheroids were larger than
those in the co-culture of NPF. The spheroid formed from normal Lin
-
Sca-1
+
cells
clearly developed only two layers with a few cells in the lumen. P63 positive cells
were located in the outer layer, and CK8 positive cells were in the inner-lumen layer
and the lumen. In the spheroids generated from cancer Lin
-
Sca-1
+
cells, a large
number of p63 basal cells in the outer layers expressed CK8 in cytoplasm, but only
the CK8 positive cells in the intra-lumen layer close to the basal layers expressed
p63. Other cells in the dense intra-lumen layer express CK8 only. Thus far, Lin
-
Sca-
1
+
cells in murine prostate cancer are suggested to differentiate to different

69
progenitors, develop cancer glandular structures in vitro, and may have atypical
signaling pathways controlling their differentiation.

Only LSC, not LSC- , in AD-cancer have stemness abilities. To test this
hypothesis that there is only a small subpopulation cancer cells within a prostate
tumor that is responsible for tumor formation, AD-Ca LSC and LSC- cells were
separately cultured with different stromal cells in vitro using the spheroid-forming
matrigel system. LSC- cells were collected from the region outside the gated LSC
area using FACS sorting (Fig. 10A). We next performed in vitro spheroid-forming
analyses to measure the stemness abilities of these two types of cells. Ten thousand
cells were seeded in matrigel and cultured with the same amount of stromal cells. As
shown in Fig. 10B, AD-Ca LSC cells formed a greater number of spheroids than Ad-
Ca LSC- cells did. It is noteworthy that AD-Ca LSC cells, as same as AD-Ca Lin
-
Sca-1
+
cells, formed more spheroids with the stimulations from CAF than from
UGSM and NPF. In controls with no stromal cells, AD-Ca LSC cells formed much
fewer spheroids than the co-cultures with UGSM or NPF. We also noticed that there
were only a very small number of spheroids formed from LSC
-
cells, and there was
no difference in the number of spheroids formed between co-cultures with various
sources of stromal cells, implying that LSC
-
cells did not respond to the stimulation
from either the normal stromal cells or CAF.

70
A
B
C

71
Fig. 10. Characterization of LSC cell subpopulation isolated from
AD-Ca.
A, the FACS plot shows the gate drawn for sorting LSC+ and LSC- cells. B,
comparing the spheroid forming ability of the LSC+ and LSC- cells with different
sources of stromal cells. LSC+ (10,000) and LSC- (10,000) cells were sorted from an
AD-Ca prostate tissue dissected from a 10 month old cPten
-/-
L mouse and then
separately cultured with the same amount of UGSM, NPF, and CAF cells for 12
days. C, Spheroids generated from LSC cells were passaged for three generations
with different sources of stromal cells. This graph shows the number of spheroids
formed at each passage.

72
We also passaged the spheroids formed from LSC cells to determine the self-renewal
ability of these cells. Ten thousand LSC cells isolated from AD-Ca were co-cultured
with different stromal cells (10,000 cells). We serially passaged these LSC cells in
three generations in the in vitro co-culture system (Fig. 10C). In the first two
passages all LSC cells in cultures with various stromal cells formed greater numbers
of spheroids than that was found in the previous passage. However, only the LSC
cells co-cultured with CAF had more spheroids formed in passage 3 than in passage
2. Thus it seems that AD-Ca LSC cells are able to keep their self-renewal and
differentiation properties for several generations, and CAF cells may further enhance
the stemness properties in AD-Ca LSC cells.

Discussion
In this report we have identified a subpopulation of cancer cells within murine
prostate adenocarcinomas, which have the capacity to form tumor-like structures in
spheroids in vitro. This subpopulation of cancer cells is marked by lack of expression
of the cell surface markers Lin and expression of Sca-1 and CD49f. These cells
display several features typically seen in stem cells, including the ability to both self-
renew and generate differentiated progeny, to differentiate to recapitulate the
henotype of the tumor, and to activate the developmental signaling pathways
regulated by BMI1.

73
It has been shown previously that cancer stem cells associated with other types of
cancers have aberrant activation of developmental signaling pathways, such as
hedgehog, the polycomb (PcG) family, Wnt, and Notch. To determine if our putative
prostate cancer stem cell population has enhanced expression of developmental
genes, we chose to examine the expression of BMI1, based on the previous reports
linking polycomb group genes to prostate cancer (van Leenders et al., 2007). PcG
genes play a role in the adult organism and have been linked to the regulation of
various biological processes, including lymphopoiesis and the cell cycle (Pasini et
al., 2004; Raaphorst et al., 2001). The vital role of PcG genes in maintenance of cell
identity and regulation of the cell cycle is underscored by the association between
development of cancer and abnormal expression of PcG genes (Gil et al., 2005). The
BMI1 gene is essential for the self-renewing capacity of normal and tumor stem cells
(Liu et al., 2006). In humans, expression of BMI1 has been linked to malignant
lymphomas, lung cancer, hepatomas, penile carcinomas, and leukemia (Dukers et al.,
2004; Ferreux et al., 2003; Lessard and Sauvageau, 2003; Neo et al., 2004;
Vonlanthen et al., 2001). Recent studies have shown that BMI1 is up-regulated in
primary and metastatic prostate cancer in humans (Berezovska et al., 2006; Glinsky
et al., 2005; van Leenders et al., 2007). We find that BMI1 is markedly up-regulated
in tumor LSC cells compared with normal LSC cells, suggesting that BMI1 may be
highly up-regulated in prostate cancer stem cells. It is also interesting to examine the

74
expression of other PcG genes, such as EZH2, which is also overexpressed in human
prostate cancer (Bryant et al., 2007), in the AD-Ca LSC cell subpopulation.
It is becoming increasingly clear that tissue stem cells are localized in defined
microenvironments, which are referred to as “niches”. The existence of a niche
probably provides stem cells with specific factors necessary for their maintenance of
the stem cell properties via a combination of intra- and inter-cellular signaling which
regulate the balance between proliferation, differentiation and quiescence in stem
cell populations (Spradling et al., 2001; Whetton and Graham, 1999). Signals from
the niche that control the stemness properties of stem cells may include the Wnt
pathway (Verras et al., 2004; Yardy and Brewster, 2005), Delta/Notch1 pathway
(Lowell et al., 2000; Shou et al., 2001; Wang et al., 2004) , hedgehog (Berman et al.,
2004; Karhadkar et al., 2004; Stecca et al., 2005), and transforming growth factor β
(TGF-β) (Bruckheimer and Kyprianou, 2002; Nemeth et al., 1997; Tsujimura et al.,
2002). Those that may be involved in the regulating processes within stem cells may
be p27
kip1
(De Marzo et al., 1998) and the polycomb group proteins (Di Cristofano et
al., 2001). However, whether the cancer stem cell niche can control and regulate the
self-renewal and differentiation properties in cancer stem cells is still not clear. In
this study, we demonstrate that LSC cells isolated from prostate AD-Ca are able to
respond to stimulation from CAF and result in the generation of tumor-like glandular
structures and increased spheroid numbers, suggesting that signals released from

