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A novel design of integrin α2β1 targeting peptide probe for molecular imaging in prostate cancer

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A novel design of integrin α2β1 targeting peptide probe for molecular imaging in prostate cancer

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A NOVEL DESIGN OF INTEGRIN α2β1 TARGETING
PEPTIDE PROBE FOR MOLECULAR IMAGING IN
PROSTATE CANCER

by

Chiun-Wei Huang

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(BIOMEDICAL ENGINEERING)

May 2011

Copyright 2011 Chiun-Wei Huang
ii

Table of Contents
List of Figures ....................................................................................................... v

Abstract ................................................................................................................ ix

Chapter 1: Introduction ......................................................................................... 1
1.1 Disease Background ................................................................................... 1
1.1.1 Prostate Cancer: A Complex Disease ..................................................... 1
1.1.2 Prostate cancer initiation ......................................................................... 2
1.1.3 Prostate cancer development and metastasis ......................................... 4
1.2 Prostate Cancer Therapies ......................................................................... 7
1.2.1 Surgery and radiation therapy ................................................................. 7
1.2.2 Hormonal therapy .................................................................................... 8
1.2.3 Chemotherapy ......................................................................................... 8
1.2.4 New molecular targeting therapies ........................................................ 10
1.3 Integrin Severed as Molecular Imaging and Therapy Target ..................... 12
1.3.2 Integrin Signaling ................................................................................... 13
1.3.3 αvβ3 Integrin as a Cellular Target Molecule .......................................... 15
1.4 Integrin α2β1 ............................................................................................. 17
1.4.1 Integrin α2β1: Relations with Prostate Cancer ...................................... 17
1.4.2 Specific Collagen Recognition Motif Sequence ..................................... 19
1.5 The Objective, Hypothesis, and Specific Aims .......................................... 21
1.5.1 The Objective and Hypothesis ............................................................... 21
1.5.2 The Specific Aims .................................................................................. 22

iii

Chapter 2: Near-Infrared Fluorescent Imaging of Prostate Cancer Using Integrin
α
2
β
1
Targeted DGEA Peptides ........................................................................... 25
2.1 Summary: .................................................................................................. 25
2.2 Introduction: .............................................................................................. 27
2.3 Materials and Methods: ............................................................................. 29
2.4 Results ...................................................................................................... 36
2.5 Discussion: ................................................................................................ 47
2.6 Conclusion: ............................................................................................... 52

Chapter 3: Cell Penetrating Peptide Sequence (CPPs) Served as the potential
Therapeutic Delivery Modality ............................................................................ 53
3.1 Summary: .................................................................................................. 53
3.2 Introduction: .............................................................................................. 55
3.3 Materials and Methods: ............................................................................. 56
3.4 Results: ..................................................................................................... 64
3.5 Discussion: ................................................................................................ 89
3.6 Conclusion: ............................................................................................... 94

Chapter 4: microPET Tracer Design and Biological Evaluation .......................... 95
4.1 Summary: .................................................................................................. 95
4.2 Introduction: .............................................................................................. 97
4.3 Materials and Methods: ............................................................................. 99
4.4 Results: ................................................................................................... 107
4.5 Discussion: .............................................................................................. 115
4.6 Conclusion: ............................................................................................. 122

Chapter 5: Biological Stability Evaluation of the α2β1 Receptor Imaging Agents:
Diamsar and DOTA Conjugated DGEA Peptide ............................................... 123
5.1 Summary: ................................................................................................ 123
5.2 Introduction: ............................................................................................ 125
iv

5.3 Materials and Methods: ........................................................................... 127
5.4 Results: ................................................................................................... 134
Table 5-1. Solution Stability Data for the HPLC-purified 64Cu-Labeled
DGEA Tracers in Phosphate Buffer (pH=7.4) ............................................... 139
5.5 Discussion: .............................................................................................. 142
5.6 Conclusion: ............................................................................................. 150

Chapter 6: Summary and Future Goals ............................................................ 151
6.1 Summary: ................................................................................................ 151
6.2 Future Goals: .......................................................................................... 153

References ....................................................................................................... 156

v

List of Figures
Figure 1-1: The general chemical structure of integrin α2β1 targeted
probe design. .................................................................................... 23

Figure 2-1: Schematic structure of FAM- and Cy 5.5-conjugated DGEA peptide
probes ............................................................................................... 37

Figure 2-2: Histograms of typical flow cytometry results of cell lines (PC-3,
CWR-22 and LNCaP) treated with FAM-DGEA peptides. ............... 39

Figure 2-3: Representative fluorescent microscopy imaging of FAM-DGEA
binding in prostate cancer cell lines in vitro. .................................... 41

Figure 2-4: In vivo near-infrared imaging of subcutaneous PC-3 prostate
tumor bearing nude mice after i.v. injection of Cy5.5-DGEA
and the qualification analysis plot. .................................................... 43

Figure 2-5: Representative blocking experiment imaging of mice bearing
subcutaneous PC-3 tumor and ex vivo imaging of major organs
and quantification analysis plot. ...................................................... 45

Figure 2-6: Immunohistochemical staining imaging results of PC-3 tumor
tissue slides. ..................................................................................... 46

Figure 3-1: Schematic structure of branched poly arginine (R4) conjugated
DGEA peptide probe ......................................................................... 65

Figure 3-2: Flow cytometry analysis of integrin α2β1 binding in prostate
cancer cell lines which were incubated with FAM-DGEA,
R4-(FAM)–DGEA and nonsense FAM-AAAA peptides. .................... 66

vi

Figure 3-3: Fluorescent microscopy images of prostate cancer cell
lines (PC-3,CWR-22 and LNCaP) incubated with of R4
-FAM-DGEA peptides .................................................................... 67

Figure 3-4: The confocal imaging of PC-3 cells incubated with integrin
α2β1 targeted FAM-DGEA peptides and the cell penetrating
R4-FAM-DGEA peptide. .................................................................. 69

Figure 3-5: The confocal imaging of CWR-22 cells incubated with nucleic
acid stain DAPI (Ex/Em=358/461nm) and the R4-FAM-DGEA
peptides (Ex/Em= 492/518 nm). ..................................................... 70

Figure 3-6: In vivo fluorescence imaging of athymic nude mice bearing
subcutaneous PC-3 or CWR-22 xenografts after i.v. injection
of R4-(Cy5.5)-DGEA. ........................................................................ 72

Figure 3-7: The schematic structure of the potential probe design which
consisted of the brush border peptidase sensitive linker (Gly-
Lys-OH), DGEA targeting sequence and CPPs. ............................. 75

Figure 3-8: In vivo fluorescence imaging and quantification analysis of
athymic nude mice bearing PC-3 or CWR-22 xenografts
after i.v. injection of R4-DGEAGK(Cy 5.5)-OH. ............................... 78

Figure 3-9: In vivo fluorescence imaging and quantification analysis of
athymic nude mice bearing subcutaneous PC-3 or CWR-22
xenografts after i.v. injection of Cy5.5-DGEAGK(R4)-OH ............... 80

Figure 3-10: Schematic structure of Cy5.5-conjugated DGEAGK(R8)-OH
peptide. ......................................................................................... 82

Figure 3-11: In vivo fluorescence imaging and quantification analysis of
athymic nude mice bearing subcutaneous PC-3 or CWR-22
xenografts after i.v. injection of Cy5.5-DGEAGK(R8)-OH. ............ 83

vii

Figure 3-12: Representative blocking experiment NIR images of mice
bearing subcutaneous PC-3 tumor by co-injection with
Cy5.5-DGEAGK(R8)-OH and 10mg/kg of unlabeled DGEA
peptides. ....................................................................................... 85

Figure 3-13: Representative images and quantification analysis of dis-
-sected organs of mice bearing PC-3 tumor sacrificed 4h
after i.v. injection of Cy5.5-DGEAGK(R8)-OH. .............................. 86

Figure 3-14: Fluorescent microscopy imaging of Cy5.5-DGEAGK(R8-OH
distribution in frozen PC-3 tumor tissue slides. ............................. 88

Figure 4-1: Schematic structure of the Z-E(diamsar)-Ahx-DGEA and
DOTA-K(Cy5.5)-Ahx-DGEA peptides ............................................ 108

Figure 4-2: In vitro stability of
64
Cu-DOTA-K(Cy5.5)-Ahx-DGEA and

64
Cu-Z-E(diamsar)-Ahx-DGEA. ..................................................... 109

Figure 4-3: Cell uptake assay and cell efflux assay of Z-E(diamsar)-
Ahx-DGEA and DOTA-K(Cy5.5)-Ahx-DGEA on PC-3 cells ............ 111

Figure 4-4: Decay-corrected whole-body coronal small-animal PET
Images of PC-3 tumor-bearing mice after injection of

64
Cu-(Z-E(diamsar)-Ahx-DGEA). .................................................. 112

Figure 4-5: Decay-corrected whole-body coronal microPET images of
blocking experiments of PC-3 and CWR22tumor-bearing
mice. ............................................................................................... 114

Figure 4-6: Representative near-infrared images of athymic nude mice
bearing subcutaneous PC-3 tumor after intravenous injection
of
64
Cu-DOTA-K(Cy5.5)-Ahx-DGEA. ............................................... 116

viii

Figure 4-7: Representative microPET images of PC-3 tumor-bearing
mice post injection of
64
Cu -DOTA-K(Cy5.5)-Ahx-DGEA .............. 117

Figure 4-8: Representative ex vivo microPET and fluorescence imaging
of dissected major organs of a PC-3 tumor bearing nude
mouse. .......................................................................................... 118

Figure 5-1: Schematic structure of DOTA-DGEA, DOTA-D(tBu)GE(tBu)A,
DOTA-Ahx-DGEA and Z-E(diamsar)-Ahx-DGEA .......................... 135

Figure 5-2: The representative radio-HPLC chromatogram of
64
Cu-
DOTA-DGEA and
64
Cu-Z-E(diamsar)-Ahx-DGEA. ........................ 138

Figure 5-3: Decay-corrected whole-body coronal small-animal PET images
of PC-3 tumor-bearing mice at 30 min post injection of
64
Cu-Z
-E(diamsar)-Ahx-DGEA (left) and
64
Cu-DOTA-DGEA (right) ......... 140

Figure 5-4: Comparison of the major organs uptake and tumor contrast
of
64
Cu-DOTA-DGEA and
64
Cu-Z-E(diamsar)-Ahx-DGEA. ............ 141

Figure 5-5: Schematic structure of the potential interfere interactions
between the carboxylic acid groups of DGEA peptide and
DOTA chelator. ............................................................................ 146

ix

Abstract
Prostate cancer is one of the leading causes of cancer-related deaths in the
United States and Europe. Despite the fact that prostate-specific antigen (PSA)
screening has greatly increased the number of patients with early stage prostate
cancer who can be cured by radical prostatectomy, about 40% of prostate cancers
are first detected at an advanced stage and half of these are found to be
extracapsular at pathologic staging. Therefore, development of an accurate
noninvasive imaging technique to detect primary, recurrent and residual prostate
cancer is critical for the effective management of this group of patients.
Accumulating experimental evidence indicates that integrins presented on various
tumor types are differentially expressed during tumor transformation, progression,
and metastasis. Development of integrin cell expression profiles of individual
tumors may have further potential in identifying a cell surface signature for a
specific tumor type and/or stage. Multiple lines of literature studies indicated that
the upregulation or overexpression of α2β1integrin may correlate with tumor
progression in human prostate cancer. For example, the more aggressive PC-3
cell line has the highest expression of α2β1 integrin compared with other less
aggressive cell lines, such as CWR-22 (medium expression of α
2
β
1
) and LNCap
(low expression of α2β1). Noninvasive imaging of this receptor with radiolabeled
peptides that specifically target α2β1 integrin could therefore be useful to decipher
the invasive potential of prostate cancers.
x

High sensitivity positron emission tomography coupled with computed
tomography for anatomical evaluation, PET/CT, has become a critical diagnostic
imaging tool in the identification of a diverse group of malignancies. In this study,
we use the knowledge of chemistry, radiochemistry, biochemistry, biology and
molecular imaging to develop clinically translatable α2β1 integrin targeting PET
probes for prostate cancer imaging. A series of α2β1 integrin targeting peptides
were synthesized and their specificity was verified both in vitro and in vivo at the
molecular level in preclinical prostate tumor models. The success of the proposed
study may provide a better understanding of basic biological mechanisms of
prostate cancer, help us more appropriately select patients considered for
anti-integrin α2β1

treatment, and allow the evaluation of disease course and
therapeutic efficacy at the earliest stages of treatment.

1

Chapter 1: Introduction
1.1 Disease Background
1.1.1 Prostate Cancer: A Complex Disease
Prostate cancer is the most common malignancy diagnosed in men and the
metastatic forms represent the second cause of mortality (1) in United States.
According to the American Cancer Society‘s most recent estimates, 217,730
American men will be diagnosed with prostate cancer in 2010 and 32,050 would
die from the disease. The chances of developing prostate cancer increase with
age. The estimated lifetime probability of developing the disease is one-sixth.
Over the years, however, the precise causes of prostate cancer remain poorly
understood. In general, the prostate cancer grows slowly, remains confined to the
gland and produces little or no symptoms or outward signs. However, some
aggressive types of prostate cancer can grow and spread beyond the prostate
into the surrounding tissues or even metastasize throughout to other distance
tissues, such as bones, lymph nodes, lungs and liver. It is this advanced
phenotype of prostate cancer can cause a significant shortening of life expectancy
in men affected by the disease. Numerous gene products and growth factors have
shown deregulated activities and are over expressed during the progression of
this heterogeneous disease. (2-11) These specific changes of gene expression
during the different developmental stages of prostate cancer notably contribute in
enhancing the tumor cell growth, survival, migration and invasiveness.
2

Deciding on treatment for an individual patient can be difficult, because not
enough reliable guiding or screening data are available on which to base the
decisions. Accordingly, carefully designed and controlled, long-term studies are
still needed to compare the benefits and risks of the various treatments. The
current treatment options for prostate cancer, consisting of radical prostatectomy
(RP), radiation therapy, hormonal therapy, cryotherapy and/or neo-adjuvant
chemotherapy, are generally curative for the majority of patients diagnosed with
localized and androgen-dependent prostate cancer forms; however, progression
to androgen-independent and metastatic disease states is often accompanied by
a recurrence of prostate cancer.(12,13) A cure for patients with
hormone-refractory prostate cancer (HRPC) is, unfortunately, unattainable at
present time. The combination of hormonal therapy and chemotherapy is rather
palliative and remain mostly ineffective with a poor prognosis. (14,15) The
prognosis is associated with a median survival rate of about 12 months after
diagnosis. Therefore, the development of a novel treatment which is more
effective against anti-androgen and chemotherapy refractory prostate cancer
forms is highly desirable.

1.1.2 Prostate cancer initiation
Although prostate cancer is now the most commonly diagnosed cancer in
American men, the etiology of this disease remains unclear. Prostate cancer is
similarly a disease of aging that is thought to begin with the chronic accumulation
3

of prostatic intraepithelial neoplasia (PIN) lesions that may eventually develop into
adenocarcinoma, although the events associated with the initiation of prostate
cancer are not precisely known, some recent lines of evidence suggest that
prostate cancer could be derived from these precancerous lesions occurring
during prostate tissue injury. (7, 16-18) On the other hand, the role of stem cells is
now being addressed in many solid tissue cancers. As a matter of fact, a pool of
prostate specific stem cells which are implicated in cell renewal during the
prostate regeneration process, has been proposed to represent the minority of
epithelial cells which could give the prostate cancer progenitor cells following the
sustained activation of different growth-factor signaling cascades. (7, 16, 17) In
support of this model, certain prostate progenitor cells have recently been isolated
from proximal regions of prostatic ducts. (19,20) Prostate progenitor cells have
some of the properties associated with stem cells due to their striking plasticity;
these properties include the ability to show unlimited growth in a specific
microenvironment and generate multiple, more differentiated prostate cells. In fact,
the niche of prostatic stem cells, which represents approximately 1-2% of basal
epithelial cells, appears to be localized at the basement membrane of the
prostatic gland. (21-24) More specifically, the prostatic adult stem cells are
characterized by specific markers such as α2β1 (hi)-integrin, CD133, stem cell
antigen-1 (Sca-1), prostate stem cell antigen (PSCA) and cytokeratin 6a (K6a).
(20,22,24-27)
There are recent advances in the identification of putative prostatic stem cells;
4

however the molecular and cellular mechanisms of the oncogenic transformation
of prostate progenitor epithelial cells and the changes in stromal-epithelial cell
interactions mediating initiative events are still not precisely known. Among the
models of prostate cancer initiation, there is the possibility that prostate dysplastic
lesions may derive from deregulated mitogenic signaling cascades in either basal
multipotent stem cells and/or transit amplifying/intermediate cells. Prostate
dysplastic lesions, in turn, may subsequently give rise to a heterogeneous
population of cancer epithelial cells showing aberrant differentiation, unlimited
division and a decreased rate of apoptotic death. Altogether, these observations
suggest that the sustained activation of androgens, estrogens and distinct growth
factor signaling cascades in prostate progenitor epithelial cells may lead to the
generation of a heterogeneous population of cancer progenitor cells showing
uncontrolled growth and altered differentiation. These cancer progenitor cells, in
turn, may induce the formation of PIN-like lesions and, ultimately, prostate cancer
development.

1.1.3 Prostate cancer development and metastasis

Almost all prostate cancers initially develop from secretary epithelial cells of the
prostate gland and generally grow slowly within the gland. When the tumor cells
penetrate the outside of the prostate gland they may spread to tissues near the
prostate, first to the pelvic lymph nodes and eventually to distant lymph nodes,
bones and organs such as the brain, liver and lungs. The in vitro and in vivo
5

characterization of behavior of numerous human prostate cancer cell lines as
compared to the normal prostatic epithelial cells has notably indicated that several
oncogenic signaling cascades are involved in regulating the progression from
localized and androgen-dependent prostate cancer forms into aggressive and
androgen-independent states. (4,7,8,10,11) In fact, the enhanced expression of a
variety of growth factors, including EGF, TGF-α, fibroblast growth factor (FGF),
hepathocyte growth factor (HGF), nerve growth factor (NGF), insulin-like growth
factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) and their cognate
receptors concomitant with the alterations in TGF-β signaling elements, appears
to assume a critical role in inducing changes in stromal-epithelial cell interactions
during prostate cancer development. (7,9,28-30)

More specifically, the enhanced VEGF expression induced by several growth
factors in tumor epithelial cells seems to contribute to the angiogenesis process of
paracrine fashion during the early stages of prostate cancer. The subsequent
expression of the VEGF receptor (VEGFR) on the tumor epithelial cells at late
stages may participate in the autocrine and paracrine regulation of the
invasiveness of tumor cells. In this matter, a model has been proposed to explain
the high frequency of bone metastasis of prostate cancer cells.(31-34)
According to this model, the molecular mechanism at the basis of osteotropism of
prostate cancer metastasis could implicate an enhanced expression of VEGF and
VEGFR-2 on the prostate cancer cells. This, in turn, could subsequently result
6

through an autocrine loop, in activating α2β1, α1β1, αvβ3 and αvβ5-integrins at
the surface of prostate cancer cells. Hence, the occurrence of these specific
changes in the prostate cancer cells could preferentially lead to their migration
and adhesion at a component of bone matrix. Hence, the oncogenic changes in
the tumor stromal epithelial cells, which may be induced by activation of distinct
growth factor signaling cascades, may confer a more malignant behavior to
cancer progenitor cells during the progression from localized prostate cancer
forms into metastatic states.

7

1.2 Prostate Cancer Therapies
1.2.1 Surgery and radiation therapy

The surgical treatment for prostate cancer patient is usually referred to radical
prostatectomy (RP), which consists of removing the entire prostate gland, seminal
vesicles, and ampulla of the vas deferens, represents the most common
treatment for patients with organ confined or localized prostate cancer. In general,
patients with confined prostate cancer and the entire gland is removed usually
have a biochemical relapse-free survival rate of five (84%) and nine (76%) years.
(35,36) The recurrence of prostate cancer at distant sites following surgery may
occur, resulting from some undetected prostate cancer lesions extended to the
surgical margins. A PSA values greater than 0.2 ng/ml detected after RP is
usually considered as evidence of cancer recurrence. (35)

Radiation therapy by external beam radiation or an implant of radioactive seeds
directly into prostate designated as brachytherapy may also be used as a
treatment option for the localized prostate cancer or when the prostate cancer
only spreads to near tissues. The goal of radiotherapy is to damage the cancer
cells or kill them, because the rapidly dividing cancer cells are more vulnerable to
destruction by the radiation than are the neighboring normal cells. The outcome in
men treated with permanent prostate brachytherapy presents a 12-year span
before recurrence in patients with localized prostate cancer. (12,37) In addition,
8

surgery and radiotherapy are also often used in combination with hormone
therapy and/ or chemotherapy in an effort to improve the long-term relapse free
treatment results.

1.2.2 Hormonal therapy

The androgenic hormone stimulates the growth of cancerous prostatic cells and,
therefore, is the primary fuel for the growth of prostate cancer. The idea of the
hormonal treatments is to decrease or block the stimulation of the androgens to
the cancerous prostatic cells and usually used in combination with surgery for
patients with prostate cancer which has spread beyond the prostate or recurred
after treatment. (38) Although patients with prostate cancer initially respond to
hormone therapy for a few years, the development of androgen-independent
states usually results in resistance to this type of treatment. It has been reported
that the treatment of long-term deprived LNCaP-abl by androgen receptor
antagonist bicalutamide leads to the stimulation of androgen receptor and cell
proliferation of LNCaP cells. (39)

1.2.3 Chemotherapy

Chemotherapy are usually used as a palliative treatment for patients with HRPC
which has spread outside of prostate gland, because a cure is unachievable at
present. The androgen-independent (hormone resistant) metastatic prostate
9

cancer cells are rare types of prostate cancer that respond better to
chemotherapy than hormone therapy. These tumors are usually treated with
etoposide and cisplatin. For locally advanced stages of prostate cancer,
preclinical trials have revealed the potential benefits of the use of cytotoxic drugs
prior to surgery or as adjuvant therapy with the anti-androgen treatment after
surgery.

The common used chemotherapeutic agents for patients with HRPC include a
combination of either mitoxantrone and prednisone, or taxols such as docetaxel
and prednisone or estramustine. These combinations have been reported to
improve the quality of life for patients with pain, offering pain relief instead of just
treatment alone. These drugs, however, showed low survival benefits. (40, 41)
Orally bioavailable platinum (IV) complex, satraplatin (as known as JM-216 or
BMS-182751), which shows an anti-tumoral activity superior to cisplatin and
carboplatin, is undergoing preclinical trials to estimate its efficacy as a second-line
treatment for HRPC. Moreover, it has recently been reported that the combination
of estramustine, docetaxel and suramin could represent a highly effective regimen
for HRPC with modest toxicities, (42) mainly due to hematology and
gastrointestinal toxicities. Of clinical interest, the data from a phase II study
carried out with a long-term five-year follow-up have also revealed that 34.8 % of
patients with HRPC survived more than two years with a treatment consisting of
oral estramustine plus oral etoposide.(43)
10

The combination of different chemotherapy medicines has shown some promise
in prolonging the survival of some patients with advanced prostate cancer. They
may also decrease the pain related to widespread cancer. However, this comes at
the cost of significant side effects that may impact quality of life. Altogether, these
observations indicated that the current chemotherapeutic agents used remain
ineffective against HRPC forms, due in part, to the dose-limiting toxicities (DLT) of
drugs. Therefore, this underlines the importance of undertaking additional
preclinical trials for optimizing the administration modes and regimen options of
conventional chemotherapeutic drugs. The establishment of new combinations of
cytotoxic drugs also seems essential for the development of a more effective
treatment against metastatic and androgen independent prostate cancer forms.

1.2.4 New molecular targeting therapies

Since the progression from the androgen-dependent prostate cancer into more
aggressive and metastatic forms often leads to disease relapse, several novel
therapeutic strategies have been investigated for improving treatments against
metastatic HRPCs. One new approach is molecular targeting of distinct
deregulated signaling elements in prostate cancer cells whose tumorigenic
products are involved in the development of from localized androgen-dependent
states into androgen-independent and metastatic forms of prostate cancer. The
identification of distinct deregulated cellular targets in prostate cancer cells
11

directly involved in prostatic carcinogenesis will allow us to target several
signaling elements in tumor cells to counteract prostate cancer progression.

12

1.3 Integrin Severed as Molecular Imaging and Therapy Target
1.3.1 Integrin Structure and Function
Integrins are transmembrane receptors in the immunoglobulin (Ig) superfamily,
which are composed of alpha and beta subunits; and have been shown to
mediate cellular adhesion as well as entry and withdrawal from the cell cycle. So
far, at least 18 α and 8 β glycoprotein subunits of the integrin family have been
discovered. (44) These subunits are expressed on cell surfaces in 24 different
heterodimeric combinations, allowing them to respond to and modulate a broad
array of extracellular matrix (ECM) proteins. The alpha subunit contains 4 binding
sites for divalent cations. Some, but not all, integrin heterodimers bind the aspartic
acid-glycinearginine (RGD) consensus sequence, first identified by Rouslahti
and Pierschbacher in 1984.(45) Other integrin binding motifs have been identified,
many by phage display, including LDV (α4β1), (46) KQAGDV (αIIbβ3)(47) and
KRLDGS (αmβ2).(48) The alpha and beta subunits, as well as the divalent metal
cations, are required for integrin binding to these ligands.

One of the best characterized integrin ligands is fibronectin. Fibronectin consists
of three repeated domains whose ligands including cell-surface integrins,
heparin, gelatin, collagen, and fibrin. The structure of fibronectin elucidates one of
the major physiologic roles of integrins, which is in mediating cellular interactions
with the extracellular matrix. Integrins bind to the extracellular matrix either
through multifunctional ligands such as fibronectin or vitronectin or through direct
13

binding to collagen and laminin, as determined by the ligand specificities of each
alpha-beta heterodimer. In general, integrin-ligand attachments tend to be of low
affinity; however, the high density of integrins on the cell surface, and their
recruitment by cytoskeletal proteins to localized plaques, increases the avidity of
cell binding to the basement membrane. (49) Along with their role in mediating cell
adhesion to the ECM, integrins also regulate cell growth and survival,
differentiation, morphology, and migration.(50) Consistent with this, the
engagement of cell surface integrins is known to trigger the assembly of the
focal assembly complex, which results in the formation of complex interactions
between the cytoplasmic domains of integrins, the actin cytoskeleton and
membrane-proximal kinases such as the focal assembly kinase.

Integrin activation/deactivation leads to a complex range of biological effects,
including but not limited to hyptotaxis (adhesive response to ECM binding),
chemotaxis (migration toward a biochemical stimulus), and anoikis (apoptosis
on loss of ECM adhesion). The multiple roles of integrin have been extensively
studied in tumor biology, where modulation of integrin profiles has been shown to
dramatically alter phenotype and prognosis.

1.3.2 Integrin Signaling
Integrin signaling is characterized by two types of events: ―inside-out‖ signaling,
in which intracellular events influence the activation state (ligand affinity) of the
14

cell surface integrins, and ―outside-in signaling‖, in which the overall state of
integrin enegagement influences intracellular signalling events and may result in
the induction of cell cycle progression and apoptosis. In ―outside-in‖ signaling,
ligand binding leads to activation of Src and FAK tyrosine kinases at the focal
adhesion complex. (51) These in turn activate Ras and Rho family GTPases,
which initiate diverse downstream events including cell cycle progression,
increased intracellular calcium, and, in the case of αvβ3, PI3K-Akt
pathway-triggered cell migration and NFkB-dependent transcription .(52-56)

In addition, un-ligated integrins recruit caspase-8 to their cytoplasmic tails,
triggering apoptosis; ligation of integrins reverses this process. (57) This
explains in part the cell survival-promoting effects of ECM attachment for many
cell types. ―Outside-in‖ signaling controls integrin expression and activation in
response to cellular events. For example, the modulation of beta3 integrin (αvβ3
and αIIbβ3) activation has been shown to be a direct result of Raf-MEK-ERK
pathway activation.(58) Pharmacological inhibition of MEK1 has been shown to
lead to down regulation of αvβ3 integrin expression in several cell lines whereas
the activation of Raf leads to up-regulation of αvβ3 expression.(59) This is
important because αvβ3-specific functions such as cell migration and bone
adhesion may be controlled both through integrin expression as well as
activation—Rac1 signaling is implicated in the incorporation of αvβ3 into the
lamellipodia of migrating cells. The role of αvβ3 in adhesion elucidates the
15

migration-specific activity of this integrin, since stationary adhesion plaques
contain static αvβ3 expression while sliding contacts show rapid focal up- and
down- regulation of beta3 expression.

1.3.3 αvβ3 Integrin as a Cellular Target Molecule
The metastatic process is the major cause of mortality in cancer patients. This
process involves tumor cell adhesion to other cells and ECM glycoproteins, and
eventual invasion through basement membranes. Such interactions may be
mediated by a variety of cell surface biomolecules, including integrins. Integrins
are essential for cell attachment and control cell migration, cell cycle progression,
and programmed cell death, which they regulate in synergy with other signal
transduction pathways. The observation that integrins present on various tumor
types are differentially expressed during tumor transformation, progression, and
metastasis, suggests that integrins may also be useful as prognostic markers.
Therapies directed at influencing integrin cell expression and function are
presently being explored for inhibition of tumor growth, development of metastase,
and angiogenesis.

Integrin αvβ3 is currently the most widely and intensely studied integrin
heterodimer of all 24 combinations discovered. Expression of αvβ3 on tumors is
generally associated with an invasive and metastatic phenotype. The four
binding sites for divalent cations that are found on the αv integrin must all be
16

filled in order for ligand binding to occur. Recent crystal structure data suggests
that these divalent cations may interact directly with ligands during receptor
binding events. Other binding sites, which preferentially bind calcium, appear to
inhibit integrin binding. The requirement of divalent cations for integrin adhesion is
an important aspect of the regulation of integrin function. Depending on the
integrin heterodimer and divalent cation, triggering of cell adhesion or cell
detachment is possible. Several antibodies, Arg-Gly-Asp (RGD) peptide and
peptidomimetic antagonists for αvβ3 are now in clinical trials for use as
antiangiogenic treatment.

17

1.4 Integrin α2β1

1.4.1 Integrin α2β1: Relations with Prostate Cancer
The precise causes of prostate cancer remain poorly understood. Numerous
growth factors and their receptors are over expressed during the progression of
this hyper proliferative disease. These specific changes of protein expression in
epithelial and stromal tumor cells during the different developmental stages of
prostate cancer notably contribute in enhancing the tumor cell growth, survival,
migration and invasiveness. From a clinical perspective, metastatic bone disease
is the major cause of mortality in prostate cancer patients. (60,61) Therefore, the
high incidence of skeletal metastasis has been suggested to be a reflection of
favorable reciprocal interactions between the bone microenvironment and
disseminated prostate cancer cells.(62-64) This process involves tumor cell
adhesion to other cells and extra-cellular matrix glycoproteins, and eventual
invasion through basement membranes. Recent advances on
differently-expressed gene products and their functions during the progression
from localized androgen-dependent states into androgen-independent and
metastatic forms of prostate cancer have been reported. The expression levels of
numerous products of oncogenes and tumor suppressor genes in distinct
prostatic cancer epithelial cell lines and tissues relative to normal prostate cells
are described to identify the signaling elements that are altered during the
initiation, progression and metastatic process of prostate cancer.
18

Integrins are essential for cell attachment and control cell migration, cell cycle
progression, and programmed cell death, which they regulate in synergy with
other signal transduction pathways. The observation that integrins present on
various tumor types are differentially expressed during tumor transformation,
progression, and metastasis, suggests that integrins may also be useful as
prognostic markers. (65) In particular, the α2β1 integrin, a receptor mainly for type
I collagens, laminins, E-cadherin, matrix metalloproteinase-1 (MMP-1), and
several viruses, (66,67) has been implicated in multiple aspects of tumor
progression and metastasis. Many tumors that have high expression of α2β1
correlate with the tumor progression. (68-73)

Moreover, recent studies indicate that high expression of α2β1 integrin is one of
the specific markers that could be used to characterize prostatic tumor stem cells,
which could be associated with the initiation of prostate cancer. Therefore, the
possible correlation between α2β1 integrin expression level and tumor
invasiveness makes α2β1 integrin a potential biomarker for cancer progression
diagnosis and treatment monitoring. The development of α2β1 integrin targeted
tracer to elucidate the cell expression profiles or ‗‗fingerprints‘‘ of individual tumors
may have further potential in identifying a specific tumor type and/or stage,
potentially leading to customized treatment options for patients.

19

1.4.2 Specific Collagen Recognition Motif Sequence
The α2β1 integrin serves as either a specific cell surface receptor for collagen or
as both a collagen and laminin receptor depending upon the cell type. It had been
found that the α2β1 integrin binds to a site within the αl (I)-CB3 fragment of type I
collagen (74). To define the α2β1 recognition sequence further they had prepared
an overlapping set of synthetic peptides which completely spans the 148-amino
acid αl(1)-CB3 fragment and tested the peptides for ability to inhibit cell adhesion
to collagen and laminin substrates. The minimal active recognition sequence
defined by these experiments is a tetrapeptide of the sequence Asp-Gly-Glu-Ala
(DGEA) corresponding to residues 435-438 of the type I collagen sequence. The
DGEA-containing peptides effectively inhibited α2β1-mediated Mg
2+
-dependent
adhesion of platelets, which use the α2β1 integrin as a collagen-specific receptor,
to collagen but had no effect on α5β1-mediated platelet adhesion to fibronectin or
α6β1-mediated platelet adhesion to laminin.

In contrast, with T47D breast adenocarcinoma cells, which use α2β1 as a
collagen /laminin receptor, adhesion to both collagen and laminin was inhibited by
DGEA containing peptides. Deletion of the alanine residue or substitution of
alanine for either the glutamic or aspartic acid residues in DGEA-containing
peptides resulted in marked loss of inhibitory activity. These results indicate that

20

the amino acid sequence DGEA serves as a recognition site for the α2β1ntegrin
on platelets and other cells. Based on this finding, we chose the DGEA sequence
as the starting point of our prototype α2β1 integrin targeting tracer design.

21

1.5 The Objective, Hypothesis, and Specific Aims

1.5.1 The Objective and Hypothesis
The development of integrin antagonists offers a promising strategy to inhibit
tumor growth, development of metastases and angiogenesis. Potential
integrin-based therapeutics includes synthetic peptides that have been modified
via cyclization, or inclusion of all-D amino acids, as well as a variety of organic
molecules. Issues of specificity have been addressed and ligands that bind to only
one particular integrin have been isolated. Here we propose a series of the
α2β1integrin targeting peptide tracer designs that can target or identify the
up-regulated expression of integrin α2β1 which may initiate the attachment to
ECM components, signal events leading to proliferation, protease induction, cell
migration/invasion, or angiogenesis. We will verify the integrin specific uptake of
these peptide traces in three different prostate cancer cell lines (PC-3, CWR-22
and LNCaP) both in vitro and in vivo at the molecular level in preclinical prostate
tumor models.

We hypothesize that imaging of the integrin receptor system will likely provide a
better understanding of basic biological mechanisms of cancer, and to evaluate
disease course and therapeutic efficacy at the earliest stages of treatment

22

1.5.2 The Specific Aims

Specific Aim 1: To develop the first generation of α
2
β
1
integrin specific peptide
ligands.
Rationale and Research Design: The general chemical structure of α
2
β
1
integrin
targeted molecular probe could be divided into three parts: the integrin targeting
ligand, bifunctional linker, and the imaging moieties which could be the NIR dyes
or radionuclide component: bifunctional chelators (BFCs) for
64
Cu labeling (Fig.
1-1). The DGEA peptide will be employed as an antagonist of the α
2
β
1
integrin to
carry the desired targeting image probes to α
2
β
1
integrin receptors at the tumor
site. The linker could have profound influence on the in vivo targeting capabilities
of these bioconjugates by altering their size, hydrophilicity, steric hindrance,
biodistribution and pharmacokinetics. In this project, a series of DGEA containing
peptide tracers with various cell penetrating penetrating peptide sequence (CPPs)
linkers will be synthesized to test the binding affinity and specificity. These newly
developed DGEA probes will be tested in vitro and in vivo for their tumor targeting
efficacy and pharmacokinetics.

23

Figure 1-1 The prototype peptide structure which consisted of the DGEA α2β1 integrin
targeting sequence and linker was labeled with Imaging moiety (such as NIR dye or
radioisotope) for in vivo imaging verification.

Specific Aim 2: To establish a series of prostate cancer models and characterize
the α
2
β
1
tumor integrin expression levels.
Rationale and Research Design: Although the literature survey have
demonstrated that the more aggressive PC-3 cell line has the highest expression
of α
2
β
1
integrin followed by less aggressive cell lines CWR-22 and LNCap,
additional in vitro studies and in vivo xenograft models would be needed to further
confirm this correlation. We hypothesize that subcutaneous tumors will have the
same α
2
β
1
integrin expression profile as the corresponding cell lines. The in vivo
tumor models will be established and the actual α
2
β
1
integrin expression in these
models will be quantified using immunofluorescence.
24

Specific Aim 3: To evaluate the capabilities of these probes to visualize and
quantify α
2
β
1
integrin expression in vivo in subcutaneous prostate tumor models.
Rationale and Research Design: An ideal molecular probe will produce tumor
contrast and a reflection of the target protein expression level in vivo through
target specific activity accumulation/retention in the prostate tumor and rapid
clearance from non-targeted normal organs and tissues. In this aim, we expect to
test these DGEA derivatives for their metabolic stability, tumor targeting, and
pharmacokinetics. The best probe would then be further tested for its ability to
quantify α
2
β
1
integrin expression in vivo in different prostate cancer models (PC-3,
CWR-22, and LNCap). The tumor uptake and tumor/muscle ratio will be
correlated with α
2
β
1
integrin expression levels.

25

Chapter 2: Near-Infrared Fluorescent Imaging of Prostate
Cancer Using Integrin α
2
β
1
Targeted DGEA Peptides

2.1 Summary:
Objectives: Accumulating experimental evidence indicates that the up-regulation
or over-expression of α
2
β
1
integrin may correlate with tumor progression in human
prostate cancer. In this study, a novel class of imaging probe based on DGEA
peptides was designed for near-infrared-fluorescent (NIRF) imaging of α2β1
integrin expression in prostate cancers.
Methods: The peptides containing the DGEA for α2β1 integrin targeting were
prepared through Fmoc Solid Phase Peptide Synthesis (SPPS). After conjugation
with appropriate fluorescent dyes, these tracers were evaluated for NIFR imaging
of α2β1 integrin expression in three human prostate xenograft models (PC-3,
CWR-22 and LNCaP). In vitro experiments and immunofluorescence staining
were carried out to confirm the α2β1 integrin expression level in the tumor cells
and tumor tissue.
Results: Flow cytometric analysis on prostate cancer cells showed that the α2β1
integrin expression followed the order of PC-3>CWR-22 >LNCaP. PC-3 tumor cell
showed the highest tracer uptake and contrast followed by CWR-22 and LNCaP
tumor cells. In the subcutaneous PC-3 model, the tumor has prominent and
specific uptake with good tumor to background contrast. Immunofluorescence
26

staining also supported the in vivo optical imaging results.
Conclusions: The DGEA based optical agents have been developed for specific
imaging of the integrin α2β1 expression in individual tumors. In vitro and in vivo
localization demonstrate the potential of this agent to help us more appropriately
select patients considered for anti-integrin α2β1 treatment, and allow the
evaluation of disease course and therapeutic efficacy at the earliest stages of
treatment.

27

2.2 Introduction:
Prostate cancer is the most common malignancy diagnosed in men and the
metastatic prostate cancer forms represent the second cause of mortality in US
and European.(1) Despite the fact that prostate-specific antigen (PSA) screening
has greatly increased the number of patients with early stage prostate cancer,
about 40% of prostate cancers are first detected at an advanced stage and half of
these are found to be extracapsular at pathologic staging. (75-77) Therefore,
development of an accurate noninvasive imaging technique to detect primary,
recurrent and residual prostate cancer is critical for the effective management of
this group of patients.

In this report we chose integrin α2β1 as our potential targeting subject. It is
implicated that α2β1 integrin, a collagen I receptor, may facilitate migration and
metastatic spread of prostate cancer cells. It had been found that the α2β1
integrin binds to a site within the α1(I)-CB3 fragment of type I collagen (Staatz, W.
D.) (74) and the minimal active recognition sequence for integrin is a tetrapeptide
of the sequence Asp-Gly-Glu-Ala (DGEA) corresponding to residues 435-438 of
the type I collagen sequence. Although DGEA has been used for various
purposes by different investigators, there are no reports on the use of DGEA for in
vivo imaging of α2β1integrin expression to our best knowledge. In the present
study, we explored the possibility for specific in vivo imaging of α2β1 integrin

28

expression in xenotransplanted prostate cancer models in mice with the optical
system. We also performed in vitro and ex vivo experiments to confirm our in vivo
imaging results.

29

2.3 Materials and Methods:

General
All commercially available chemical reagents were used without further
purification. 5(6)-Carboxyfluorescein (FAM), 9-fluorenylmethoxycarbonyl (Fmoc)
amino acids, and Wang resin preloaded with the Fmoc-Ala amino acid were
purchased from Novabiochem (San Diego, CA). Aspartic acid and glutamic acid
were all protected as the tert-butyl ester. Cy5.5 monofunctional
N-hydroxysuccinimide (NHS) ester (Cy5.5-NHS) was purchased from Amersham
Biosciences (Piscataway, NJ). The purification of the crude product was carried
out on a analytical reverse-phase high-performance liquid chromatography
(HPLC) system equipped with a dual ulraviolet absorbance detector (Waters
2487,Waters, Milford, MA) using a Phenomenex (Torrance, CA) synergi 4µ
Hydro-RP 80Å (150 x 4.6 mm, 4 microns). The flow was 1 mL/min, with the mobile
phase starting from 98% solvent A (0.1% Trifluoroacetic acid (TFA) in water) and
2% solvent B (0.1% TFA in acetonitrile) (0–2 minutes) to 50% solvent A and 50%
solvent B at 40 minutes.

Synthesis of Peptides
All linear DGEA peptides were synthesized by a standard Fmoc solid-phase
peptide synthesis method with fourfold excess amounts of benzotriazole-1-yl-
oxy-tris-pyrrolidino-phosphonium-hexafluorophosphate(PyBOP),1-hydroxy-
benzotriazole (HOBt) and an eightfold molar excess of diisopropylethylamine
30

(DIPEA; Sigma, St. Louis, MO). Acylation was carried out for 60 minutes, and
complete reaction was confirmed by the trinitrobenzene sulfonic acid test [25].
Removal of the Fmoc protective group on the N-terminal group was achieved with
20% piperidine in dimethylformamide (DMF) (v/v) (Sigma). DMF was used to
wash the resin between each acylation and deprotection step.