75
CAF regulate the self-renewal potential of prostate cancer stem cells and promote
differentiation of these cells to more terminal-differentiated luminal cells.
The origin of prostate cancer stem cells is still unclear. Several studies support the
concept that cancer stem cells may be derived from transformed normal tissue stem
cells by accumulating mutations in the self-renewal pathway (Ayyanan et al., 2006;
Bonnet and Dick, 1997; Liu et al., 2006). Although we have isolated a subpopulation
of tumorigenic cancer cells from murine prostate tumors and have characterized
them, we cannot state at present whether these prostate cancer stem cells are derived
from a mutated normal prostate epithelial stem cell, or a downstream progenitor or
terminal-differentiated tumor cell that has regained stem cell-like properties because
of genetic alterations. Determination of the cell of origin in prostate cancer will be
greatly enhanced by the development of novel experimental methods that can detect
and distinguish the difference between normal and cancer stem cells.
The results from this in vitro study have significant implications for the treatment
of prostate cancer. The increased percentage of LSC cells in the cancer relative to
normal counterpart tissues argues for an important role for LSC cells in tumor
growth, and describes Sca-1 as a prominent cell surface marker of stem / progenitor
cells in both normal and neoplastic context of the mouse prostate. Continued
presence or even further enrichment of LSC cells in ADI cancer might serve as a
clue for future strategies to better understand the development of recurrent prostate
cancer. A better understanding of prostate cancer stem cells will not only affect our

76
ability to better understand the effect of current therapeutics, but additionally will
help us identify novel diagnostic markers and therapeutic targets, especially taking
into account the useful information that can be obtained form expression studies of
prostate cancer tem cells.

77
CHAPTER 4
Conclusions and Future Directions
Conclusions
The goal of the work described in this dissertation was to study prostate cancer
cells collected from our mouse models of prostate adenocarcinoma to identify cancer
stem cells.
First, we introduced two novel mouse lines for enhancing the feasibility of the
study. Mouse models of prostate adenocarcinoma provide an alternative to human
prostatic tumor samples to understand the role of prostate cancer stem cells in the
temporal stages of tumor growth and regrowth. The application of our prostate
epithelium-specific Cre/loxP system to inactivate one or more tumor suppressor
genes had resulted in successful development of mouse models of prostate cancer.
The efficiency of the Cre recombinase is determined to cause recombination in at
least four alleles in a single cell in the PB-Cre4 mouse line developed in our
laboratory. Among these Cre/loxP models, the conditional Pten mouse line (cPten
-/-
)
has been proven to efficiently generate murine prostatic primary cancer at a
reasonably early age that in time can develop into metastatic cancer. Because the rate
of growth and progression of prostate cancer may vary among individual mice, large
cohorts of animals are required to derive statistically meaningful data. To increase
the efficiency of this mouse line, we combined it with Cre mediated reporter genes,
luciferase and enhanced green fluorescence protein (EGFP). Recently developed

78
non-invasive imaging techniques have been applied to monitor tumor information in
individual living animals. Among the different techniques, bioluminescence imaging
(BLI) has been widely used in cancer studies. In the luciferase reporter model, the
growth of prostatic cancers could be followed non-invasively in live animals by BLI.
Similarly, the Rosa26-EGFP loxP line offered an opportunity to improve
fluorescence imaging of the cPten
-/-
model and to isolate tumor cells by cell sorting
(Liao et al., 2007). What we will derive in benefit from these models is not only in
studies of prostate tumorigenesis but also in the development of novel therapies, such
as immunotherapy (Haga et al., (in press)).
We then evaluated and attempted to characterize the stem / progenitor cells
isolated from these mouse models mentioned above. In this study, we demonstrate
that Sca-1 is an important cell surface marker for identifying not only normal stem
cells but also cancer stem cells in prostate tissues. Lin
+
Sca-1
+
cells isolated from
AD-Ca are capable of differentiating to different progenitors in spheroids formed.
Importantly, AD-Ca Lin
-
Sca-1
+
spheroids can develop prostate tumor-like structures
by stimulation from CAF. Our findings further show that the Lin
-
Sca-1
+
CD49f
+
cell
subpopulation is the only cell group in prostate tumors that have the potential to
generate spheroids, and these spheroids could be serially passaged. The percentage
of this cell subpopulation is also increased in AD- and ADI-cancer relative to the
normal prostate tissues. Taken together, these in vitro results suggest that cancer

79
LSC cells should be the putative cancer stem cells in murine prostate that have the
stemness properties and that may maintain the homeostasis of prostate cancer.
It is becoming increasingly clear that prostate cancer may be derived from the
differentiation and proliferation of cancer stem cells (Kelly and Yin, 2008; Lawson
et al., 2005; Nikitin et al., 2007). The origin of these cells is still a debate. Cancer
stem cells may derive from the normal somatic stem cells by accumulating
mutations; alternatively, they may originate from tumorous progenitors via de-
differentiation. A better understanding of the differences and similarities of the
normal and cancer stem cells in prostate tissues will help to discover specific cell
surface markers or develop novel therapies particularly targeting the cancer stem
cells inside prostate cancers.

Future Directions
The studies described in this dissertation broadened our understanding of the role
of cancer stem cells in prostate oncogenesis; however, many more interesting and
important questions do arise from our findings.
First, a specific stem cell surface marker will be required for a better isolation of
cancer stem cells from murine prostate tumor. In this report we describe that the LSC
cell subpopulation isolated from AD-Ca contains a small fraction of cancer stem-like
cells, which can form the spheroids with tumor-like prostatic glandular structure.

80
This prostate cancer stem cell subpopulation is only 0.1% within AD-Ca LSC cells,
suggesting that most of the LSC cells are transit-amplifying (TA) cells which
coexpress Sca-1 and CD49f but do not have self-renewal and differentiation abilities.
To approach the real prostate cancer stem cell subpopulation, it is important to
identify new specific surface markers and combine these markers with Lin, Sca-1
and CD49f to obtain prostate cancer stem cells from TA cell. These cells will
provide more information and improve our knowledge in the prostate cancer research
field.
Second, identification of the signaling molecules and pathways from the cancer
microenvironment, which regulate cancer stem cells, will be important. Published
data have shown that the normal stem cell niche regulates the self-renewal and
differentiation properties of stem and progenitor cells in adult tissues (Fuchs et al.,
2004). Cancer stem cells may also be controlled by their niches. Here, we have
shown that the in vitro prostate spheroid-forming analysis is a powerful method to
facilitate the studies of interactions between CAF and cancer stem cells in vitro.
Because CAF cells are in the insert above the matrigel containing LSC cells, there is
no cell-cell contact between two cell groups in this system. With this advantage, it
should be possible to use siRNA to knock down the genes of the signaling molecules
emitted from CAF, or antibodies to block receptors located on LSC cells to test
whether LSC cells still maintain the same number of spheroids formed and the
morphology change in spheroid structures. If the signaling pathways that are