FAM Conjugation
The N-terminal Fmoc groups were removed after coupling the last amino acid.
FAM was then coupled onto the exposed amino group at threefold excess in the
presence of equimolar amounts of PyBOP and a sixfold molar excess of DIPEA
for 2 hours in the dark. Following the acylation, unbound FAM was removed by
washing the resin with DMF, and then the conjugated peptide FAM-DGEA was
cleaved from the resin by treating with cleavage solution (95% TFA and 5% water)
for 3 hours. The desired conjugated peptides were purified and characterized by
analytical HPLC.

Conjugation and Purification of Cy5.5-DGEA Conjugates
The synthesis of Cy5.5-DGEA conjugates was achieved through conjugation of
Cy5.5-NHS ester with the N-terminal amino group of the aspartic acid residue of
the DGEA peptides. The Cy5.5-NHS (1 mg) dissolved in DMF (77 µL) was added
to the fully protected DGEA peptide, which was still on the resin, followed by
DIPEA (3.3 µL). The reaction mixture was stirred overnight in the dark at room
31

temperature. All conjugated peptides and side chain protecting groups were
simultaneously removed by treating with cleavage solution (95% TFA and 5%
water) for 3 hours. Peptide-containing supernatants were separated from the solid
support by filtration and concentrated under a stream of nitrogen. Crude peptide
was precipitated and washed twice with ice-cold diethylether and dissolved in
10% acetic acid in water before lyophilization. The desired products were purified
and characterized by analytical HPLC. The purity of Cy5.5-labeled peptides was
over 95% from analytical HPLC analysis. The retention times on analytical HPLC
for unlabeled and labeled DGEA peptides were 7 and 30 minutes, respectively.
Fractions containing Cy5.5-DGEA conjugates were collected, lyophilized, and
stored in the dark at -20˚C until use. The purified Cy5.5-DGEA conjugates were
characterized by LTQ Orbitrap (Thermo Scientific, West Palm Beach, FL) hybrid
mass spectrometry.

Cell Lines
The human prostate cancer cell line PC-3 was obtained from American Type
Culture Collection (Manassas, VA) and was maintained at 37 ˚C in a humidified
atmosphere containing 5% CO2 in F-12K medium and 10% fetal bovine serum
(Life Technologies, Inc., Grand Island, NY). CWR-22 and LNCaP cell lines were
also from American Type Culture Collection and were grown in RPMI-1640 with
10% fetal bovine serum in 5% CO2 at 37 ˚C.

32

Flow Cytometry Analysis of the Cell Binding
Human prostate PC-3, CWR-22, and LNCaP cancer cells were used to assess
the cell binding and internalization efficiencies of FAM-DGEA-targeting peptide
probe. To minimize the nonspecific uptake of peptides by pinocytosis,
incubations were performed on ice followed by flow cytometry analysis to allow
rapid quantization of fluorescence. For the quantification of fluorescence by flow
cytometry (FACScan, Becton, Dickinson, Franklin Lakes, NJ), 5,000 cells were
counted and viable cells with similar size and granularity in the forward and
sideways scatterplots were analyzed. The fluorescence profiles and the overall
mean fluorescence intensities of the cells within this region were obtained and
analyzed using CellQuest software (Becton, Dickinson).

Fluorescence Microscopy and Cell Uptake Studies of FAM-DGEA
For fluorescence microscopy studies, PC-3, CWR-22, and LNCaP prostate cells
(1x105) were cultured on BD Falcon four-chamber vessel culture slides (BD
Biosciences, Bedford, MA). After 24 hours, the cells were washed twice with
phosphate-buffered saline (PBS) and then incubated at 25 ˚C in the presence of 1
µM FAM-DGEA for 30 minutes. After the incubation period, cells were washed
three times with ice-cold PBS. For the blocking study, unconjugated DGEA
peptide 20 µM was added to the culture medium before the addition of
FAM-DGEA conjugates. Nuclear counterstain was performed with
4‘,6-diamidino-2-phenlindole (DAPI) in PC-3 cells. The fluorescence signals
33

from the cells were recorded using a fluorescent microscope (Carl Zeiss
Micro-Imaging, Thornwood, NY) equipped with a fluorescein isothiocyanate filter
set (exciter, HQ 475/20 nm; emitter, HQ 540/30 nm). An AttoArc HBO 100 W (Carl
Zeiss/AttoArc, Thornwood, NY) microscopic illuminator was used as a light source
for fluorescence excitation. Images were taken using a thermoelectrically cooled
charge-coupled device camera (Micromax, model RTE/CCD-576, Princeton
Instruments, Trenton, NJ).

Tumor Xenografts
Animal procedures were performed according to a protocol approved by the
University of Southern California Institutional Animal Care and Use Committee.
Male athymic nude mice (BALB/c nu/nu), obtained from Harlan (Indianapolis, IN)
at 4 to 6 weeks of age, were given injections subcutaneously in the right shoulder
with 1x106 of PC-3 human prostate cancer cells suspended in 100 mL of PBS.
When the tumors reached 0.4 to 0.6 cm in diameter (14–21 days after
implantation), the tumor-bearing mice were subject to in vivo imaging studies.

In Vivo Near-Infrared Optical Imaging of Tumors
In vivo fluorescence imaging was performed with an IVIS 200 small-animal
imaging system (Xenogen, Alameda,CA). A Cy5.5 filter set was used for
acquiring the Cy5.5-conjugated DGEA peptide probes‘ fluorescence in vivo.
Identical illumination settings (lamp voltage, filters, f/stop, field of views, binning)
34

were used for acquiring all images, and fluorescence emission was normalized to
photons per second per centimeter squared per steradian (p/s/cm2/sr). Images
were acquired and analyzed using Living Image 2.5 software (Xenogen). For the
control experiment, peptide probes were injected into three mice via tail veins with
1.5 nmol Cy5.5-DGEA and subjected to optical imaging at various time points
postinjection. For the blocking experiment, the mice (n = 3) for each probe were
also injected with a mixture of 10 mg/kg of unlabeled DGEA peptide and 1.5 nmol
Cy5.5-conjugated DGEA peptide. All near-infrared fluorescence images were
acquired using a 1-second exposure time (f/stop = 4). Mice of the experimental
and blocking groups were euthanized at 28 hours postinjection. The tumor and
major tissue and organs were dissected, and ex vivo fluorescence images were
obtained.

Immunohistochemistry
For immunohistochemical examinations, the collected xenograft PC-3 tumor
tissue samples were fixed in 4% freshly prepared buffered paraformaldehyde,
embedded in paraffin according to routine histologic procedures, and sectioned at
a thickness of 5 µm. Immunohistochemical analysis of paraffin-embedded
PC-3 prostate carcinoma was done using a2/CD49b monoclonal antibody (R&D
Systems, Minneapolis, MN). The antibody was applied by a two-step peroxidase
method using the DakoEnVision+HP Mouse Kit (Dakopatts, Glosstrup, Denmark).
Briefly, deparaffinized tissue sections were rinsed in 0.1 mol/L Tris-HCl buffer, pH
35

7.6, containing 0.15mol/L NaCl (Tris-buffered saline [TBS]). Endogenous
peroxidase was inactivated by incubating the sections with 1% (v/v) hydrogen
peroxide in TBS for 20 minutes. The tissue sections were then rinsed thoroughly
in TBS and incubated for a further 10 minutes with 1% bovine serum albumin
(BSA) in TBS at room temperature before incubation with the primary
antibody overnight at 4 ˚C . The monoclonal antibody CD49b was diluted
1:200 in TBS containing 1% BSA. As negative controls, duplicate sections were
incubated with 1% BSA instead of specific primary antibodies. The sections were
washed three times for 5 minutes each time in TBS followed by 30 minutes‘
incubation with one drop of peroxidase-conjugated rabbit antimouse secondary
antibody. After a washing step in TBS, peroxidase activity was visualized by
incubation sections in TBS containing 0.06% (w/v) 3, 3‘-diamino-benzidine
tetrahydrochloride (Sigma) and 0.034% (v/v) hydrogen peroxide for 8 minutes.

Data Processing and Statistics
All of the data are given as means ± SD of three independent measurements.
Statistical analysis was performed with a Student t-test. Statistical significance
was assigned for p values of .05. For determining tumor contrast, mean
fluorescence intensities and the tumor area at the right shoulder of the animal and
of the normal tissue at the surrounding tissue were calculated by the
region-of-interest function of Living Image software.

36

2.4 Results

Synthesis and Characterization of FAM-DGEA and Cy5.5-DGEA
The schematic molecule structures of FAM-DGEA and Cy5.5-DGEA conjugates
are shown in Fig. 2-1. The fluorophores are conjugated to the N-terminal amino
group of the aspartic acid residue and purified by analytic HPLC with ultraviolet
absorption at 500 nm (to purify FAM-DGEA conjugate) and 690 nm (to purify
Cy5.5-DGEA conjugate), respectively. The retention time on analytical HPLC
was 25 and 30 minutes, respectively. The mass analysis was performed by LTQ
Orbitrap Hybrid Mass Spectrometer: m/z=1,287.3 for Cy5.5-DGEA [M +H]
+

(calculated MW= 1,286.36 for C
55
H
61
N
6
O
22
S
4
); m/z = 750.2 for FAM-DGEA
[M + H]
+
(calculated MW= 748.65 for C
35
H
32
N
4
O
15
).

37

Figure 2-0-1: Schematic structure of FAM- and Cy 5.5-conjugated DGEA peptide probes

38

In Vitro FAM-DGEA Peptide Binding Specificity Studies
It has been demonstrated that the most aggressive PC-3 cells displayed the
highest surface expression of integrin α2β1 compared to two other less
aggressive prostate cancer cell lines (CWR-22 and LNCap). Fig. 2-2 shows
typical fluorescence histograms of FAM dye–conjugated DGEA peptide probes in
three prostate cancer cell lines. A comparison of the histograms resulting from
this binding experiment suggests that there is little or no nonspecific binding of the
LNCaP cell line (with low integrin α2β1 expression level) at the concentrations
used. Given that the histograms are of a relatively normal distribution, the mean
fluorescence intensity correlates well with the expression level of the integrin
α2β1in these cell lines. Although we cannot obtain an exact measure of the
number of probes binding to the cell from these data, fluorescent peak shifts in
these histogram profiles under a given set of conditions will reflect the binding
affinity differences in the corresponding mean fluorescence intensity. Correlations
between cell lines and individual experiments are dependent on maintaining the
same average surface area for the cells. Fortunately, we have found through the
observation of channel settings on the Coulter counter, forward and sideways
scatter on the flow cytometer, and visual observation that all of the cell lines used
here have very similar volumes and hence surface areas. From the flow cytometry
results, we can validate that the expression level detected with FAM-conjugated
peptide probe was consistent with previous reports determined by the antibody
labeling. (78-81) It was found that PC-3 cells incubated with the targeting DGEA
39

peptide displayed the highest mean fluorescence intensity followed by CWR-22
and LNCaP (see Figure 2-2).

Figure 2--2: Histograms of typical flow cytometry results. Each cell line was treated with a
FAM-DGEA probe as described in Materials and Methods. Fluorescence histograms
were produced and the results plotted. The binding percentages of each cell line are
99.7% (PC-3), 51.4% (CWR-22), and 15.6% (LNCaP), respectively.

40

In addition to the flow cytometry data, intracellular localization of the fluorescent
constructs was also examined using fluorescence microscopy. In Fig. 2-3, the
labeled peptide bound distinctly to PC-3 cells, whereas the CWR-22 revealed
substantially lower levels of cellular fluorescence signals and LNCaP cells
showed almost no specific binding of the probes. In addition, binding of the
FAM-DGEA could be blocked with the unlabeled DGEA peptide, which
demonstrated that the binding is specific. Like many integrin-targeted probes,
such as cyclic RGD peptide, internalization of the ligand after binding does occur,
but to a limited extent. (82,83) Radiolabeled RGD peptides also demonstrate
limited cell uptake (less than 1%).(84,85) However, it is also possible that the
fluorescent dye motif in the conjugates may facilitate ligand internalization after
binding owing to increased lipophilicity of the conjugate after attaching the
fluorescent tags.(86)

Allosteric cooperation of the targeting motif and fluorochrome may further
facilitate the internalization of the imaging probe, as we observed in this study.
However, the mechanism of internalization of these ligand conjugates after
receptor binding remains unclear and requires further investigation.

41

Figure 2-0-3
Figure 2-3. Binding of FAM-DGEA to prostate tumor cell lines with different levels of
integrin α2β1 expression. PC-3 cells (A), CWR-22 cells (E), and LNCaP cells (F) were
incubated with 1µM FAM-DGEA to assess the target selectivity of the probe. In
accordance with the FACS analysis data, PC-3 cells overexpress integrin α2β1 revealed
strong cellular fluorescence. (A.FAM-DGEA; B. DAPI; C. merge image), which could be
selectively blocked by co-incubation with 20µM unlabeled DGEA peptides (D).

42

In Vivo Fluorescence Imaging with Cy5.5-DGEA
Figure 4 shows typical NIRF images of athymic nude mice bearing a
subcutaneous human prostate PC-3 tumor after intravenous injection of 1.5 nmol
of Cy5.5-DGEA. The whole animal became fluorescent immediately after
injection, and the subcutaneous PC-3 tumor could be clearly delineated from the
surrounding background tissue from 30 minutes to 24 hours postinjection, with
maximum contrast occurring around 2 hours postinjection. Significantly, the
amount of fluorescence was still detectable in the tumor at 48 hours postinjection.
The fluorescence intensities defined as photons per second per centimeter
squared per steradian (p/s/cm2/sr) in the tumor and the normal tissues as a
function of time are depicted in Fig. 2-4. The tumor uptake reached a maximum at
2 hours postinjection and slowly washed out over time. On the other hand, normal
tissue had rapid uptake but overall lower uptake compared to tumor throughout
the time period studied. To validate the targeting specificity of the DGEA peptide
probe, we performed a blocking experiment. The control mice were each given
injections of 1.5 nmol of Cy5.5-DGEA, and those in the blocking experiment were
each given co-injections of 1.5 nmol of Cy5.5-DGEA and 10 mg/kg unlabeled
DGEA peptide (300 nmol). The typical NIRF images of PC-3 tumor–bearing
mice of both groups are shown in Fig. 2-5. The pseudocolored fluorescence
images were acquired 4 hours after intravenous injection. At this time point, the
contrast of tumor to normal tissue was maximal as the nonspecific binding had
washed out.
43

Figure 2-0-4
Figure 2-4. (Top), In vivo near-infrared imaging of subcutaneous PC-3 prostate tumor
bearing nude mice after intravenous injection of 1.5 nmol of Cy5.5-DGEA. The position of
the tumor is indicated by arrows. The fluorescence signal from probes is seudocolored
red. The tumor can be clearly visualized from 30 minutes to 24 hours postinjection.
(Bottom), The fluorescence intensity was recorded as per second per centimeter squared
per steradian (P/s/cm2/sr). Tumor fluorescence was higher than that in the normal tissue
(muscle) through 24 hours.

44

Unlabeled DGEA peptide successfully reduced tumor uptake compared to the
unblocked imaging result. Furthermore, ex vivo evaluation of excised organs at 28
hours postinjection (see Fig. 2- 5) showed that the compound was predominantly
taken up by the PC-3 tumor, which correlated well with our in vivo imaging results.
We also noticed that the tumor fluorescence intensity and contrast on this ex vivo
experiment were significantly higher than those obtained from in vivo imaging,
which could be attributed to the difference in tissue penetrations. The
immunohistochemistry staining results are shown in Fig. 2- 6. The xenograft PC-3
tumor tissue did have a strong positive staining in accordance with the in vivo and
ex vivo imaging results.

45

Figure 2-0-5
Figure 2-5. Left, Representative blocking experiment imaging (acquired 4 hours
postinjection) of mice bearing subcutaneous PC-3 tumor on the right shoulder
demonstrating blocking of Cy5.5-DGEA (1.5 nmol) uptake by coinjection with unlabeled
DGEA (10 mg/kg). Right top row, Representative images of dissected organs of mice
bearing PC-3 prostate tumor, A (experiment) and B (block), sacrificed 28 hours after
intravenous injection of Cy-5.5-DGEA. (1: muscle; 2 : heart; 3: tumor; 4 : liver; 5 : lung; 6 :
spleen; 7 : brain; 8 : pancreas; 9 : kidney). Right bottom row, Region of interest analysis
of fluorescence intensity ex vivo of major organs with (block) and without (experiment)
coinjection of a blocking dose of DGEA was plotted. Strong fluorescence signal could be
detected in tumor and kidney tissue.

46

Figure 2-0-6
Figure 2- 6. Expression of integrin α2β1 in PC-3 prostate tumor xenografts. Tissue
sections were preincubated without (negative control, left) or with the integrin antibody
CD49b directed against integrin α2β1 receptor (positive staining, right). Note the strong
integrin α2β1 expression in the PC-3 tumor tissue. (objective magnification x 20).

47

2.5 Discussion:

The current treatments for prostate cancer, consisting of malignant prostate
ablation by radical prostatectomy, radiotherapy, hormonal therapy, and/ or
neo-adjuvant chemotherapy, (87, 88) are generally curative for the majority of
patients diagnosed with localized and androgen-dependent prostate cancer forms.
However, progression to androgen-independent and metastatic disease states is
often accompanied by a recurrence of prostate cancer. (89-91) The available
chemotherapeutic treatment options for patients with hormone-refractory prostate
cancer are rather palliative and remain mostly ineffective, with a poor prognosis.
The prognosis is associated with a median survival rate of about 12 months after
diagnosis. (1) Therefore, development of an accurate noninvasive imaging
technique to detect primary, recurrent, and residual prostate cancer is critical for
the effective management of this group of patients. Given that the progression
from the androgen-dependent prostate cancer into more aggressive and
metastatic forms often leads to disease relapse, several novel therapeutic
strategies have been investigated for improving treatments against metastatic
prostate cancer.

The progression of prostate cancer primarily involves the formation of secondary
metastatic lesions to bone, a process partially mediated by integrin cell adhesion
proteins. Although molecular events responsible for the differences remain
largely unclear, a number of studies have described changes in integrin
48

expression in relation to the metastatic progression of prostate cancer. It has
been demonstrated that the more aggressive PC-3 cells display increased
surface expression of an integrin α2β1 compared to two other less aggressive
prostate cancer cell lines (CWR-22 and LNCap). Interactions of integrin α2β1 with
type I collagen have also been implicated in the formation of bone metastasis. All
of these results suggest that the overexpression of integrin α2β1 may facilitate
migration and metastatic spread of prostate cancer cells. The possible correlation
between integrin α2β1 expression level and tumor invasiveness makes integrin
α2β1 a potential biomarker for cancer progression diagnosis and treatment
monitoring.

In this study, we focused on the development of imaging agents that can probe
the integrin α2β1 cell expression profiles of individual tumors. Furthermore,
identification of a cell surface signature for a specific tumor type and/or stage may
potentially lead to customized treatment options for patients. After successful
demonstration of the specificity of our novel integrin α2β1 targeting peptide probe
in three different human prostate cancer cell lines, including PC-3 and CWR-22
(both are integrin α2β1positive) and LNCaP (integrin α2β1 negative) in vitro, we
further tested the PC-3 tumor model in vivo. Coupling of a NIRF dye to the DGEA
peptide allowed optical imaging of the PC-3 human prostate cancer cell line
model. The in vivo imaging demonstrated prominent uptake of the Cy5.5-DGEA
with the receptor specificity confirmed in a blocking experiment. The expression of
49

integrin α2β1 in PC-3 tumors was also confirmed with immunohistochemistry
antibody staining. In the ex vivo biodistribution semiquantitative data (see Figure
2-5), we found that the fluorescence intensity of the dissected tumor or tissue was
significantly higher than that measured by region of interest analysis of the
noninvasive images, which may be explained by the more effective fluorescence
detection of excised organs and tissues without attenuation of the excitation and
emission light in and out of the skin, as well as the scattering caused by the skin.
However, the residual tumor contrast in the blocking experiment was not
completely blocked by unconjugated DGEA peptide as only a 40% reduction was
observed. This may due to the nonspecific binding, accumulation in extracellular
space, or autofluorescence of the tissue itself. In addition, it has been reported
that these cyanine dyes have tumor-targeting capability even without any specific
targeting moiety conjugated; perhaps explaining why some fluorescence signals
can still be observed in the blocking experiment. We also observed similar results
for the optical imaging with dye-RGD conjugates. It should be noted that some
organs showed the signal reduction in the blocking experiment, such as liver, lung,
spleen, and muscle. This phenomenon is most likely due to the normal expression
of integrin α2β1 in these organs. In normal liver tissue, integrin α2β1 is expressed
in vascular endothelia, bile duct epithelium, connective tissue stroma, and
sinusoidal lining cells.(92) Integrin α2β1 is diffusely expressed around surface
airway epithelial cells.(93) Decreased expression of integrin α2β1 has been
reported in lung adenocarcinoma. (94)
50

Despite the success of this proof-of-principle optical imaging study in small-animal
models, there are some barriers to overcome before the eventual clinical
translation of this DGEA-based imaging agent for noninvasive imaging of tumor
integrin α2β1 expression and in development of integrin α2β1–targeted drugs.
The major drawback of optical imaging is the poor tissue penetration and intense
light scattering, which allows only for demarcation of superficial tumors and
tissues accessible by endoscopy, as well as intraoperative imaging. Another
concern is the use of different fluorochromes for in vitro and in vivo imaging
studies. The chemical property of each dye may have not only a different impact
on the binding affinity of the probe but also varying steric hindrance. Compared to
the small peptide structure, the size of the fluorochrome is relatively large, which
may also influence the binding affinity of the probe. To obtain quantitative tumor
targeting and distribution patterns of the DGEA-based probes, radionuclide
imaging modalities such as positron emission tomography and single-photon
emission computed tomography will be required for further quantitative studies.
Moreover, it is important to have high tumor to kidney ratios, as well as high
absolute tumor uptake and longer retention, for both imaging and therapeutic
applications. Based on our optical imaging results, this DGEA-based probe clears
rapidly from the body. Thus, further modification is needed to improve the
pharmacokinetics and binding affinity of these integrin α2β1–targeted probes.