81
participating in the interactions between CAF and cancer stem cells are revealed,
then new therapeutic methods would be developed to target these signal molecules
and stop tumorigenesis from cancer stem cells to terminal differentiated tumor cells
in prostate cancer.
Finally, and most importantly, the origin of ADI-Ca prostate cancer must be
resolved. After androgen depletion treatment, the prostate AD-cancer will go through
regression, latency, and recurrence to become ADI-cancer. The ADI-Ca may come
from two sources: one is from mutations of normal prostate epithelial stem cells; the
other one is the dedifferentiation of terminal differentiated AD-Ca tumor cells. In our
work, with luciferase expression from the newly developed cPten
-/-
L mice (Liao et
al., 2007) we can track the growth and progression of prostate tumors burden in mice
and collect samples at different stages. We can even determine the castration-
induced regression of tumors in living animals and the relapse of ADI-cancer,
thereby facilitating the ability to accurately collect prostate tumor tissues when the
tumors are in either growth or latent or regrowth phase. Because prostate epithelial
luminal cells and a fraction of basal cells respond to androgen (Wang et al., 2006),
these cells can not survive without androgen and their death after castration will
trigger the regression of AD-Ca. However, prostate stem cells do not express
androgen receptor and, therefore, do not respond to androgen ablation (Collins et al.,
2005). A major fraction of epithelial cells in the prostate tissue at the latency stage
after castration should be stem cells. This stem cell population may be composed of

82
normal and cancer stem cells or only the cancer ones. Isolation and characterization
of this LSC cell subpopulation in prostate tissues collected from the mice at the
latency stage should provide suggestions about of the origin of recurrent ADI-Ca
and, thereby, to strategies to prevent the initiation of ADI-Ca, a major problem in the
management of this cancer in humans.

83
Reference
Abbas, F., and Scardino, P.T. (1997). The natural history of clinical prostate
carcinoma. Cancer 80, 827-833.

Abdulkadir, S.A., Magee, J.A., Peters, T.J., Kaleem, Z., Naughton, C.K., Humphrey,
P.A., and Milbrandt, J. (2002). Conditional loss of Nkx3.1 in adult mice induces
prostatic intraepithelial neoplasia. Mol Cell Biol 22, 1495-1503.

Abrahamsson, P.A. (1999). Neuroendocrine differentiation in prostatic carcinoma.
Prostate 39, 135-148.

Abrahamsson, P.A., and di Sant'Agnese, P.A. (1993). Neuroendocrine cells in the
human prostate gland. J Androl 14, 307-309.

Adams, J.Y., Johnson, M., Sato, M., Berger, F., Gambhir, S.S., Carey, M., Iruela-
Arispe, M.L., and Wu, L. (2002). Visualization of advanced human prostate cancer
lesions in living mice by a targeted gene transfer vector and optical imaging. Nat
Med 8, 891-897.

Al-Hajj, M., Becker, M.W., Wicha, M., Weissman, I., and Clarke, M.F. (2004).
Therapeutic implications of cancer stem cells. Curr Opin Genet Dev 14, 43-47.

Al-Hajj, M., and Clarke, M.F. (2004). Self-renewal and solid tumor stem cells.
Oncogene 23, 7274-7282.

Al-Hajj, M., Wicha, M.S., Benito-Hernandez, A., Morrison, S.J., and Clarke, M.F.
(2003). Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad
Sci U S A 100, 3983-3988.

Askenasy, N., and Koretsky, A.P. (2002). Transgenic livers expressing
mitochondrial and cytosolic CK: mitochondrial CK modulates free ADP levels. Am
J Physiol Cell Physiol 282, C338-346.

Ayyanan, A., Civenni, G., Ciarloni, L., Morel, C., Mueller, N., Lefort, K.,
Mandinova, A., Raffoul, W., Fiche, M., Dotto, G.P., et al. (2006). Increased Wnt
signaling triggers oncogenic conversion of human breast epithelial cells by a Notch-
dependent mechanism. Proc Natl Acad Sci U S A 103, 3799-3804.

84
Berezovska, O.P., Glinskii, A.B., Yang, Z., Li, X.M., Hoffman, R.M., and Glinsky,
G.V. (2006). Essential role for activation of the Polycomb group (PcG) protein
chromatin silencing pathway in metastatic prostate cancer. Cell Cycle 5, 1886-1901.

Bergers, G., and Coussens, L.M. (2000). Extrinsic regulators of epithelial tumor
progression: metalloproteinases. Curr Opin Genet Dev 10, 120-127.

Berman, D.M., Desai, N., Wang, X., Karhadkar, S.S., Reynon, M., Abate-Shen, C.,
Beachy, P.A., and Shen, M.M. (2004). Roles for Hedgehog signaling in androgen
production and prostate ductal morphogenesis. Dev Biol 267, 387-398.

Bonkhoff, H., and Remberger, K. (1996). Differentiation pathways and histogenetic
aspects of normal and abnormal prostatic growth: a stem cell model. Prostate 28, 98-
106.

Bonkhoff, H., Stein, U., and Remberger, K. (1995). Endocrine-paracrine cell types in
the prostate and prostatic adenocarcinoma are postmitotic cells. Hum Pathol 26, 167-
170.

Bonkhoff, H., Wernert, N., Dhom, G., and Remberger, K. (1991). Relation of
endocrine-paracrine cells to cell proliferation in normal, hyperplastic, and neoplastic
human prostate. Prostate 19, 91-98.

Bonnet, D., and Dick, J.E. (1997). Human acute myeloid leukemia is organized as a
hierarchy that originates from a primitive hematopoietic cell. Nat Med 3, 730-737.

Bruckheimer, E.M., and Kyprianou, N. (2002). Bcl-2 antagonizes the combined
apoptotic effect of transforming growth factor-beta and dihydrotestosterone in
prostate cancer cells. Prostate 53, 133-142.

Bryant, R.J., Cross, N.A., Eaton, C.L., Hamdy, F.C., and Cunliffe, V.T. (2007).
EZH2 promotes proliferation and invasiveness of prostate cancer cells. Prostate 67,
547-556.

Burger, P.E., Xiong, X., Coetzee, S., Salm, S.N., Moscatelli, D., Goto, K., and
Wilson, E.L. (2005). Sca-1 expression identifies stem cells in the proximal region of
prostatic ducts with high capacity to reconstitute prostatic tissue. Proc Natl Acad Sci
U S A 102, 7180-7185.

Carver, B.S., and Pandolfi, P.P. (2006). Mouse modeling in oncologic preclinical and
translational research. Clin Cancer Res 12, 5305-5311.

85
Chen, Z., Trotman, L.C., Shaffer, D., Lin, H.K., Dotan, Z.A., Niki, M., Koutcher,
J.A., Scher, H.I., Ludwig, T., Gerald, W., et al. (2005). Crucial role of p53-
dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Nature
436, 725-730.

Chow, S., Hedley, D., Grom, P., Magari, R., Jacobberger, J.W., and Shankey, T.V.
(2005). Whole blood fixation and permeabilization protocol with red blood cell lysis
for flow cytometry of intracellular phosphorylated epitopes in leukocyte
subpopulations. Cytometry A 67, 4-17.

Collins, A.T., Berry, P.A., Hyde, C., Stower, M.J., and Maitland, N.J. (2005).
Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res 65,
10946-10951.

Collins, A.T., Habib, F.K., Maitland, N.J., and Neal, D.E. (2001). Identification and
isolation of human prostate epithelial stem cells based on alpha(2)beta(1)-integrin
expression. J Cell Sci 114, 3865-3872.

Collins, A.T., and Maitland, N.J. (2006). Prostate cancer stem cells. Eur J Cancer 42,
1213-1218.
Cunha, G.R., Reese, B.A., and Sekkingstad, M. (1980). Induction of nuclear
androgen-binding sites in epithelium of the embryonic urinary bladder by
mesenchyme of the urogenital sinus of embryonic mice. Endocrinology 107, 1767-
1770.

De Marzo, A.M., Meeker, A.K., Epstein, J.I., and Coffey, D.S. (1998). Prostate stem
cell compartments: expression of the cell cycle inhibitor p27Kip1 in normal,
hyperplastic, and neoplastic cells. Am J Pathol 153, 911-919.

Dehm, S.M., and Tindall, D.J. (2006). Molecular regulation of androgen action in
prostate cancer. J Cell Biochem 99, 333-344.

DeMarzo, A.M., Nelson, W.G., Isaacs, W.B., and Epstein, J.I. (2003). Pathological
and molecular aspects of prostate cancer. Lancet 361, 955-964.

Denis, L., and Murphy, G.P. (1993). Overview of phase III trials on combined
androgen treatment in patients with metastatic prostate cancer. Cancer 72, 3888-
3895.

Di Cristofano, A., De Acetis, M., Koff, A., Cordon-Cardo, C., and Pandolfi, P.P.
(2001). Pten and p27KIP1 cooperate in prostate cancer tumor suppression in the
mouse. Nat Genet 27, 222-224.

86

di Sant'Agnese, P.A. (1992a). Neuroendocrine differentiation in carcinoma of the
prostate. Diagnostic, prognostic, and therapeutic implications. Cancer 70, 254-268.

di Sant'Agnese, P.A. (1992b). Neuroendocrine differentiation in human prostatic
carcinoma. Hum Pathol 23, 287-296.

di Sant'Agnese, P.A. (2001). Neuroendocrine differentiation in prostatic carcinoma:
an update on recent developments. Ann Oncol 12 Suppl 2, S135-140.

Dukers, D.F., van Galen, J.C., Giroth, C., Jansen, P., Sewalt, R.G., Otte, A.P., Kluin-
Nelemans, H.C., Meijer, C.J., and Raaphorst, F.M. (2004). Unique polycomb gene
expression pattern in Hodgkin's lymphoma and Hodgkin's lymphoma-derived cell
lines. Am J Pathol 164, 873-881.

El Hilali, N., Rubio, N., Martinez-Villacampa, M., and Blanco, J. (2002). Combined
noninvasive imaging and luminometric quantification of luciferase-labeled human
prostate tumors and metastases. Lab Invest 82, 1563-1571.

Ellwood-Yen, K., Wongvipat, J., and Sawyers, C. (2006). Transgenic mouse model
for rapid pharmacodynamic evaluation of antiandrogens. Cancer Res 66, 10513-
10516.

English, H.F., Santen, R.J., and Isaacs, J.T. (1987). Response of glandular versus
basal rat ventral prostatic epithelial cells to androgen withdrawal and replacement.
Prostate 11, 229-242.

Evans, G.S., and Chandler, J.A. (1987). Cell proliferation studies in the rat prostate:
II. The effects of castration and androgen-induced regeneration upon basal and
secretory cell proliferation. Prostate 11, 339-351.

Falciatori, I., Borsellino, G., Haliassos, N., Boitani, C., Corallini, S., Battistini, L.,
Bernardi, G., Stefanini, M., and Vicini, E. (2004). Identification and enrichment of
spermatogonial stem cells displaying side-population phenotype in immature mouse
testis. Faseb J 18, 376-378.

Feinberg, A.P., Ohlsson, R., and Henikoff, S. (2006). The epigenetic progenitor
origin of human cancer. Nat Rev Genet 7, 21-33.

87
Ferreux, E., Lont, A.P., Horenblas, S., Gallee, M.P., Raaphorst, F.M., von Knebel
Doeberitz, M., Meijer, C.J., and Snijders, P.J. (2003). Evidence for at least three
alternative mechanisms targeting the p16INK4A/cyclin D/Rb pathway in penile
carcinoma, one of which is mediated by high-risk human papillomavirus. J Pathol
201, 109-118.

Fuchs, E., Tumbar, T., and Guasch, G. (2004). Socializing with the neighbors: stem
cells and their niche. Cell 116, 769-778.

Gil, J., Bernard, D., and Peters, G. (2005). Role of polycomb group proteins in stem
cell self-renewal and cancer. DNA Cell Biol 24, 117-125.

Gingrich, J.R., Barrios, R.J., Foster, B.A., and Greenberg, N.M. (1999). Pathologic
progression of autochthonous prostate cancer in the TRAMP model. Prostate Cancer
Prostatic Dis 2, 70-75.

Gingrich, J.R., Barrios, R.J., Morton, R.A., Boyce, B.F., DeMayo, F.J., Finegold,
M.J., Angelopoulou, R., Rosen, J.M., and Greenberg, N.M. (1996). Metastatic
prostate cancer in a transgenic mouse. Cancer Res 56, 4096-4102.

Gittes, R.F. (1991). Carcinoma of the prostate. N Engl J Med 324, 236-245.

Glinsky, G.V., Berezovska, O., and Glinskii, A.B. (2005). Microarray analysis
identifies a death-from-cancer signature predicting therapy failure in patients with
multiple types of cancer. J Clin Invest 115, 1503-1521.

Goldie, J.H., and Coldman, A.J. (1979). A mathematic model for relating the drug
sensitivity of tumors to their spontaneous mutation rate. Cancer Treat Rep 63, 1727-
1733.

Goto, K., Salm, S.N., Coetzee, S., Xiong, X., Burger, P.E., Shapiro, E., Lepor, H.,
Moscatelli, D., and Wilson, E.L. (2006). Proximal prostatic stem cells are
programmed to regenerate a proximal-distal ductal axis. Stem Cells 24, 1859-1868.

Guasch, G., and Fuchs, E. (2005). Mice in the world of stem cell biology. Nat Genet
37, 1201-1206.

Haga, K., Tomioka, A., Liao, C.P., Kimura, T., Matsumoto, H., Ohno, I., Hermann,
K., Loog, C.R., Tanaka, M., Hirao, Y., et al. ((in press)). Effective Tumor Growth
Inhibition of PTEN-Knockout Prostate Cancer by Tumor Vaccine and Adoptive
Immunotherapy Strategies. Journal of Urology.

88
Hill, R., Song, Y., Cardiff, R.D., and Van Dyke, T. (2005). Selective evolution of
stromal mesenchyme with p53 loss in response to epithelial tumorigenesis. Cell 123,
1001-1011.

Holmes, C., and Stanford, W.L. (2007). Concise review: stem cell antigen-1:
expression, function, and enigma. Stem Cells 25, 1339-1347.