Our future work will also focus on the structure-activity relationship to develop
51

various high binding affinity ligands for integrin α2β1, including the construction of
DGEA multimers to enhance binding affinity through the multivalency effect.

52

2.6 Conclusion:

It has been shown that the up-regulation or overexpression of integrin α2β1 may
correlate with tumor progression in human prostate cancer. Noninvasive imaging
of integrin α2β1 expression may therefore play a key role in detecting prostate
cancer progression, perhaps leading to treatment modification. The studies
described above demonstrate that it is feasible to detect and semiquantify tumor
integrin α2β1 status by noninvasive NIRF imaging with the DGEA-based optical
agents. Despite the limited penetration of light through tissue, this
proof-of-principle approach provides opportunities for rapid and cost-effective
preclinical evaluation in animal models before the more costly radionuclide-based
imaging techniques are applied.

53

Chapter 3: Cell Penetrating Peptide Sequence (CPPs)
Served as the potential Therapeutic Delivery Modality

3.1 Summary:
Objectives: The over-expression of integrin α2β1 has been shown to correlate
with prostate tumor aggressiveness and metastatic potential. A series of peptides
containing the peptide sequence DGEA and a cell penetrating peptide sequence
(CPPs) were synthesized for near-infrared fluorescent (NIRF) imaging of Integrin
α2β1 expression. In this study, the kidney uptake of these NIRF imaging probes
were successfully reduced using the peptide linker strategy.
Methods: After conjugation with appropriate fluorescent dyes, various
DGEA-CPP peptides were evaluated in different human prostate cell lines (PC-3,
CWR-22 and LNCaP), which contain different expression levels of α2β1 integrin.
In addition, a specific carboxypeptidase recognized short sequence Gly-Lys was
incorporated into the probe structure to reduce the excess kidney uptake. The
lysine specific carboxypeptidase activity of the kidney brush border enzymes can
cleave the peptide linker from the imaging probe prior to uptake by the proximal
tubule cells.
Results: Although the CPPs motif greatly facilitated the translocation of tracers
without affecting the binding specificity in vitro, fluorescent dye labeled
DGEA-CPP demonstrated extremely high kidney uptake in vivo. Using the
54

specific peptide linker strategy (Gly-Lys-), kidney uptake was dramatically
decreased. For the optimized probe, subcutaneous PC-3 xenograft showed a fast
and time-dependent activity accumulation in tumor tissue and lower signals in
most normal tissues. Tumor xenografts were clearly visualized by Xenogen
system up to 24 h post injection. Receptor specificity was confirmed by a blocking
experiment and evaluation in the CWR-22 negative control tumor model.
Conclusions: This study demonstrated that the use of cleavable peptide linker
between imaging probe fragments is a feasible approach to lower kidney uptake
while preserving good tumor uptake.

55

3.2 Introduction:
In previous studies, we have developed α2β1 integrin targeted optical tracers for
lesion detection and molecular profiling of different human prostate cancer cell
lines. We have obtained promising results in these preclinical tumor model studies.
The targeting imaging results of optical imaging modality do demonstrated
prominent and specific tumor uptake in vivo. However, the low binding affinity of
the integrins makes the tracers clears quickly through the renal pathway. In order
to enhance the binding affinity and retention time, an attractive solution is to
facilitate the targeting peptide to α2β1 integrin positive cells using cell-penetrating
peptides (CPPs) as a delivery moiety.
A common property shared amongst CPPs is the abundance of arginine residues
within their sequences and there is a general consensus that these cationic
residues are critical for membrane translocating abilities (95,96). This attribute of
arginine containing peptides has been exploited by a number of groups and it has
been shown that simple arginine oligomers of varying lengths can effectively
translocate across cell membranes of a variety of cell types facilitating transport of
peptide derivatives (95,97-102).
In this study, a series of CPPs conjugated α2β1 integrin targeted peptide probes
were designed for optical imaging applications in prostate cancer. We hypothesize
that these specific α2β1 integrin targeting peptide tracers can be used not only as
a useful diagnostic imaging agent, but also serve as an efficient drug delivery
carrier for prostate cancer therapy.
56

3.3 Materials and Methods:

General
All commercially available chemical reagents were used without further
purification. 5(6)-carboxyfluorescein(FAM),9-Fluorenylmethoxycarbonyl (Fmoc)
amino acids and Wang resin preloaded with the Fmoc-Ala amino acid were
purchased from Novabiochem (San Diego, CA). Aspartic acid and glutamic acid
were all protected as the tert-butyl (tBu) ester. Cy5.5 monofunctional
N-hydroxysuccinimide (NHS) ester (Cy5.5-NHS) was purchased from Amersham
Biosciences (Piscataway, NJ). The purification of the crude product was carried out
on a analytical reversed-phase high performance liquid chromatography (HPLC)
system equipped with a dual UV absorbance detector (Waters 2487) using a
Phenomenex synergi 4μ Hydro-RP 80Å (150 x 4.6 mm 4 micron). The flow was 1
mL/min, with the mobile phase starting from 98% solvent A (0.1% TFA in water)
and 2% solvent B (0.1% TFA in acetonitrile) (0-2 min) to 50% solvent A and 50%
solvent B at 40 min.

Synthesis of Peptides
All of the peptides used in this study were chemically synthesized by Fmoc
(9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis on a Wang resin.
Fmoc-Lys(Fmoc) was employed as a building block for branching poly-arginine
peptide structures. Four equivalents of Fmoc-amino acid derivatives,
benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium-hexafluorophosphate
57

(PyBOP), HOBt and a 8-fold molar excess of diisopropylethylamine (DIPEA;
Sigma) per amino function were used for each coupling. Acylation was carried out
for 60 min and complete reaction was confirmed by the trinitrobenezene sulfonic
acid test. Removal of the Fmoc protective group on the -amino group was
achieved with 20% piperidine in DMF (v/v) (Sigma). Dimethylformamide (DMF)
was used to wash the resin between each acylation and deprotection step.
Deprotection of the peptide and cleavage from the resin were conducted by
treatment with a trifluoroacetic acid/H
2
O mixture (95:5) at room temperature for 3
h.

Carboxyfluorescein (FAM) Conjugation
5(6)-Carboxyfluorescein (FAM) was coupled onto the exposed amino group at
3-fold excess in the presence of equimolar amounts of PyBOP and a 6 fold molar
excess of DIPEA for 2h in the dark. Following the acylation, unbound FAM was
removed by washing the resin with DMF, and then the conjugated peptide DGEA
was cleaved from the resin by treating with cleavage solution (95% TFA, and 5%
water) for 3 h. The desired conjugated peptides were purified and characterized
by analytical HPLC. The fidelity of the products was ascertained by LTQ Orbitrap
Hybrid Mass Spectrometry.

58

Conjugation and Purification of Cy5.5 Conjugates
The synthesis of Cy5.5 conjugates was achieved through conjugation of
Cy5.5-NHS ester with the exposed amino group of the DGEA peptide probes. The
Cy5.5-NHS dissolved in DMF was added to the fully protected DGEA peptide,
which was still on the resin, followed by DIPEA to do the conjugation reaction. The
reaction mixture was stirred overnight in the dark at room temperature. All
conjugated peptides and side chain protecting groups were simultaneously
removed by treating with cleavage solution (95% TFA, and 5% water) for 3 h.
Peptide-containing supernatants were separated from the solid support by
filtration and concentrated under a stream of nitrogen. Crude peptide was
precipitated and washed twice with ice-cold diethylether and dissolved in 10%
acetic acid in water before lyophilization. The desired products were purified and
characterized by analytical HPLC. The purity of Cy5.5 labeled peptides were over
95% from analytical HPLC analysis. Fractions containing Cy5.5 conjugates were
collected, lyophilized, and stored in the dark at -20 °C until use. The purified Cy5.5
labeled peptide probes were characterized by LTQ Orbitrap Hybrid Mass
Spectrometry.

Cell Lines
The human prostate cancer cell line PC-3 was obtained from American Type
Culture Collection (Manassas, VA) and was maintained at 37°C in a humidified
atmosphere containing 5% CO
2
in F-12K medium and 10% fetal bovine serum
59

(Life Technologies, Inc., Grand island, NY). CWR-22 and LNCaP cell lines were
also from American Type Culture Collection (Manassas, VA) and grown in
RPMI-1640 with 10% fetal bovine serum in 5% CO
2
at 37°C. The LNCaP and
CWR-22 human prostate cancer cell lines express androgen receptors and
prostate specific antigens are stimulated by dihydroxytestosterone. The PC-3
human prostate cancer cell line is initiated from a bone metastasis of a grade IV
prostatic adenocarcinoma and displays low testosterone-5-alpha reductase
activity.

Flow Cytometry Analysis of the Cell binding
Human prostate PC-3, CWR-22 and LNCaP cancer cells were used to assess cell
binding and internalization efficiencies of α2β1 targeting peptide probes. To
minimize the non-specific uptake of peptides by pinocytosis, incubations were
performed on ice followed by flow cytometric analysis to allow rapid quantization
of fluorescence. To acquire the quantification of fluorescence by flow cytometry
(FACScan, Becton Dickinson, USA), a total of 10,000

cells were counted and
viable cells with similar size and granularity in the forward and side-scatter plots
were analyzed. The fluorescence profiles and the overall mean fluorescence
intensities of the cells within this region were obtained and analyzed using
CellQuest Software (Becton Dickinson, USA).

60

Fluorescence Microscopy and Cell-Uptake Studies
For fluorescence microscopy studies, PC-3, CWR-22 and LNCaP prostate cells (1
x 10
5
) were cultured on BD Falcon 4 chamber vessel culture slides (Bedford, MA).
After 24 h, the cells were washed twice with PBS and then incubated at 25°C in
the presence of equimolar amount of fluorescenated peptide for 30 min. After the
incubation period, cells were washed 3 times with ice-cold PBS. For the blocking
study, excess of unconjugated DGEA peptide was added to the binding medium
before the addition of fluorescenated DGEA peptide conjugates. The fluorescence
signals from the cells was recorded using an Axioskop 40 (Carl Zeiss
Micro-Imaging, Inc., Thornwood, NY) equipped with a FITC filter set (exciter, HQ
475/20 nm; emitter, HQ 540/30 nm). An AttoArc HBO 100 W microscopic
illuminator was used as a light source for fluorescence excitation. Images were
taken using a thermoelectrically cooled charged-coupled device (CCD)
(Micromax,model RTE/CCD-576, Princeton Instruments, Inc., Trenton, NJ)

Confocal Microscopic Studies
The human prostate cancer cells, PC-3 and CWR-22, were obtained by washing
the confluent monolayer of cells with PBS before gently dislodging the adherent
cells by treating with 2 % trypsin. Cells (1x10
5
) were then incubated for 30 min at
25°C with equimolar amounts of fluorescenated peptides dissolved in 200 uL of
PBS. Samples were gently mixed every 15 min. Cells were washed twice with
cold PBS .Cells were examined by confocal microscopy using a Bio-Rad 1024
61

MRC instrument. Green fluorescence was induced at a wavelength of 494 nm
with a krypton/ argon laser and detected at 550 nm. The photomultiplier gain
and laser power levels used were set by adjusting their levels such that the
background fluorescence of cells incubated with PBS was not visible. These
settings were then used to obtain confocal sections of cells incubated with various
fluorescenated peptides.

Tumor Xenografts
Animal procedures were performed according to a protocol approved by the
University of Southern California Institutional Animal Care and Use Committee. In
the procedure, mice of 4-6 week old, 20-30 g, non-castrated male athymic mice
(BALB/c nu/nu) will be injected subcutaneously of PC-3 and CWR-22 human
prostate cancer cells (American Type Culture Collection, Manassas, VA) at a
concentration of 1 x 10
6
cells per 0.1 mL in the shoulder, and allowing enough
time for tumors to grow to at least 3 mm in diameter (14–21 days after implant).
Tumor volume is calculated using the formula S
2
x L/2 where S and L represent
the small and large diameters of the lesion. The mice will be fed with Teklad
Global 18% Protein Rodent Diet (Ralston Purina Co., St. Louis, MO) to reduce the
autofluorescent signal at least 11 days before taking any optical imaging, and
maintained on 12-h light, 12-h dark cycle. These tumor-bearing mice were subject
to in vivo imaging studies. The three-tailed test was used for statistical analysis of

62

the differences between two prostate tumor models for the uptake levels of the
peptide tracer.

In Vivo NIR Optical Imaging of Tumors
In vivo fluorescence imaging was performed with an IVIS 200 small animal
imaging system (Xenogen, Alameda, CA). A Cy5.5 filter set was used for
acquiring the Cy5.5-conjugated DGEA peptide probes‘ fluorescence in vivo.
Identical illumination settings (lamp voltage, filters, f/stop, field of views, binning)
were used for acquiring all images, and fluorescence emission was normalized to
photons per second per centimeter squared per steradian (p/s/cm
2
/sr). Images
were acquired and analyzed using Living Image 2.5 software (Xenogen, Alameda,
CA). For the control experiment, peptide probes were injected to 3 mice via tail
vein with 1.5 nmol Cy5.5 conjugated DGEA peptide probes and subjected to
optical imaging at various time points post injection (p.i.). For the blocking
experiment, the mice (n= 3) for each probe were also injected with a mixture of 10
mg/kg of unlabeled DGEA peptide and 1.5 nmol Cy5.5-conjugated DGEA peptide
probes. All NIRF images were acquired using 1 s exposure time (f/stop= 4). Mice
of the experimental and blocking groups were euthanized at 28 hours p.i. The
tumor and major tissue and organs were dissected, and ex vivo fluorescence
images were obtained. To determine the localization and distribution of the probe
in the tumor, tumor was dissected, placed in tissue holders, and then filled with
Tissue-Tec Optimal Cutting Temperature Compound (Sakura Finetek USA, Inc.,
63

Torrence, CA), and immediately frozen in dry ice. The fresh tumor tissue slides
(5µm) were obtained with a tissue slicer. Fluorescence microscopy imaging of
these tissue slides was taken.

Data Processing and Statistics
All of the data are given as means ± SD of 3 independent measurements.
Statistical analysis was performed with a Student‘s t test. Statistical significance
was assigned for P values 0.05. For determining tumor contrast, mean
fluorescence intensities and mean fluorescence intensities of the tumor (T) area
at the right shoulder of the animal and of the normal tissue (N) at the surrounding
tissue were calculated by the region-of-interest function of Living Image software
(Xenogen 4.0).

64

3.4 Results:
Assessing the Binding and Internalization of Cationic Peptide Constructs in
Human Prostate cancer cell lines
To acquire the quantification of fluorescence by flow cytometry (FACScan, Becton
Dickinson, USA), a total of 10,000 cells were counted and viable cells with similar
size and granularity in the forward and side-scatter plots were analysed. The
fluorescence profiles and the overall mean fluorescence intensities of the cells
within this region were obtained and analysed using CellQuest Software (Becton
Dickinson, USA).

From the flow cytometry results, the expression levels detected with two peptide
tracer (FAM-DGEA and R4-(FAM)-DGEA) were compared and which were
consistent with our previous studies. The fluorescence signals only apparent in
the α2β1 integrin positive cells (PC-3) that were incubated with fluorescent
peptide tracers. Besides, the mean cell fluorescence intensities of cells incubated
with R4-(FAM)-DGEA were almost twice as high than when FAM-DGEA was used
indicating that binding is more efficient when the branched structure of arginine
residues are used. Based on these flow cytometry data, the CPPs structure can
enhance the binding affinity of DGEA peptide probes without compromising the
binding specificity among these prostate cancer cell lines.

65

Figure 3-0-1
Figure 3-1. The prototype peptide structure which consisted of the DGEA α2β1 integrin
targeting sequence and branched poly arginine (R4) was labeled with FAM dye for in vitro
and Cy 5.5 for in vivo imaging experiments.

Dye

KKDGEA

K

K

R
R
R

R

66

Figure 3-0-2
Figure 3-2. Flow cytometry analysis of integrin α2β1 binding in prostate cancer cell lines
which were incubated with FAM-DGEA, R4-(FAM)–DGEA and nonsense FAM-AAAA
peptides and the mean fluorescence intensity of each prostate cell line was calculated.

67

The branched R4 structure is smaller in size than those previously described in
studies by Buschle et al. (103) who reported that poly-arginine chains of at least
15 residues were required to significantly enhance peptide uptake. In another
report by Mitchell et al.(104), cellular uptake was only apparent when polymers
containing six or more arginine residues were used which is consisted with the
FACS results from our FAM-DGEA-(R)n (n=4,6,8) peptide tracers (data not
showed). Only when the linear arginine number exceeds 6, then the dramatically
improvement uptake by α2β1 positive PC-3 cell line was observed. Mitchell et al.
(104) and others (95,100) have also demonstrated that it is the guanidine group
that is responsible for efficient binding of arginine-containing constructs.

Figure 3-0-3
Figure 3-3. Fluorescent microscopy images of prostate cancer cell lines (PC-3, CWR-22
and LNCaP) incubated at 25°C in the presence of equimolar amount of R4-FAM-DGEA
peptide for 30 min.

68

In addition to the flow cytometric data, the binding specificities of the poly arginine
R4 constructed fluorescent DGEA peptides were also examined using
fluorescence microscopy. From Fig.3-3 the labeled peptides bound distinctly to
PC-3 cells, while the CWR-22 and LNCaP revealed substantially lower levels of
cellular fluorescence signals. In addition, binding of the fluorescent DGEA probes
could be blocked with the unlabeled DGEA peptide, which further confirmed the
DGEA peptide binding is α2β1 integrin specific. In these microscopy images,
unexpectedly, the PC-3 cells incubated with R4-FAM-DGEA peptide seemed to
show strong signal accumulation from the nucleus site while in the other cell lines,
such as the CWR-22 and LNCaP cell lines the fluorescent signals were mostly
confined to the cytosol and cell membrane.

Intracellular localization of fluorescenated R4-DGEA construct was further
examined by using confocal microscopy. From Fig. 3-4, only PC-3 cells that were
incubated with R4-FAM-DGEA exhibited a strong fluorescent signal inside the
nucleus, but not CWR-22 cells. (Fig. 3-5 ); and the signals were mostly confined
to the cytosol and cell membrane when PC-3 cells incubated with
FAM-DGEA.(Fig. 3-4) Usually, the integrin targeted probes, such as cyclic RGD
peptides, the internalization of the ligand after binding does occur but to a limited
extent.(83) It is possible that the fluorescent dye motif in the conjugates may
facilitate ligand internalization after binding due to increased lipophilicity of the
conjugate after attaching the fluorescent tags.(86) Allosteric cooperation of the
69

targeting motif and CPPs structure may further facilitate the internalization of the
imaging probe. However, the mechanism of nucleus penetration after receptor
binding is not fully understood at this time, but may rely on α2β1 integrin
correlated delivery system to the nuclear membrane in this cell line. The further
more sophisticated biological investigations are required. The nucleus penetrating
property of this peptide tracer not only improves the α2β1 binding affinity but also
provides an opportunity for radiotherapy with isotopes.

Figure 3-0-4

Figure 3-4. The confocal imaging of PC-3 cells incubated with integrin α2β1 targeting
FAM-DGEA peptides and the cell penetrating R4-FAM-DGEA peptide.

70

Figure 3-0-5

Figure 3-5. The CWR-22 cells were incubated with classic nucleic acid stain DAPI
(Ex/Em=358/461nm) and the R4-FAM-DGEA peptides (Ex/Em= 492/518 nm), we can
see the signal from the peptide was more spread out instead of aggregating in the
nucleus area comparing with the PC-3 cells.

In vivo Fluorescence Imaging
In vivo fluorescence imaging of athymic nude mice bearing subcutaneous PC-3
and CWR-22 prostate cancer xenografts after intravenous injection of 1.5 nmol
R4-(Cy5.5)-DGEA peptide probe were shown in Fig.3-6 and the fluorescence
intensities had been quantified. The fluorescence intensities defined as photons
per second per centimeter squared per steradian (p/s/cm2/sr) in the tumors and
the normal tissues as a function of time are depicted. The tumor uptake reached a
maximum at 2 hours post-injection and slowly washed out over time. On the other
hand, normal tissue had rapid uptake but overall lower uptake compared to tumor
throughout the time period studied.
71

The whole animal became fluorescent immediately after injection, and the
subcutaneous PC-3 tumor could be clearly delineated from the surrounding
background tissue from 10 minutes to 24 hours p.i. which proves that the peptide
could be a very potent targeting agent.