Hoosein, N.M., Logothetis, C.J., and Chung, L.W. (1993). Differential effects of
peptide hormones bombesin, vasoactive intestinal polypeptide and somatostatin
analog RC-160 on the invasive capacity of human prostatic carcinoma cells. J Urol
149, 1209-1213.

Hsieh, C.L., Xie, Z., Liu, Z.Y., Green, J.E., Martin, W.D., Datta, M.W., Yeung, F.,
Pan, D., and Chung, L.W. (2005). A luciferase transgenic mouse model:
visualization of prostate development and its androgen responsiveness in live
animals. J Mol Endocrinol 35, 293-304.

Huang, J., Powell, W.C., Khodavirdi, A.C., Wu, J., Makita, T., Cardiff, R.D., Cohen,
M.B., Sucov, H.M., and Roy-Burman, P. (2002). Prostatic intraepithelial neoplasia in
mice with conditional disruption of the retinoid X receptor alpha allele in the prostate
epithelium. Cancer Res 62, 4812-4819.

Huang, J., Yao, J.L., di Sant'Agnese, P.A., Yang, Q., Bourne, P.A., and Na, Y.
(2006). Immunohistochemical characterization of neuroendocrine cells in prostate
cancer. Prostate 66, 1399-1406.

Hudson, D.L., O'Hare, M., Watt, F.M., and Masters, J.R. (2000). Proliferative
heterogeneity in the human prostate: evidence for epithelial stem cells. Lab Invest
80, 1243-1250.

Isaacs, J.T. (1999). The biology of hormone refractory prostate cancer. Why does it
develop? Urol Clin North Am 26, 263-273.

Isaacs, J.T., and Coffey, D.S. (1989). Etiology and disease process of benign
prostatic hyperplasia. Prostate Suppl 2, 33-50.

Isaacs, J.T., Schulze, H., and Coffey, D.S. (1987). Development of androgen
resistance in prostatic cancer. Prog Clin Biol Res 243A, 21-31.

Isaacs, W., De Marzo, A., and Nelson, W.G. (2002). Focus on prostate cancer.
Cancer Cell 2, 113-116.

89
Ito, T., Yamamoto, S., Ohno, Y., Namiki, K., Aizawa, T., Akiyama, A., and
Tachibana, M. (2001). Up-regulation of neuroendocrine differentiation in prostate
cancer after androgen deprivation therapy, degree and androgen independence.
Oncol Rep 8, 1221-1224.

Jemal, A., Murray, T., Samuels, A., Ghafoor, A., Ward, E., and Thun, M.J. (2003).
Cancer statistics, 2003. CA Cancer J Clin 53, 5-26.

Jenkins, D.E., Oei, Y., Hornig, Y.S., Yu, S.F., Dusich, J., Purchio, T., and Contag,
P.R. (2003). Bioluminescent imaging (BLI) to improve and refine traditional murine
models of tumor growth and metastasis. Clin Exp Metastasis 20, 733-744.

Jongsma, J., Oomen, M.H., Noordzij, M.A., Van Weerden, W.M., Martens, G.J., van
der Kwast, T.H., Schroder, F.H., and van Steenbrugge, G.J. (2000). Androgen
deprivation of the PC-310 [correction of prohormone convertase-310] human
prostate cancer model system induces neuroendocrine differentiation. Cancer Res 60,
741-748.

Kamiura, S., Nolan, C.M., and Meruelo, D. (1992). Long-range physical map of the
Ly-6 complex: mapping the Ly-6 multigene family by field-inversion and two-
dimensional gel electrophoresis. Genomics 12, 89-105.

Karhadkar, S.S., Bova, G.S., Abdallah, N., Dhara, S., Gardner, D., Maitra, A., Isaacs,
J.T., Berman, D.M., and Beachy, P.A. (2004). Hedgehog signalling in prostate
regeneration, neoplasia and metastasis. Nature 431, 707-712.

Kelly, K., and Yin, J.J. (2008). Prostate cancer and metastasis initiating stem cells.
Cell Res 18, 528-537.

Khodavirdi, A.C., Song, Z., Yang, S., Zhong, C., Wang, S., Wu, H., Pritchard, C.,
Nelson, P.S., and Roy-Burman, P. (2006). Increased expression of osteopontin
contributes to the progression of prostate cancer. Cancer Res 66, 883-888.

Knudson, A.G., Jr., Strong, L.C., and Anderson, D.E. (1973). Heredity and cancer in
man. Prog Med Genet 9, 113-158.

Lang, S.H., Stark, M., Collins, A., Paul, A.B., Stower, M.J., and Maitland, N.J.
(2001). Experimental prostate epithelial morphogenesis in response to stroma and
three-dimensional matrigel culture. Cell Growth Differ 12, 631-640.

90
Lawson, D.A., Xin, L., Lukacs, R., Xu, Q., Cheng, D., and Witte, O.N. (2005).
Prostate stem cells and prostate cancer. Cold Spring Harb Symp Quant Biol 70, 187-
196.

Lawson, D.A., Xin, L., Lukacs, R.U., Cheng, D., and Witte, O.N. (2007). Isolation
and functional characterization of murine prostate stem cells. Proc Natl Acad Sci U S
A 104, 181-186.

Lesche, R., Groszer, M., Gao, J., Wang, Y., Messing, A., Sun, H., Liu, X., and Wu,
H. (2002). Cre/loxP-mediated inactivation of the murine Pten tumor suppressor gene.
Genesis 32, 148-149.

Lessard, J., and Sauvageau, G. (2003). Polycomb group genes as epigenetic
regulators of normal and leukemic hemopoiesis. Exp Hematol 31, 567-585.

Li, C., Heidt, D.G., Dalerba, P., Burant, C.F., Zhang, L., Adsay, V., Wicha, M.,
Clarke, M.F., and Simeone, D.M. (2007). Identification of pancreatic cancer stem
cells. Cancer Res 67, 1030-1037.

Liao, C.P., Zhong, C., Saribekyan, G., Bading, J., Park, R., Conti, P.S., Moats, R.,
Berns, A., Shi, W., Zhou, Z., et al. (2007). Mouse models of prostate
adenocarcinoma with the capacity to monitor spontaneous carcinogenesis by
bioluminescence or fluorescence. Cancer Res 67, 7525-7533.

Liu, S., Dontu, G., Mantle, I.D., Patel, S., Ahn, N.S., Jackson, K.W., Suri, P., and
Wicha, M.S. (2006). Hedgehog signaling and Bmi-1 regulate self-renewal of normal
and malignant human mammary stem cells. Cancer Res 66, 6063-6071.

Lowell, S., Jones, P., Le Roux, I., Dunne, J., and Watt, F.M. (2000). Stimulation of
human epidermal differentiation by delta-notch signalling at the boundaries of stem-
cell clusters. Curr Biol 10, 491-500.

Lyons, S.K. (2005). Advances in imaging mouse tumour models in vivo. J Pathol
205, 194-205.

Lyons, S.K., Lim, E., Clermont, A.O., Dusich, J., Zhu, L., Campbell, K.D., Coffee,
R.J., Grass, D.S., Hunter, J., Purchio, T., et al. (2006). Noninvasive bioluminescence
imaging of normal and spontaneously transformed prostate tissue in mice. Cancer
Res 66, 4701-4707.

91
Lyons, S.K., Meuwissen, R., Krimpenfort, P., and Berns, A. (2003). The generation
of a conditional reporter that enables bioluminescence imaging of Cre/loxP-
dependent tumorigenesis in mice. Cancer Res 63, 7042-7046.

Ma, X., Ziel-van der Made, A.C., Autar, B., van der Korput, H.A., Vermeij, M., van
Duijn, P., Cleutjens, K.B., de Krijger, R., Krimpenfort, P., Berns, A., et al. (2005).
Targeted biallelic inactivation of Pten in the mouse prostate leads to prostate cancer
accompanied by increased epithelial cell proliferation but not by reduced apoptosis.
Cancer Res 65, 5730-5739.

Mackillop, W.J., Trent, J.M., Stewart, S.S., and Buick, R.N. (1983). Tumor
progression studied by analysis of cellular features of serial ascitic ovarian
carcinoma tumors. Cancer Res 43, 874-878.

Maddison, L.A., Nahm, H., DeMayo, F., and Greenberg, N.M. (2000). Prostate
specific expression of Cre recombinase in transgenic mice. Genesis 26, 154-156.

Mao, X., Fujiwara, Y., Chapdelaine, A., Yang, H., and Orkin, S.H. (2001).
Activation of EGFP expression by Cre-mediated excision in a new ROSA26 reporter
mouse strain. Blood 97, 324-326.

Massoud, T.F., and Gambhir, S.S. (2003). Molecular imaging in living subjects:
seeing fundamental biological processes in a new light. Genes Dev 17, 545-580.

Matsuura, K., Nagai, T., Nishigaki, N., Oyama, T., Nishi, J., Wada, H., Sano, M.,
Toko, H., Akazawa, H., Sato, T., et al. (2004). Adult cardiac Sca-1-positive cells
differentiate into beating cardiomyocytes. J Biol Chem 279, 11384-11391.

McNeal, J.E., Villers, A., Redwine, E.A., Freiha, F.S., and Stamey, T.A. (1991).
Microcarcinoma in the prostate: its association with duct-acinar dysplasia. Hum
Pathol 22, 644-652.

Montanaro, F., Liadaki, K., Volinski, J., Flint, A., and Kunkel, L.M. (2003). Skeletal
muscle engraftment potential of adult mouse skin side population cells. Proc Natl
Acad Sci U S A 100, 9336-9341.

Morrison, S.J., Qian, D., Jerabek, L., Thiel, B.A., Park, I.K., Ford, P.S., Kiel, M.J.,
Schork, N.J., Weissman, I.L., and Clarke, M.F. (2002). A genetic determinant that
specifically regulates the frequency of hematopoietic stem cells. J Immunol 168,
635-642.

92
Nemeth, J.A., Sensibar, J.A., White, R.R., Zelner, D.J., Kim, I.Y., and Lee, C.
(1997). Prostatic ductal system in rats: tissue-specific expression and regional
variation in stromal distribution of transforming growth factor-beta 1. Prostate 33,
64-71.

Neo, S.Y., Leow, C.K., Vega, V.B., Long, P.M., Islam, A.F., Lai, P.B., Liu, E.T.,
and Ren, E.C. (2004). Identification of discriminators of hepatoma by gene
expression profiling using a minimal dataset approach. Hepatology 39, 944-953.

Nikitin, A.Y., Matoso, A., and Roy-Burman, P. (2007). Prostate stem cells and
cancer. Histol Histopathol 22, 1043-1049.

Nikitin, A.Y., Nafus, M.G., Zhou, Z., Liao, C.P., and Roy-Burman, P. ((In Press)).
Stem Cells and Cancer (Springer Science & Humana Press, Inc.).

O'Brien, C.A., Pollett, A., Gallinger, S., and Dick, J.E. (2007). A human colon
cancer cell capable of initiating tumour growth in immunodeficient mice. Nature
445, 106-110.

Olumi, A.F., Grossfeld, G.D., Hayward, S.W., Carroll, P.R., Tlsty, T.D., and Cunha,
G.R. (1999). Carcinoma-associated fibroblasts direct tumor progression of initiated
human prostatic epithelium. Cancer Res 59, 5002-5011.

Pasini, D., Bracken, A.P., and Helin, K. (2004). Polycomb group proteins in cell
cycle progression and cancer. Cell Cycle 3, 396-400.

Patrawala, L., Calhoun, T., Schneider-Broussard, R., Li, H., Bhatia, B., Tang, S.,
Reilly, J.G., Chandra, D., Zhou, J., Claypool, K., et al. (2006). Highly purified
CD44+ prostate cancer cells from xenograft human tumors are enriched in
tumorigenic and metastatic progenitor cells. Oncogene 25, 1696-1708.

Patterson, J.M., Johnson, M.H., Zimonjic, D.B., and Graubert, T.A. (2000).
Characterization of Ly-6M, a novel member of the Ly-6 family of hematopoietic
proteins. Blood 95, 3125-3132.

Podsypanina, K., Ellenson, L.H., Nemes, A., Gu, J., Tamura, M., Yamada, K.M.,
Cordon-Cardo, C., Catoretti, G., Fisher, P.E., and Parsons, R. (1999). Mutation of
Pten/Mmac1 in mice causes neoplasia in multiple organ systems. Proc Natl Acad Sci
U S A 96, 1563-1568.

93
Prince, M.E., Sivanandan, R., Kaczorowski, A., Wolf, G.T., Kaplan, M.J., Dalerba,
P., Weissman, I.L., Clarke, M.F., and Ailles, L.E. (2007). Identification of a
subpopulation of cells with cancer stem cell properties in head and neck squamous
cell carcinoma. Proc Natl Acad Sci U S A 104, 973-978.

Raaphorst, F.M., Otte, A.P., and Meijer, C.J. (2001). Polycomb-group genes as
regulators of mammalian lymphopoiesis. Trends Immunol 22, 682-690.

Reiner, T., de Las Pozas, A., Parrondo, R., and Perez-Stable, C. (2007). Progression
of prostate cancer from a subset of p63-positive basal epithelial cells in FG/Tag
transgenic mice. Mol Cancer Res 5, 1171-1179.

Reya, T., Morrison, S.J., Clarke, M.F., and Weissman, I.L. (2001). Stem cells,
cancer, and cancer stem cells. Nature 414, 105-111.

Ricci-Vitiani, L., Lombardi, D.G., Pilozzi, E., Biffoni, M., Todaro, M., Peschle, C.,
and De Maria, R. (2007). Identification and expansion of human colon-cancer-
initiating cells. Nature 445, 111-115.

Rice, B.W., Cable, M.D., and Nelson, M.B. (2001). In vivo imaging of light-emitting
probes. J Biomed Opt 6, 432-440.

Richardson, G.D., Robson, C.N., Lang, S.H., Neal, D.E., Maitland, N.J., and Collins,
A.T. (2004). CD133, a novel marker for human prostatic epithelial stem cells. J Cell
Sci 117, 3539-3545.