But the kidney uptake remained very high during the whole
scanning period.(Fig. 3-6) Even though this is very common phenomena in
peptide probes, because the kidney usually is the dose limited organ. The higher
binding affinity of the R4 conjugated DGEA derivatives is offset by the
unacceptable high kidney uptake due to rapid filtration of the probes, reabsorption
by the kidney tubules in preclinical xenograft mice models. Thus, the potential
clinical application is limited, so the strategy is needed to further improve
metabolic stability as well as pharmacokinetic behavior to make the CPPs
conjugated DGEA peptide probe suitable for imaging application.

72

Figure 3-0-6

Figure 3-6. (Top) In vivo fluorescence imaging of athymic nude mice bearing
subcutaneous PC-3 or CWR-22 xenografts after intravenous injection of 1.5 nmol
R4-(Cy5.5)-DGEA. The location of the tumor was indicated by an arrow. (bottom) The
PC-3 tumor display higher peptide uptake than that of CWR-22 tumor from 10 min to 4 h
pi. However, the kidney contrasts in both tumor models are significantly higher than that
of tumors.
73

Reduce Kidney Uptake Peptide Sequence Design Concepts
Several possible approaches to the kidney uptake problem have been
investigated in several research groups. The first is the co-administration of L- or
D-lysine as originally described by Solling and Morgenson (105) and more
recently reviewed (106,107). This approach has been applied to human studies
but the results were minor compared to the animal studies, perhaps because the
amount of lysine administered was less on a per weight basis.

A second approach to the problem is the use of organ specific cleavable linkers
design. Arano and coworkers (106) have synthesized C-terminal Lys linkers for a
radioiodinated hippuric acid-Fab conjugate that lowered kidney uptake by up to
50%. In this study they demonstrated the critical importance of the α-carboxyl
group of the C-terminal lysine residue, presumably confirming the role of brush
border carboxypeptidase activity in the kidney. They have hypothesized that
cleavage of the antibody fragment from the radiolabel prior to uptake by the
proximal tubule cells allows excretion of the residualized radiolabel. When the
peptide linker-radiolabeled fragment was compared to fragment radioiodinated by
standard chemistry in a tumor bearing nude mouse model, no difference was
observed in tumor uptake, but kidney uptake was reduced by 25% at 3 h. Thus,
the peptide linker played a critical role in reducing kidney uptake while preserving
tumor uptake.

74

On the basis of Arano and co-workers approach, we synthesized a DGEA-GK
peptide as a new targeting probe structure which composed with N-terminal
amino group and a ε-amino group of C-terminal lysine that can be regioselectviely
conjugated to CPPs for cell penetration function or the Cy 5.5 dye for imaging
detection. (Fig.3-7) This approach takes advantage of the lysine specific
carboxypeptidase activity of the kidney brush border enzymes that cleave off the
peptide linker from the DGEA peptide probe prior to uptake by proximal tubule
cells.

75

Figure 3-0-7

Figure 3-7. The schematic structure of the potential probe design which consisted of the
brush border peptidase sensitive linker (Gly-Lys-OH), DGEA targeting sequence and the
relative coupling positions of image agents and CPPs. (N-terminal or ε-amino group of
C-terminal lysine)

76

To circumvent high kidney uptake problems, we first try to change the Cy 5.5 NIR
dye conjugation site from the N-terminal to the ε-amino group of C-terminal lysine
without changing the original branched R4-DGEA peptide structure. Hypothetically,
once the kidney carboxypeptidase recognized the Gly-Lys sequence, the Cy5.5
dye can be released from the major peptide probe structure. To investigate the
biodistribution behavior of the R4-DGEAGK(Cy5.5)-OH probe, the whole-body
imaging of the subcutaneous PC-3 and CWR-22 tumor xenogrfted mice was
performed with IVIS 200 system and monitoring the distribution at serial time
points.

Fig. 3-8 shows typical NIRF images of athymic nude mice bearing subcutaneous
PC-3 and CWR-22 tumor after intravenous (iv) injection of 1.5 nmol of
R4-DGEAGK (Cy5.5)-OH probe, respectively. During this in vivo optical imaging,
the subcutaneous PC-3 tumor could be clearly visualized from the surrounding
background tissue from early time points and the contrast was growing with time.
Quantification analysis of these images was performed using software Living
Image 3.2. The fluorescence intensities in the tumor and the kidney of the both
tumor models as a function of time are also depicted in Fig. 3-8.

The tumor uptake was growing with time and almost equates with the kidney
peptide uptake at 4h pi and slowly washed out over time. The differences in tumor
uptakes among two prostate cancer models confirm the binding specificity of the
77

probe and the difference still remained at 24 h time point. Kidneys were still clearly
visualized in two tumor models but fluorescent intensity was dramatically
decreased even though still higher than PC-3 tumor uptake, indicating the Gly-Lys
linker design is feasible strategy to reduce the kidney uptake of CPPs conjugates.

78

Figure 3-0-8

Figure 3-8. (Top) In vivo fluorescence imaging of athymic nude mice bearing
subcutaneous PC-3 or CWR-22 xenografts after intravenous injection of 1.5 nmol
R4-DGEAGK(Cy 5.5)-OH. (bottom) The specificity of the peptide uptake among these
two tumor models is not compromised. The PC-3 tumor display higher peptide uptake
and tumor/normal contrast than that of CWR-22 tumor and the kidney accumulation also
demonstrate less peptide uptake even though still little higher than that of tumors.
0.00E+00
2.00E+08
4.00E+08
6.00E+08
8.00E+08
1.00E+09
1.20E+09
1.40E+09
0 1 2 3 4
PC3 kidney PC3 tumor CWR 22 tumor
Branch R4-DGEAGK(Cy 5.5)-OH
79

To further characterized the linker design, we reversed the conjugation sites of R4
and Cy5.5 to prepare the Cy5.5-DGEAGK(R4)-OH peptide probe for the in vivo
imaging evaluation. After injection of the peptide probe, the fluorescent signals
cleared rapidly from the normal tissue, but the tumor uptake was also rapid and
high. The highest uptake in tumor was observed at 1 h p.i. whereas tumor wash
out rate was also fairly rapid over time. Tumor uptake was higher for PC-3
xenograft mice model than CWR-22 model at all-time points examined, but the
difference between the two models was marginal at 24 h p.i. The clearest
difference between the previous probe designs was the kidney uptake. The
kidney uptake reached maximal at 1h p.i., but was lower than the PC-3 tumor
uptake and remained relatively low at all examined time points.

Despite the improved lower kidney uptake, the tumor uptake and retention time
was also compromised by this probe structure design. We assumed that this may
be due to the bulky structure of the branched R4 structure which might interfere
the binding between the DGEA sequence and integrin α
2
β
1
.

80

Figure 3-0-9

Figure 3-9. (Top) In vivo fluorescence imaging of athymic nude mice bearing
subcutaneous PC-3 or CWR-22 xenografts after intravenous injection of 1.5 nmol
Cy5.5-DGEAGK(R4)-OH. (Bottom) The specificity of the peptide uptake among these
two tumor models is not compromised. The PC-3 tumor showed higher peptide uptake
than that of CWR-22 tumor and the kidney uptake was decreased dramatically due to the
linker strategy and the selection of conjugated position of the CPPs motif.
0.00E+00
2.00E+08
4.00E+08
6.00E+08
8.00E+08
1.00E+09
1.20E+09
0 1 2 3 4
PC3 kidney PC3 tumor CWR 22 tumor
Cy5.5 -DGEAGK(R4)-OH
81

The kidney uptake seems to be influenced by the relative conjugation position of
the CPPs construct and NIR Cy5.5 dye. Only when the CPPs structure installed
on the ε-amino group of C-terminal lysine residue can the optimal kidney
reduction was obtained. But in the distribution imaging study of
Cy5.5-DGEAGK(R4)-OH, the binding affinity also was decreased, probably due to
the steric hindrance of the R4 branch structure. To verify the hypothesis, a linear
R8 CPPs structure was used instead. (Fig. 3-10) The Cy5.5-DGEAGK(R8)-OH
peptide probe was synthesized for the further evaluation in vivo imaging of the
same established PC-3 and CWR-22 tumor models which were served as integrin
α
2
β
1
positive and negative control.

82

Figure 3-0-10

Figure 3-10. Schematic structure of Cy5.5-conjugated DGEAGK(R8)-OH peptide.

83

Cy5.5-DGEAGK(R8)-OH

Figure 3-0-11

Figure 3-11. (Top) In vivo fluorescence imaging of athymic nude mice bearing
subcutaneous PC-3 or CWR-22 xenografts after intravenous injection of 1.5 nmol
Cy5.5-DGEAGK(R8)-OH. (Bottom) The specificity of the peptide uptake among these
two tumor models is not compromised. The PC-3 tumor showed higher peptide uptake
than that of CWR-22 tumor and the kidney uptake was decreased dramatically due to the
linker strategy and the binding affinity is enhanced by choosing the linear R8 instead of
the branch R4 structure.
0.00E+00
2.00E+08
4.00E+08
6.00E+08
8.00E+08
1.00E+09
1.20E+09
1.40E+09
1.60E+09
1.80E+09
0 1 2 3 4
PC3
kidney
PC3
tumor
CWR 22
tumor
Tumor Uptake Vs. Renal Clearance
84

The localizations of Cy5.5-DGEAGK(R8)-OH in PC-3 and CWR-22 tumor-bearing
mice as determined by Xenogen optical imaging were shown in Fig. 3-11.
On the top panel was the representative imaging of PC-3 and CWR-22
tumor-bearing mouse, at different time points. (0.5h,1h,2h,4h,8h and 24h p.i ).
Tumor was clearly visualized at the early time points, indicating the excellent
targeting capability of the probe. Most importantly, the tumor/kidney ratio was
significantly higher than the previous probe designs. In addition to the kidney
reduction, the specificity of the peptide probe was maintained, because the
imaging of CWR-22 tumor model shown the much lower uptake compared with
the PC-3 tumor xenografted model.

To further validate the targeting specificity of the Cy5.5-DGEAGK(R8)-OH peptide
probe, a blocking experiment was performed. The control mice were each given
injections of 1.5 nmol of Cy5.5-DGEAGK(R8)-OH, and those in the blocking
experiment were co-injections of 10 mg/kg unlabeled DGEA peptide (300 nmol).
The typical NIR fluorescence images of PC-3-tumor-bearing mice of both groups
are shown in Fig.3-12. The pseudo-color fluorescence images were acquired 2
hours after intravenous injection. (Left, experiment; Right, block). At this time point
the contrast of tumor to normal tissue was maximal, as the non-specific binding
had washed out. Unlabeled DGEA peptide successfully reduced tumor uptake
compared with the unblocked imaging result.

85

Figure 3-0-12

Figure 3-12. Representative NIR images of mice bearing subcutaneous PC-3 tumor on
the right foreleg demonstrating blocking of Cy5.5-DGEAGK(R8)-OH conjugates (1.5 nmol)
uptake in the tumors by coinjection with 10mg/kg of unlabeled DGEA peptides.

Furthermore, ex vivo evaluation of excised organs at 4 hours p.i. (Fig.3-13.)
showed that the compound was predominantly taken up by the PC-3 tumor.
Regions of interest (ROIs) were placed on tumors as well as other organs of
interest to semi-quantify the imaging data. The fluorescence intensities defined as
photons per second per centimeter squared per steradian (p/s/cm2/sr) in the
tumor and the normal tissues are depicted. The ex vivo imaging was concord well
with the in vivo imaging results. The biodistribution profile was in good agreement
with that obtained from the in vivo imaging.

86

Figure 3-0-13
B

Figure 3-13. (A) Representative images of dissected organs of mice bearing PC-3 tumor
sacrificed 4h after intravenous injection of Cy5.5-DGEAGK(R8)-OH. (B) Biodistribution of
the probes at 4h postinjection.
To determine if the Cy5.5-DGEAGK(R8)-OH really can translocate into the tumor
87

cells after specific binding with integrin α2β1 in vivo. The fluorescence microscopy
imaging of fresh tissue slides was performed. (Fig. 3-14) The spatial distribution of
fluorescence for Cy5.5-DGEAGK(R8)-OH peptide (shown in green) in frozen
tissue slices 4 hours after injection of peptide demonstrating nicely that the
peptide really can accumulate inside the tumor cells but not just outside of the
vascular space in stroma and tumor cells. DAPI was used for nucleus staining,
and shown in blue.

88

Figure 3-0-14
Figure 3-14. Validation of the distribution of DGEAGK(R8) in PC-3 tumor. The spatial
distribution of the peptides in frozen tumor tissue slices (5µm) two hours after injection of
peptide was observed under fluorescent microscopy. (A:Cy5.5-DGEAGK(R8)-OH peptide
is shown in green. B: DAPI, C: merge). The images demonstrate that CPPs constructed
DGEA peptide really can accumulated inside the tumor cells.

89

3.5 Discussion:
The progression of prostate cancer primarily involves the formation of secondary
metastatic lesions to bone, a process partially mediated by integrin cell adhesion
proteins. Although molecular events responsible for the differences remain largely
unclear, a number of studies have described changes in integrin expression in
relation to the metastatic progression of prostate cancer. It has been
demonstrated that the more aggressive PC-3 cells display increased surface
expression of α2β1 integrin compared with two other less aggressive prostate
cancer cell lines (CWR-22 and LNCap). Interactions of α2β1 integrin with type-I
collagen have also been implicated in the formation of bone metastasis. All these
results suggest that the over-expression of α2β1 integrin may facilitate migration
and metastatic spread of prostate cancer cells. The possible correlation between
α2β1 integrin expression level and tumor invasiveness makes α2β1

integrin a
potential biomarker for cancer progression diagnosis and treatment monitoring. In
previous sections, we had discussed the synthesis and biological evaluation of Cy
5.5 conjugated DGEA peptide as a NIFR optical probe for integrin α2β1 targeting
imaging study using athymic nude mice bearing subcutaneous PC-3 human
prostate cancer xenografts. It was concluded that the DGEA based peptide is a
very promising targeting ligand that can be used to image integrin α2β1
expression in vivo. However, the quick renal clearance rate and short retention
time of integrins binding remain an imperfection for it to be clinically useful as an
imaging agent in prostate cancer.
90

Basic peptide-mediated protein delivery into living cells has been attracting great
attention in the last decades as a novel technology having potential both for basic
research in cellular biology and for therapeutic application. The peptides
corresponding to the human immunodeficiency virus type 1 Tat-(48-60) (108,109)
and Antennapedia-(43-58)(110) are among the most well-known peptides for
these purposes. By hybridization of these carrier peptides genetically or
chemically, efficient intracellular delivery of various oligopeptides and proteins in
vitro and in vivo was achieved successfully to modulate cellular events such as
cell cycles and apoptosis(111-115).

If the arginine-rich polymers are able to go across the membranes, this will offer
great versatility in the design of our integrin α2β1 targeting imaging agent. The
peptide probe can possess high binding efficiency and cell or organ specificity by
conjugation of the targeting motif DGEA sequence with a variety of CPPs
constructs, more sophisticated means of delivery may be realized.

With these hopes in mind, we synthesized several CPPs conjugated DGEA
peptides for the peptide probes characterization in vivo. We observed a similar
tendency in the prostate tumor targeting efficiency of peptides according to the
position and structure (linear or branched) of poly-arginine residues. The imaging
results validated that a certain cluster of arginine residues is important for
improvement of the targeting affinity. Whereas, the difference in the structure and
91

the conjugation position of the CPPs resulted in different spectra of biodistribution,
especially for the kidney accumulation.

In this study we had demonstrated and compared the tumor targeting capability
and in vivo kinetics of a serial of CPPs modified DGEA peptide probes in murine
subcutaneous PC-3 and CWR-22 human prostate cancer models. Among all the
CPPs conjugated peptides, Cy5.5-DGEAGGK(R8)-OH demonstrates the highest
tumor uptake and low kidney uptake. The linear DGEA peptide sequence was first
been found to fit the binding pocket of the integrin α2β1receptor. Due to its low
molecular mass and small size, the introduction of any labeling groups or
additional sequences is likely to result in a loss of the integrin binding affinity and
specificity. Adding CPPs construct and Cy5.5 dye to the distant N-terminal of the
R4-K(Cy5.5)-DGEA peptide is expected to have minimal impact of binding
characteristics of the DGEA peptide and thus allows high receptor-specific tumor
uptake which is confirmed in vitro flow cytometry and fluorescent microscopy
imaging studies. However, having a bulky and highly positive charged CPPs
sequence greatly influences the in vivo biodistribution behavior of the probe which
was demonstrated by the unacceptable high kidney uptake. The prolonged kidney
uptake of CPPs modified peptide probe is problematic for this probe, suggesting
limited potential clinical applications. It has been demonstrated that the high renal
uptake of some imaging agents is related to the overall molecular positive charge
of the probes, and the relatively low renal uptake of our previous reported Cy5.5
92

conjugated DGEA probe is probably due to the overall negative charge character
under physiological condition. In addition, persistent localization of the probe in
the kidney would also be attributed to the internalization of the probes into the
renal cells by specific receptor coordinated mechanism or just through lipophilic
interactions with cell membrane. Therefore, the optimization of more
pharmacokinetic suitable CPPs conjugated DGEA peptide probes, therefore, was
needed for creation of the novel integrin α2β1 targeted delivery carrier for
diagnosis and therapy application.

The follow up modifications adopting the peptide linker strategy greatly reduce the
kidney uptake were successfully applied to our α2β1 targeting peptide design with
that the Cy 5.5 be located at the N-terminus of the peptide and the poly arginine
cell penetration fragment attachment site on the ε-amino group of the lysine.
The kidney uptake was dramatically decreased and without affecting the peptide
uptake specificity of different prostate cancer xenogrft models. This study
demonstrates that the use of cleavable peptide linkers between the fluorescent
dye labeled DGEA tracer and poly arginine (CPPs) is a feasible approach to lower
kidney uptake while preserving clear tumor contrast. Although the kidney uptake
is significantly reduced by the use of an appropriate linker, the chemical linkage
between the linker and the poly arginine construct may also play a large role, and
if properly manipulated, has the potential to further reduce kidney uptake. The
ultimate goal of this work is to reduce maximal kidney uptake, a level shown to be
93

safe in the future studies with radiometal labeled peptide tracer. The incorporation
of additional strategies, such as administration of L- or D-lysine may further lower
kidney uptake, allowing the future routine use of radiometal labeled DGEAGK
(R8)-OH tracers in the clinical application.

94

3.6 Conclusion:

The success of this research could lead to a selective and highly effective
diagnostic (imaging) agent to evaluate prostate cancer stage, help us more
appropriately select patients considered for potential anti-integrin α2β1

based
treatment, and allow the evaluation of disease course and therapeutic efficacy at
the earliest stages of treatment. Moreover, the lead compound holds great
potential for peptide receptor based radionuclide therapy in near future.

95

Chapter 4: microPET Tracer Design and Biological
Evaluation

4.1 Summary:
Objectives:The ability of PET to aid in diagnosis and management of recurrent
and/or the disseminated metastatic prostate cancer may be enhanced by the
development of novel prognostic imaging probes. Accumulating experimental
evidence indicates that overexpression of integrin α2β1 may correlate with
progression in human prostate cancer. In this study, copper-64 labeled integrin
α2β1 targeted PET probes have been designed and evaluated for the imaging of
prostate cancer.
Methods: The bifunctional chelator (BFC) conjugated DGEA peptides were
developed to image integrin α2β1 expression with PET in a subcutaneous PC-3
xenograft model. The microPET images were reconstructed by a two dimensional
ordered subsets expectation maximum (2D-OSEM) algorithm. The average
radioactivity accumulation within a tumor or an organ was quantified from the
multiple regions of interest (ROI) volumes.
Results: The PET tracer demonstrated prominent tumor uptake in PC-3 xenograft
(integrin α
2
β
1
positive). The receptor specificity was confirmed in a blocking
experiment. Moreover, the low tracer uptake in CWR-22 tumor model (negative
control) further confirmed the receptor specificity.
96

Conclusion: The Sarcophagine conjugated DGEA peptide allows non-invasive
imaging of tumor associated α2β1 expression, which may be a useful PET probe
for evaluating the metastatic potential of prostate cancer.

97

4.2 Introduction:
High sensitivity positron emission tomography (PET) coupled with computed
tomography (CT) for anatomical evaluation has become a critical diagnostic
imaging tool in the identification of a diverse group of malignancies in clinical
practice. PET scanning with
18
F-fluorodeoxyglucose (FDG) is the most common
diagnostic imaging tool in the identification of a diverse group of malignancies.
Despite great interest in and success of FDG-PET scanning of many cancers,
FDG may not accumulate sufficiently in the primary and recurrent prostate tumor
in part due to the slow growing nature of the disease. Therefore, development of
an accurate noninvasive imaging agent to identify primary, recurrent and residual
prostate cancer is critical for the effective management of this group of patients.

In previous studies, we had made the synthesis and biological evaluation of Cy
5.5 conjugated DGEA peptide as a NIFR optical probe for integrin α2β1 targeted
imaging using athymic nude mice bearing subcutaneous PC-3 human prostate
cancer xenografts. It was concluded that the DGEA based peptide is a very
promising targeting ligand that can be used to image integrin α2β1 expression in
vivo. However, the lack of quantitative information and deep tissue penetration
capability remain as significant challenges for optical probes being considered for
clinically useful imaging agents.

98

In this study, we will assess the usefulness of the DGEA peptide as a PET
imaging agent for α2β1

integrin. We hypothesize that the invasive potential of
prostate cancers is correlated with their α2β1

integrin expression. Appropriately
labeled DGEA probes may bind to α2β1

integrin and therefore allow us visualize
and quantify α2β1

integrin levels in prostate cancer models using non-invasive
imaging techniques. To our best knowledge, this is the first in vivo microPET
imaging study to noninvasively quantify the integrin α2β1 expression with DGEA
based peptide tracer. If successful, the resulting probe would be useful in cancer
detection, patient stratification, and treatment monitoring based on an anti-integrin
mechanism.

99

4.3 Materials and Methods:

General
All commercially available chemical reagents were purchased from Aldrich (St.
Louis, MO) and used without further purification. (Fmoc) amino acids and Wang
resin preloaded with the Fmoc-Ala amino acid were purchased from
Novabiochem (San Diego, CA). Aspartic acid and glutamic acid were all protected
by the tert-butyl (tBu) ester protecting groups. DOTA (OBu-t)3
-NHS(1,4,7,10-tetraazacyclododecane-1-(N-hydroxysuccinimideacetate)-4,7,10-t
ris(tert-butyl acetate)) was purchased from Macrocyclics Inc. (Dallas,TX).Cy5.5
monofunctional N-hydroxysuccinimide (NHS) ester (Cy5.5-NHS) was purchased
from Amersham Biosciences (Piscataway, NJ).
64
CuCl
2
solution, produced
through the
64
Ni (p, n)
64
Cu nuclear reaction, was purchased from University of
Wisconsin at Madison.

HPLC Methods
The purification of the crude product was carried out on an analytical
reversed-phase high performance liquid chromatography (HPLC) system
equipped with a dual UV absorbance detector (Waters 2487, Milford, MA) using
a Phenomenex C18 RP column (250 x 4.6 mm 5 micron ) (Torrance, CA). The
flow was 1 mL/min, with the mobile phase starting from 98% solvent A (0.1% TFA
in water) and 2% solvent B (0.1% TFA in acetonitrile) (0-2 min), followed by a
gradient mobile phase to 40% solvent A and 60% solvent B at 32 min. The
100

radioactivity was detected by a model of Ludlum 2200 single-channel radiation
detector.

Synthesis of Peptides
All linear DGEA peptides were synthesized by a standard Fmoc solid phase
peptide synthesis method with 4-fold excess amounts of
benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluoro phosphate
(PyBOP), HOBt and a 8-fold molar excess of diisopropylethylamine (DIPEA).
Acylation was carried out for 60 min and complete reaction was confirmed by the
trinitrobenezene sulfonic acid test (TNBS) [17]. Removal of the Fmoc protective
group on the -amino group was achieved with 20% piperidine in DMF (v/v).
Dimethylformamide (DMF) was used to wash the resin between each acylation
and deprotection step.

Synthesis and Purification of Z-E(diamsar)-Ahx-DGEA Conjugates
The synthesis of Z-E(diamsar)-Ahx-DGEA conjugates was achieved through
conjugation of diamsar chelator with the exposed carboxylic group of N-terminal
protected glutamic acid at 3-fold excess in the presence of equimolar amounts of
PyBOP and a 6-fold molar excess of DIPEA for 3h at room temperature. Following
the acylation, unbound diamsar chelators were removed by washing the resin with
DMF, and then the conjugated peptide was cleaved from the resin. All desired
conjugated peptides were cleaved from the resin and side chain protecting groups
101

were simultaneously removed by treating with cleavage solution (95% TFA, and
5% water) for 3 h. Peptide-containing supernatants were separated from the solid
support by filtration and concentrated under a stream of nitrogen. Crude peptide
was precipitated and washed twice with ice-cold diethylether and dissolved in
10% acetic acid in water before lyophilization. The identities of peptides were
characterized by analytical HPLC and high resolution LTQ Orbitrap hybrid mass
spectrometry (Thermo Scientific, West Palm Beach, FL) with a nanospray
capillary inlet. The analytic data for the peptide is as follows:
Z-E(diamsar)-Ahx-DGEA, mass spectrometry (MS) (positive ion spray)
m/z[M+H]
+
=1064.5 (calculatedC
47
H
78
N
14
O
14
:1063.21).

Synthesis and Purification of DOTA-K(Cy5.5)-Ahx-DGEA Conjugates
The synthesis of DOTA-K(Cy5.5)-Ahx-DGEA conjugates were achieved through
the conjugation of DOTA-mono-NHS-tris(t-Bu) ester with the N-terminal amino
group of lysine residue of the NH
2
-K-Ahx-DGEA peptides. The DMF solution of
DOTA-mono-NHS-tris (t-Bu) ester (3 eq.) was added to the fully protected DGEA
peptide (1 eq.) that was still on the resin in the presence of DIPEA (10 eq.). The
reaction mixture was stirred overnight in dark at room temperature. The lysine
side chain protecting group 4-Methyltrityl (mtt) was removed with 1% TFA solution,
for the conjugation with Cy5.5-NHS ester. The Cy5.5-NHS (1 mg) dissolved in
DMF (100 μL) was added to the resin solution in the presence of DIPEA (10eq.)
and stirred overnight in the dark at room temperature. Eventually, all conjugated
102

peptides and side chain protecting groups were simultaneously removed by
treating with cleavage solution (95% TFA and 5% water) for 3 hours. Crude
peptide was precipitated and washed twice with ice-cold diethylether and
dissolved in 10% acetic acid in water before lyophilization. The desired products
were purified and characterized by analytical HPLC and mass spectrometry. The
analytic data for the peptides are as follows: DOTA-K(Cy5.5)-Ahx-DGEA, MS m/z
[M+H]
+
=1915.3 (calculated C
83
H
110
N
13
O
31
S
4
3-
: 1914.09).

Copper-64 Labeling
[
64
Cu]Acetate (
64
Cu (OAc)
2
) was prepared by adding 37-111 MBq of
64
CuCl
2
into
300 µL of 0.1 N ammonium acetate buffer (pH 5.5), which was then added to the
DGEA peptide solution (about 5-10µg in 100 µL of 0.1 N ammonium acetate
buffer). The reaction mixture was incubated at 40 °C for 1 h. The labeled peptides
were purified and analyzed by analytical radio-HPLC. The radiochemical yield
was > 90% and radiochemical purity (RCP) was >95% for both radiotracers,
which have the estimated specific activity of 0.29 ± 0.04 Ci/µmol. The radioactive
peak containing the desired product was collected and rotary-evaporated to
remove the solvent. The HPLC-purified radiotracers were then reconstituted in
phosphate-buffered saline to 1 mCi/mL and passed through a 0.22-mm Millipore
filter into a sterile multidose vial for in vitro and in vivo experiments.

103

Cell Lines
The human prostate cancer cell line PC-3 was obtained from American Type
Culture Collection (Manassas, VA) and was maintained at 37°C in a humidified
atmosphere containing 5% CO
2
in F-12K medium and 10% fetal bovine serum
(Life Technologies, Inc., Grand island, NY). The CWR-22 cell line, obtained from
American Type Culture Collection (Manassas, VA), was grown in RPMI-1640 with
10% fetal bovine serum in 5% CO
2
at 37°C.

In Vitro Stability Assay
The in vitro stability of
64
Cu-(Z-E(diamsar)-Ahx-DGEA) and
64
Cu-DOTA-K(Cy5.5)-
Ahx-DGEA in PBS solution was studied by measuring the radiochemical purity at
different time points at 37°C. Samples of the resulting solution were analyzed by
radio-HPLC at 0, 4, 12, and 24 h post-purification

Cell Uptake and Efflux Studies
The cell uptake and efflux studies were performed to quantify the internalization
level of peptides. Experiments were performed with triplicate wells. Uptake and
efflux of
64
Cu-(Z-E(diamsar)-Ahx-DGEA) and
64
Cu-DOTA-K(Cy5.5)-Ahx-DGEA
into PC-3 cells were examined according to the following protocol. In
the cell uptake experiment, PC-3 cells were seeded into 24-well plates at a
density of 7x10
4
cells per well for overnight incubation. Cells were rinsed 3 times
with phosphate buffered saline (PBS), followed by the addition of
104

64
Cu-(Z-E(diamsar)-Ahx-DGEA) and
64
Cu-DOTA-K(Cy5.5)-Ahx-DGEA to the
cultured wells in triplicate (~4 μCi/well). After incubation at 37°C for 15, 30, 60,
and 120 min, cells were rinsed 3 times with PBS and lysed with NaOH-sodium
dodecyl sulfate (SDS) (0.2 M NaOH, 1% SDS). The cell lysate was collected in
measurement tubes for counting. The cell uptake was normalized in terms of
added radioactivity. In the cell efflux experiment, PC-3 cells were seeded into
24-well plates at a density of 7x10
4
cells per well for overnight incubation. Cells
were rinsed 3 times with PBS and then
64
Cu-labeled peptide tracers were added
(~4 μCi/well). The cells were incubated at 37°C for 2 h, then washed with PBS,
and re-incubated with serum free medium. Then, the cells were washed at
different time points (0, 15, 30, 60 and 120 min post re-incubated with medium
(p.r.i.)) with PBS and lysed with NaOH-SDS (0.2 M NaOH, 1% SDS). The cell
lysate was collected in measurement tubes for counting. Efflux values at different
time points were calculated by subtracting retention from 0-min retention and
normalized by dividing the total counts at 0 min (p.r.i.).

Tumor Xenografts
Animal procedures were performed according to a protocol approved by the
University of Southern California Institutional Animal Care and Use Committee.
Male athymic nude mice (BALB/c nu/nu), obtained from Harlan (Indianapolis, IN)
at 4 to 6 weeks of age, were given injections subcutaneously in the right shoulder
with 1 X 10
6
of PC-3 or CWR-22 human prostate cancer cells suspended in 100 u
105

L of PBS. When the tumors reached 0.4 to 0.6 cm in diameter (14–21 days after
implant), the tumor-bearing mice were subject to in vivo microPET imaging
studies.

microPET imaging study
microPET imaging of the tumor-bearing mice was performed using a microPET
R4 rodent model scanner (Concorde Microsystems, Knoxville, TN). The PC-3
tumor-bearing mice (n = 3) were imaged in the prone position in the microPET
scanner. The tumor-bearing mice were injected with 250-300 µCi of
64
Cu-labeled
DGEA peptide via the tail vein, then anesthetized with 2% isoflurane and placed
near the center of the FOV, where the highest resolution and sensitivity are
obtained. Multiple static scans were obtained at 0.5, 1 and 2h p.i. The images
were reconstructed by a two-dimensional ordered subsets expectation maximum
(2D-OSEM) algorithm. After each microPET scan, the regions of interest (ROIs)
were drawn over the tumor and major organs on decay-corrected whole body
coronal images. The average radioactivity concentration within the tumor or an
organ was obtained from mean pixel values within the multiple ROI volume, which
were converted to µCi/g (or MBq/g) by using the calibration constant C. Assuming
a tissue density of 1 g/mL, the ROIs were converted to µCi/g (or MBq/g) and were
then divided by the total administered activity to obtain an imaging ROI-derived
percentage administered activity per gram of tissue (%ID/g).

106

In Vivo Near-Infrared Optical Imaging of Tumors
For the cross-evaluation of the tumor uptake of dual modality tracer
DOTA-K(Cy5.5)-Ahx-DGEA in microPET imaging. In vivo fluorescence imaging
was performed with an IVIS 200 small animal imaging system (Xenogen,
Alameda, CA). A Cy5.5 filter set was used for acquiring the Cy5.5-conjugated
DGEA peptide probes‘ fluorescence in vivo. Identical illumination settings (lamp
voltage, filters, f/stop, field of views, binning) were used for acquiring all images,
and fluorescence emission was normalized to photons per second per centimeter
squared per steradian (p/s/cm
2
/sr). Images were acquired and analyzed using
Living Image 2.5 software (Xenogen). Mice were given injections via tail vein with
1.5 nmol of DOTA-K(Cy5.5)-Ahx-DGEA and were anesthetized with 2% isoflurane
before subjected to optical imaging at various time points post-injection. All
near-infrared fluorescence images were acquired using a 1 second exposure time
(f/stop= 4).

Data Processing and Statistics
All of the data are given as means ± SD of 3 independent measurements.
Statistical analysis was performed with a Student‘s t test. Statistical significance
was assigned for P values < 0.05. For determining tumor contrast,
target-to-background (T/B) ratios were reported as an average plus the standard
variation based on results from three tumor-bearing mice at each time point.

107

4.4 Results:

Chemistry and Radiochemistry
The diamsar conjugated DGEA peptide and DOTA & Cy5.5 dual conjugated
DGEA peptide were analyzed by HPLC and mass spectroscopy to confirm the
identity of the products. The schematic molecular structures of these tracers were
shown in Fig. 4-1. The purity of conjugated peptides was over 95% from analytical
HPLC analysis. The retention times on analytical HPLC for
Z-E(diamsar)-Ahx-DGEA and DOTA-K(Cy5.5)-Ahx-DGEA were found to be 32
min and 26.5 min, respectively. Fractions containing desired DGEA conjugates
were collected, lyophilized, and stored in the dark at -20 °C until used. On the
analytical HPLC, no significant difference in retention time was observed between
64
Cu-labeled tracers and the unlabeled conjugates. For in vitro and in vivo studies,
the specific activity of the
64
Cu after labeling and purification was estimated to be
0.29 ± 0.04 Ci/µmol, with radiochemical purity greater than 98% as determined by
analytical radio-HPLC.

108

Figure 4-0-1
Figure 4-1. Schematic structure of the Z-E(diamsar)-Ahx-DGEA (top) and
DOTA-K(Cy5.5)-Ahx-DGEA (bottom) peptides.

109

In vitro Stability
In vitro stability of
64
Cu-(Z-E(diamsar)-Ahx-DGEA) and
64
Cu-DOTA-K(Cy5.5)-
Ahx-DGEA were determined in PBS using radio-HPLC after incubation at different
time intervals (0, 4, 12 and 24h).The stability results throughout the whole
experimental course are shown in Fig.4-2. The radiochemical purity of
64
Cu-DOTA-K(Cy5.5)-Ahx-DGEA in PBS is less than 20% after 24h of incubation.
On the other hand, over 98% of
64
Cu-(Z-E(diamsar)-Ahx-DGEA) still remained its
integrity after the incubation in PBS for 24 h. These in vitro stability studies
demonstrated that DOTA as a BFC is very unstable even in PBS solution at
physiological pH.
Figure 4-0-2

Figure 4-2. In vitro stability of
64
Cu-DOTA-K(Cy5.5)-Ahx-DGEA and
64
Cu-Z-E(diamsar)
-Ahx-DGEA in PBS (pH=7.4) at 37°C

110

Cell Uptake and Efflux Studies
Cell uptake studies were performed using human prostate cancer PC-3 (α2β1
positive) cells. The amount of internalized activity was found to be very low for
receptor-positive PC-3 cells. As shown in Fig. 4-3, the cell uptake reached the
plateau at 120min. The limited internalization of the DGEA tracers after binding
may be the main reason for the relatively low cell uptake (< 0.4%). In fact, we also
observed low cell uptake value for the well-established integrin αvβ3 and RGD
tracers (84,85).
64
Cu-DOTA-K(Cy5.5)-Ahx-DGEA demonstated a slightly higher
uptake in comparison with
64
Cu-(Z-E(diamsar)-Ahx-DGEA) and this probably due
to the higher hydrophobic characteristics of the Cy5.5 dye structure. Because
these integrin targeted tracers do not appear to be internalized into the cells, the
cell efflux reflected mainly dissociation of
64
Cu-(Z-E(diamsar)-Ahx-DGEA) and
64
Cu-DOTA-K(Cy5.5)-Ahx-DGEA from the tumor cells. After 1 h, the efflux of
tracers reached a plateau, indicating that the cells maintained similar activity after
1h.

111

Figure 4-0-3

Figure 4-3. Cell uptake assay (left) and cell efflux assay (right) of Z-E(diamsar)-
Ahx-DGEA and DOTA-K(Cy5.5)-Ahx-DGEA on PC-3 cells (n=3,mean±SD).

In vivo microPET imaging study
The microPET imaging study was performed on
64
Cu-(Z-E(diamsar)-Ahx-DGEA)
using athymic nude mice (n = 3) bearing integrin α2β1-positive PC-3 human
prostate cancer xenografts at multiple time points (30 min, 1 h and 2 h). Fig.4-4
showed the representative decay-corrected coronal images and the activity
accumulation quantification in several organs of the tumor-bearing mice after
administered with 250-300 µCi of
64
Cu-(Z-E(diamsar)-Ahx-DGEA).
112

Figure 4-0-4

Figure 4-4. (A) Decay-corrected whole-body coronal small-animal PET images of PC-3
tumor-bearing mice at 30 min, 1 h and 2 h after injection of approximately 9.16 MBq (250
µCi) of
64
Cu-(Z-E(diamsar)-Ahx-DGEA). Images shown are static scans of a single
mouse, which are representive of the 3 mice tested in each group. Tumors were indicated
by arrows. (B) The tumor, muscle, kidney, and liver uptake of
64
Cu-(Z-E(diamsar)-Ahx-
DGEA) at 30 min, 1 h and 2 h post injection (p.i.) in PC-3 tumor bearing mice (n = 3,
mean ± SD).

113

The tumor was clearly visualized as early as 30 min p.i. with high
tumor-to-background contrast. No significant radioactivity accumulation was
detected in the brain. The radioactivity accumulation in the heart and muscle was
also low. After normalization, the tumor uptake of
64
Cu-(Z-E(diamsar)-Ahx-DGEA)
is 2.28 ± 0.47, 0.94 ± 0.53, and 0.36 ± 0.55%ID/g at 30 min, 1 h and 2 h p.i.,
respectively. The tracer uptake was also calculated for kidney (2.8±0.18, 1.5±0.23
and 1.06±0.14%ID/g at 30,1 h and 2 h after injection), liver (1.25±0.18, 0.65±0.22
and 0.33±0.14%ID/g at 30 min,1 h and 2 h after injection) and muscle (0.38 ± 0.35,
0.24±0.28 and 0.16±0.22%ID/g at 30 min,1 h and 2 h after injection), respectively.

Receptor-specificity was confirmed by blocking experiments and the negative
control tumor CWR-22 (Fig.4-5). The tumor uptake was reduced to
0.9±0.31%ID/g at 30min p.i. in the blocking experiment. In the CWR-22 control
tumor, the uptake was 0.3±0.2 %ID/g which is around 8-fold lower compared with
the α2β1-positive PC-3 tumor. Activity distribution in major organs was
comparable in mice either bearing the receptor-positive or the negative control
tumor. Nonspecific tracer uptake in the contralateral negative control tumor was
low, and circulation elimination kinetics was comparable between two models.

114

Figure 4-0-5
Figure 4-5. (A) Decay-corrected whole-body coronal microPET images of PC-3
tumor-bearing mice, PC-3 model with a blocking dose of linear DGEA (10 mg/kg of
mouse body weight), and a CWR-22 control model at 30 min p.i. of
64
Cu-(Z-E(diamsar)-Ahx–DGEA. Tumors are indicated by arrows. (B) The uptake of
64
Cu-(Z-E(diamsar)-Ahx-DGEA) in PC-3 tumor with or without pre-injection of blocking
dose of DGEA peptides , and the negative control CWR-22 tumor model. ROIs are shown
as %ID/g± SD (n = 3).

115

4.5 Discussion:

In this study, novel
64
Cu-labeled DGEA peptide tracers were synthesized and
characterized to demonstrate that imaging integrin α2β1 expression with PET is
possible. The
64
Cu labeling can be carried out in high radiochemical purity and
good radiochemical yields. The overall synthesis is straightforward that can be
easily be carried out in short time with only one HPLC separation step.

However, radiometals require BFCs with different donor atoms and chelator
frameworks. The main goal in choosing a successful BFC is to minimize the in
vivo dissociation of radionuclide from the radiometal chelate in
radiopharmaceuticals. In our first approach, we synthesized a series of
DOTA-DGEA compounds that were labeled with Cu-64. However, these tracers
demonstrated predominant hepatobiliary clearance with unfavorable activity
accumulation in the liver and intestine. This unexpected distribution pattern is
significantly different from our DGEA based optical probes that demonstrated
mainly renal clearance. In order to verify whether the difference was caused by
the presence of a fluorescent dye, we designed a dual modality tracer
DOTA-K(Cy5.5)-Ahx-DGEA (Fig.4-1) to evaluate the targeting efficacy and
pharmacokinetic behavior of the DGEA peptide. Similar to our previously reported
optical probe, DOTA-K(Cy5.5)-Ahx-DGEA demonstrated prominent tumor uptake
and favorable in vivo distribution pattern in optical imaging (Fig.4-6).

116

Figure 4-0-6

Figure 4-6. Representative near-infrared images of athymic nude mice bearing
subcutaneous PC-3 tumor on the left foreleg after intravenous injection of 1.5 nmol of
64
Cu-DOTA-K(Cy5.5)-Ahx-DGEA. The location of the tumor was indicated by an arrow.

As shown in Fig.4-7, after injected with 1.5 nmol of DOTA-K(Cy5.5)-Ahx-DGEA
probe, the subcutaneous PC-3 tumor could be clearly visualized from the
surrounding background tissue at early time point and reached the highest
contrast at 2 h post-injection. After obtaining this encouraging result, microPET
imaging was then performed using the same dual modality tracer and the results
were compared with optical imaging. Unlike the optical imaging, we only observed
moderate tumor uptake but high activity concentration in liver and abdomen area
when it came to PET imaging as shown in Fig 4-7.These controversial results
suggested that other factors may cause the discrepancy between optical and PET
imaging.

117

Figure 4-0-7

Figure 4-7. Representative microPET images of PC-3 tumor-bearing mice at 2 h, 4 h and
16 h post injection of approximately 5.5 MBq (150 µCi) of
64
Cu
-DOTA-K(Cy5.5)-Ahx-DGEA. The PC-3 tumors are indicated with arrows.

In vivo stable attachment of
64
Cu
2+
to targeted biomolecules requires the use of
BFCs. The stability of the radio-copper complex in vivo is critical to achieving high
uptake of the copper radionuclide in the tissue or organ of interest while
minimizing the non-selective binding or incorporation into non-target organs or
tissues. It is known that the DOTA-
64
Cu
2+
complexes have limited stability in vivo
and the dissociation of
64
Cu
2+
from this BFC could lead to high retention in liver in
some cases. Based on these facts, we believe that the high liver uptake observed
in our case could be partially caused by the dissociation of the
64
Cu from the
DOTA-DGEA complex. Therefore, we performed the ex-vivo PET/optical imaging
studies to validate the assumption. The PC-3 tumor bearing mouse, after
intravenous injection of 250µCi of
64
Cu- DOTA-K(Cy5.5)-Ahx-DGEA with carrier,
was euthanized at 4 hours p.i. The tumor and major tissue and organs were
dissected, and ex vivo PET/fluorescence images were obtained. (Fig.4-8)
118

Figure 4-0-8

Figure 4-8. Representative ex vivo microPET (left) and fluorescence imaging (right) of
dissected major organs of a PC-3 tumor bearing nude mouse. Animal was sacrificed 4 h
after intravenous injection of 250µCi 64Cu-DOTA-K(Cy5.5)-Ahx-DGEA. Dramatic
biodistribution differences were observed. The predominant liver uptake in microPET
imaging indicated the labile chelation of DOTA. (A: kidney, B: heart, C: liver, D: Spleen, E:
lung, F: tumor)

As shown in Fig.4-8, the ex vivo PET/optical imaging clearly demonstrated the
dramatic discrepancy between these two imaging modalities. During optical
imaging, the tumor is well seen and the major excretion route is kidneys. Liver has
much lower uptake compared with tumor. These results correlate well with our
previous studies. In contrast, the liver demonstrated the highest uptake followed
by tumor and kidney during microPET imaging. The most plausible explanation for
the PBS stability study results and high liver uptake in microPET imaging is
dissociation of Cu from the DOTA chelator, although further confirmatory studies
are required.

119

It is expected that a more stable Cu-chelator complex might improve the
pharmacokinetic and pharmacodynamic value of the tracer. In our laboratory, a
new class of cage type hexaazamacrobicyclic sarcophagine (Sar) chelators and
derivatives have been investigated for improving the in vivo stability of the
64
Cu
complex. (116) Furthermore, the Sar chelator has been conjugated to the well
characterized cyclic RGDyK peptide for integrin αvβ3 imaging. (117,118)
64
Cu-Labeled Sar conjugated RGD peptides were shown to have higher stability
in vivo and much lower liver uptake. Therefore, in our sequent studies, a
64
Cu-labeled DGEA conjugate with diamsar chelator was synthesized for integrin
α2β1 imaging. microPET imaging studies demonstrated high tumor uptake, high
tumor-to-backgroud ratio, and rapid renal clearance for this diamsar-DGEA
complex. High tumor uptake and rapid renal clearance are important to ensure
high tumor/background ratio and to reduce radiation burden to non-target organs.
Nonetheless, whether the distribution difference could be attributed to the
chelating stability differences between these two tracers still requires further
investigation.

In this study, the tumor uptake specificity was confirmed by the blocking
experiments in the PC3 tumor and the negative control tumor model CWR-22.
microPET imaging showed that the activity distribution in organs was comparable
between mice bearing the receptor-positive tumor and the negative control
CWR-22 tumor. The activity accumulation in the receptor-positive PC-3 tumor was
120

approximately 8-fold higher than in control. In contrast, approximately 60%
reduction of tumor uptake was found in the blocking group.

In comparison with our previously reported NIR dye conjugated Cy5.5-DGEA
peptides,
64
Cu-(Z-E(diamsar)-Ahx-DGEA) clears more rapidly in the tumor than
Cy5.5-DGEA as observed in the optical system. Based on the microPET scan, the
64
Cu-(Z-E(diamsar)-Ahx-DGEA) was almost completely washed out through the
urinary pathway within 2 h, while the tumor is still clearly visible at 24 h p.i. by
optical imaging with our previous Cy5.5-DGEA peptide probe. The difference in
clearance rate could be caused by the circulation half-life in. We presume that the
relatively bulky structure of the fluorescent dye may affect the peptide resistance
for the enzymatic degradation, thus prolonging the circulation time. Furthermore,
It was been reported that NIR dyes may be able to facilitate the internalization of
some receptor specific peptides. For example, Lucie et al,(83) reported that more
than 10% of Cy5 conjugated Cyclo-RGD peptides can be internalized rapidly in 10
min, but radiolabeled Cu-cyclo-RGD peptides only demonstrated limited cell
uptake (less than 1%).(84,85) Nonetheless, further experiments are needed
before a conclusion is drawn.

The high background signal coming from the bladder and the rapid clearance of
peptide from tumor will limit further applications of this prototype tracer, especially
if an attempt to detect lesions in the primary lesion site is made. To further
121

improve the tumor targeting affinity, more potent cyclized peptide structures and
DGEA multimers are now under the investigation. It is expected that improved
targeting efficacy could lead to longer tumor retention time and better tumor to
background contrast. Moreover, the integrin α2β1 binding peptides could be
conjugated to nanoparticles and HSA protein to reduce the renal clearance. The
ultimate goal of this research is to obtain an optimized PET probe to image
integrin α2β1 expression level in prostate cancer. Despite the success of this
proof-of-principle PET imaging study in small-animal models, further
understanding of the in vivo pharmacokinetic behavior of the DGEA peptide is
needed prior to its clinical translation.

In summary, we have developed a prototype integrin α2β1 targeted tracer that
may serve as a candidate for imaging integrin α2β1-positive tumors. The possible
correlation between integrin α2β1 expression level and tumor invasiveness
makes this DGEA imaging probe potentially useful for detecting metastatic
potential and prognosis in prostate cancer. Correlations with other parameters
such as hormone sensitivity and other integrin expression levels also will be
considered in the future.

122

4.6 Conclusion:

The current study demonstrated that
64
Cu-labeled diamsar-DGEA peptide
64
Cu-(Z-E(diamsar)-Ahx-DGEA) could allow non-invasive imaging of tumor
associated α2β1 expression, which may be a useful PET probe for evaluating the
metastatic potential of prostate cancer. The research also indicates that chelator
selection could have significant effect on the in vivo distribution pattern of the final
imaging probes.

123

Chapter 5: Biological Stability Evaluation of the α2β1
Receptor Imaging Agents: Diamsar and DOTA
Conjugated DGEA Peptide

5.1 Summary:

Objectives: Robust chelating stability under biological conditions is critical for the
design of copper-based radiopharmaceuticals. In this study, the stabilities of
64
Cu-DOTA and diamsar (two bifunctional Cu-64 chelators (BFCs)) conjugated
DGEA peptides were evaluated.
Methods: The in vitro stabilities of
64
Cu-DOTA-DGEA,
64
Cu-DOTA-Ahx-DGEA
and
64
Cu-Z-E(diamsar)-Ahx-DGEA were evaluated in PBS. A carboxyl protected
DOTA-DGEA was also synthesized to study the potential inter- and intramolecular
interactions between DOTA and the carboxylate groups of DGEA peptide.
microPET imaging of
64
Cu-DOTA-DGEA and
64
Cu-Z-E(diamsar)-Ahx-DGEA were
performed in PC-3 prostate tumor model to further investigate the in vivo behavior
of the tracers.
Results: DOTA-DGEA, DOTA-Ahx-DGEA, Z-E(diamsar)-Ahx-DGEA, and
protected DOTA-DGEA peptides were readily obtained and their identities were
confirmed by MS. The
64
Cu
2+
labeling was performed with high radiochemical
yields (>98%) for all tracers after 1 h incubation. Stability experiments revealed
that
64
Cu-DOTA-DGEA had unexpectedly high
64
Cu
2+
dissociation when
124

incubated in PBS (>55% free
64
Cu
2+
was observed at 48 h time point). The
64
Cu
2+
dissociation was significantly reduced in the carboxyl protected
64
Cu-DOTA-DGEA complex but not in the
64
Cu-DOTA-Ahx-DGEA complex, which
suggests the presence of competitive binding for
64
Cu
2+
between DOTA and the
carboxyl groups of the DGEA peptide. In contrast, no significant
64
Cu
2+

dissociation was observed for
64
Cu-Z-E(diamsar)-Ahx-DGEA in PBS. For
microPET imaging, the PC-3 tumors were clearly visualized with both
64
Cu-DOTA-DGEA and
64
Cu-Z-E(diamsar)-Ahx-DGEA tracers. However,
64
Cu-DOTA-DGEA demonstrated 5 times higher liver uptake than
64
Cu-Z-E(diamsar)-Ahx-DGEA. This biodistribution variance could be attributed to
the chelating stability difference between these two tracers, which correlated well
with the PBS stability experiments.
Conclusions: In summary, the in vitro and in vivo evaluations of
64
Cu-Z-E
(diamsar)-Ahx-DGEA and
64
Cu-DOTA-DGEA have demonstrated the significantly
superior Cu-chelation stability for the diamsar derivative compared with the
established DOTA chelator. The results also suggest that diamsar may be
preferred for Cu chelation especially when multiple carboxylic acid groups are
present. Free Carboxyl groups may naturally compete with DOTA for
64
Cu
2+

binding and therefore reduce the complex stability.

125

5.2 Introduction:

Positron emission tomography (PET) is a non-invasive functional imaging
technique with good resolution, high sensitivity, and accurate quantification. An
important advantage of PET is that it provides quantitative information of
physiological, biochemical and pharmacological processes in living subjects at
high sensitivity. Recently, the development of PET imaging agents based on
transition metal radionuclides is gaining considerable attention due to their
increased production at low cost and availability with high specific activity. For
peptide and antibody based imaging, it is highly desirable that the complex of
radiometal isotopes is stable in vivo in order to minimize the radio-metal release,
thus reducing the background signals and minimizing radiation exposure to
normal tissues. Among all the metallic radionuclides,
64
Cu has been one of the
most promising ones. The decay characteristics of
64
Cu (t
1/2
=12.7 h; 38% β
−
,
E
β-max
=573 keV; 19% β
+
, E
β+ max
=656 keV) make it an attractive radionuclide for
both PET imaging and radiotherapy. (117,119,120)

Stable attachment of
64
Cu
2+
to a targeting molecule usually requires the use of a
bifunctional chelator (BFC). One of the most commonly used chelators is the
macrocyclic ligand 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''tetraacetic acid
(DOTA). Although most DOTA-Cu complexes demonstrate good stability in vitro,
significant loss of
64
Cu
2+
from the DOTA conjugates in vivo can lead to high uptake
in the liver, impairing the use of this chelator in preparing
64
Cu-
126

radiopharmaceuticals. It has been reported that Sarcophagine (Sar) based
ligands with multiple macrocyclic rings comprising the cage structure can rapidly
coordinate
64
Cu
2+
under mild conditions, providing high stability in vivo.(121-123)
In our laboratory, the Sar chelator has been conjugated to the well characterized
cyclic RGDyK peptide and shown to be superior to DOTA in terms of in vivo
stability. (116-118)

In the current study, we synthesized
64
Cu-DOTA and
64
Cu-diamsar
(1,8-diamino-3,6,10,13,16,19-hexaazabicyclo(6,6,6)eicosane) conjugated DGEA
peptides, and evaluated their stability in vitro and in vivo. A carboxylic protected
DOTA-D(tBu)GE(tBu)A peptide was also synthesized to study the unexpected
high level of dissociation of
64
Cu
2+
from
64
Cu-DOTA-DGEA in PBS solution.

127

5.3 Materials and Methods:

General
All commercially available chemical reagents were purchased from Aldrich (St.
Louis, MO) and used without further purification. The Wang resin (or Tritry resin)
preloaded with the Fmoc-Ala and all the Fmoc conjugated amino acids,
benzyloxycarbonyl protected glutamic acid (Z-E-OH), and short linker
aminohexanoic acid (Ahx) were all purchased from Novabiochem (San Diego,CA).
Aspartic acid and glutamic acid were all protected by the tert-butyl (tBu) ester
protecting groups. DOTA(OBu-t)3-NHS(1,4,7,10-tetraazacyclododecane-1-
(N-hydroxysuccinimideacetate)-4,7,10-tris(tert-butyl acetate)) was purchased
from Macrocyclics Inc. (Dallas,TX). The chelator diamsar was prepared following
a method described in our published study. (116)
. 64
Cu
2+
was purchased from
Washington University School of Medicine, where using a CS-15 biomedical
cyclotron by the
64
Ni(p,n)
64
Cu nuclear reaction. Water was purified using a Milli-Q
ultrapure water system from Millipore (Milford, MA, USA), followed by passing
through a Chelex 100 resin before bioconjugation and radiolabeling.

HPLC Methods
The purification of the crude product was carried out on a analytical
reversed-phase high performance liquid chromatography (HPLC) system
equipped with Water 2487, a dual UV absorbance detector (Waters, Milford, MA)
using a phenomenex C18 RP (250 x 4.6 mm 5 micron ). The flow was 1 mL/min,
128

with the mobile phase starting from 98% solvent A (0.1% TFA in water) and 2%
solvent B (0.1% TFA in acetonitrile) (0-2 min), followed by a gradient mobile
phase to 40% solvent A and 60% solvent B at 32 min. The radioactivity was
detected by a model of Ludlum 2200 single-channel radiation detector.

Synthesis of Peptides
Peptides were synthesized on Wang resin or 2-chlorotrityl resin preloaded with
Fmoc-Ala by a standard Fmoc solid phase peptide synthesis method with 4-fold
excess amounts of benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate (PyBOP) and 1-hydroxybenzotriazole (HOBt) and a 8-fold
molar excess of diisopropylethylamine (DIPEA; Sigma). Acylation was carried out
for 60 min and reaction completion was confirmed by the trinitrobenezene sulfonic
acid test (TNBS) (25). Removal of the Fmoc protective group on the -amino
group was achieved with 20% piperidine in dimethylformamide (DMF) (v/v)
(Sigma). DMF was used to wash the resin between each acylation and
deprotection step.

Synthesis of DOTA-DGEA,

DOTA-Ahx-DGEA and Z-E(diamsar)-Ahx-DGEA
The synthesis of DOTA-DGEA and DOTA-Ahx-DGEA conjugates were achieved
through the conjugation of DOTA-mono-NHS-tris (t-Bu) ester with the N-terminal
amino group of aspartic acid residue of the DGEA peptides or the aminohexanoic
acid of Ahx-DGEA peptides. The DOTA-mono-NHS-tris (t-Bu) ester (3 eq.)
129

dissolved in DMF was added to the fully protected DGEA peptide (1 eq.), which
was still on the resin, followed by DIPEA (10 eq.). The reaction mixture was stirred
overnight in dark at room temperature.

Diamsar was coupled onto the exposed carboxy group of glutamic acid at 3-fold
excess in the presence of equimolar amounts of PyBOP and 6-fold excess of
DIPEA for 3 h at room temperature. Following the acylation, unbound diamsar
chelators were removed by washing the resin with DMF, and then the cleavage of
the conjugated peptide from the resin was performed.

All desired conjugated peptides were cleaved from the resin and side chain
protecting groups were simultaneously removed by treating with cleavage solution
(95% TFA, and 5% water) for 3 h. Peptide-containing supernatants were
separated from the solid support by filtration and concentrated under a stream of
nitrogen. Crude peptide was precipitated out and washed twice with ice-cold
diethylether, which was then dissolved in 10% acetic acid in water before
lyophilization. The desired products were purified and characterized by HPLC.
The purity of DOTA and diamsar conjugated peptides were greater than 95%,
based on analytical HPLC result. Fractions containing DOTA-DGEA,
DOTA-Ahx-DGEA and Z-E(diamsar)-Ahx-DGEA conjugates were collected,
lyophilized, and stored in dark at -20 °C until used. The purified DGEA conjugates
were characterized by LTQ Orbitrap Hybrid Mass Spectrometer.
130

Synthesis of DOTA-D(tBu)GE(tBu)A
DOTA-D(tBu)GE(tBu)A was synthesized through a similar procedure as that of
DOTA-DGEA except 2-chlorotrityl resin and DOTA-mono-NHS ester were used
instead. Briefly, the orthogonally protected linear peptide
[H-Asp(tBu)-Gly-Glu(tBu)-Ala-OH] was prepared from a Fmoc-Ala-2-chlorotrityl
resin, followed by DOTA–mono-NHS ester conjugation with the N-terminal of the
partially protected DGEA at 3-fold excess. The product was then cleaved from the
resin under mild condition with 1% TFA in dichloromethane.

64
Cu Labeling
DOTA-DGEA, DOTA-Ahx-DGEA and DOTA-D(tBu)GE(tBu)A were radiolabeled
with
64
Cu
2+
according to our previously reported method.(116,117) Briefly,
64
CuCl
2
was added to DOTA conjugated peptides in 0.1 M sodium acetate (pH 5.5)
and incubated at 40°C for 1 h. The radiochemical purity of the final products was
>95% as confirmed by radio-HPLC. Diamsar-conjugated DGEA peptide was
labeled with
64
Cu
2+
by using the literature reported condition (116). Radio-HPLC
assessment showed that the radiochemical purity of
64
Cu-Z-E(diamsar)-Ahx-DGEA was > 98%. The radioactive peak containing the
desired product was collected and rotary-evaporated to remove the solvent. The
HPLC-purified products were then reconstituted in phosphate-buffered saline to 1
mCi/mL and passed through a 0.22-mm Millipore filter into a sterile multidose vial
for in vitro and in vivo experiments.
131

Cell Lines
The human prostate cancer cell line PC-3 was obtained from American Type
Culture Collection (Manassas, VA) and was maintained at 37°C in a humidified
atmosphere containing 5% CO
2
in F-12K medium and 10% fetal bovine serum
(Life Technologies, Inc., Grand island, NY).

Tumor Xenografts
Animal procedures were performed according to a protocol approved by the
University of Southern California Institutional Animal Care and Use Committee.
Male athymic nude mice (BALB/c nu/nu), obtained from Harlan (Indianapolis, IN)
at 4 to 6 weeks of age, were given injections subcutaneously in the right shoulder
with 1 × 10
6
of PC-3 human prostate cancer cells suspended in 100 µL of PBS.
When the tumors reached 0.4 to 0.6 cm in diameter (14–21 days after implant),
the tumor-bearing mice were subjected to in vivo microPET imaging studies.

Solution Stability
The solution stability of
64
Cu-DOTA-DGEA,
64
Cu-DOTA-Ahx-DGEA,
64
Cu-DOTA-D(tBu)GE(tBu)A and
64
Cu-Z-E(diamsar)-Ahx-DGEA were studied by
measuring the radiochemical purity with HPLC after PBS incubation at 40°C. The
64
Cu-radiotracers were first purified by HPLC. The radioactive peaks containing
the desired products were collected and rotary-evaporated to remove the solvent.
The HPLC-purified products were then reconstituted in phosphate-buffered saline
132

to 1 mCi/mL. Samples of the resulting solution were analyzed by radio-HPLC at 0,
4, 12, 24 and 48 h post-purification.

MicroPET Imaging Study
microPET imaging of the tumor-bearing mice were performed using a microPET
R4 rodent model scanner (Concorde Microsystems, Knoxville, TN). The PC-3
tumor-bearing mice (n = 3) were imaged in the prone position in the microPET
scanner. The tumor-bearing mice were injected with 250-300 µCi of
64
Cu-labeled
DGEA peptide via tail vein. For imaging, the mice were anesthetized with 2%
isoflurane and placed near the center of the FOV, where the highest resolution
and sensitivity are obtained. Multiple static scans were obtained at 0.5 h p.i. The
images were reconstructed by a two-dimensional ordered subsets expectation
maximum (2D-OSEM) algorithm. For each microPET scan, the regions of interest
(ROIs) were drawn over the tumor and major organs on decay-corrected whole
body coronal images. The average radioactivity concentration within the tumor or
an organ was obtained from mean pixel values within the multiple ROI volume,
which were converted to counts/mL/min by using the calibration constant C.
Assuming a tissue density of 1 g/mL, the ROIs were converted to counts/g/min
and were then divided by the total administered activity to obtain an imaging
ROI-derived percentage administered activity per gram of tissue (%ID/g).

133

Data Processing and Statistics
All of the data are given as means ± SD of 3 independent measurements.
Statistical analysis was performed with a Student‘s t test. Statistical significance
was assigned for P values less than 0.05.

134

5.4 Results:

Nonradioactive Peptides
The compounds were prepared as described above and obtained in high purity.
The structures of DOTA-DGEA, DOTA-D(tBu)G-E(tBu)A, DOTA-Ahx-DGEA and
Z-E(diamsar)-Ahx-DGEA are shown in Fig. 5-1. The analytic data for the peptides
are as follows: DOTA-DGEA, HPLC retention time (R
t
) = 13.8 minutes, mass
spectrometry (MS) (positive ion spray)m/z[M+H]
+
= 778 (calculated C
30
H
48
N
8
O
16
:
776.75); DOTA-D(tBu)GE(tBu)A, R
t
= 23.2 minutes, MS m/z [M + H]
+
=889.2
(calculated C
38
H
64
N
8
O
16
: 888.96); DOTA-Ahx-DGEA, R
t
= 16.05 minutes, MS m/z
[M + H]
+
= 890.6 (calculated C
36
H
59
N
9
O
17
: 889.9); Z-E(diamsar)-Ahx-DGEA, R
t
=
19.5 minutes; MS m/z [M + H]
+
= 1064 (calculated C
47
H
78
N
14
O
14
: 1063.21).

Radiochemistry
The
64
Cu
2+
labeled tracers were prepared as described before. The overall
synthesis time is approximately 2 hours. On the analytic HPLC, no significant
difference in retention time was observed between
64
Cu-labeled tracers and the
unlabeled DOTA and diamsar conjugates. The radiochemical yield was
determined by radio-HPLC. The radiochemical yield for all the tracers was > 98%,
with specific activity of 0.29 ± 0.04 Ci/µmol. The radiochemical purity (RCP) was
>98% as determined by HPLC.
135

Figure 5-0-1

Figure 5-1. Schematic structure of DOTA-DGEA, DOTA-D(tBu)GE(tBu)A,
DOTA-Ahx-DGEA and Z-E(diamsar)-Ahx-DGEA

136

Solution Stability
The solution stability experiments were first performed for
64
Cu-DOTA-DGEA, and
64
Cu-Z-E(diamsar)-Ahx-DGEA (Fig.5-2). The purpose of these studies was to
demonstrate that the
64
Cu radiotracer remains intact before being injected into the
tumor-bearing mice. It is clear that diamsar conjugated radiotracers remained
stable for >48 h. On the other hand,
64
Cu-DOTA-DGEA was extremely unstable in
PBS, which is unusual as most Cu-DOTA complexes are stable even in serum
(23). Up to 55%
64
Cu
2+
was released after 48 h incubation.

A six carbon linker-aminohexanoic acid structure was introduced to increase the
distance between DOTA and DGEA motifs. The stability of the resulting
64
Cu-DOTA-Ahx-DGEA tracer was not improved as free Cu was still observed in
its radio-HPLC profiles. For
64
Cu-DOTA-D(tBu)-GE(tBu)A (two of the carboxyl
groups in DGEA were protected), the dissociation of
64
Cu was significantly
reduced throughout the experiment period (only 3%
64
Cu
2+
was dissociated from
the DOTA complex after 48h), which indicated the carboxyl groups of the DGEA
peptide may compete, either between molecules or within the same molecule,
with the DOTA chelator for
64
Cu binding.

Table 5-1 summarized the solution stability data for HPLC purified
64
Cu-DOTA-DGEA,
64
Cu-DOTA-Ahx-DGEA,
64
Cu-DOTA-D(tBu) GE(tBu)A, and
64
Cu-Z-E(diamsar)-Ahx-DGEA in the PBS solution ( pH 7.4) throughout the whole
137

course of the experiment period. Base on this solution stability results, we
conclude that the unique feature of the DGEA sequence makes diamsar a most
suitable chelator option for our integrin α2β1 targeting tracer design.

\

138

Figure 5-0-2

Figure 5-2. The representative radio-HPLC chromatogram of
64
Cu-DOTA-DGEA and
64
Cu-Z-E(diamsar)-Ahx-DGEA after incubation in
PBS at various time points.

139

Table 5-1. Solution Stability Data for the HPLC-purified 64Cu-Labeled DGEA
Tracers in Phosphate Buffer (pH=7.4)

In vivo microPET imaging study
The stability difference between
64
Cu-DOTA-DGEA and
64
Cu-Z-E(diamsar)-Ahx-DGEA was also reflected in microPET imaging. Static
microPET scans were performed on a PC-3 xenograft model (n=3, integrin α2β1
positive), and selected coronal images at 30 min after injection of
64
Cu-DOTA-DGEA and
64
Cu-Z-E(diamsar)-Ahx-DGEA are shown in Fig.5-3. The
activity distribution in major organs and the tumor contrast ratios were quantified
and compared for both tracers (Fig.5-4). The tumors were clearly visualized for
both tracers, but with lower tumor uptake for
64
Cu-DOTA-DGEA. After
normalization, the tumor uptake of
64
Cu-Z-E(diamsar)-Ahx-DGEA and
64
Cu-DOTA-DGEA is 2.08 ± 0.47%ID/g, 0.92 ± 0.53%ID/g at 30 min p.i.,
140

respectively. However,
64
Cu-DOTA-DGEA demonstrated five times higher liver
uptake compared with the
64
Cu-Z-E(diamsar)-Ahx-DGEA ( 5.89±0.42%ID/g for
64
Cu-DOTA-DGEA and 1.25±0.25%ID/g for
64
Cu-Z-E(diamsar)-Ahx-DGEA.) The
predominantly hepatobiliary elimination leads to unfavorable activity accumulation
in liver and intestine, which may limit further applications of
64
Cu-DOTA-DGEA,
especially in an attempt to detect lesions in the lower abdomen.

Figure 5-0-3

Figure 5-3. Decay-corrected whole-body coronal small-animal PET images of
PC-3 tumor-bearing mice at 30 min post injection of approximately 9.25 MBq (250
µCi) of
64
Cu-Z-E(diamsar)-Ahx-DGEA (left) and
64
Cu-DOTA-DGEA (right). The
PC-3 tumors are indicated with arrows.

DOTA
141

Figure 5-0-4

Figure 5-4.(A)Comparison of the major organs uptake of
64
Cu-DOTA-DGEA and
64
Cu-Z-E(diamsar)-Ahx-DGEA at 30 min p.i. in athymic male nude mice bearing
PC-3 tumor (n = 3, mean ± SD).(B)Tumor contrast (tumor-to-normal tissue ratio)
were also compared in both tracers. Diamsar conjugated DGEA showed much
favorable biodistribution, indicating the better chelating stability in vivo.

142

5.5 Discussion:

The advancement of PET depends on the development of new radiotracers that
will complement
18
F-FDG. Copper-64 (t
1/2
= 12.7 h) decays by β
+
(20%) and β
-

emission (37%), as well as electron capture (43%), making it well suited for
radiolabeling proteins, antibodies and peptides, both for PET imaging (β
+
) and
therapy (β
+
and β
-
). In vivo stable attachment of
64
Cu
2+
to targeted biomolecules
generally requires the use of a bifunctional chelator (BFC). The stability of the
radio-copper complex in vivo is critical to achieve high uptake of the copper
radionuclide in the tissue or organ of interest while minimizing the non-selective
binding or incorporation into non-target organs or tissues. Acyclic BFCs, such as
Ethylenediaminetetraacetic acid (EDTA), Diethylene triamine pentaacetic acid
(DTPA), and their derivatives have been used for labeling Cu-64 (124-127). Most
of these complexes showed low stability in vitro and in vivo. To improve the in vivo
stability of
64
Cu
2+
complexes, researchers turned to various chelators that utilize
both the macrocyclic and chelate effects to enhance stability. Today, three of the
most commonly used chelators for
64
Cu
2+
labeling are DOTA,
1,4,8,11-tetraazacyclotetradecane N,N′,N′′,N′′′-tetraacetic acid (TETA) and
1,4,7-triazacyclononane-N,N′,N′′-triacetic acid (NOTA). However, it is well known
that the DOTA, TETA, NOTA and their derivatives still have limited stability in vivo
due to dissociation of
64
Cu
2+
from these BFCs, leading to high retention in liver
(128-130). Therefore, more effective BFCs for
64
Cu
2+
are required if these
copper-based imaging agents are to be successfully applied in the clinical setting.
143

Recently the hexaazamacrobicyclic cage type ligands have gained significant
attention as potential
64
Cu
2+
chelators (121-123). These ligands can be prepared
in high purity at low cost. It is hypothesized that when one of the chelating
nitrogen atoms of the cage dissociates from the metal center, the topological
constraint induced by the bicyclic cage does not allow it to move very far away
from the metal center, which will effectively ensure its facile re-coordination. In
addition to the improved stability reported in our published works (116-118), the
64
Cu
2+
complexation can be completed under mild conditions to nearly 100%
within several minutes at 40 °C.

In this study, we performed the stability comparisons between
64
Cu-Z-E
(diamsar)-Ahx-DGEA and
64
Cu-DOTA-DGEA tracers. In solution stability
experiments, unexpected high dissociation of
64
Cu
2+
from the
64
Cu-DOTA-DGEA
complex at early time points was observed. As early as 1 h post purification, 3% of
64
Cu
2+
had been disassociated from the complex in PBS solution (pH=7.4). At the
48 h time point, DOTA-DGEA retained only 45 % of the
64
Cu
2+
originally
incorporated. This phenomenon is unusual as most of the known DOTA-Cu
complexes have been shown to be very stable in PBS, serum, and even blood.
On the other hand, Woodin et al. (131) had reported that proton- assisted
decomplexation is a convenient indicator for solution kinetic inertness of several
copper chelators, with the relative inertness order: Cu-CB-TETA>>
Cu-DOTA~Cu-TETA>Cu-CB-DO2A. The DOTA chelator showed the medium in
144

vitro inertness in high acid concentration (e.g. 5M HCl, 90°C). Furthermore, EDTA
possesses four carboxylates that can form strong complexes with metal cations,
such as, Mn(II), Cu(II), Fe(III), Pb (II) and Co(III). The EDTA challenge competition,
therefore, has been utilized to evaluate the thermodynamic stability of several
copper-based radiopharmaceutical tracers.(132-134) Those BFC conjugates
tested were stable over 12 h after purification and remained intact without any
decomposition in the presence of EDTA (1 mg/mL in 25 mM phosphate buffer, pH
7.4).

All above stability studies of DOTA chelator are contrary to our in vitro stability
profile of
64
Cu-DOTA-DGEA in PBS solution. Even in our recently reported study,
we had evaluated the biological property of another Sar based BFC, AmBaSar,
compared the stability with DOTA by using
64
Cu-AmBaSar-RGD and
64
Cu-DOTA-RGD as the model compounds. For in vitro stability studies, the
radiochemical purity of
64
Cu-AmBaSar-RGD and
64
Cu-DOTA-RGD was
comparable, showing more than 97% in PBS or FBS and 95% in mouse serum
after 24h of incubation. In comparison,
64
Cu-DOTA-DGEA was much less stable in
PBS than
64
Cu-DOTA-RGD, which may indicate the stability difference could be
caused by the DGEA peptide as all other segments are basically the same. The
major difference between DGEA and RGD peptide is the presence of multiple
carboxyl groups on the peptide. As the integrin α2β1 targeted DGEA peptides
possess three carboxylate groups, we speculated that the carboxylate groups
145

might interfere or compete with the DOTA chelation with
64
Cu
2+
, just as EDTA and
DTPA can form the coordination bond with metals with their multiple carboxylate
groups. We believe that the observed high level of free
64
Cu
2+
dissociation could
be a result of the potential inter- or intra-molecular interactions of the carboxylate
groups. In addition, the Gly residue in this DGEA peptide might provide some
degree of the flexibility of the DGEA structure to further enhance the internal
competing opportunities (Fig.5-5).

146

Figure 5-5
Figure 5-5. Schematic structure of the potential interfere interactions between the
carboxylic acid groups of DGEA peptide and DOTA chelator. Intermolecular
interaction is also possible

147

In order to investigate this unexpectedly high dissociation of
64
Cu
2+
from DOTA
and confirm our assumption, we first introduced a short linker-amonohexanoic
acid to separate the DOTA chelator and DGEA peptide structure. However, the
instability of the DOTA chelating with
64
Cu
2+
was still observed only with the
marginal improvement. Then, two tBu protecting groups were introduced to the
Asp and Glu carboxylic side chains of DOTA-DGEA to block the potential
interference on Cu-DOTA complexing from the carboxylic groups. As expected,
we did not observe significant amount of
64
Cu
2+
dissociation from the protected
complex,
64
Cu-DOTA-D(tBu)GE(tBu)A, throughout the course of the experiment
period (48h). This result suggests that the carboxyl groups of the DGEA peptide
did interfere with the DOTA-Cu coordination stability. Nonetheless, as the detailed
decomplexation mechanisms have not yet been elucidated, further investigations
with X-ray crystallography of
64
Cu-DOTA-DGEA or computer modeling may be
needed to confirm these interactions of DGEA carboxyl groups with
64
Cu
2+
. In
contrast,
64
Cu-Z-E(diamsar)-DGEA is very stable in PBS at all-time points studied
which is consistent with the bio-stability shown in microPET imaging studies.

The stability difference between
64
Cu-Z-E(diamsar)-Ahx-DGEA and
64
Cu-DOTA-
DGEA also had significant impact on the microPET imaging results. Although both
tracers demonstrated prominent tumor contrast, predominant hepatobiliary
elimination with unfavorable activity accumulation in the liver and intestine was
observed for
64
Cu-DOTA-DGEA (5.89±0.42%ID/g liver uptake). This unexpected
148

distribution pattern is significantly different from our previously DGEA based
optical probes that demonstrated mainly renal clearance. On the other hand, the
liver uptake of
64
Cu-Z-E(diamsar)-Ahx-DGEA was significantly lower (1.25±0.25
%ID/g; 5 times lower) and had a comparable biodistribution pattern with optical
DGEA probes.

It was reported that the borderline softness of copper (II) might favor amines,
imines and bidentate ligands to form a stable chelation,(135) which could partially
explain the superior
64
Cu
2+
chelate stability of diamsar when compared with DOTA
chelator (117,118). It is most likely that the discrepancy in imaging results
between diamsar and DOTA conjugated are related in part to the stability of the
64
Cu
2+
complex in each agent. The carboxylate groups in the targeting ligand
likely play some roles in chelation stability; although further investigation is
needed before a conclusion is drawn. In any case, the imaging results clearly
demonstrated the selection of chelator could have an important impact on the
biodistribution characterization of
64
Cu

based radiopharmaceuticals.

In summary, DOTA has been widely used to modify proteins, polymers, and
nanoparticles; this study, however, has demonstrated that the small terapeptide
DGEA can dramatically influence the
64
Cu
2+
complexation of DOTA likely due to
the presence of multiple carboxylic acid groups. The results from this study
suggest that special consideration should be given during ligand design if the
149

conjugation site has multiple carboxylic acid groups nearby when DOTA will be
used for
64
Cu
2+
chelation.

150

5.6 Conclusion:

In vivo application of copper-64 based radiopharmaceuticals depends highly upon
the radioligand‘s delivery specificity and stability. Therefore, the development of
stable bifunctional chelators is essential. The in vitro and in vivo evaluations of the
64
Cu-diamsar-DGEA and
64
Cu-DOTA-DGEA have demonstrated significantly
improved Cu-chelation stability for diamsar compared with the established
chelator DOTA. The research also indicates that diamsar may be a preferred
ligand for
64
Cu
2+
chelation especially when multiple carboxylate acid groups are
present in the targeting ligand, which may compete with DOTA for
64
Cu
2+
binding.

151

Chapter 6: Summary and Future Goals

6.1 Summary:

Tumor cells are usually characterized by uncontrolled growth, invasion to
surrounding tissues, and metastatic spread to distant sites. Tumor progression
leading to metastasis appears to involve equipping cancer cells with the
appropriate adhesive phenotype for interaction with the ECM. Therefore,
adhesion molecules from the integrin family and components of angiogenesis
might be useful as tumor progression markers for prognostic and for diagnostic
purposes. Therefore, imaging tracers that can target these biomarkers may play a
crucial role in helping oncologists meet several goals: detection of solid tumors;
detection of recurrence; and evaluation of the success of a treatment regimen. At
the moment, most of the work is concentrated on the development of
radiolabelled αvβ3

–antagonists and MMP inhibitors. A series of radiolabeled RGD
peptides for αvβ3 integrin imaging has gained a significant progress and some of
them are already in clinical trials (24-26).

In this research, we have developed DGEA based optical and microPET imaging
agents for specific imaging of the integrin α2β1 expression in individual prostate
tumors. To our best knowledge, this is the first in vivo optical imaging study about
noninvasive quantification of α2β1 integrin expression with DGEA based peptide
tracers. The novelties of this research include the identification of α2β1 integrin as
152

a potential metastasis marker for prostate cancer and the development of imaging
probes to image its expression in vivo.

It is essential to identify new prognostic and therapeutic options for advanced
prostate cancer. In hormone-refractory prostate cancers, the α2β1 integrin has
been implicated in multiple aspects of tumor progression and metastasis. Due to
the possible correlation between upregulation of α2β1 integrin and tumor
progression in human prostate cancer, α2β1 integrin will serve as both an imaging
biomarker to study the extent of disease and response to treatment, and as a
useful therapeutic target for advanced prostate cancer patients. The findings in
this research could lead to α2β1 integrin targeted imaging probes as a selective
and highly effective diagnostic (imaging) agent to evaluate prostate cancer stage,
help us more appropriately select patients considered for anti-integrin α2β1
treatment, allow the evaluation of disease course and therapeutic efficacy at the
earliest stages of treatment. Moreover, the lead compound holds great potential
for peptide receptor based radionuclide therapy. All these will lead to a better
treatment arrangement decision for prostate cancer patients and finally lead to
personalized medicine.

Our study may also provide new insights into the molecular events involved in
prostate carcinogenesis that are controlled by α2β1 integrin associated signaling
cascades, which are altered during prostate cancer initiation and progression.
153

6.2 Future Goals:

The success of this approach will have significant impacts on my long-term
scientific research plan in the following aspects:

1) Prostate cancer diagnosis: The possible correlation between α
2
β
1
integrin
expression level and tumor invasiveness makes these imaging probes
useful for prostate cancer progression diagnosis and treatment monitoring.
Correlations with other parameters such as hormone sensitivity and other
integrin expression levels can be considered in the future.

2) A further integrin structure and ligand binding activity studies led to highly
potent and selective peptide structure are needed. Therefore, our next
research goal is to shed light on the binding mechanism of α2β1 integrins
which containing I-domains, a less well-studied class of 9 integrins.
I-domain-containing integrins differ from other integrins by virtue of having
an inserted domain in their subunit that is responsible for binding
extracellular ligands. No crystallographic or NMR structures, however,
have been solved for intact I-domain-containing α2β1 integrin, although
crystal structures for the isolated I-domains are available.(136) The
mechanism of activation of the ligand-binding I-domain could be inferred
through comparison with better-known non-I-domain integrins such as
αIIbβ3 and αvβ3, for which more high-resolution structural information is
available.
154

3) Multimeric DGEA probes: For integrin binding, multimeric peptides might
be superior to the monomeric counterparts in terms of receptor-binding
affinity in vitro and tumor-targeting efficacy in vivo, presumably due to
multivalency effect (24, 27). After the success of this proposal, multimeric
DGEA peptides can be designed to improve the tumor uptake/retention as
well as the in vivo kinetics as compared with monomeric DGEA peptides.

4) Other cancer types: In addition to being seen in prostate cancer, α
2
β
1

integrin is also an important receptor in primary breast carcinoma cases
(28, 29). The imaging probes indentified in this study will thus be applicable
to many cancer types that express α
2
β
1
integrin.

5) Therapy applications: Although the newly developed DGEA probes were
only labeled with NIR dye or
64
Cu for optical and PET imaging in this
project, the same peptide construct can be labeled with other tags for other
imaging modalities. It may also be coupled with chemo- and
radiotherapeutics for peptide receptor targeted therapy.

6) Multimodality strategy:
64
Cu has been shown to dissociate from DOTA
conjugates and undergo transchelation to liver superoxide dismutase. In
this project, we have developed a sarcophagine derivative with high
binding affinity to
64
Cu. This stable bifunctional chelator would also allow us
155

to develop PET/MRI, PET/Optical, or MRI/Optical dual modality probes for
α
2
β
1
integrin imaging.

7) Clinical translation: It is our expectation that the success of this proposal
will eventually help us identify the right DGEA peptide probes with high and
persistent tumor uptake and favorable pharmacokinetics for clinical
translation for lesion detection and molecular profiling of prostate cancer.

156

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Abstract (if available)

Abstract Prostate cancer is one of the leading causes of cancer-related deaths in the United States and Europe. Despite the fact that prostate-specific antigen (PSA) screening has greatly increased the number of patients with early stage prostate cancer who can be cured by radical prostatectomy, about 40% of prostate cancers are first detected at an advanced stage and half of these are found to be extracapsular at pathologic staging. Therefore, development of an accurate noninvasive imaging technique to detect primary, recurrent and residual prostate cancer is critical for the effective management of this group of patients.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Huang, Chiun-Wei (author)

Core Title A novel design of integrin α2β1 targeting peptide probe for molecular imaging in prostate cancer

School Viterbi School of Engineering

Degree Doctor of Philosophy

Degree Program Biomedical Engineering

Publication Date 01/11/2011

Defense Date 12/20/2010

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

Tag integrin α2β1,molecular imaging,OAI-PMH Harvest,PET,prostate cancer

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Conti, Peter S. (committee chair), Jadvar, Hossein (committee member), Mackay, J. Andrew (committee member), Neamati, Nouri (committee member), Shung, Kirk K. (committee member)

Creator Email chiunweh@usc.edu,steincafe77@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3610

Unique identifier UC188018

Identifier etd-HUANG-4270 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-432536 (legacy record id),usctheses-m3610 (legacy record id)

Legacy Identifier etd-HUANG-4270.pdf

Dmrecord 432536

Document Type Dissertation

Rights Huang, Chiun-Wei

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