Roy-Burman, P., Tindall, D.J., Robins, D.M., Greenberg, N.M., Hendrix, M.J.,
Mohla, S., Getzenberg, R.H., Isaacs, J.T., and Pienta, K.J. (2005). Androgens and
prostate cancer: are the descriptors valid? Cancer Biol Ther 4, 4-5.

Roy-Burman, P., Wu, H., Powell, W.C., Hagenkord, J., and Cohen, M.B. (2004).
Genetically defined mouse models that mimic natural aspects of human prostate
cancer development. Endocr Relat Cancer 11, 225-254.

Roy-Burman, P., Zheng, J., and Miller, G.J. (1997). Molecular heterogeneity in
prostate cancer: can TP53 mutation unravel tumorigenesis? Mol Med Today 3, 476-
482.

Rubio, N., Villacampa, M.M., El Hilali, N., and Blanco, J. (2000). Metastatic burden
in nude mice organs measured using prostate tumor PC-3 cells expressing the
luciferase gene as a quantifiable tumor cell marker. Prostate 44, 133-143.

94
Scatena, C.D., Hepner, M.A., Oei, Y.A., Dusich, J.M., Yu, S.F., Purchio, T., Contag,
P.R., and Jenkins, D.E. (2004). Imaging of bioluminescent LNCaP-luc-M6 tumors: a
new animal model for the study of metastatic human prostate cancer. Prostate 59,
292-303.

Shariatmadari, R., Sipila, P.P., Huhtaniemi, I.T., and Poutanen, M. (2001). Improved
technique for detection of enhanced green fluorescent protein in transgenic mice.
Biotechniques 30, 1282-1285.

Sharifi, N., Kawasaki, B.T., Hurt, E.M., and Farrar, W.L. (2006). Stem cells in
prostate cancer: resolving the castrate-resistant conundrum and implications for
hormonal therapy. Cancer Biol Ther 5, 901-906.

Shen, M.M., and Abate-Shen, C. (2007). Pten inactivation and the emergence of
androgen-independent prostate cancer. Cancer Res 67, 6535-6538.
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.

Signoretti, S., Pires, M.M., Lindauer, M., Horner, J.W., Grisanzio, C., Dhar, S.,
Majumder, P., McKeon, F., Kantoff, P.W., Sellers, W.R., et al. (2005). p63 regulates
commitment to the prostate cell lineage. Proc Natl Acad Sci U S A 102, 11355-
11360.

Sneddon, J.B., Zhen, H.H., Montgomery, K., van de Rijn, M., Tward, A.D., West,
R., Gladstone, H., Chang, H.Y., Morganroth, G.S., Oro, A.E., et al. (2006). Bone
morphogenetic protein antagonist gremlin 1 is widely expressed by cancer-associated
stromal cells and can promote tumor cell proliferation. Proc Natl Acad Sci U S A
103, 14842-14847.

Spangrude, G.J., Heimfeld, S., and Weissman, I.L. (1988). Purification and
characterization of mouse hematopoietic stem cells. Science 241, 58-62.

Spradling, A., Drummond-Barbosa, D., and Kai, T. (2001). Stem cells find their
niche. Nature 414, 98-104.

Stecca, B., Mas, C., and Ruiz i Altaba, A. (2005). Interference with HH-GLI
signaling inhibits prostate cancer. Trends Mol Med 11, 199-203.

Trotman, L.C., Niki, M., Dotan, Z.A., Koutcher, J.A., Di Cristofano, A., Xiao, A.,
Khoo, A.S., Roy-Burman, P., Greenberg, N.M., Van Dyke, T., et al. (2003). Pten
dose dictates cancer progression in the prostate. PLoS Biol 1, 385-396.

95

Tsujimura, A., Koikawa, Y., Salm, S., Takao, T., Coetzee, S., Moscatelli, D.,
Shapiro, E., Lepor, H., Sun, T.T., and Wilson, E.L. (2002). Proximal location of
mouse prostate epithelial stem cells: a model of prostatic homeostasis. J Cell Biol
157, 1257-1265.

van de Rijn, M., Heimfeld, S., Spangrude, G.J., and Weissman, I.L. (1989). Mouse
hematopoietic stem-cell antigen Sca-1 is a member of the Ly-6 antigen family. Proc
Natl Acad Sci U S A 86, 4634-4638.

van Leenders, G.J., Dukers, D., Hessels, D., van den Kieboom, S.W., Hulsbergen,
C.A., Witjes, J.A., Otte, A.P., Meijer, C.J., and Raaphorst, F.M. (2007). Polycomb-
group oncogenes EZH2, BMI1, and RING1 are overexpressed in prostate cancer
with adverse pathologic and clinical features. Eur Urol 52, 455-463.

Vashchenko, N., and Abrahamsson, P.A. (2005). Neuroendocrine differentiation in
prostate cancer: implications for new treatment modalities. Eur Urol 47, 147-155.

Verras, M., Brown, J., Li, X., Nusse, R., and Sun, Z. (2004). Wnt3a growth factor
induces androgen receptor-mediated transcription and enhances cell growth in
human prostate cancer cells. Cancer Res 64, 8860-8866.

Vonlanthen, S., Heighway, J., Altermatt, H.J., Gugger, M., Kappeler, A., Borner,
M.M., van Lohuizen, M., and Betticher, D.C. (2001). The bmi-1 oncoprotein is
differentially expressed in non-small cell lung cancer and correlates with INK4A-
ARF locus expression. Br J Cancer 84, 1372-1376.

Vooijs, M., Jonkers, J., and Berns, A. (2001). A highly efficient ligand-regulated Cre
recombinase mouse line shows that LoxP recombination is position dependent.
EMBO Rep 2, 292-297.

Wang, S., Gao, J., Lei, Q., Rozengurt, N., Pritchard, C., Jiao, J., Thomas, G.V., Li,
G., Roy-Burman, P., Nelson, P.S., et al. (2003). Prostate-specific deletion of the
murine Pten tumor suppressor gene leads to metastatic prostate cancer. Cancer Cell
4, 209-221.

Wang, S., Garcia, A.J., Wu, M., Lawson, D.A., Witte, O.N., and Wu, H. (2006). Pten
deletion leads to the expansion of a prostatic stem/progenitor cell subpopulation and
tumor initiation. Proc Natl Acad Sci U S A 103, 1480-1485.

Wang, X.D., Shou, J., Wong, P., French, D.M., and Gao, W.Q. (2004). Notch1-
expressing cells are indispensable for prostatic branching morphogenesis during

96
development and re-growth following castration and androgen replacement. J Biol
Chem 279, 24733-24744.

Weinstein, M.H., Partin, A.W., Veltri, R.W., and Epstein, J.I. (1996).
Neuroendocrine differentiation in prostate cancer: enhanced prediction of
progression after radical prostatectomy. Hum Pathol 27, 683-687.

Whetton, A.D., and Graham, G.J. (1999). Homing and mobilization in the stem cell
niche. Trends Cell Biol 9, 233-238.

Wu, X., Wu, J., Huang, J., Powell, W.C., Zhang, J., Matusik, R.J., Sangiorgi, F.O.,
Maxson, R.E., Sucov, H.M., and Roy-Burman, P. (2001). Generation of a prostate
epithelial cell-specific Cre transgenic mouse model for tissue-specific gene ablation.
Mech Dev 101, 61-69.

Xie, X., Luo, Z., Slawin, K.M., and Spencer, D.M. (2004). The EZC-prostate model:
noninvasive prostate imaging in living mice. Mol Endocrinol 18, 722-732.

Xin, L., Ide, H., Kim, Y., Dubey, P., and Witte, O.N. (2003). In vivo regeneration of
murine prostate from dissociated cell populations of postnatal epithelia and
urogenital sinus mesenchyme. Proc Natl Acad Sci U S A 100 Suppl 1, 11896-11903.

Xin, L., Lawson, D.A., and Witte, O.N. (2005). The Sca-1 cell surface marker
enriches for a prostate-regenerating cell subpopulation that can initiate prostate
tumorigenesis. Proc Natl Acad Sci U S A 102, 6942-6947.

Xin, L., Lukacs, R.U., Lawson, D.A., Cheng, D., and Witte, O.N. (2007). Self-
renewal and multilineage differentiation in vitro from murine prostate stem cells.
Stem Cells 25, 2760-2769.

Yang, S., Pham, L.K., Liao, C.P., Frenkel, B., Reddi, A.H., and Roy-Burman, P.
(2008). A novel bone morphogenetic protein signaling in heterotypic cell interactions
in prostate cancer. Cancer Res 68, 198-205.

Yardy, G.W., and Brewster, S.F. (2005). Wnt signalling and prostate cancer. Prostate
Cancer Prostatic Dis 8, 119-126.

Young, R.H., Srigley, J. R., Amin, M. B., Ulbright, T. M., and Cubilla, A. L. (2000).
Tumors of the prostate gland, seminal vesicles, male urethra, and penis, Vol 28
(Washington, Armed Forces Institute of Pathology).

97
Zhang, J., Thomas, T.Z., Kasper, S., and Matusik, R.J. (2000). A small composite
probasin promoter confers high levels of prostate-specific gene expression through
regulation by androgens and glucocorticoids in vitro and in vivo. Endocrinology 141,
4698-4710.

Zhong, C., Saribekyan, G., Liao, C.P., Cohen, M.B., and Roy-Burman, P. (2006).
Cooperation between FGF8b overexpression and PTEN deficiency in prostate
tumorigenesis. Cancer Res 66, 2188-2194.

Zhou, Z., Flesken-Nikitin, A., Corney, D.C., Wang, W., Goodrich, D.W., Roy-
Burman, P., and Nikitin, A.Y. (2006). Synergy of p53 and Rb Deficiency in a
Conditional Mouse Model for Metastatic Prostate Cancer. Cancer Res 66, 7889-
7898.

Zhou, Z., Flesken-Nikitin, A., and Nikitin, A.Y. (2007). Prostate cancer associated
with p53 and Rb deficiency arises from the stem/progenitor cell-enriched proximal
region of prostatic ducts. Cancer Res 67, 5683-5690.

Abstract (if available)

Abstract The application of our prostate epithelium-specific Cre/loxP system to inactivate tumor suppressor genes had resulted in successful development of mouse models of prostate cancer. We further increased the efficiency of the conditional Pten deletion model of prostate adenocarcinoma by combining it with either a conditional luciferase or EGFP reporter line. The growth kinetics of the androgen dependent cancer (AD-Ca) and androgen-depletion independent recurrent cancer (ADI-Ca) could be followed non-invasively in live animals by bioluminescence imaging. We expect that such an investigation will be facilitated by timing the collection of tumorsat specific growth or re-growth points, an advantage that is provided by the model. The EGFP model can provide an opportunity to locate tumors or to isolate enriched populations of cancer cells from tumor tissues via fluorescence-based technologies. Previous studies have shown a small cell subpopulation with Lin-Sca-1+CD49f+ (LSC) cell surface marker phenotypes in the normal murine prostate to have the capacities of stemness. We examined the presence of such cells in our mouse models of prostate cancer, and found a much higher percentage of the LSC cellsubpopulation among the Lin- cells in AD-Ca and ADI-Ca compared to that in the proximal region of the normal counterparts.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Exploration of the roles of cancer stem cells and survivin in the pathogenesis and progression of prostate cancer

PDF

Homologous cell systems for the study of progression of androgen-dependent prostate cancer to castration-resistant prostate cancer

PDF

Study of the role of bone morphogenetic proteins in prostate cancer progression

PDF

Study of bone morphogenetic protein-2 and stromal cell derived factor-1 in prostate cancer

PDF

Role of cancer-associated fibroblast secreted annexin A1 in generation and maintenance of prostate cancer stem cells

PDF

Bimodal effects of bone morphogenetic proteins in prostate cancer

PDF

The effect of tumor-mediated immune suppression on prostate cancer immunotherapy

PDF

The noncanonical role of telomerase in prostate cancer cells: exploring a non-telomeric signaling role for telomerase protein (TERT) in a cancer cell line

PDF

Identification of novel androgen receptor target genes in prostate cancer

PDF

The role of GRP78 in the regulation of apoptosis and prostate cancer progression

PDF

Harnessing the power of stem cell self-renewal pathways in cancer: dissecting the role of BMI-1 in Ewing’s sarcoma initiation and maintenance

PDF

Targeting glioma cancer stem cells for the treatment of glioblastoma multiforme

PDF

Molecular mechanisms of chemoresistance in breast cancer

PDF

The cancer stem-like phenotype: therapeutics, phenotypic plasticity and mechanistic studies

PDF

Co-expression of monoamine oxidase A and prostate cancer stem cell markers in Pten knockout mice

PDF

Targeting molecular signals involved in the development of castration resistant prostate cancer

PDF

Role of beta-catenin in mouse epiblast stem cell, embryonic stem cell self-renewal and differentiation

PDF

PTEN deletion induced tumor initiating cells: Strategies to accelerate the disease progression of liver cancer

PDF

Immune signature of murine solid tumor models

PDF

Ancestral inference and cancer stem cell dynamics in colorectal tumors

Asset Metadata

Creator Liao, Chun-Peng (author)

Core Title Studies of murine prostate cancer stem / progenitor cells

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Biochemistry and Molecular Biology

Degree Conferral Date 2008-08

Publication Date 07/09/2008

Defense Date 05/29/2008

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cancer,cell,mouse,OAI-PMH Harvest,progenitor,prostate,STEM

Language English

Advisor Roy-Burman, Pradip (committee chair), Dubeau, Louis (committee member), Frenkel, Baruch (committee member)

Creator Email chunpenl@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m1326

Unique identifier UC1218192

Identifier etd-Liao-20080709 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-80120 (legacy record id),usctheses-m1326 (legacy record id)

Legacy Identifier etd-Liao-20080709.pdf

Dmrecord 80120

Document Type Dissertation

Rights Liao, Chun-Peng

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu