Thesis Final Submission 22 August 2013

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AN IMMUNOHISTOPATHOLOGICAL AND FUNCTIONAL INVESTIGATION
OF β3 INTEGRIN ANTAGONISM AS A THERAPEUTIC STRATEGY IN
CANCER
F O F ALSHAMMARI
PhD
2013
I
AN IMMUNOHISTOPATHOLOGICAL AND FUNCTIONAL INVESTIGATION
OF β3 INTEGRIN ANTAGONISM AS A THERAPEUTIC STRATEGY IN
CANCER
Characterisation, development, and utilisation of preclinical cancer
models to investigate novel β3 integrin anatgonists
FATEMAH O F O ALSHAMMARI
Submitted for the degree of Doctor of Philosophy
Institute of Cancer Therapeutics
University of Bradford
2013
II
FATEMAH O F O ALSHAMMARI
Title: An immunohistopathological and functional investigation of β3
integrin antagonism as a therapeutic strategy in cancer
Key words: integrins, αIIbβ3, αVβ3, integrin antagonists, tumour cell migration,
histopathology of integrin expression, cancer therapy.
Abstract
Tumour cell dissemination is a major issue with the treatment of cancer, thus
new therapeutic strategies which can control this process are needed.
Antagonism of integrins highly expressed in tumours is one potential
strategy.
The integrins are transmembrane glycoprotein adhesive receptors. Two of
the integrins, αVβ3 and αIIbβ3, are highly expressed in a number of tumours
and induce bi-directional signalling through their interaction with extracellular
matrix proteins, and growth factor receptors. Through this signalling they play
an important role in a number of cellular processes that are involved in
tumour dissemination such as tumour growth, migration, invasion, metastasis
and angiogenesis. Dual αIIbβ3 and αVβ3 integrin antagonism will have a direct
effect on β3-expressing tumour cells that leads to the inhibition of cell
migration and dissemination. Furthermore, through targeting tumour cell
interaction with endothelial cells and platelets, this will also lead to inhibition
of angiogenesis and metastasis.
The aim of this project was to characterise the expression of αVβ3 and αIIbβ3
integrin in a panel of tumour cell lines and in human tumour xenograft
samples, and to develop and utilise cell-based models to investigate potential
novel β3 antagonists.
The expression of αV and β3 subunits was detected in xenograft tissue using
immunoblotting techniques. A panel of cell lines of different tumour types
including melanoma, prostate, breast, colon and non small cell lung
carcinoma was then characterised for αVβ3 and αIIbβ3 integrin expression
using immunoblotting and immunocytochemistry. Melanoma cell lines
demonstrated the strongest αVβ3 expression. No αIIbβ3 integrin expression
was seen in any of the cell lines evaluated. A selection of cell lines with
varying αVβ3 expression were then used to develop a functional test for cell
migration, the scratch wound healing assay. Migration of tumour cells that
expressed αVβ3 integrin was inhibited by the known β3 antagonists, cRGDfV
peptide and LM609 antibody. A panel of 12 potential novel β3 integrin
antagonists was screened for cytotoxicity and activity in the validated scratch
assay. ICT9055 was the most effective antagonist in inhibition of M14 cell
migration as determined by the scratch assay, with an IC50 of < 0.1 µM.
Therefore the work presented in this thesis has established models and tools
for evaluating potential novel β3 integrin antagonists, and identified a
promising molecule to progress for further preclinical evaluation.
i
Contents
Abstract ......................................................................................................... i
Contents ....................................................................................................... ii
List of Figures ........................................................................................... viii
List of Tables ............................................................................................... xi
Acknowledgement ..................................................................................... xii
Abbreviations ............................................................................................ xiii
1 Chapter 1: Introduction ........................................................................ 1
1.1 Cancer background........................................................................... 1

1.1.1 Definition and aetiology of cancer ................................................ 1
1.1.2 Incidence of cancer ...................................................................... 2
1.1.3 Treatment of cancer ..................................................................... 3
1.1.3.1 Surgery ..................................................................................... 3
1.1.3.2 Radiotherapy ............................................................................ 3
1.1.3.3 Biological therapy ..................................................................... 4
1.1.1.1.1 Endocrine therapy .............................................................. 4
1.1.3.3.2 Gene therapy ..................................................................... 4
1.1.3.3.3 Antibodies .......................................................................... 5
1.1.3.4 Chemotherapy .......................................................................... 6
1.1.3.4.1 Conventional chemotherapy .............................................. 6
1.1.3.5 New strategies for cancer therapy ............................................ 7
1.1.4 The general pathobiology of cancer ............................................. 9
1.2 The Integrins.................................................................................... 15

1.2.1 Introduction ................................................................................ 15
1.2.2 Structure of integrins .................................................................. 15
1.2.3 Types of integrins ....................................................................... 19
1.2.4 Function of integrins ................................................................... 24
1.2.4.1 Function of αVβ3 integrin ......................................................... 27
1.2.4.1.1 Role of αvβ3 integrin in angiogenesis .............................. 28
1.2.4.2 Function of αIIbβ3 integrin ........................................................ 30
1.2.5 Role of αVβ3 and αIIbβ3 integrins in cancer development ............ 31
1.2.5.1 Role of αVβ3 integrin in cancer development .......................... 31
1.2.5.1.1 Role of αVβ3 integrin in breast cancer .............................. 33
1.2.5.1.2 Role of αVβ3 integrin in prostate cancer ........................... 35
1.2.5.1.3 Role of αVβ3 integrin in melanoma ................................... 36
1.2.5.1.4 αVβ3 integrin in glioma ...................................................... 38
ii
1.2.5.2 Role of αIIbβ3 integrin in cancer development ......................... 38
1.2.5.2.1 Role of αIIbβ3 integrin in melanoma .................................. 38
1.2.5.2.2 Role of αIIbβ3 integrin in prostate cancer .......................... 40
1.2.5.3 αVβ3 and αIIbβ3 integrin antagonism in cancer therapeutics .... 41
1.2.5.3.1 Anti-αVβ3 antibodies as cancer therapeutics .................... 41
1.2.5.3.2 Integrin binding peptides as cancer therapeutics ............. 42
1.2.5.3.3 Small molecule integrin antagonists as cancer therapeutics
………………………………………………………………….43
1.3.5.1.1 Dual αVβ3/αVβ5 integrin antagonism in cancer therapy ..... 45
1.2.5.3.4.1 Cilengitide, a dual αVβ3/αVβ5 integrin antagonist, in
cancer therapy………………………………………………………….47
1.2.5.3.5 Dual αVβ3/ αIIbβ3 integrin antagonism ............................... 49
1.3 Aims and objectives........................................................................ 57
2 Chapter 2: Characterisation of integrin αIIbβ3 and αVβ3 expression in

human xenograft tissue in mice using immunohistochemical and
immunoblotting techniques ...................................................................... 59
2.1 Introduction ..................................................................................... 60

2.1.1 Aims and objectives ................................................................... 63
2.2 Materials and methods ................................................................... 64

2.2.1 Materials .................................................................................... 64
2.2.2 Cell lines .................................................................................... 64
2.2.3 Methods ..................................................................................... 66
2.2.3.1 Collection of xenograft materials ............................................ 66
2.2.3.2 Slide coating ........................................................................... 66
2.2.3.3 Tissue fixation, processing, embedding and paraffin sectioning
………………………………………………………………………66
2.2.3.4 Haematoxylin and Eosin staining............................................ 68
2.2.3.5 Immunohistochemistry............................................................ 69
2.2.3.5.1 Immunodetection of αIIb and αVβ3 integrins expression in
FFPE human tumour xenograft sections ........................................... 69
2.2.3.5.1.1 Sample preparation ................................................... 69
2.2.3.5.1.2 Antigen retrieval......................................................... 69
A. Citrate buffer antigen retrieval ............................................................. 69
B. Pre-warmed pepsin digestion method antigen retrieval .................... 70
C. Proteinase K method antigen retrieval ................................................ 70

2.2.3.5.1.3 Antibody incubation ................................................... 70
2.2.3.5.2 Immunodetection of αIIbβ3 and αVβ3 integrins expression in
frozen human xenograft tumour ........................................................ 71
iii
2.2.3.5.2.2 Labelling procedure ................................................... 72
2.2.3.5.3 Immunodetection of β3 subunits and αVβ3 integrin
expression in FFPE and frozen human tumour xenograft sections by
using a mouse on mouse (M.O.M.) kit .............................................. 72
2.2.3.5.3.2 Preparation of M.O.M. kit working solutions .............. 73
2.2.3.5.3.3 Antibody incubations ................................................. 73
2.2.3.6 Immunoblotting (Western Blotting) ......................................... 74
2.2.3.6.1 Materials .......................................................................... 74
2.2.3.6.2 Tissue homogenisation .................................................... 74
2.2.3.6.2.1 Bradford assay .......................................................... 75
2.2.3.6.2.2 SDS-Polyacrylamide Gel Electrophoresis ................. 76
2.2.3.6.2.3 Western Blot for αV and β3 Integrins .......................... 77
2.2.3.6.2.4 Ponceau S Stain ........................................................ 77
2.2.3.6.2.5 Coomassie Brilliant Blue Staining .............................. 77
2.2.3.6.2.6 Immunoblotting of nitrocellulose membrane with anti-β3
and anti-αV integrin subunits .......................................................... 78
2.3 Results ............................................................................................. 80

2.3.1 Detection of the expression of αIIbβ3 and αVβ3 integrin in human
tumour xenograft tissue by IHC ................................................................ 80
2.3.1.1 Tumour xenograft ................................................................... 80
2.3.1.2 Studies on FFPE tissue samples ............................................ 81
2.3.1.3 IHC studies on Frozen Sections. ............................................ 84
2.3.2 Immunodetection of β3 and αVβ3 integrin subunits using an
M.O.M. kit. ………………………………………………………………………87
2.3.2.1 M.O.M. Immunodetection of β3 and αVβ3 integrins in FFPE
sections. ………………………………………………………………………87
2.3.2.2 M.O.M. Immunodetection of β3 and αVβ3 integrins in frozen
sections. ………………………………………………………………………92
2.3.3 Detection of the expression of αIIb, αV, and β3 subunits in
homogenised tumour xenograft mouse tissue by immunoblotting ............ 95
2.3.3.1 Expression of αV and β3 integrin in homogenised human
xenograft mouse tissue compared to negative control tissue and human
tumour cells .......................................................................................... 98
2.4 Discussion ..................................................................................... 100
2.5 Conclusion..................................................................................... 107

a panel of human tumour cell lines ........................................................ 108
3.1 Introduction ................................................................................... 109

3.1.1 Aims and objectives ................................................................. 110
3.2 Materials and Methods.................................................................. 112

3.2.1 Materials .................................................................................. 112
iv
3.2.2 Methods ................................................................................... 112
3.2.2.1 Cell maintenance .................................................................. 112
3.2.2.2 Tumour cell line growth kinetics............................................ 115
3.2.2.3 Immunoblotting ..................................................................... 115
3.2.2.3.1 Cell Lysis........................................................................ 116
3.2.2.3.2 IMB of nitrocellulose membrane with different anti αV, αIIb
and β3 integrin antibodies ................................................................ 116
3.2.2.4 Immunocytochemistry (ICC) ................................................. 118
3.2.2.4.1 Detection of αV, αIIb and β3 subunits, and αVβ3 integrin
expression in different tumour cell lines by ICC .............................. 118
3.2.2.4.1.1 Materials .................................................................. 118
3.2.2.4.1.2 Cell preparation ....................................................... 118
3.2.2.4.1.3 Confocal microscopy ............................................... 119
3.2.2.4.1.4 ICC analysis ............................................................ 119
3.3 Results ........................................................................................... 121

3.3.1 Characterisation of cellular growth kinetics .............................. 121
3.3.2 Detection of αV, αIIb, and β3 integrins using IMB ....................... 123
3.3.2.1 Optimisation of IMB .............................................................. 123
3.3.2.2 Screening different tumour cell lines for αV and β3 integrin
expression by IMB technique using the optimised conditions for anti-αV,
Q20, and anti-β3, B7, antibodies ......................................................... 126
3.3.3 Detection of αV, αIIb, β3 and αVβ3 integrins using ICC ............... 129
3.3.3.1 Optimisation of ICC .............................................................. 129
3.3.3.2 Evaluation of the effect of method of cell harvesting on integrin
expression .......................................................................................... 132
3.3.3.3 Screening different cell lines for expression of α V, β3, αIIb and
αVβ3 integrins by ICC technique. ......................................................... 133
3.3.3.4 Analysis of the expression of αV, αIIb, β3 and αVβ3 integrin in
different human tumour cell lines detected by ICC. ............................ 136
3.3.3.5 Confirmation of sub-cellular immunolocalisation of αIIb, β3 and
αVβ3 integrin using confocal microscopy. ............................................ 137
3.3.4 Comparison of result of IMB and ICC....................................... 139
3.4 Discussion ..................................................................................... 140
3.5 Conclusion..................................................................................... 148
4 Chapter 4: Investigation of the effect of αVβ3 integrin inhibition on

tumour cell migration .............................................................................. 149
4.1 Introduction ................................................................................... 150

4.2 Materials and methods ................................................................. 154

4.2.1 Materials .................................................................................. 154
4.2.2 Methods ................................................................................... 154
4.2.2.1 Wound healing migration assay............................................ 154
v
4.2.2.1.1 Characterisation of the phenotype of the migrated cells in
terms of proliferation and integrin expression .................................. 156
4.2.2.2 Evaluation of the cytotoxicity of cRGDfV and LM609 using the
MTT assay .......................................................................................... 157
4.2.2.3 Validation of the scratch assay using the integrin antagonists
cRGDfV and LM609 ............................................................................ 158
4.2.2.4 Statistical analysis ................................................................ 160
4.3 Results ........................................................................................... 161

4.3.1 Development of the wound healing migration assay ................ 161
4.3.1.1 Seeding density .................................................................... 161
4.3.1.2 Healing time ......................................................................... 163
4.3.2 Evaluation of the cytotoxicity of cRGDfV and LM609 using the
MTT assay.............................................................................................. 167
4.3.3 The effect of cRGDfV on the migration of human tumour cell lines
………………………………………………………………………..168
4.3.4 The effect of LM609 on human tumour cell migration .............. 171
4.4 Discussion ..................................................................................... 173
4.5 Conclusion..................................................................................... 177
5 Chapter 5: Investigation of the cytotoxicity of potential novel β3

integrin antagonists and their effect on tumour cell migration ........... 178
5.1 Introduction ................................................................................... 179

5.2 Materials and methods ................................................................. 181

5.2.1 Materials .................................................................................. 181
Table 5.1 Compounds used .................................................................... 181

5.2.2 Methods ................................................................................... 182
5.2.2.1 Evaluation of the cytotoxicity of potential novel β3 integrin
antagonists using the MTT assay ....................................................... 182
5.2.2.2 Wound healing migration assay............................................ 182
5.2.2.3 Statistical analysis ................................................................ 182
5.3 Results ........................................................................................... 183

5.3.1 Evaluation of the cytotoxicity of potential novel β 3 integrin
antagonists ............................................................................................. 183
5.3.2 Characterisation of the anti-migratory effect of potential novel β3
integrin antagonists using the scratch assay .......................................... 185
5.4 Discussion ..................................................................................... 192
5.5 Conclusion..................................................................................... 194
vi
6 Chapter 6: Discussion and future work .......................................... 195
6.1 General discussion and future perspective ................................ 196
6.2 Conclusion..................................................................................... 206
7 Chapter 7: References and Appendixes ......................................... 207
7.1 References ..................................................................................... 208
7.2 Appendices .................................................................................... 280

7.2.1 Appendix 1 ............................................................................... 280
7.2.2 Appendix 2 ............................................................................... 281
vii
List of Figures
Figure 1.1 The basic steps of the cancer metastatic pathway (Bellahcene et
al. 2008) ....................................................................................................... 11
Figure 1.2 Role of integrins in cancer development ..................................... 14
Figure 1.3 The structure of αIIbβ3 integrin in its active and inactive state ..... 18
Figure 1.4 The integrin family....................................................................... 19
Figure 1.5 inside-out-signalling pathways for integrins (Askari et al. 2009) . 26
Figure 1.6 Interaction of active integrin with signalling molecules regulates
cellular function (Hynes 2002)...................................................................... 27
Figure 1.7 Interaction of αVβ3 integrin and VEGFR-2 (Somanath et al. 2009).
..................................................................................................................... 30
Figure 1.8 Active αIIbβ3 integrin in platelet activation and thrombus formation
(Millard, Odde & Neamati 2011)................................................................... 31
Figure 1.9 Anti-integrin antagonists ............................................................. 52
Figure 2.1 Representative calibration curve using Bradford assay. ............. 76
Figure 2.2 Confirmation of successful protein transfer ................................. 78
Figure 2.3 Haematoxylin and Eosin stain in M14 human melanoma xenograft
paraffin embedded tissues ........................................................................... 80
Figure 2.4 Optimisation of IHC to detect αIIbβ3 and αVβ3 integrin expression in
FFPE human xenografts, using different methods ....................................... 82
Figure 2.5 Optimisation of IHC to detect αV and β3 integrin expression in
FFPE M14 human melanoma xenografts using Q20 and B7 ....................... 83
Figure 2.6 Optimisation of the protocol used to characterise expression of
αIIbβ3 and αVβ3 integrin in frozen human xenograft mouse tissue by IHC ..... 85
Figure 2.7 IHC of αV integrin expression in human xenograft mouse frozen
tissue using Q20 .......................................................................................... 86
Figure 2.8 IHC detection of αVβ3 and β3 integrin in human FFPE tumours with
an M.O.M. kit................................................................................................ 89
Figure 2.9 IHC detection of β3 integrin expression using B7 with M.O.M. kit in
FFPE M14 melanoma human tumour xenografts in mouse ......................... 90
Figure 2.10 IHC detection of β3 integrin expression in FFPE human tumour
xenografts in mouse using B7 with M.O.M. kit ............................................. 91
Figure 2.11: IHC optimisation for αVβ3 and β3 integrin expression in frozen
sections using the M.O.M. detection kit. ...................................................... 93
Figure 2.12 Expression of β3 integrin in human tumour xenograft frozen
tissues immunolabelled with BV4 (anti-β3) using M.O.M. detection kit ......... 94
viii
Figure 2.13 Optimisation steps for extraction of protein from human xenograft
mouse tissue. ............................................................................................... 96
Figure 2.14 Expression of β3 subunit in M14 human xenograft mouse tissue
using B7 by IMB ........................................................................................... 97
Figure 2.15 Expression of αV and β3 integrin in homogenised tissue ........... 99
Figure 3.1 Growth parameters of human tumour cell lines ........................ 122
Figure 3.2 Optimisation of IMB protocol. .................................................... 124
Figure 3.3 Expression of αV and β3 integrin in a panel of human tumour cell
lines from a supernatant ............................................................................ 127
Figure 3.4 Expression of αV and β3 integrin protein in a panel of human
tumour cell lines from a pellet resuspended in lysis buffer ......................... 128
Figure 3.5 Detection of αV, αIIb, β3 and αVβ3 integrin in PC-3 using ICC. .... 130
Figure 3.6 Comparison of cell harvesting techniques ................................ 132
Figure 3.7 Screening of the first part of the cell line panel with anti-αV (Q20),
anti-αIIb (C20), anti-β3 (B7), and anti-αVβ3 (LM609) using ICC. ............... 134
Figure 3.8 Screening of the second part of the cell line panel with anti-αV
(Q20), anti-αIIb (C20), anti-β3 (B7), and anti-αVβ3 (LM609) using ICC. ....... 135
Figure 3.9 The expression of αIIb, β3, and αVβ3 integrin in human tumour cell
lines by confocal microscopy. .................................................................... 138
Figure 4.1 Wound healing assay steps (Hulkower 2011) ........................... 156
Figure 4.2 Scratch assay analysis ............................................................. 159
Figure 4.3 Formation of confluent monolayers for the cell line panel (cell
density per ml and time in hours) ............................................................... 162
Figure 4.4 Migration of different tumour cell lines as assessed by the scratch
assay ......................................................................................................... 164
Figure 4.5 Labelling M14 cells at initial wound perimeter (T=0) and after 24
hours of wound healing with LM609 and Ki-67 using ICC.......................... 166
Figure 4.6 Evaluation of cRGDfV and LM609 cytotoxicity against human
tumour cell lines M14 and HT-29 ............................................................... 167
Figure 4.7 Effects of cRGDfV on tumour cell migration in the cell panel using
the scratch assay ....................................................................................... 170
Figure 4.8 The effect of LM609 on tumour cell migration ........................... 172
Figure 5.1 Evaluation of the cytotoxicity of the potential novel β 3 integrin
antagonists in the tumour cell line panel .................................................... 184
Figure 5.2 Effect of potential β3 integrin antagonists on M14 cell migration 186
Figure 5.3 Effect of 10 µM β3 antagonists on the inhibition of M14 cell
migration by the scratch assay................................................................... 188
ix
Figure 5.4 Effect of potential β3 integrin antagonists in inhibition M14 cell. 190
x
List of Tables
Table 1.1 Drugs currently used for targeted therapy ...................................... 8
Table 1.2 Functions and ligands of different types of integrins .................... 20
Table 1.3 Further anti-integrin agents in clinical use and advanced
development ................................................................................................ 53
Table 2.1 Primary antibodies and related secondary antibodies used in
immunohistochemistry (IHC) and immunoblotting (IMB) .............................. 65
immunocytochemistry (ICC) and IMB techniques. ..................................... 113
Table 3.2 Cell lines .................................................................................... 115
Table 3.3 Conditions investigated while optimising detection of expression of
αV, αIIb, β3 and αVβ3 integrin in human tumour cell lines using ICC. ........... 120
Table 3.4 The optimised protocol for all antibodies used to detect the
expression of αV, αIIb, and β3 subunit and β-actin using IMB technique. .... 125
Table 3.5 Optimised protocols for all antibodies used to detect the expression
of αV, αIIb, β3 subunits, and αVβ3 integrin in cell membrane using ICC ....... 131
Table 3.6 Screening different tumour cell lines for the membranous
expression of αV, β3, αIIb and αVβ3 by ICC .................................................. 136
Table 3.7 Comparison of integrin expression in different tumour cell lines
using specific antibodies for αV and β3 integrin subunits using ICC and IMB.
................................................................................................................... 139
Table 4.1 Comparison of cell migration assays (Kramer et al. 2013) ......... 153
Table 5.1 Compounds used ....................................................................... 181
Table 5.2 IC50 values for the potential novel β3 integrin antagonists in the
tumour cell line panel ................................................................................. 185
Table 5.3 A summary of effect of the potential novel β3 integrin antagonists in
inhibition of M14 cell migration after 24 hours treatment with the scratch
assay ......................................................................................................... 191
xi
Acknowledgement
Firstly, I would like to express my profound gratitude to my supervisors Dr.
Steve Shnyder, Dr. Helen Sheldrake and Professor Laurence Patterson for
providing me with an exciting and challenging research project. I would like to
thank them for help and guidance and illustrious advice which they given me,
throughout my PhD studies.
I am extremely thankful to the staff of Institute of Cancer Therapeutics who
had helped in this research either by their support or encouragement. I would
like also to thank the staff in the lab: in the histology lab I would like to thank
Beryl Cronin and Patricia Cooper, in cell culture I would like to thank Dave
Healey and in the molecular lab I would like to thank Dr. Mark Sutherland and
Andrew Gordon (who provided PCR data shown in the appendix).
I would also like to thank the Public Authority for Applied Education and
Training (PAAET) for their funding.
My special thanks must also go to my dear parents (Khairyah Al Shammari
and Owayed Al Shammari), my husband Abdul Mohsen Al Shammari and for
my four daughters Sarah, Nourah, Noof and Shaikha.
xii
Abbreviations
ABC: Avidin biotin complex
ADMIDAS: Adjacent to metal-ion-dependent adhesion site
ADP: Adenosine diphosphate
Akt/PKB: Protein kinase B
Angpt13: Angiopoietin-like 3
APES: 3-aminopropyltriethoxysilane
ARMD: Age-related macular degeneration
ATCC: American Type Culture Collection
bFGF: basic fibroblast growth factor
BME: Basal membrane extract
BSA: Bovine serum albumin
BSP: Bone sialoprotein
CAMs: cell adhesion molecules
CD3: Cluster of differentiation 3
CMRIT: Combined radioimmunotherapy with cilengitide
cRGDfV : Arg-Gly-Asp-phe-Val
CTGF: Connective tissue growth factor
CTP: Cyclotetrapeptide
CYR61: Cysteine-rich angiogenic inducer 61
2D: Two dimensional
xiii
3D: Three dimensional
DAB: 3,3-diaminobenzidine tetrahydrochloride
DAPI: 4,6-Diamidino-2-phenylindole
Db: Double bands
dH2O: Distilled water
D/I: Dilution/incubation
DMSO: Dimethyl sulphoxide
DNA: Deoxyribonucleic acid
DPX: Distyrene/plasticiser/xylene
ECM: Extracellular matrix
EDTA: Ethylenediamine tetraacetic acid
EGF: Epidermal growth factor
EGFR: Epidermal Growth Factor Receptor
EMT: Epithelial mesenchymal transmission
EP: Endogenous peroxidase
ERK: Extracellular Signal-regulated Kinase
FAK: Focal adhesion kinase
FBS: Foetal bovine serum
FFPE: Formalin fixed paraffin embedded
FGFR: Fibroblast growth factor receptor
FITC: Fluorescein isothiocyanate
g: Gram
xiv
GAR: Goat anti rabbit
GIST: Gastrointestinal stromal tumour
GTPase: Guanosine triphosphate
H2O2: Hydrogen peroxide
HBSS: Hanks’s balanced salt solution
H&E: Haematoxylin and Eosin
HER2: Human Epidermal Growth Factor Receptor 2
HPV: Human papilloma virus
HRP: Horse-radish peroxidase
hrs: hours
HTS: High throughput screening
HUVECs: Human umbilical vein endothelial cells
IARC: International Agency for Research on Cancer
iC3b: Inactivated C3b
IC50: Inhibitory concentration
ICA: Ice pre-cooled acetone
ICAM: Interacellular Adhesion Molecule
ICC: Immunocytochemistry
ICM: Ice pre-cooled methanol
IF: Immunofluorescence
Ig: Immunoglobulin
IgG: Immunoglobulin g
xv
IHC: Immunohistochemistry
IMB: Immunoblotting
ISH: In-situ hybridisation
isoDGR: iso-Aspartic acid-Glycine-Arginine
kDa: Kilodalton
kg: Kilogram
LAP-TGF- β: Latency associated peptide-Transforming growth factor B
LIMBS: Ligand induced metal binding site
mA: Milliampere
MAdCAM-1: Mucosal vascular addressin cell adhesion molecule 1
MAPK: Mitogen-activated protein kinase
Mb: Multiple bands
MIDAS: Metal-Ion-Dependent Adhesion Site
ml: Milliliter
mM: Millimolar
MMAb: Monoclonal mouse antibody
MMPs: Matrix metalloproteinases
MMP-9: Matrix metalloproteinase-9
M.O.M.: Mouse on mouse
mTOR: mammalian Target Of Rapamycin
MTT: 3-(4,5-Dimethylthiazole-2-y1)-2,5-diphenyl tetrazolium bromide
NaCl: Sodium chloride

xvi
Nc: No clear band
NCI: National Cancer research
n.d.: Not done
NF-Kb: Nuclear factor kappa-light chain enhancer of activated B cells
NGS: Normal goat serum
NHS: Normal horse serum
nM: Nanomolar
NRS: Normal rabbit serum
NS: Normal serum
NSCLC: Non small cell lung cancer
NSP: Non specific binding
OPN: Osteopontin
P13k/Akt: Phosphotidylinositide 3-kinase/protein kinase B
PAb: Primary antibody
PBS: Phosphate buffered saline
PC: Polycarbonate
PCI: Percutaneous coronary intervention
PDGFR: Platelet Derived Growth Factor Receptor
PET: Polyethyleneterphthalate
PFA: Paraformaldehyde
PGAb: Polyclonal goat antibody
PGAR: Polyclonal goat anti rabbit

xvii
PKC: Protein kinase C
PKPD: Pharmacokinetic pharmacodynamic
PML: progressive multifocal leukoencephalopathy
PRAb: Polyclonal rabbit antibody
PRAG: Polyclonal rabbit anti goat
PRAM: Polyclonal rabbit anti mouse
PSI: Plexin-Semaphorin-integrin
RCF: Relative centrifugal force
RGD: Arginine-Glycine-Aspartic acid
RGP: Radial growth phase
RPM: Revolution per minute
RPMI: Roswell Park Memorial Institute
R.T.: Room temperature
RT-PCR: Reverse transcription-polymerase chain reaction
Sb: Single band
SD: Standard deviation
SDS: Sodium dodecyl sulphate
siRNA: small interfering RNA
SM: Skimmed milk
SU24: Untreated scratch after 24 hours
ST24: Treated scratch after 24 hours
TBS: Tris buffered saline

xviii
TEM: Transendothelial migration
TGF-β1: Tissue growth factor β1
THN: Tetramethylethylenediamine
TNF: Tumour necrosis factor
TRITC: Tetramethylrhodamin isothiocyanate
µg: Microgram
µl: Microliter
µM: Micromolar
VCAM-1: Vascular cell adhesion protein 1
VEGF: Vascular Endothelial Growth Factor
VEGFR-2: Vascular Endothelial Growth Factor receptor 2
VEGFR-A: Vascular Endothelial Growth Factor receptor A
VGP: Vertical growth phase
vWF: von Willebrand factor
xix
1 Chapter 1: Introduction
1.1 Cancer background
1.1.1 Definition and aetiology of cancer
Cancer is a complex family of diseases characterised by uncontrolled growth
of cells and spread from the primary tumour site to other sites (Varmus
2006). The formation of cancer is a multistep process that originates from a
single cell in which vital regulatory pathways have been disturbed. Cancer
has multiple causes, with a wide range of genetic, physiological and
histological features. However, genetic and environmental factors such as
chemical exposure, ionizing radiation exposure, and certain viruses (such as
human papilloma virus; HPV) are the main causes (Fearon 1997).
Clinically, tumours include two main types: benign and malignant. A benign
tumour grows locally, with a gradual increase in size, and causes local
pressure or obstruction within the surrounding tissue. In contrast, a malignant
tumour grows rapidly, metastasises via the lymph or blood vessels to distant
sites, and is dangerous due to its potential for invasion and destruction of
normal tissues (van Slooten et al. 1985).
1
1.1.2 Incidence of cancer
A wide variation is reported for the incidence and prevalence of cancer at
different anatomical sites, and for patients of different ages, sex and
geographical locations. The overall trend is for an increase in cancer
incidence and death annually. Statistics obtained from the International
Agency for Research on Cancer (IARC), showed that the incidence of cancer
in the world reached 12.7 million cases in 2008 with a mortality rate of 7.6
million. The incidence of cancer was estimated to increase to reach 13.2
million cases for both sexes in 2011 (Jemal et al. 2011). Europe estimated
1.3 million deaths from cancer in 2013 (Malvezzi et al. 2013). In the United
Kingdom, the cancer incidence reported in 2008 and cancer mortality in 2009
was 408,381 new cancer cases and 156,090 cancer deaths (CRUK 2012).
Moller indicated that the incidence rate of all cancers in the English
population will rise from 224,000 in 2001 to around 299,000 in 2020, or an
increase of 33 per cent (Moller et al. 2007). In the United States, cancer is
the second most common cause of death after heart disease and it is
estimated that one in four people will die from cancer. The American Cancer
Society estimates that in 2013 there will be 1.7 million new cancer cases
diagnosed and about 580,350 Americans are expected to die of cancer
(Siegel et al. 2013). These statistics support the need for continued research
into the causes of cancer and the development of effective preventive
methods and effective treatments.
2
1.1.3 Treatment of cancer
Treatment of cancer is a challenge. The choice of treatment depends on
tumour type, histological grade and stage of the tumour at the time of
diagnosis. The overall aim of the treatment is to remove all cancerous cells
without affecting normal tissue (Rosenberg 2001). The major types of
treatment for cancer are surgery, radiotherapy, biological therapy and
chemotherapy.
1.1.3.1 Surgery
In most cases, surgery is the first line of cancer treatment. However, before
diagnosis, the cancer may have already metastasised to another area of the
body; therefore, chemotherapy and/or radiotherapy must be used in
conjunction with surgical removal in order to slow tumour growth and prevent
further metastasis. In addition, some primary tumours, such as brain
tumours, are not easily removed by surgery. Therefore, neoadjuvant
chemotherapy (the use of chemotherapy prior to surgery or radiation) can
also play an important role. This treatment can reduce the tumour load
thereby making surgery or radiotherapy easier. The removal of residual
tumour after surgery can also be done by adjuvant treatment (Rosenberg
2001).
1.1.3.2 Radiotherapy
Radiotherapy is used to reduce the size of tumour before surgery
(Camporeale 2008), ensures that all cancer cells are destroyed following
3
surgery and slows down the progression of the disease. For example,
radiotherapy can be used in conjunction with surgery for treatment of
carcinomas of the pancreas (Alfieri et al. 2001), larynx, other head and neck
sites, cervix, breast, bladder and prostate. However, several studies have
shown that prostate cancer patients who undergo radiotherapy treatment can
have recurrence of the disease after a few months (Tefilli et al. 1998; Tefilli et
al. 1998; Tefilli et al. 1998). Furthermore, radiotherapy is not efficient at
curing metastasis.
1.1.3.3 Biological therapy
1.1.3.3.1 Endocrine therapy
The success of endocrine therapy depends on its ability to suppress further
growth of the tumour rather than its ability to kill cells. It can be used to treat
hormone dependent tumours such as breast and prostate cancer (Labrie
2004). The treatment depends on lowering the plasma concentration of a
particular hormone (e.g. by removing the producing organ such as by
orchidectomy (removal of the testes) (Suzuki et al. 2010) or by antagonising
its action at its receptor (e.g. tamoxifen, which binds to oestrogen receptors
on the mammary epithelium and thereby blocks the proliferative actions of
oestrogen) (Criscitiello et al. 2011).
1.1.3.3.2 Gene therapy
The principle of gene therapy involves changing the actual gene responsible
for the neoplastic processes. This therapy requires that the gene must
4
access to the cell via a vector. The vector may be a virus that is defective in
replication and into which the therapeutic gene has been inserted, along with
the promoter that will activate the gene once inside the cell. Gene therapy
may have one of the following aims: restoring the function of a defective
tumour-suppressor gene such as p53 to inhibit tumour growth; blocking the
action of a mutated or over-expressed oncogene or insertion of a gene that
activates a prodrug or produces other cell-killing effects thereby eliminating
cancer cells near the cell into which the gene has penetrated (Brown &
Lillicrap 2002; Edelstein et al. 2007). However, one type of gene therapy,
ganciclovir/Hstk, reached phase III clinical trials but did not show any
significant effect on malignant brain tumours due to low transduction of the
vector and immune response to vector-producing cells (Rainov 2000). Gene
therapy can cause damage to the immune system (Brown & Lillicrap 2002).
1.1.3.3.3 Antibodies
Monoclonal antibodies can disrupt cancer cells through a variety of
mechanisms (Strome et al. 2007; Trikha et al. 2002). Monoclonal antibodies
can trigger the immune system to attack cancer cells (e.g. Rituximab and
Alemtuzumab) (Murakami et al. 2011; Schweighofer & Wendtner 2010),
prevent the cancer cells from taking up proteins (e.g. Transtuzumab), block
angiogenesis through inhibition of VEGF-A (e.g. Bevacizumab) or carry
cancer drugs or radiation to cancer cells (e.g. Zevalin, Bexxar and Mylotarg).
However, a recent study reported that Bevacizumab administration led to
increased treatment-related mortality (Ranpura et al. 2011). Several
5
limitations also make antibodies unattractive for use as therapeutics,
including production costs, pharmacokinetics versus tissue penetration and
modes of action (Chames et al. 2009).
1.1.3.4 Chemotherapy
1.1.3.4.1 Conventional chemotherapy
Cancer chemotherapy is a systemic treatment and its selectivity depends on
the ability of the drugs to target the tumour. However, cancer chemotherapy
drugs also affect normal cells that replicate rapidly, such as hair follicles
(Botchkarev 2003), bone marrow, intestinal mucosa and gonads (Meirow &
Nugent 2001; Vijayalaxmi 2011). Chemotherapy works mainly by disrupting
cell proliferation, especially targeting DNA synthesis and cell division.
Traditional chemotherapy drugs include: anti metabolites such as 5-
fluorouracil (inhibits DNA synthesis), alkylating agents such as cisplatin
(blocks DNA replication), anti microtubule agents such as taxanes (blocks
microtubule depolymerisation) and anthracyclin antibiotics like doxorubicin
(interacts with topoisomerase II, intercalates between DNA nucleotides and
blocks DNA synthesis) (Kaye 1998).
Tumour cells may be resistant to cytotoxic drugs because of extracellular
circumstances that limit drug access to the tumour or due to defects in the
p53 gene that render the tumour cell resistant to the cytotoxic drug. Tumour
cell resistance to chemotherapy can also arise due to DNA damage,
decreased uptake of drug by the cell and increased drug efflux by a p-
6
glycoprotein that acts as a drug efflux pump and reduces the intracellular
concentrations of some cytotoxic agents. Another problem with
chemotherapy is that the drug may reach the target cells at insufficient
concentrations as a result of inactivation or removal of the drug from the
circulatory system (Moxley & McMeekin 2010).
1.1.3.5 New strategies for cancer therapy
While the increased incidence and mortality of cancer and the inadequacy of
the different kinds of therapy already mentioned, a clear need thus exists for
the development of treatments with greater effectiveness. The development
of targeted therapies has the aim of improving differentiation between normal
and neoplastic cells. The discoveries in molecular biology about the control of
cell cycle events highlighted that certain signalling pathways that control the
growth and differentiation were inappropriately activated in various tumours
(Hersey et al. 2009). Targeted agents can be used against cell adhesion
molecules, growth factors, intracellular signalling and angiogenesis (Gerber
2008; Korpanty et al. 2010; Lord & Ashworth 2010). Table 1.1 summarises
the targeted therapy drugs currently in clinical use (Collins & Workman 2006;
Malinowsky et al. 2010).
7
Table 1.1 Drugs currently used for targeted therapy
Drug Tumour type Target Ref
Trastuzumab Metastatic breast cancer, HER2 (Farolfi et
gastric cancer al. 2013)
Cetuximab Metastatic colorectal cancer EGFR (Stintzing
et al. 2013)
Imatinib Chronic myeloid leukkaemia Bcr/abl, c-kit, (Spiera et
Mesylate and Gastrointestinal stromal PDGFR, al. 2011)
(Gleevec) tumours with activated c-kit
receptor tyrosine kinase, other
sarcomas
Bevacizumab Colorectal cancer VEGF (Bennouna
et al. 2013)
Gefitinib Non-small-cell lung cancer mutant EGFR (Sugiura et
(Iressa) al. 2013)
Erlotinib Non-small-cell lung cancer mutant EGFR (Stevenson
(Tarveca) & El-Modir
2011)
Rapamycin Breast, prostate and renal mTOR (Armstrong
RAD001 cancer et al. 2010)
Sorafinib Melanoma RAF kinase (Panka et
BAY 43-9006 Renal carcinoma al. 2006;
Procopio et
al. 2013)
Vemurafenib Melanoma BRAF (Gonzalez
mutation et al. 2013)
Dasatinib Gastrointestinal stromal Kit, PDGFR (Caenepeel
BMS354825 tumour Dual Src/Abl et al. 2010;
Chronic myeloid leukemia kinase Quintas-
Cardama
et al. 2007)
Lapatinib Breast cancer EGFR, HER2 (Bachelot
et al. 2013)
Sunitinib Renal cell cancer VEGFR, (Blagoev et
PDGFR, cKit, al. 2013;
Flt-3 Huang et
al. 2010)
Pertuzumab Breast cancer HER2 (O'Sullivan
& Swain
2013)
Dasatinib Breast cancer Bcr/abl (Somlo et
al. 2013)
Key: HER2: Human Epidermal Growth Factor Receptor 2. EGFR: Epidermal Growth Factor
Receptor. PDGFR: Platelet Derived Growth Factor. VEGF: Vascular Epithelial Growth
Factor. mTOR: mammalian Target of Rapamycin GIST: Gastrointestinal stromal tumour.
8
1.1.4 The general pathobiology of cancer
Cell growth is usually well organised and controlled to meet the needs of the
body. In contrast, cancer cell division is uncontrolled. Growth factors such as
tumour necrosis factor (TNF) and extracellular matrix (ECM) components that
interact with cell surface receptors can lead to changes in cell division which
activate different protein pathways inside the cell (Jinka et al. 2012). With the
help of signal transducers inside the cell, these pathways will lead to changes
in gene expression and induce cell proliferation (Radeff-Huang et al. 2007).
Apoptosis is the process of programmed cell death. Apoptosis involves
biochemical events that lead to cell morphology changes and ultimately cell
death. These changes include blebbing, loss of cell membrane asymmetry
and attachment, cell shrinkage, nuclear fragmentation, chromatin
condensation and chromosomal DNA fragmentation. Tumour cells can
survive and proliferate if they can escape from undergoing apoptosis (Fulda
2009). Apoptosis can be mediated by both extrinsic and intrinsic pathways.
The extrinsic pathway is initiated by extracellular death ligand such as tumour
necrosis factor (TNF) and tumour necrosis factor-related apoptosis inducing
ligand (TRAIL), which leads to the recruitment of caspase-8 to form a death-
inducing signal complex. The extrinsic pathway can activate caspase-3 and
cleave BID to tBID which facilitates the release of the cytochrome c from the
mitochondria. The intrinsic pathway is mediated by BAX, BAK and BID
proapoptotic proteins which promote release of cytochrome c from the
9
mitochondria leading to activation of caspase-9 ultimately leading to
apoptosis.
The tumour suppressor gene p53 can bind to DNA and activates genes that
control the cell cycle and programmed cell death. If the cell has damaged
DNA, p53 can either repair the damage or induce apoptosis to prevent
proliferation of the defective cell and potential cancer development.
Therefore, inactivation of p53 can also play a role in the survival of tumour
cells. Cell adhesion molecules can also control cell apoptosis via anoikis
(apoptosis resulting from loss of interactions with the extracellular matrix),
direct induction of caspases and increased bcl-2 expression, p53 inactivation
and several other pathways related to survival, such as Extracellular Signal-
regulated Kinase (ERK) and protein kinase B/Akt (Guadamillas et al. 2011;
Stiewe 2007; Tan et al. 2004; Vachon 2011; Zhong & Rescorla 2012).
As a malignant tumour progresses it will metastasise (Bozzuto et al. 2010).
Metastasis is the process whereby the cancer cells migrate from the primary
tumour site to other parts of the body (Geho et al. 2005). In essence, it is the
fundamental difference between benign and malignant tumours. The ability of
cancer cells to metastasise depends on the interaction of their cell surface
molecules with the microenvironment including the ECM and neighbouring
cells. Several steps are involved in metastasis (Figure 1.1) including
migration, intravasation, transportation, extravasation and metastatic
colonisation (Hanahan & Weinberg 2000; Hanahan & Weinberg 2011;
Talmadge & Fidler 2010; Williams 1974).
10
Figure 1.1 The basic steps of the cancer metastatic pathway
(Bellahcene et al. 2008)
Normal cells are transformed into cancer cells (a). Cancer cells proliferate
(b), detach from each other, penetrate the basement membrane, migrate and
invade the surrounding tissues (c). Cells intravasate into blood vessels, travel
in the circulation to distant sites. They then extravasate through the vessel
wall (d) and migrate (e) to nearby tissue, where they may form distant
metastases (f).
The tools of cell migration are the cell adhesion molecules (CAMs) (Hood &
Cheresh 2002) and protease enzymes. Cell adhesion molecules such as
cadherins and integrins play a role in the interactions of similar and different
cells together. Cell adhesion molecules can also induce gene expression in
the nucleus by binding with transcription factors. Integrins can interact with
actin binding proteins and specific kinases such as focal adhesion kinase
(FAK) (Jockusch et al. 1995). FAK mediates cell motility through Src (Cary et
11
al. 1996) and activation of the RAS pathway (Figure 1.2) (Schlaepfer et al.
1994).
Further, integrins can promote the movement of metastatic cells by allowing
the cell to adhere to different ECM components. Cell migration starts with cell
polarization in the direction of movement and the formation of a protrusion.
Integrins serve as points of contact with the ECM and stabilise the protrusion
via connections to actin filaments. At the leading edge adhesion of the
protrusion to the substratum allows migration through enhancement of focal
adhesion formation (Gawecka et al. 2010; Wozniak et al. 2005). Finally, the
detachment of the cell from the previous attachment happens through
contraction of the cell from the edge toward the nucleus and the adhesion
receptor released from the cytoskeleton. This detachment is associated with
the release of cell vesicles and fragments (Lock et al. 2008; Sahai 2007).
The last factor that has a role in cell migration is the protease. Proteases
degrade a path through the ECM and stroma. The cancer cells in a tumour
can synthesise matrix metalloproteinases (MMPs); these MMPs also have an
association with integrins (Brooks et al. 1996; Jin et al. 2011), since integrins
induce the surrounding stromal cells to produce MMPs (Brooks et al. 1996).
Different integrin signalling pathways control MMP expression, thereby
controlling tumour cell invasion (Kubota et al. 1997; Westermarck & Kahari
1999). MMPs are involved in integrin processing and integrins can activate
MMPs in the cell. MMP activity can lead to proteolytic cleavage of the
12
extracellular domain of E-cadherin, which can lead to disruption of the ability
of E-cadherin to control cellular interactions (David & Rajasekaran 2012).
Migrating tumour cells will intravasate into the blood and lymphatic vessels.
During this process, tumour cells attach to the stromal face of the blood
vessel and degrade its basement membrane, passing between the
endothelial cells into the bloodstream. The tumour cells are then transported
with platelets in the direction of blood flow (Felding-Habermann et al. 2001;
Weis & Cheresh 2011). The role of integrins in this step will be discussed in
section 1.2.4.1.1
When a tumour cell reaches the metastatic site, it will escape from the blood
or lymph vessel (extravasation) by attaching to endothelial cells lining the
vessel and passing through them into the surrounding stroma. In this step,
another type of cell adhesion molecule can also play a role. Selectins, which
are expressed on endothelial cells, also help the attachment of cancer cells
to the endothelium (Barthel et al. 2013).
The last stage of metastasis is the colonisation of the tumour at a distant site.
With the help of angiogenesis (formation of new blood vessels), the tumour
can grow (Weis & Cheresh 2011). Vascular endothelial growth factor (VEGF)
and basic fibroblast growth factor (bFGF) induce and regulate angiogenesis
(Hanahan & Weinberg 2011; Weis & Cheresh 2011).
13
Figure 1.2 Role of integrins in cancer development
14
1.2 The Integrins
1.2.1 Introduction
Adhesion receptor molecules on the cell surface became attractive targets for
cancer therapy due to their important role in the control of tumour cell
dissemination (Okegawa et al. 2004; Simmons 2005). Many types of
adhesion cellular receptors (Shimaoka et al. 2002) are recognised including
cadherins, selectins, immunoglobulins and integrins (Mousa 2002). The
integrins are transmembrane glycoprotein adhesive receptors found on the
cell surface; they bind to the extracellular matrix components at the outer
membrane surface and interact with the cytoskeletal components at the inner
membrane surface. Integrins induce signals (Dedhar & Hannigan 1996;
Huveneers et al. 2007; Qin et al. 2004; Stupack 2005; Stupack & Cheresh
2002) through interaction with the extracellular matrix proteins (Brakebusch
et al. 2002) and growth factor receptors (Hynes 2002); these are important in
cell migration (Felding-Habermann 2003), cell cycle regulation (Fahraeus &
Lane 1999), gene expression and apoptosis (Jinka et al. 2012).
1.2.2 Structure of integrins
The integrins are heterodimers consisting of α and β chains that are non
covalently associated (Hynes 2002). The two chains each have a large
extracellular portion comprising multiple domains, a single transmembrane
domain and a short cytoplasmic tail (Figure 1.3) (Shimaoka et al. 2002;
Srichai 2010; Ulmer 2010).
15
The N-terminus of the α subunit consists of seven segments folded into a
single compact domain, forming a structure called the β-propeller. The α
subunit may also contain another domain that is inserted between blades 2
and 3 of the β propeller; this is known as the I domain, or von Willebrand
factor type A domain (Shimaoka et al. 2002). The I-domain is the key ligand-
binding domain in those integrins where it is present. The β subunit has a
highly conserved N-terminus region, which is similar to the I domain and
therefore named the β I-like domain; this domain is responsible for ligand
binding in integrins that do not contain an I domain (Figure 1.3) (Humphries
2000; Humphries 2004; Humphries et al. 2004; Takada et al. 2007; Takagi
2007).
The N-terminus of the integrin has a divalent cation binding site known as
MIDAS (Metal-Ion-Dependent Adhesion Site) located in each I and I-like
domain. Divalent cations bind here, as well as at other sites such as the
ligand induced metal binding site (LIMBS) and ADMIDAS, and serve a
central function in ligand recognition by the I domain and I like domain
(Humphries 2002; Lee et al. 1995; Shimaoka & Springer 2003).
The C-terminus of the α subunit includes three β sandwich domains as a
thigh, and two calf regions (calf 1, calf 2). In the β subunit, the C-terminus is
composed of the PSI (Plexin-Semaphorin-Integrin) domain and four
epidermal growth factor domains (EGF1, EGF2, EGF3 and EGF4). The
region where the integrin is bent in the resting state is termed the knee
region. In the β subunit, it forms a junction between the hybrid domain and
16
two EGF and PSI, while in the α subunit, it is found between the thigh and
the two calf regions (Figure 1.3). The terminal end of the C-terminus of both
subunits spans the plasma membrane to interact with the intracellular
cytoskeleton. During inside-out signalling (the regulation of integrin function
from within the cell) (Hynes & Lander 1992), the integrin undergoes a
structural change from the bent, closed or inactive conformation (Kalli et al.
2011), which has a low affinity for ligand binding, to an extended, open or
active form that has a high affinity for ligand binding (Chen et al. 2011; Hynes
2002). After activation, the signals are transmitted to the extracellular
domains of the integrin, which then alters the conformational of the ligand
binding site and hence the affinity for the ligand (Figure 1.3) (Hynes 2002;
Kim et al. 2011).
17
Figure 1.3 The structure of αIIbβ3 integrin in its active and inactive state
The resting inactive integrin (left), has a closed bent structure. The active
form (right) is open. The N-terminus includes the β-propeller and βA-domain
in αIIb and β3 subunits respectively. It has an activation-dependent ligand-
binding domain (indicated by the red arrow) for ECM proteins,
macromolecules or receptors on the surface of opposing cells. The C
terminus includes the thigh domain and two calf regions in αIIb and the hybrid
domain, PSI domain and β-tail domain in β3 region. The tails of both subunits
span the plasma membrane to receive signals from inside the cell (Shattil et
al. 2010).
18
1.2.3 Types of integrins
Humans have 24 integrins made up of a combination of 18 α and 8 β
subunits. As shown in Figure 1.4, the different types of integrins can be
classified according to specificity of ligand binding, and to the presence or
absence of the I domain. Each integrin has a different function (Table 1.2),
although a number of integrins can bind the same ECM proteins, providing
redundancy in cell signalling and adhesion pathways.
αIIb
α8 α5
β3
β5
β8 αV
α1
β6
α2
β1 α4 β7
α10
α9 αE
α11
αL αM
α3 α6 α7
β2
β4 αX αD
Figure 1.4 The integrin family
Different kinds of integrin subunits can bind together to form an integrin sub
family. There are 5 subfamilies: Red indicates the RGD receptors, yellow the
laminin the receptors, blue the collagen receptors and green the leukocyte
receptors. The black frame indicates an I-domain integrin.
19
Table 1.2 Functions and ligands of different types of integrins
Integrin Function Ligands Ref
(Bodary
&
McLean
Regulates cell adhesion, 1990;
migration and controls matrix Henry et
Laminin E1
accumulation. al. 2001;
α1β1 fragment,
Rossino
Collagen
Regulates epidermal growth et al.
factor receptors. 1991;
Tomaselli
et al.
1993)
Mediates the adhesion of
platelets to collagen. (Klein et
Collagen I-IV, al. 1991;
Used by fibroblasts for tissue Laminin, Schiro et
α2β1
collagen remodelling during Echovirus-1 al. 1991;
wound repair. Thrombospondin Staatz et
al. 1991)
Involved in tumour progression.
(Coopma
n et al.
Laminin5, 1996;
Regulates expression of MMP-9.
Epithelial Manohar
basement et al.
Mediates survival of
α3β1 membrane 2004;
keratinocytes.
protein,Epiligrin Pulido et
Thrombospondin al. 1991;
Participates in phagocytosis.
Wang et
al. 2004)
(Hsia et
al. 2005;
Maintains the structural integrity
VCAM-1, Moyano
of the placenta and heart during
Fibronectin, et al.
embryogenesis.
α4β1 Thrombospondin, 1997;
Osteopontin, Springer
Mediates adhesion of inflamed
Mad CAM-1 et al.
vascular endothelial cells to T
1994; Wu
cells.
et al.
1995)
20
Mediates lymphocyte adhesion to Mad CAM-1,
MAd CAM-1 on endothelial VCAM-1, (Erle et
α4β7
specific Peyer’s patches in the Fibronectin, al. 1994)
gut. Osteopontin
Mediates fibronectin assembly
into extracellular matrix,
fibronectin dependent cell
adhesion, spreading, migration
(Hynes
and signal transduction.
1992;
Fibronectin, Varner et
α5β1 Acts as a co-stimulatory
Osteopontin al. 1995;
molecule to the T cell receptor
Wu et al.
CD3 complex in T-cell activation
1993)
and enhanced phagocytosis of
opsonised particles by
monocytes.
Angiogenesis.
Platelet interaction with sub
endothelial basement membrane
(Leu et
laminin exposed on blood vessel
al. 2003;
damage.
α6β1 Laminin Sonnenb
erg et al.
Activation of T-cells.
1990)
Angiogenesis
Sonnenb
Adhesion to basement
α6β4 Laminin 5 erg et al.
membranes and matrix protein
1990
Involved in muscle development,
Laminin 1,2,4, E8 (Song et
α7β1 linkage between muscle fibre and
region al. 1992)
extracellular matrix.
(Bossy et
Osteopontin,
Kidney morphogenesis. al. 1991;
Fibronectin,
α8β1 Muller et
Vitronectin,
Expressed in neural tissues al. 1995)
Tenascin
(Desloge
s et al.
Involved in osteoclast formation
Osteopontin, 1998;
and function.
Tenascin-c Oommen
α9β1
VCAM-1 et al.
Cell migration and adhesion via
VEGF A,C,D 2010;
direct binding of VEGF.
Rao et al.
2006)
Expressed on chondrocytes, (Bengtss
Collagen II
α10β1 deficiency leads to on et al.
Laminin
chondrodysplasia. 2005)
21
Required on periodontal ligament (Carrace
fibroblasts for cell migration and do et al. ;
α11β1 collagen reorganisation to help Collagen I Popova
generate the forces needed for et al.
axial tooth movement. 2007)
Intestinal targeting of lymphocyte (Cepek et
αE β 7 E cadherin
subpopulations. al. 1994)
(Brockba
nk et al.
2005; Hu
et al.
Fibronectin, 1995;
Cell matrix and intracellular
αV β 1 Osteopontin Marshall
interaction.
LAP-TGF-β et al.
1995;
Vogel et
al. 1990)
Vitronectin, (Gladson
Vitronectin endocytosis and
αV β 5 Osteopontin, et al.
angiogenesis.
BSP 1997)
Response of epithelium to (Breuss
inflammatory stimuli and injury et al.
such as in asthma. 1995;
Fibronectin Erikson
αV β 6 Regulation of epithelial Osteopontin, et al.
proliferation in vitro and wound LAP-TGF-β 2009;
healing. Munger
et al.
Activation of LAP-TGF- β 1999)
(Chernou
sov &
Carey
2003;
Jackson
Neural function.
et al.
LAP-TGF-β
αV β 8 2004;
Receptor for foot and mouth Vitronectin
Nishimur
disease virus.
a et al.
1994;
Pozzi &
Zent
2011)
22
Involved in immune function, cell-
cell interactions during
(Springer
lymphocyte transit through
1990;
αLβ2 endothelium to sites of ICAM 1,2,3
Springer
inflammation
1990)
and during recirculation through
the lymph node.
Has a role in inflammation due to
its expression on many
leukocytes involved in immune
system.
Mediates adhesion to and

Complement
transmigration through vascular
fragment iC3b,
endothelium of monocytes and (Springer
αMβ2 ICAM 1,2,4,
granulocytes. 1990)
Fibrinogen,
Factor X
Participates in leucocyte
aggregation.
Involved in chemotaxis,
apoptosis and other phagocytic
activities.
(Keizer et
Complement
Cytotoxic T cell killing and al. 1987;
fragment iC3b,
αXβ2 monocyte and granulocyte Vorup-
Fibrinogen,
adhesion to endothelium. Jensen et
ICAM-1, Collagen
al. 2003)
(Grayson
Has role in phagocytosis by
αDβ2 ICAM-3, VCAM-1 et al.
macrophages.
1998)
(Buensuc
eso et al.
Fibrinogen, 2005;
Essential for normal platelet
Fibronectin, Muller et
adhesion to damaged vascular
αIIbβ3 Vitronectin, al. 1993;
endothelium and normal platelet
Thrombospondin, Podolniko
aggregation.
VWF va et al.
2003)
23
(Bader et
al. 1998;
Gavrilovs
kaya et
al. 1999;
Regulates angiogenesis, Humphrie
Endothelial cell adhesion to s et al.
ECM proteins. 2006;
Tumour progression and Vitronectin, Leavesle
invasion. Activation of MMPs. Fibronectin, y et al.
Fibrinogen, 1992;
αV β 3 Osteoclast mediated bone Thrombospondin, Nelsen-
resorption. Osteopontin, Salz et al.
BSP, 1999;
T cell activation. VWF Varner &
Cheresh
Act as a receptor for viruses like 1996;
adenovirus, Echo virus. Wadehra
et al.
2005;
Wickham
et al.
1993)
1.2.4 Function of integrins
As can be seen in Table 1.2, the integrins have multiple functions, including
the adhesion between the cell and the surrounding tissue (Hynes 1999;
Niland & Eble 2012). The integrins help the membrane to adhere to the
extracellular sites and create an attractive force that adheres the cells to their
surroundings (Schwartz 2010). Integrins are important in the regulation of cell
shape, migration (Schneider et al. 2011) and cell cycle regulation. They are
also involved in signalling pathways (Scales & Parsons 2011) that regulate
cell growth, proliferation, survival, division, differentiation and apoptosis (Guo
& Giancotti 2004).
24
The regulation of integrin function can occur internally within the cell through
signalling complexes pathways (Hynes 2002), where the activation of
integrins can result from binding to certain ligands or by inside-out-signalling
(Figure 1.5). Cell-matrix contact induces a wide range of signalling pathways
mediated by adaptor proteins and enzymes (Takada, Ye & Simon 2007). For
example, integrins can be activated intracellularly by signals from G-protein
coupled receptors that lead to phosphorylation of the cytoplasmic domain of
the β subunit. The cytoskeletal adaptor proteins such as talin, skelemin,
filamin, α-actinin, myosin and tensin can regulate integrin affinity, changing
the integrin into its active conformation. The adaptor protein head binds to
the β tail and induces conformational changes that are accompanied by
dissociation of α and β subunits and increased affinity for the ligand binding,
so that inside-out signals regulate the cell adhesion. When the integrins bind
to their ligands in the ECM, they cluster to form a focal adhesion, which
affects the organisation of the cytoskeleton underlying the plasma membrane
(Humphries 2000).
In the extracellular region, ligand binding transfers a signal to the interior of
the cell (outside-in signalling) and can induce conformational changes,
including separation of the α and β legs (Humphries 2000; Humphries et al.
2003; Legate et al. 2009). This separation allows the interaction of the
cytoplasmic tail with intracellular signalling molecules such as FAK/c-Src
(Huveneers et al. 2007), the small GTPases Ras & Rho and adaptors like
cas/crk and paxillin (Guo & Giancotti 2004; Takada, Ye & Simon 2007;
25
Yamada et al. 2003). These signals then regulate cell function (Figure 1.6)
(Millard et al. 2011).
This project will concentrate on the study of the αVβ3 and αIIbβ3 integrins (β3
subfamily). The subsequent sections will describe their role in cancer
development, survival, metastasis and invasion.
Figure 1.5 inside-out-signalling pathways for integrins (Askari et al.

2009)
Inside-out signals occur through binding of β integrin tail with cellular proteins
(B). The inside signals leads to integrin changing from inactive (A and B) to
active form, which is able to bind to ECM proteins (C). Binding ECM proteins
like fibrinogen, vitronectin, fibronectin, thrombospondin, von Willebrand factor
and osteopontin, and interaction with other proteins and receptors induce
various outside-in signals (D) which begin signalling cascades controlling
cellular processes (E).
26
Figure 1.6 Interaction of active integrin with signalling molecules
regulates cellular function (Hynes 2002).
1.2.4.1 Function of αVβ3 integrin
The αVβ3 integrin can recognise several ligands such as vitronectin,
fibronectin, fibrinogen, proteolysed collagen and other proteins. It does this
by recognising the tripeptide sequence Arginine-Glycine-Aspartic acid (RGD)
which is common to the above ligands. The αVβ3 integrin can also recognise
WGD, and isoDGR (iso-Aspartic acid-Glycine-Arginine) (Spitaleri et al. 2008)
and contains another non-RGD binding site for MMP-2 and MMP-9 (Brooks
et al. 1996).
αVβ3 is widely expressed on the endothelial cells (Takada et al. 2007),
epithelial cells, smooth muscle cells and monocytes. It is expressed on
activated endothelial cells and new vessels but it is absent in resting
endothelial cells and normal organ systems (Rusnati et al. 1997). It regulates
27
both angiogenesis in endothelial cells and cell adhesion to the extracellular
matrix (Soldi et al. 1999). It also has a role in osteoclast activity where it
mediates osteoclast adhesion and regulates the cytoskeletal organisation
required for cell migration (Duong et al. 2000; Nakamura et al. 1999; Srivatsa
et al. 1997; Tolar et al. 2004).
1.2.4.1.1 Role of αvβ3 integrin in angiogenesis
Angiogenesis is the physiological process involving the growth of new blood
vessels. Angiogenic blood vessels express αVβ3 at elevated level, whereas
αVβ3 expression is low in quiescent endothelial cells (Leu et al. 2002; Weis &
Cheresh 2011). Interaction between αVβ3 and the ECM can control
angiogenesis through pro-angiogenic and anti-angiogenic molecules that
originate from ECM (Robinson & Hodivala-Dilke 2011). The pro-angiogenic
function of αVβ3 is promoted through its co-operation with factors that
enhance angiogenesis such as VEGFR-2, vitronectin, fibronectin, Del1,
Angptl3, CYR61, bone sialoprotein (BSP) and thrombin. In contrast, the anti-
angiogenic effect occurs through its interaction with thrombospondin,
angiostatin and tumstatin (Hodivala-Dilke et al. 2003).
For example, adhesion of endothelial cells (expressing αVβ3) to vitronectin
leads to tyrosine phosphorylation of VEGFR-2, indicating an involvement of
αVβ3 in VEGFR-2 activation (Soldi et al. 1999). The αVβ3 integrin binds
directly to VEGFR-2, activating VEGFR-2 and leading to β3 subunit
phosphorylation. In turn, the phosphorylated αVβ3 will phosphorylate VEGFR-
2 in the presence of VEGF, indicating that the association of αVβ3 integrin

28
with VEGFR-2 promotes the activation of each receptor. Experiments using
mutant integrin have shown that the cytoplasmic tyrosine residues of β3
integrin (Y747 and Y759) are necessary for successful functional
communication between β3 integrin and VEGFR-2 (Figure 1.7) (Robinson &
Hodivala-Dilke 2011; Robinson et al. 2004). Studies using the same model
found that a mutant β3 (model had mutant β3 with two tyrosine residue Tyr747
and Tyr759 which were unable to induce signalling) integrin is expressed on
the cell surface but it is defective in signalling (Mahabeleshwar et al. 2006).
However, a study using a β3 knockout mouse model showed an induction of
angiogenesis and tumour growth, indicating role for the αV integrin as well as
in angiogenesis such as αVβ3 integrin (Reynolds et al. 2002).
The ability of αVβ3 integrin antagonists to block in vivo angiogenesis supports
a role for αVβ3 integrin in angiogenesis. LM609 anti αVβ3 integrin, a specific
antibody that targets αVβ3 integrin, has been used but LM609 cannot react
with mouse αVβ3 integrin. Thus, the effect of LM609 on angiogenesis was
studied by Brook et al., who used human breast carcinoma cells injected into
chimeric human/mouse model (this is a mouse model in which a piece of skin
is removed and replaced with a sutured piece of human skin). When MCF-
7PB cells were injected into the human skin, LM609 was able to block tumour
growth and angiogenesis in the human skin microenvironment (Brooks et al.
1995).
29
Figure 1.7 Interaction of αVβ3 integrin and VEGFR-2 (Somanath et al.
2009).
1.2.4.2 Function of αIIbβ3 integrin
The αIIbβ3 integrin (also known as GPIIb/IIIa) is expressed on the surface of
platelets, megakaryocytes, monocytes, granulocytes and lymphocytes. It is
inactive in resting platelets but is rapidly activated by thrombogenic stimuli
and promotes binding to fibrinogen (Huang et al. 1993; O'Toole et al. 1990),
von Willebrand factor and fibronectin. This integrin is necessary for platelet
aggregation (Bennett 2005).
Platelet aggregation occurs through the activation of platelets by
physiological molecules such as thrombin (Hodivala-Dilke et al. 1999),
collagen or ADP (Bhavaraju et al. 2011; Gay & Felding-Habermann 2011).
The activation of αIIbβ3 is mediated by a G-protein that acts through protein
kinase C (PKC) to cause a conformational change. Active αIIbβ3 binds to
fibrinogen, a multivalent ligand, through RGD and KQAGDV recognition
motifs, resulting in clustering of the integrin-ligand complex and formation of
adhesive patches on the surface of platelets, thereby allowing platelet cross-
30
linking and thrombus formation (Figure 1.8) (Geiger et al. 2009; Giuliano et
al. 2003; Quinn et al. 2003; Rocco et al. 1993; Ruoslahti 1996; Springer et al.
2008).
Figure 1.8 Active αIIbβ3 integrin in platelet activation and thrombus

formation (Millard, Odde & Neamati 2011)
1.2.5 Role of αVβ3 and αIIbβ3 integrins in cancer development
1.2.5.1 Role of αVβ3 integrin in cancer development
Alteration in the pattern of the αVβ3 integrin receptor is observed in tumour
cells (Arosio et al. 2009; Gasparini et al. 1998; Gladson & Cheresh 1991;
Landen et al. 2008; Max et al. 1997). The expression of αVβ3 integrin has
been detected in many cancer cell lines such as breast (Verbisck et al.
2009), ovarian (Landen et al. 2008) cervical carcinoma (Shen et al. 2006),
glioblastoma (Gladson & Cheresh 1991), melanoma (Petitclerc et al. 1999;
Seftor et al. 1992), prostate carcinoma (Chatterjee et al. 2001), kidney cancer
cells (Arosio et al. 2009) and lung cancer cells (Tsai et al. 2008). The β3 and
αVβ3 integrin receptor are also detected in clinical samples of melanoma
31
(Albelda et al. 1990; Neto et al. 2007) and ovarian carcinoma (Partheen et al.
2009). Breast cancer (Gasprini et al. 1998) and colon cancer show
expression of αVβ3 integrin (Reinmuth et al. 2003).
The αVβ3 integrin plays an important role in a number of cellular process that
lead to cancer development including malignant transformation, tumour
growth and progression, invasion, metastasis (Byzova et al. 2000), apoptosis
(Desgrosellier & Cheresh 2010; Jin & Varner 2004; Li et al. 2001; Lu et al.
2008; Mizejewski 1999; Mousa 2002) cell surface localisation of
metalloproteinases and angiogenesis (Jin & Varner 2004; Varner & Cheresh
1996). Consequently, it plays a key role in the regulation of tumour cell
survival and apoptosis. Signalling by αVβ3 integrin regulates both the
expression and activity of bcl-2 and suppresses the expression of the pro-
apoptotic protein Bax by down regulation of p53. αVβ3 increases the bcl-2:
BAX ratio, which leads to increased tumour cell survival by preventing the
mitochondrial apoptotic pathway that involves the release of cytochrome c
and caspase activation (Martin & Vuori 2004; Uhm et al. 1999). Crosstalk
between αVβ3 and fibroblast growth factor receptor (FGFR) also prevents
apoptosis through the mitogen-activated protein kinase (MAPK) pathway.
αVβ3 also enhances tumour progression by activation of the tyrosine kinase
SRC, which leads to activation of the FAK-independent survival pathway and
promotion of tumour growth (Desgrosellier & Cheresh 2010).
Increased expression of αVβ3 allows tumour cells to bind to extracellular
matrix proteins such as fibronectin, fibrinogen, vitronectin, von Willebrand
32
factor and osteopontin that are present in the tumour microenvironment.
These adhesive interactions provide survival signals for invading endothelial
cells (Desgrosellies & Cheresh 2010). MMP-2 binds to αVβ3 on the cell
surface and this interaction leads to activation of MMP-2 and localizes its
proteolytic activity to the invasive front of the cell (Varner & Cheresh 1996).
Breast, prostate, lung and thyroid tumour cell lines became more invasive
when αVβ3 forms a link with bone sialoprotein and MMP-2 (Karadag et al.
2004). The αVβ3-ERK1/2 pathway is essential for connective tissue growth
factor (CTGF) induced ERK1/2 activation and cellular migration in the MCF-7
breast carcinoma cell line (Chen et al. 2007).
1.2.5.1.1 Role of αVβ3 integrin in breast cancer
αVβ3 is expressed in human breast cancer tissue (Zhao et al. 2007), as well
as in breast cancer cell lines such as MDA-MB-231(Cai et al. 2006; Liu et al.
2009; Manzoni et al. 2009; Takayama et al. 2005). αVβ3 expression
correlates with the metastasis of MDA-MB-435 human breast cancer cell line,
which interacts with platelets to promote arrest of tumour cells during blood
flow and leads to haematogenous metastasis (Felding-Habermann 2003).
Expression of αVβ3 has been shown on cancer cells in the active
conformation and it activates platelets, leading to increased metastatic
activity to the lung. The activated state of αVβ3 in breast cancer cells may be
responsible for the tumour cell-platelet interaction that leads to arrest MDA-
MB-435 tumour cell during blood flow, resulting in increased binding of a
ligand mimetic antibody and increased cell migration toward vitronectin. The
33
metastasis of breast cancer cells was seen to be strongly inhibited by LM609
anti-αVβ3 antibody (Felding-Habermann et al. 2001).
Sloan et al. detected expression of αVβ3 on the cell surface of the 66c14
mammary cell line, which led to metastasis of breast tumour from the
mammary gland to the bone. The increase in metastasis resulted from the
ability of αVβ3 to adhere to vitronectin and invade the basement membrane
and the surrounding stroma, which depended on protease and MMP activity.
αVβ3 expression can induce haptotactic migration towards osteopontin (Sloan
et al. 2006).
Other studies have shown that αVβ3 is important for the development of bone
metastasis; expression of β3 was detected in human breast carcinoma with
bone metastasis and was stronger in tissue with bone metastasis than in
primary tissue. Tumour cells expressing αVβ3 integrin stimulate bone
destruction and osteoclast-mediated bone resorption once they are located in
the bone marrow (Zhao et al. 2007). Transfection of breast carcinoma cells
MDA-MB-231 with αVβ3 integrin or using cells from primary site of bone
metastasis led to increased number and area of osteolytic bone metastases
in vivo, due to expression of active αVβ3 integrin on the cell surface. Inhibition
of αVβ3 function by LM609 inhibited tumour cell invasion and adhesion to
cortical bone (Pecheur et al. 2002).
34
1.2.5.1.2 Role of αVβ3 integrin in prostate cancer
Prostate cancer cells express both αVβ3 and αV (Cooper et al. 2002; van der
Horst et al. 2011; Wang et al. 2005). The expression of αVβ3 may differ
between different prostate carcinoma cell lines and between labs (Saxena et
al. 2012; Zheng et al. 1999). A recent study that examined the expression of
αV and β3 integrin under different conditions reported the expression of αV
and β3 in the prostate carcinoma cell line, PC-3 and in human xenograft
mouse tissue. The expression differed in the in vitro and in vivo environments
(Taylor et al. 2011).
The expression of αVβ3 was correlated with the ability for adherence and
migration of the cells on vitronectin. The PC-3 cells expressing αV and αVβ3
integrin migrated on vitronectin and fibronectin whereas LNCaP cells, which
do not express αVβ3, migrated only on fibronectin. Transfection of LNCaP
cells with the β3 subunit resulted in adherence to vitronectin, which was
inhibited by LM609 (Zheng et al. 1999). In contrast, an immunocytochemistry
study using LM609 found higher expression of αVβ3 in LNCaP than PC-3
cells. After expression was characterised, the cells were treated with cRGDfV
(cyclic peptide: Arg-Gly-Asp-phe-Val) which was more effective in LNCaP
and had no effect on PC-3 cells. Blocking of αvβ3 integrin on LNCaP
membrane by cRGDfV treatment caused FAK cleavage, and reduced
Akt/PKB phosphorylation, which led to activation of caspase-9 and caspase-
3, indicating that cRGDfV induced programmed cell death in the LNCaP cells
(Chatterjee et al. 2001).
35
αVβ3 plays an important role in migration of prostate cancer to the bone;
expression of αVβ3 in PC-3 promoted cancer migration to the bone matrix
(McCabe et al. 2007). Barthel et al. found that prostate cancer extravasation
into bone marrow endothelium under physiological blood flow conditions
depended on the presence of E-selectin, αVβ3 integrin, β1, and Rac1/Rap1
GTPase activity (Barthel et al. 2012).
1.2.5.1.3 Role of αVβ3 integrin in melanoma
Melanoma cells and tissue express αVβ3, with the highest expression in
metastatic melanoma tissue (Hofmann et al. 2000). Using frozen melanoma
sections, Albelda et al. found that the expression of β3 integrin was more
common in invasive melanoma than in primary melanoma: vertical growth
phase (VGP) showed immunoreactivity with β3 integrin while radial growth
phase (RGP) failed to show any reactivity. The expression of β3 integrin was
detected only in metastatic melanoma cells and not in benign melanocytes.
These results suggested that the expression of β 3 integrin could be important
for the development of tumour invasiveness and could be useful as a marker
for melanoma cells entering the aggressive phase of the malignancy
process(Albelda et al. 1990).
Li et al. reported that high expression of αVβ3 integrin in vitro and in vivo can
increase metastasis of melanoma cells by mediating adhesion to vitronectin
(Li et al. 2001). The adhesion of αVβ3-expressing A375M melanoma cells to
vitronectin was inhibited by LM609, whereas the same antibody had no effect
on melanoma cell adhesion to fibronectin and laminin. However, an increase

36
in melanoma cells invasion through the basement membrane was not upon
exposure to either anti αVβ3 antibody or vitronectin. Exposure of melanoma
cells to vitronectin was associated with an increased production of type IV
collagenase as a result of αVβ3-initiated signalling indicated that αVβ3 integrin
had a role in melanoma cell invasion through modulation of protease levels
(Seftor et al. 1992).
Denatured collagen colocalised with αVβ3 integrin in melanoma cells and
attachment of M21 cells to vitronectin and denatured collagen led to an
increase in the bcl-2: BAX ratio. The αVβ3 antagonist LM609 could inhibit M21
cell growth in vivo. This antagonism inhibits tumour growth by inducing
apoptosis through the regulation of bcl-2 and BAX expression, indicating that
the role of αVβ3 integrin in tumour survival involves interaction with the ECM
(Petitclerc et al. 1999).
Voura et al. localised αVβ3 integrin expression on the cell surface of WM293
melanoma cells using immunofluorescence. The expression was localised in
cell-to-cell contact regions when the WM293 cells were co-cultured with
HUVECs and was diffuse before the cells started extravasation, increasing to
be strongest during extravasation. The study reported a 40% inhibition of
melanoma cell transmigration in response to the linear RGD peptide at
concentration 100 µM, whereas a cyclic RGD peptide (c-RGDfV) showed
50% inhibition of melanoma cell transmigration at concentration 5 µM and
had its greatest effect at 5 hours co-culture. Treatment with LM609 at
concentration 40 µg/ml led to 40-50% inhibition, indicating that this
37
melanoma extravasation is at least partially mediated by αVβ3 integrin (Voura
et al. 2001).
1.2.5.1.4 αVβ3 integrin in glioma
αVβ3 is overexpressed in glioblastoma tumour cells such as U-87MG (Cheng
et al. 2005), U-373MG and U-251MG (Mattern et al. 2005) and in endothelial
cells of tumour-associated proliferating microvessels. αVβ3 activation was
related to tumour cell invasion through an enhancement of angiogenesis that
prevents development of hypoxia in brain lesions (Lorger et al. 2009).
Glioblastoma cell line U-251MG also showed expression of αVβ3, which was
involved in cell adhesion to vitronectin (Gladson & Cheresh 1991).
αVβ3 is expressed on clinical glioma tumour samples and its expression
correlates with histological grade (Bello et al. 2001). Different glioblastoma
samples showed heterogenous expression of αVβ3 integrin and most of the
expression of αVβ3 was derived from glial tumour cells (Schnell et al. 2008). A
higher expression of αVβ3 was seen in grade III tumours than in grade I&II
tumours for glioblastoma.
1.2.5.2 Role of αIIbβ3 integrin in cancer development
1.2.5.2.1 Role of αIIbβ3 integrin in melanoma
The expression of αIIbβ3 integrin has been detected in tumour cells or tissues
(Chen et al. 1997; Trikha et al. 1998; Trikha et al. 1997). αIIb expression was
first seen in malignant melanoma by Puerschel et al. by
immunohistochemistry using the anti-αIIb monoclonal antibody sz22 (Santa

38
Cruz). In contrast, none of the tested tissue samples from patients with non-
metastatic melanoma showed αIIb immunoreactivity (Puerschel et al. 1996).
Chen et al. detected the expression of αIIbβ3 in melanoma cell line WM35 by
reverse transcription-polymerase chain reaction (RT-PCR) (Chen et al.
1997). Trikha et al. showed melanoma cells and tissue expressed high
affinity αIIbβ3 (Trikha et al. 1997).
The expression of αIIbβ3 was low in thin melanoma tissue (early stage
tumours) but increased in thick melanoma tissue (later stage disease),
whereas the expression of αVβ3 integrin was intense in thin melanoma. The
importance of αIIbβ3 integrin in human melanoma cell invasion, spreading and
migration was demonstrated using isogenic cell lines. The αIIbβ3 expressing
cells adhered to fibrinogen but couldnot adhere to vitronectin, whereas αIIbβ3
negative cells (expressing only αVβ3 integrin) adhered to vitronectin but
couldnot adhere to fibrinogen, suggesting that expression of αIIbβ3 integrin
suppresses the function of αVβ3 integrin. No difference was observed in the in
vitro growth between αIIbβ3 expressing cells and mock-transfected cells, but
αIIbβ3 expressing cells grew larger tumours in vivo due to a reduction in the in
vivo rate of apoptosis (Trikha et al. 2002). Dome et al. found that expression
of αIIbβ3 integrin in the vertical growth phase upregulate bFGF expression,
thereby providing an angiogenic growth factor for melanoma cells and
promoting tumour growth (Dome et al. 2005).
39
1.2.5.2.2 Role of αIIbβ3 integrin in prostate cancer
The expression of αIIb has been reported in human prostate tumour tissue
(Trikha et al. 1996), as well as in cell lines such as such as PC-3 and DU-145
(Chen et al. 1997).
Trikha et al. detected the expression of αIIb DNA/RNA in prostate carcinoma
tissue using an in situ hybridisation (ISH) technique using an antisense
riboprobe to αIIb mRNA. Prostate cancer tissues produced both αIIb and β3
integrin proteins. The presence of intact αIIbβ3 integrin was confirmed by flow
cytometry, dot blotting and cell adhesion assays. αIIbβ3 was stored
intracellularly and translocated to the cell surface in response to PKC
activation. Monoclonal antibodies directed to αIIbβ3 integrin blocked DU-145
cell invasion (Trikha et al. 1996).
Expression of αIIbβ3 differed between PC-3 and DU-145 cell lines. Although
both expressed αIIbβ3, the expression was intracellular in PC-3 whereas αIIbβ3
was localised on the cell surface of DU-145 cells and was involved in the
focal adhesion sites. DU-145 cells were also more invasive than PC-3. Mice
injected with PC-3 cells developed intraprostatic tumours whereas mice
injected with DU-145 developed tumours that infiltrated the surrounding
tissue and metastasised to the lymph nodes. The function blocking
antibodies, 10E5 and PAC-1 directed to αIIbβ3, inhibited lung colonization in a
tail vein injection model. These antibodies can bind to platelet αIIbβ3 to inhibit
platelet function and therefore platelet tumour interaction. Antibodies can also
block the interaction of DU-145 cells with host tissues by binding to the αIIbβ3
40
integrin expressed by tumour cells. Consequently, combined blockage of
αIIbβ3 integrins on multiple cell types can lead to a blockade of lung
colonisation by DU-145 cells (Trikha et al. 1998).
1.2.5.3 αVβ3 and αIIbβ3 integrin antagonism in cancer therapeutics
The role of integrins in cancer development, metastasis and angiogenesis
discussed in the previous sections suggests that the β3 subfamily may
represent attractive therapeutic targets (Cox et al. 2010; Desgrosellier &
Cheresh 2010; Eble & Haier 2006; Millard, Odde & Neamati 2011; Niu &
Chen 2011; Rust et al. 2002; Shimaoka & Springer 2003). Therapies based
on integrin antagonism that include antibodies, peptides, small molecules
and small interfering RNA (siRNA) are summarised in Figure 1.8 and Table
1.3 (Bisanz et al. 2005; Goodman & Picard 2012; Liu et al. 2008).
1.2.5.3.1 Anti-αVβ3 antibodies as cancer therapeutics
Abegrin (etaracizumab, previously known as vitaxin or MEDI-522) is an IgG1
humanized monoclonal antibody engineered from the murine monoclonal
antibody LM609. LM609 targets αVβ3 and has the ability to reduce tumour
growth in vivo and in vitro by inhibiting the tumour cells from interacting with
underlying extracellular matrix proteins such as fibrinogen. LM609 can block
human angiogenesis and reduce invasion; thus, it may be useful as an anti-
angiogenic approach for treatment of any cancer (Brooks et al. 1995).
Abegrin can inhibit the growth of ovarian cancer in vitro and in vivo (Landen
et al. 2008), although at high doses it can show impaired anti-tumour activity.
41
This is possibly because the high serum level might cause aggregation of the
antibody, limiting its availability (Mulgrew et al. 2006).
A phase I clinical trial conducted on advanced stage metastatic cancer found
Abegrin to have no significant toxicity (McNeel et al. 2005). Abegrin was then
used in a phase II clinical trial on stage IV melanoma, either as a single agent
or in combination with dacarbazine. However, Abegrin showed no tumour
response if used alone and the combination of Abegrin with dacarbazine had
only a low percentage of tumour response. Consequently, Abegrin is no
longer being investigated (Hersey et al. 2010).
1.2.5.3.2 Integrin binding peptides as cancer therapeutics
In addition to antibodies, peptides have been used as tool compounds in
cancer studies (Danhier et al. 2012; Millard, Odde & Neamati 2011; Temming
et al. 2005). Bello et al. examined the effects of the RGD peptide IS201 as an
inhibitor of αVβ3 integrin in vitro and in vivo on malignant glioma cells U87-
MG, 4373-MG, U118-MG and endothelial cells. The IS201 peptide inhibited
glioma growth in vivo, and its effects were associated with a decrease in
angiogenesis and cell proliferation and an increase in apoptosis (Bello et al.
2003).
Another peptide used by Chatterjee et al. who reported the effects of cyclic
cRGDfV and linear RGD peptides in glioma cells. They found that cRGDfV
blocked cell-to-cell adhesion and inhibited either survival or proliferation in
human glioma cells, whereas RGD had no inhibitory effect (Chatterjee et al.
42
2000). The same group also examined the effects of cRGDfV on prostate
cancer and found that it induced apoptosis in LNCaP prostate carcinoma
cells, whereas the linear RGD peptide caused only a partial reduction in the
number of prostate cancer cells. However, cRGDfV had a significant
cytotoxic effect on prostate cancer cells expressing αVβ3 integrin (Chatterjee
et al. 2001).
Another study by Allman et al. showed that cRGDfV inhibited melanoma cell
adhesion in vitro and prevented development of tumours in vivo. However,
the peptide was unable to induce apoptosis in malignant melanoma that
expressed high levels of bcl-2 (Allman et al. 2000). Furthermore, although
cRGDfV apparently induced melanoma cell detachment, the peptide did not
remain bound to the cell surface (Castel et al. 2001).
At present, the most advanced peptide antagonist undergoing clinical
development is Cilengitide which an N-methylated cRGDfV derivative
c(RGDf(NMe)V) that shows high affinity for αVβ3 and αVβ5 integrins. For
further details see section 1.2.5.3.4.1.
1.2.5.3.3 Small molecule integrin antagonists as cancer therapeutics
The small molecule antagonist IH0162 can induce anoikis in vitro through
inhibition of αVβ3 expressing M21 melanoma cells binding to vitronectin. It
was able to inhibit pulmonary metastasis of M21 cell in vivo. IH0162 activates
caspases 3, 8, and 9; both caspase 8 and 9 affect the intrinsic and extrinsic
43
apoptosis pathways. IH0162 can also decrease bcl-2 and survivin, extending
its effects further to the FAK pathway (Zhang et al. 2011).
The antagonism of αVβ3 can be exploited as a therapeutic target to prevent
skeletal metastasis. MDA-MB-435 breast carcinoma cells were transfected
with αVβ3 integrin and injected into tail vein of animal to determine the role of
αVβ3 integrin in development of bone metastasis in vivo. A lower bone
volume to tissue volume ratio, indicating higher bone destruction, was
observed when cells were injected directly into the tibial bone marrow cavity,
and a larger number of αVβ3 integrin expressing tumour cells were found
residing in the bone marrow; these stimulated osteoclast mediated bone
resorption. For example, long-term treatment with the αVβ3 integrin
antagonist PSK1404 resulted in multiple inhibitory effects on cancer cells,
endothelial cells, and osteoclasts and enhanced the anticancer efficiency.
while short term therapy inhibited tumour cell invasion in vivo (Zhao et al.
2007).
The effect of oral administration of XV454 (a specific αIIbβ3 antagonist) on
platelet aggregation, tumour induced thrombocytopenia and metastasis has
been investigated by Amirkhosravi et al. Oral administration of 5 mg/kg
XV545 led to complete inhibition of platelet aggregation within ten minutes.
Tumour cell induced thrombocytopenia was inhibited significantly by both oral
and intravenous administration of XV454 and it had anti-metastatic activity
with a reduced number of metastatic lung nodules seen (Amirkhosravi et al.
2003).
44
Bakewell et al. hypothesised that tumour cells require β3 integrin for
metastasis to bone and for induction of bone osteolysis. ML464 (a pro-drug
antagonist of αIIbβ3) protected mice from metastasis in vivo but it did not
disrupt platelet/tumour interactions in vitro because the pro-drug requires
activation in vivo (Bakewell et al. 2003).
Tirofiban, an αIIbβ3 antagonist, can block the interaction of αIIbβ3 integrin with
the col15 cell adhesion domain of collagen XVII, thereby blocking the
transmigration and invasion of the HSC-3 cell line (squamous cell carcinoma)
normally induced by col15. This transmigration is mediated by factors other
than αIIb. Since the blockade of α5 and αV reduced migration of HSC-3 in
response to col15 by 53%, whereas a 35% reduction in migration was
caused by tirofiban (Parikka et al. 2006).
1.2.5.3.4 Dual αVβ3/αVβ5 integrin antagonism in cancer therapy
Targeting more than one integrin with one compound is becoming an
attractive concept (Sheldrake & Patterson 2009). Dual antagonists can show
important antiangiogenic and antitumour effects, and may represent a
targeted approach for the inhibition of tumour angiogenesis and growth. One
pseudopeptide, ST1646, targets αVβ3/αVβ5 with antiangiogenic effects both in
vivo and in vitro, and has shown inhibitory effects on the growth of human
ovarian carcinoma A2780 cells injected into xenograft tissue in nude mice
(Belvisi et al. 2005).
45
S247 is a small molecule αVβ3/αVβ5 integrin antagonist that has been
investigated in both in vitro and in vivo models. Both continuous treatment
and short-term treatment with S247 were highly effective in reducing
metastasis, either by blocking early events such as stable attachment of
tumour cells to vasculature of distant organs or by blocking their
extravasation from vessels into nearby tissues evaluated by measurements
of gross lung weight, flow cytometric analysis, and histology. The effect of
S247 in inhibition of metastasis at the early stage (during stable attachment
of tumour cell to the vasculature of distant organ) was examined in another
tumour cell line because the 435/HAL cells did not colonise the lung after i.v.
injection. GFP-expressing B16-F10 murine melanoma cells were injected into
the tail vein of mice, following S247 injection just prior to tumour injection.
After first day of injection, 68% decrease in final tumour burden and an 86%
decrease in the tumour-associated gain in lung weight were observed.
Therefore, the study indicated that the use of αVβ3/αVβ5 integrin antagonists
shows promise for treatment of both early and late steps of metastasis
(Shannon et al. 2004).
Treatment with S247 prevented the development of colon cancer liver
metastasis in vivo but had no growth inhibitory effect on the primary tumour.
In vitro, S247 affected both endothelial cells and some types of tumour cell,
causing decreased cell proliferation, adhesion and migration and also
induced apoptosis (Reinmuth et al. 2003). A further study showed the effect
of S247 is increased if it is combined with radiotherapy (Abdollahi et al.
2005).
46
Kumar et al. found that a dual antagonist of αVβ3/αVβ5 may be useful in
blocking tumour induced angiogenesis by using the nonpeptide small
molecule SCH221153, which inhibits the binding of radiolabelled echistatin to
αVβ3/αVβ5 with an IC50 equal to 3.2 and 1.7 nM, respectively. SCH221153 has
the ability to block adhesion of foetal bovine aortic endothelial GM7373 cells
to vitronectin and FGF2. In addition, it inhibits HUVECs proliferation and the
mitogenic activity of FGF2 and VEGF. Testing in the human melanoma Lox
cell line (which expresses low levels of αVβ5) showed SCH221153 to be
ineffective in inhibiting cell proliferation in vitro, but it inhibited tumour growth
in vivo by decreasing the number of blood vessels surrounding a xenograft
tumour (Kumar et al. 2001).
1.2.5.3.4.1 Cilengitide, a dual αVβ3/αVβ5 integrin antagonist, in cancer
therapy
Cilengitide is another dual αVβ3/αVβ5 integrin antagonist. It is a cyclic
pentapeptide that binds to integrin αVβ3 and αVβ5 with high affinity (Alghisi et
al. 2009). Cilengitide has antiangiogenic and anti-proliferative effect in glioma
cells and xenografts (MacDonald et al. 2001; Mas-Moruno et al. 2010;
Mikkelsen et al. 2009; Tabatabai et al. 2010; Taga et al. 2002).
In phase I of a clinical trial, Cilengitide was well tolerated and showed limited
side effects, although the maximum tolerated dose in phase I was not
identified (MacDonald et al. 2008; Nabors et al. 2007). Hariharan et al.
carried out a clinical study using different doses of this peptide (600 or 1200
47
mg/ml) against different solid tumours, and concluded that Cilengitide has a
mild toxicity and can be used as antiangiogenic agent (Hariharan et al. 2007).
In a randomized phase II clinical trial by Reardon et al., patients were
assigned to receive either 500 mg or 2000 mg Cilengitide to determine
whether a higher dose would give a greater response. They recommended
Cilengitide as a safe and non-toxic agent and suggested that it could be used
in combination regimens for glioblastoma, because it had a high anti-tumour
activity (Reardon et al. 2008). Combination of Cilengitide with standard
chemotherapy such as temozolomide was safe and well tolerated (Stupp et
al. 2010).
A recent preclinical study by Mikkelsen et al. reported that treatment with
Cilengitide could lead to prolonged survival if it were given prior to
radiotherapy. Cilengitide increased the sensitivity of U-251MG cells and
HUVECs to radiation, leading to suppression of U-251MG growth and death
of the HUVECs in vitro. Cilengitide dramatically prolonged survival in vivo if it
was used with radiation rather than as a single agent. Further, Cilengitide
decreased the expression of p13k/pAkt, a gene in the NF-kB signalling
network with a recognised role in cell resistance to radiation in vivo
(Mikkelsen et al. 2009).
Cilengitide alone had no effect on breast tumour growth, whereas combined
radioimmunotherapy with Cilengitide (CMRIT) substantially increased the
cure rate (44% cure rate for CMRIT, 20% cure rate for RIT alone). CMRIT
48
increased apoptosis and decreased proliferation more than Cilengitide alone
(Burke et al. 2002).
Reynolds et al. found that nanomolar concentrations of Cilengitide enhanced
tumour growth and tumour vascularisation. When Cilengitide was
administered as a bolus at 200 mg/kg, its concentration in the plasma fell
rapidly from micromolar to nanomolar levels, which was then sustained for 16
to 24 hours after administration. This led to promotion of tumour growth by
direct stimulation of angiogenesis in vitro and in vivo. In β3/β5 deficient mice,
no increases in tumour growth or angiogenesis were observed. Cilengitide
therefore did not promote tumour growth directly but it acted in the cells that
expressed αVβ3/αVβ5 by enhancing cell migration in response to VEGF and
increased VEGFR-2/αVβ3 receptor recycling (Hodivala-Dilke 2008; Reynolds
et al. 2009).
1.2.5.3.5 Dual αVβ3/ αIIbβ3 integrin antagonism
Initial studies showed the antibody m7E3 (a monoclonal antibody created by
joining the Fab fragments of the mouse monoclonal antibody 7E3 to the FC
fragment of human Ig; it has high affinity and specificity for αIIbβ3/αVβ3)
reduced tumour growth and angiogenesis by blocking host αVβ3/αIIbβ3 in vivo
in β3 negative tumour xenografts in rat bone. m7E3 increased tumour-free
survival and decreased tumour volume and weight, but did not have any
effect on tumour metastasis (Engebraaten et al. 2009).
49
The c7E3 Fab fragment binds with equivalent affinity to αIIbβ3 and αVβ3, and
c7E3 can redistribute between αIIbβ3 and αVβ3 integrins. c7E3 completely
inhibited αVβ3-mediated cell adhesion of HUVECs and A375S2 cells to
fibrinogen and gelatin, and partially inhibited adhesion to fibrin and
vitronectin. c7E3 inhibited bFGF-mediated migration of HUVECs and A375S2
cells and also inhibited cell invasion through fibrin. c7E3 reduced proliferation
by inducing apoptosis of endothelial cells and completely inhibited
angiogenesis. Inhibition of both endothelial αVβ3 and the platelet αIIbβ3
receptor inhibited endothelial sprouting stimulated either by bFGF or by
platelets. c7E3 inhibited growth of human melanoma tumours in nude mice,
but blocking of expression of human melanoma cell-expressed αVβ3 integrin
without inhibition of mouse β3 integrin failed to completely inhibit tumour
growth in vivo. Taken together, these results suggested that a combined
blockade of both tumour cell expressed αVβ3 and host β3 integrin (endothelial
αVβ3 and platelets αIIbβ3) could enhance inhibition of tumour growth in vivo
(Trikha et al. 2002).
Another study investigated the contribution of αVβ3 and αIIbβ3 to MDA-MB-231
breast adenocarcinoma cells adhesion to endothelial cell ECM under flow
conditions. The αVβ3 antagonist (SB-273005) had no effect on tumour cell
adhesion to the ECM while αIIbβ3 antagonist lamifiban (Ro 44-9883)
decreased adhesion of tumour cell to ECM. However, the combination of two
antagonists (Ro 44-9883 and SB-273005) resulted in synergistic effect on
adhesion when compared to the effects of single antagonist. Further, the
previous finding of combining two antagonists was confirmed using c7E3

50
Fab, which targets both αVβ3 and αIIbβ3 integrins with the same affinity as
lamifiban Ro 44-9883 and SB-273005. The result was similar, indicating a co-
operation between αVβ3 and αIIbβ3 in the adhesion of MDA-MB-231 cells to
the ECM (Gomes et al. 2004).
Activation of platelets by thrombin leads to an increased capability for
migration by HeLa cells exposed to platelets. Migration and invasion of HeLa
cells decreased after inhibition of αIIbβ3 and αVβ3 integrin by eptifibatide and
RGDWE peptides respectively. HeLa cell migration depended on both
platelets and endothelial cells. Blocking αIIbβ3 on platelets and αVβ3 on HeLa
cells led to decreased adhesion of HeLa cells on monolayer HUVECs. The
study concluded that both αVβ3 and αIIbβ3 participated in the adhesion
interactions between cancer cells and endothelial cells (Liu et al. 2009).
51
Further anti-integrin agents in clinical use and advanced development
Single antagonist Dual antagonists Multiple antagonists
Anti-αVβ3/αVβ5
Anti-α2 Anti-α4 Anti-αIIbβ3 Anti-α5β1 αIIbβ3, αVβ3, αMβ2
Anti- α5β1,
Cilengitide SCH221153 S247 αVβ1, αVβ3,
22
E7820 Tysabri Abciximab αVβ5, αVβ6
22
Tirofiban 22
Eptifibatide
GLPG0187
JSM6427 Volcoximab PF-04605412
Figure 1.9 Anti-integrin antagonists
52
Table 1.3 Further anti-integrin agents in clinical use and advanced development
Agent Integrin Type of Disease indication Stage of Comments Ref
Target agent development
Abciximab αIIbβ3 Antibody Acute myocardial infarction Clinical use Adverse effect include bleeding, (Derer et al.
αV β 3 Unstable angina thrombocytopenia 2009; Jones
αMβ2 Percutaneous coronary Lead to double increase the need for red et al. 2008;
intervention (PCI) blood cells and platelets transfusion Kim et al.
2006;
Schwarz et
al. 2002)
Eptifibatide αIIbβ3 Peptide Percutaneous coronary Clinical use Adverse effect include bleeding, (Giugliano
intervention (PCI) thrombocytopenia and severe hypotension et al. 2009;
Unstable angina Pathak et
Myocardial infarction al. 2008)
Tirofiban αIIbβ3 Small Percutaneous coronary Clinical use Adverse effect includes intracranial (Valgimigli
Molecule intervention (PCI) retroperitoneal bleeding, pulmonary et al. 2010)
Unstable angina hemorrhage, spinal-epidural hematoma.
Myocardial infarction
Tysabri α4 Antibody Multiple sclerosis Suspended Inhibits immune cells from crossing blood (Gani et al.
,
Crohn s disease from clinical vessels to reach the affected organ. 2008; Ismail
use Several patients with MS in a long-term et al. 2009)
clinical trial of Tysabri recently died from
progressive multifocal leukoencephalopathy
(PML).
Treatment possibly associated with
melanoma development.
53
Cilengitide αVβ3/ Peptide Glioblastoma Phase III (Reynold
αV β 5 s et al.
2009)
CNTO-95 αV Antibody Solid tumours Phase I Safe, partial activity against angiosarcoma (Mullamit
ha et al.
2007;
Ning et
al. 2008)
Vitaxin αV β 3 Antibody Lymphoma, Leukemia, Phase II (Brooks
Rheumatoid arthritis, solid et al.
tumours 1995; Cai
et al.
2006)
Volociximab α5β1 Antibody Renal cell carcinoma Phase II Inhibits tumour angiogenesis by blocking (Bell-
Melanoma binding of activated endothelial cells to McGuinn
ARMD fibronectin present in the extracellular matrix, et al.
thereby inducing apoptosis and inhibiting 2011;
tumour growth. Ricart et
It has efficacy against kidney cancer but al. 2008)
showed poor clinical activity as a single
agent to treat melanoma.
PF-04605412 α5β1 Antibody Solid tumours Phase I Inhibits endothelial cell-cell interaction (Li et al.
It has anti-angiogenic and anti-metastatic 2010)
effects.
54
Ro 27-2441 α4β1, α4β7 Small Asthma Phase II (http://clin
molecule completed icaltrials.
gov/show
/NCT000
48022)
EMD525797 αV Antibody Colorectal and ovarian Phase I (Manfred
carcinoma with liver Wirth
metastasis 2013)
IMGN388 No target Antibody Solid tumours Phase I (Vater
revealed by conjugate 2008)
company
GLPG0187 αV β 1 Small Anti-angiogenic Phase I Inhibits osteoclast bone resorption and (van der
αV β 3 molecule Anti-metastasis of prostate angiogenesis in vitro and in vivo. Horst et
αV β 5 and breast cancer to the Inhibits the progression of bone metastasis al. 2010)
αV β 6 bone and the formation of new bone metastasis
α5β1 during the treatment.
JSM6427 α5β1 Small Proliferative Phase 1 Inhibits proliferation of retinal cells. (Zahn et
molecule vitreoretinopathy completed al. 2009)
55
ATN -161 α5β1 Peptide Lung cancer Phase II PHSCN derived from PHSRN of human (Donate
αV β 3 (linear) Renal carcinoma fibronectin in which a cysteine residue et al.
replaces the arginine in the original 2008)
sequence.
PHSRN of fibronectin interacts with α5β1
integrin and increases its avidity for RGD
PHSRN- increases tumour growth
ANT-161 inhibits angiogenesis and tumour
progression.
It inhibits tumour growth and metastasis.
Efalizumab αLβ2 Antibody Psoriasis Suspended Associated with PML fatal side effect through (Frampto
Raptiva from clinical its immune suppressive properties n &
use Plosker
2009)
E7820 α2 Small Solid tumours Phase II It can be used for treatment of advanced solid (Funahas
molecule tumour in combination with cetuximab hi et al.
2002;
Mita et al.
2011;
Semba et
al. 2004)
56
1.3 Aims and objectives
New drugs for the treatment of cancer that can control the tumour
dissemination process are required. As has been reviewed here; the β3
integrin subfamily (αIIbβ3 and αVβ3) plays an important role in this process.
The hypothesis underlying this work is that dual αIIbβ3 and αVβ3 integrin
antagonism will have an enhanced effect by having a direct effect on β3-
expressing tumour cells (compared to antagonism of αIIbβ3 and αVβ3
separately), inhibiting cell migration and dissemination. Furthermore, through
targeting tumour cell interaction with endothelial cells and platelets, this will
also lead to inhibition of angiogenesis and metastasis.
The main aims of the work described in this thesis are to assemble a panel of
human tumour cell lines characterised for their integrin expression which will
be utilised to evaluate potential novel β3 integrin antagonists.
These aims will be achieved through the following objectives:
 The characterisation of αIIbβ3 and αVβ3 integrin expression in human
tumour xenograft tissue.
 Evaluation of the expression of αIIbβ3 and αVβ3 integrins in a panel of
human tumour cell lines with different integrin expression to use in cell
based assays to investigate the effects of β3 integrin antagonists.
 Development and validation of cell based assays to investigate the
effect of β3 inhibition on tumour cell migration.

57
 Evaluation of the effect of potential novel β3 integrin antagonists in
inhibition of human tumour cell migration.
58
human xenograft tissue in mice using immunohistochemical and
immunoblotting techniques
59
Chapter 2
2.1 Introduction
The development of effective targeted therapies for cancer relies on the
availability of in vivo models that have been characterised for expression of
the target (Teicher 2006). Preparation of an in vivo model can involve
subcutaneous implantation of a tumour cell line or orthotopic grafting at a site
where the tumour commonly metastasises; for example, into the lungs.
Subcutaneous transplantation models are considered effective for
investigating integrins. A study done by Gouon et al. used a subcutaneous
model to determine the difference in αVβ3 integrin expression in
subcutaneous vs. in vitro melanoma cells using HT-144 (weak expression of
αVβ3 integrin) and SK-MEL-2 (high expression of αVβ3 integrin) cell lines. The
HT-144 human melanoma cells showed up-regulation of β3 integrin, which
was correlated with MMP-9 expression, when the tumour was grown in nude
mice (Gouon et al. 1996). Trikha et al. found that subcutaneous implantation
of PC-3 and DU-145 cells, which express αIIbβ3 integrin, was tumorigenic but
not metastatic while an orthotopic in vivo model showed that DU-145 cells
were both tumorigenic and invasive (Trikha et al. 1998).
Tumour implants at a specific site in the model (orthotopic xenograft) produce
tumours with metastasis; these models permit study of compounds which
affect the tumour dissemination process in vivo (Bibby 2004). Such models
have been useful in identifying new therapeutics (Kerbel 2003; Popkov et al.
60
2006) and in anti-integrin drug evaluation. For example, Landen et al. used
an orthotopic model to evaluate the effects of the etaracizumab (anti-αVβ3)
antibody on ovarian carcinoma cell lines such as SKOV-3, A2780, and HeyA.
Etaracizumab was effective in inhibiting tumour growth and invasion (Landen
et al. 2008). Allman et al. used an in vivo model to determine the effect of
cRGDfV peptide on melanoma cells, with A375 administered subcutaneously
into nude mice. cRGDfV was able to decrease melanoma growth and
invasion (Allman et al. 2000). Furthermore, Zhao et al. used an in vivo model
to appraise the effect of the small molecule PSK1404 (anti-αVβ3) peptide on
breast and prostate carcinoma (Zhao et al. 2007). An orthotopic model was
also used to reveal the functional state of αIIbβ3 integrin and its role in tumour
metastasis in vivo. Injection of DU-145 cells had membranous expression of
αIIbβ3 integrin in mouse led to development of tumour which infiltrated in the
surrounding tissue, and metastasised to lymph node. Inhibition of αIIbβ3
integrin with 10E5 antibody (specific for αIIbβ3 integrin) led to block interaction
of DU-145 cells with host tissue and therefore led to blockade of colonisation
of DU-145 cells in the lung (Trikha et al. 1998).
Human prostate carcinoma cell lines with a high expression of αVβ3 integrin
are more tumorigenic in vivo; for example, PC-3 cells with a high expression
of αVβ3 integrin are more tumorigenic when xenografted, and grow more
rapidly when implanted in nude mice than do LNCaP cells which have a low
expression of αVβ3 integrin (Taylor et al. 2012). Trikha et al. reported that a
tumour cell line with expressed αIIbβ3 integrin can develop a large tumour in
61
vivo due to the decreased apoptotic rate in the cells expressing αIIbβ3 integrin
(Trikha et al. 2002).
Several studies have reported a lack of expression of αVβ3/αIIbβ3 integrin in
normal tissues. For example, using immunoblotting, Zheng et al. found no
expression of β3 integrin in normal human prostate tissue, whereas human
prostate adenocarcinoma tissue expressed this protein (Zheng et al. 1999).
Immunohistochemistry of FFPE tissue samples revealed no expression of β3
integrin in normal breast tissue, while breast carcinoma tissue clearly
expressed β3 integrin (Pontes-Junior et al. 2009; Wang et al. 2010). Havaki
et al. found no expression of β3 integrin in normal breast tissue by
immunoblotting, whereas breast carcinoma tissue expressed β3 integrin
(Havaki et al. 2007).
In the present study, immunohistochemistry and immunoblotting were used
to characterise the expression of αVβ3/αIIbβ3 integrin in human tumour
xenograft mouse tissue generated in house. Previous studies have detected
integrin expression using immunohistochemistry on either fresh frozen tissue
sections (Brooks et al. 1995; Li et al. 2001) or paraffin embedded tissue
sections (Bisanz et al. 2005; Brooks et al. 1995).
The mouse LM609 monoclonal antibody that targets the αVβ3 integrin can be
used to detect expression of αVβ3 in angiogenic vessels and in the tumour
cells of many human cancers such as colorectal carcinoma, pancreatic
carcinoma, breast and glioma (Vonlaufen et al. 2001). LM609 has been
reported to successfully detect integrins in breast xenograft mouse tissue,

62
formalin-fixed, paraffin embedded tissue (Brooks et al. 1995) as well as in
frozen tissue sections. LM609 was also used in an in vivo model to detect the
expression of αVβ3 integrin in ovarian carcinoma (Landen et al. 2008). Thus
this antibody was chosen for use in this part of the study to characterise the
expression of αVβ3 integrin in human xenograft mouse tissue.
2.1.1 Aims and objectives
The overall aim of the work described in this chapter is to evaluate αIIb and
αV β 3 integrin expression in human tumour xenografts, using
immunohistochemistry and immunoblotting. The information obtained will be
of great use in guiding the selection of cell lines to be characterised for
inclusion in the in vitro screening panel and for model selection, once novel
antagonists eventually progress to in vivo screening (the latter is beyond the
scope of this thesis).
The aims will be addressed by pursuing the following objectives:
1- Characterisation of the expression of αIIb and αVβ3 integrin in formalin
fixed paraffin embedded human tumour xenograft tissue using
immunohistochemistry.
2- Characterisation of the expression of αIIb and αVβ3 integrin in fresh
frozen human tumour xenograft tissue using immunohistochemistry.
3- Characterisation of the expression of αIIb, αV and β3 integrin in fresh
frozen human tumour xenograft tissue using immunoblotting.
63
2.2 Materials and methods
2.2.1 Materials
All general chemicals were obtained from Sigma-Aldrich (Poole, UK), unless
otherwise specified. Primary antibodies and related secondary antibodies
were used in this part of the study to detect expression of α V, αIIb, β3, αVβ3
and β actin as summarised in Table 2.1. The blocking reagents were bovine
serum albumin (BSA) (Sigma-Aldrich, Poole, UK), normal rabbit serum
(NRS), normal goat serum (NGS), and normal horse serum (NHS) obtained
from Vector Laboratories Ltd (Peterborough, UK). Vectastain Avidin Biotin
Complex (ABC) and the M.O.M. immunodetection basic kit were from Vector
Laboratories Ltd (Peterborough, UK). 3,3-diaminobenzidine
tetrahydrochloride (DAB) chromogen (Sigma-Aldrich, Poole, UK).
2.2.2 Cell lines
The cell lines used to generate the tumour xenograft material: PC-3 prostate
carcinoma, DLD-1 and HCT-116 colon adenocarcinoma, M14 and UACC-62
melanoma, SKOV-3 and A2780 ovarian carcinoma, A549 and H460 human
non small cell lung carcinoma, and PANC-1 pancreatic carcinoma. These
were all obtained from American Type Culture Collection (ATCC, Middlesex,
UK) except for M14 and UACC-62, which was from the US National Cancer
Institute (NCI, USA). One xenograft was analysed per cell line.
64
Table 2.1 Primary antibodies and related secondary antibodies used in immunohistochemistry (IHC) and immunoblotting
(IMB)
Primary Receptor Type Source Secondary Source Secondary antibody Source
antibody antibody (IHC) (IMB)
BV4 Anti-β3 MMAb BV4. ab 7167, biotinylated horse BA2000, Vector - -
Abcam (Cambridge, UK) anti-mouse Laboratories Ltd
(Peterborough, UK)
B7 Anti-β3 MMAb Clone B-7, SC-46655, biotinylated horse BA2000, Vector Polyclonal rabbit anti P0260,
Santa Cruz Biotechnology anti-mouse Laboratories Ltd mouse immunoglobulins Dako Cytomation,
(Santa Cruz, USA) (Peterborough, UK) horseradish peroxidase Glostrup, Denmark
C20 Anti-αIIb PGAb SC-6602, Santa Cruz biotinylated rabbit BA500, Vector - -
Biotechnology anti-goat Laboratories Ltd
(Santa Cruz, USA) (Peterborough, UK)
Q20 Anti-αV PRAb SC-6617-R, Santa Cruz biotinylated goat BA1000, Vector Polyclonal goat rabbit P0448,
Biotechnology anti-rabbit Laboratories Ltd immunoglobulins Dako Cytomation,
(Santa Cruz, USA) (Peterborough, UK) horseradish peroxidase. Glostrup, Denmark
LM609 Anti-αVβ3 MMAb Clone LM609, MAB 1976, biotinylated horse BA2000, Vector - -
Chemicon-Millipore, anti-mouse Laboratories Ltd
Watford, UK (Peterborough, UK)
A1978 β-actin MMAb Sigma-Aldrich, Poole, UK - - Polyclonal rabbit anti P0260,
mouse immunoglobulins DAKO Cytomation,
horseradish peroxidase Glostrup, Denmark
Key: MMAb: monoclonal mouse antibody, PGAb: polyclonal goat antibody, PRAb: polyclonal rabbit antibody.
65
2.2.3 Methods
2.2.3.1 Collection of xenograft materials
Creation of in vivo tumour models was carried out in-house under a project
licence issued by the UK Home Office and in accordance with the UK
National Cancer Research Institute Guidelines for the Welfare of Animals
(Workman et al. 2010). Human tumour cell lines were xenografted in
immunodeficient mice (Harlan, Loughborough, UK) aged six to eight weeks
under general inhalation anaesthesia with recovery. Animal welfare and
tumour growth was monitored frequently and when the tumours were
approximately 500 mm3 in volume, the mice were sacrificed and the tumours
removed and processed as indicated below.
2.2.3.2 Slide coating
Adherence of tissue sections to microscope slides during
immunohistochemical procedures was improved by coating all slides with 3-
aminopropyltriethoxysilane (APES). Slides were washed for two minutes with
acetone, then immersed for two minutes in a freshly prepared solution of 2%
APES in acetone. The slides were washed for two minutes in running tap
water and left to dry at room temperature and then stored in a dust free
environment.
2.2.3.3 Tissue fixation, processing, embedding and paraffin sectioning
Harvested tissue was placed for 24 to 48 hours in 10% neutral buffered
formalin (VWR International Ltd. Poole, UK) before transferring to 70%

66
ethanol. Then tissue was placed in plastic cassettes (Raymond A. Lamb Ltd,
UK) along with labels giving histology numbers. The cassettes were
processed using an automatic tissue processor (Leica TP 1020, Leica
Microsystems, Nussloch, Germany). The tissue processing steps were
achieved by incubating the tissues in 70% ethanol (Fisher Scientific, UK) for
one hour, 80% ethanol for one hour, 95% ethanol for one hour, 100% ethanol
for one hour (twice, in different tanks), 100% ethanol for two hours, Histoclear
for one hour (twice, in different tanks), Histoclear for two hours and paraffin
wax for two hours and thirty minutes (twice, in different tanks).
Finally, the tissue was placed in labelled plastic moulds (Polysciences,
Warrington, USA) which were filled with molten paraffin wax and left to set on
a refrigerated surface. Wax blocks were stored at room temperature until
required for sectioning.
The wax rigidity was increased before sectioning by placing the wax blocks
containing tumour tissues at -20 °C for at least 30 minutes. Then, 5 µm
sections were cut from each wax block using a Leitz rotary microtome (Leica
RM2155, Leica Microsystems, Nussloch, Germany). Sections were collected,
placed in a warm water bath and collected onto APES coated slides. Slides
were placed on a hot plate for thirty minutes to one hour to increase adhesion
of tissue to the slides and then stored at room temperature.
67
2.2.3.4 Haematoxylin and Eosin staining
All steps were carried out at room temperature. Sections were de-
paraffinised prior to staining by passing the sections through xylene and
different grades of ethanol. Sections were immersed in two different changes
of 100% xylene, 50% xylene/ethanol, two changes of 100% ethanol, 90%
ethanol and 70% ethanol for five minutes in each solution.
Deparaffinised sections were stained with Harris’s Haematoxylin solution for
ten minutes. Slides were washed with running tap water, and immersed in
acidic alcohol (0.25% hydrochloric acid in 70% ethanol) for five seconds to
differentiate staining. Differentiated sections were washed in tap water and
placed for two minutes in Scott’s Tap Water (3.5 g sodium bicarbonate and
20 g magnesium sulphate dissolved in 1000 ml H2O). Sections were
counterstained with 1% aqueous Eosin for one minute and washed briefly
with running tap water. Sections were then drained to allow the excess water
to run off.
Stained sections were dehydrated through different grades of ethanol and
xylene, sections were immersed sequentially for two minutes in absolute
ethanol, three minutes in 50% xylene/ethanol and three minutes in 100%
xylene (twice, in different tanks). Finally, sections were mounted with
Distyrene-plasticiser-xylene (DPX) medium (VWR International Ltd. Poole,
UK), a coverslip was applied, and the sections were left to dry.
68
2.2.3.5 Immunohistochemistry
2.2.3.5.1 Immunodetection of αIIb and αVβ3 integrins expression in FFPE
human tumour xenograft sections
2.2.3.5.1.1 Sample preparation
Five µm thick FFPE tissue sections were mounted on APES coated slides.
Sections were deparaffinised and rehydrated by sequential incubation in
100% xylene for ten minutes (twice, in different tanks), 50% xylene/ethanol
for five minutes, absolute ethanol for five minutes (twice, in different tanks),
90% ethanol for five minutes, 70% ethanol for five minutes and distilled water
for five minutes. Sections were incubated with 1% or 3% hydrogen peroxide
(H2O2) for thirty minutes to block the endogenous peroxidase activity.
Sections were washed in PBS, or 0.15 M TBS, pH 7.35 for 3 washes for 5
minutes each.
2.2.3.5.1.2 Antigen retrieval
Using antigen retrieval, attempts were made to expose antigens masked by
fixation. In this part of the study, three types of antigen retrieval methods
were evaluated (citrate buffer, pepsin digestion and proteinase K).
A. Citrate buffer antigen retrieval
Slides were placed in a plastic microwavable container filled with citrate
buffer (10 mM citrate buffer, pH 6). The container was covered with clingfilm
and heated in a microwave (NN-E201W, Panasonic) at 600 W for 3-6 × 5
69
minutes each time. The level of the citrate buffer was checked and topped up
if needed each time. Microwaved slides were allowed to cool at room
temperature for thirty minutes then washed in PBS for 3 washes of 5 minutes
each.
B. Pre-warmed pepsin digestion method antigen retrieval
Sections were incubated at 40 ˚C with pre-warmed pepsin (0.65 mg/ml) in
PBS at for five minutes then washed with PBS for 3 washes of 3 minutes
each.
C. Proteinase K method antigen retrieval
Sections were incubated with 0.1 µg/µl Proteinase K (MB2005, Melford Lab,
Ltd, Suffolk) in distilled water (dH2O) for five minutes at room temperature
then washed in 1X Tris buffered saline (TBS, 1.2 g Tris-HCl, 0.28 g Tris
base, 4.4 g NaCl dissolved in 450 ml dH2O, adjusted to pH 7.4 then volume
adjusted to 500 ml with dH2O), for five minutes.
2.2.3.5.1.3 Antibody incubation
Sections were incubated with normal blocking serum diluted with PBS or TBS
(1.5%) for twenty minutes at room temperature in order to avoid non-specific
binding of the antibody. The blocking serum was selected according to the
species in which the secondary antibody was raised. The excess blocking
serum was carefully removed by blotting with a tissue and primary antibodies
applied (see text for antibody dilution and incubation time). Sections were
then washed in PBS (3 washes for 3 minutes each). Secondary antibody;

70
horse anti mouse, goat anti rabbit, or rabbit anti goat (dilution, 1:200) was
applied for thirty minutes at room temperature, washed as described for the
primary antibody, incubated with peroxidase-labelled streptavidin for thirty
minutes at room temperature and then washed in 3 changes of PBS for 3
minutes in each change. The immunoreactivity was visualised by incubating
sections with DAB chromogen solution for two to five minutes. Sections were
then washed in dH2O for five minutes and counterstained with Harris’s
Haematoxylin solution for twenty seconds. Sections were washed well in
running tap water, and then incubated in Scott’s Tap Water substitute for two
minutes.
Sections were dehydrated and cleared by incubation in 70%, 90%, absolute
ethanol (twice, in different tanks), 50% xylene/ethanol for three minutes each,
xylene (two different tanks for five minutes each). Finally, coverslips were
mounted with DPX medium and left to dry at room temperature. The slides
were examined by light microscope (Leica DMRB, Leica Microsystems,
Wetzlar, Germany).
2.2.3.5.2 Immunodetection of αIIbβ3 and αVβ3 integrins expression in
frozen human xenograft tumour
Fresh frozen human tumour xenografts (section 2.2.2) were sectioned (5 µm)
using a Cryostat (Leica CM1100, Leica Microsystems, Nussloch Germany)
71
and mounted on APES coated slides. Slides were air-dried at room
temperature then stored at -20 °C.
2.2.3.5.2.2 Labelling procedure
Slides were removed from the freezer and allowed to equilibrate at room
temperature for about twenty minutes. Sections were fixed with acetone,
methanol or 4% paraformaldehyde (PFA) for ten minutes, air-dried for ten
minutes then rehydrated in PBS for ten minutes. Sections were placed in a
humidified chamber and the non-specific binding of antibodies was blocked
by incubating with either normal serum (NS) diluted in PBS (1.5%) or 5%
BSA for twenty minutes at room temperature. The excess blocking solution
was removed and the primary antibody was applied (Table 2.1). Sections
were then washed in PBS (3 washes of 3 minutes each), incubated with
1:200 secondary antibody (Table 2.1) and washed as described for primary
antibody. Sections were incubated for thirty minutes with ABC peroxidase
labelled streptavidin at room temperature. The immunoreactivity was
visualised with DAB, counterstained with Harris’s Haematoxylin and mounted
with DPX as in section 2.2.3.5.1.3.
2.2.3.5.3 Immunodetection of β3 subunits and αVβ3 integrin expression
in FFPE and frozen human tumour xenograft sections by using
a mouse on mouse (M.O.M.) kit
The M.O.M. kit is specifically designed to localise mouse primary antibodies
on mouse tissues and should eliminate background staining in mouse tissue.
72
The M.O.M. kit was used during immunolocalisation for mouse primary
antibodies using FFPE and fresh frozen sections.
FFPE tissue sections 5 µm-thick were de-waxed and rehydrated as stated in
section 2.2.3.5.1.1. Following rehydration and antigen retrieval sections were
treated with 3% hydrogen peroxide (H2O2) in order to block endogenous
peroxidase activity and then washed twice in PBS for two minutes.
Frozen sections were prepared as in section 2.2.3.5.2.1.
2.2.3.5.3.2 Preparation of M.O.M. kit working solutions
The mouse Ig blocking reagent was prepared by adding two drops of stock
solution to 2.5 ml of PBS. The diluent solution was prepared by diluting 600
µl of protein stock solution with 7.5 ml of PBS. Biotinylated anti-mouse IgG
reagent was prepared by mixing 10 µl of stock solution with 2.5 ml of diluent
solution prepared as above.
2.2.3.5.3.3 Antibody incubations
Blocking reagent was applied to the sections before the application of the
primary antibody in order to avoid non-specific binding, and incubated for one
to two hours followed by washing with PBS (2 washes for 3 minutes each).
Sections were incubated with diluent for five minutes, the excess diluent
removed, and the primary antibody directly applied. All incubations were
undertaken in a humid chamber at room temperature for all the
73
immunolabelling steps, except for primary antibody, which was either at room
temperature or at 4 °C depending on the optimised conditions for each
antibody. The negative control was incubated with M.O.M. mouse blocking
solution for the same period of time. Sections were then washed in PBS (2
washes for 3 minutes each). M.O.M. biotinylated anti-mouse IgG reagent was
applied for ten minutes then sections were washed in PBS (2 washes for 3
minutes each). Sections were incubated with the peroxidase-labelled
streptavidin for thirty minutes at room temperature and then washed in PBS
(2 washes for 3 minutes each). Sections were counterstained with Harris’s
haematoxylin, differentiated in Scott’s Tap Water, dehydrated and mounted
as described in section 2.2.3.5.1.3.
2.2.3.6 Immunoblotting (Western Blotting)
2.2.3.6.1 Materials
Primary and secondary antibodies used in this part of the study are
summarised in Table 2.1 and section 2.2.1.
2.2.3.6.2 Tissue homogenisation
Human xenograft mouse frozen tissue samples (-80°C) were weighed and
cut into approximately 2 mm3 pieces. Each piece was placed in a 1.5 ml
microfuge tube with lysis buffer (1:3 w/v compared to the weight of tissue to
be lysed). The lysis buffer was either PBS buffer alone, or was assembled as
follows: 2x lysis buffer [100 mM Tris pH 8.8, 5 mM EDTA, 300 mM NaCl, 2%
Triton X-100], protease inhibitor cocktail (Roche, Mannheim, Germany) and
74
water. The samples were incubated on ice for thirty minutes, and then
homogenised using an Ultra Turax homogeniser (IKA Labortechnik,
Germany), for 3 pulses of 5 seconds with cooling on ice between each
homogenisation to avoid overheating. The homogenate was centrifuged for
different speeds and times, and both the supernatant and pellet collected.
The samples were aliquoted and stored at -80 °C for later use.
2.2.3.6.2.1 Bradford assay
Protein concentration was measured using the Bradford Assay (Bradford
1976). Serial dilutions for six protein standards (0.0625 - 1.0 mg/ml) were
prepared using 2 mg/ml of BSA dissolved in dH2O. After preparing serial
dilutions, 50 µl from each BSA dilution was added to 1.5 ml of Bradford
reagent. All tubes were vortexed and left to stand at room temperature for ten
minutes to allow Coomassie Brilliant Blue dye to bind to protein. Dilutions of
tissue lysates were prepared in dH2O (50 µl in total) and added to 1.5 ml of
Bradford reagent. 1 ml from each BSA dilution (standard) and lysate were
vortexed and transferred to cuvettes. Absorbance readings were recorded at
595 nm using a spectrophotometer (Thermo Scientific Multiskan Spectrum,
1500, Thermo Fisher Scientific, Finland) and a standard curve plotted. If the
sample absorbance was outside of the range of the standard curve, then
appropriate dilutions were carried out to bring the reading within the range of
standard curve (Figure 2.1).
75
1
0.9
Absorbance at 595 nm 0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0 0.2 0.4 0.6 0.8 1
Protein concentration mg/ml
Figure 2.1 Representative calibration curve using Bradford assay.

Each data point represents the mean +/- SD of 3 readings at 595 nm. Linear
regression analysis gives a straight line with r2= 0.99.
2.2.3.6.2.2 SDS-Polyacrylamide Gel Electrophoresis
Proteins were separated using pre-cast 8% polyacrylamide SDS gels (Bio-
Rad, Hertfordshire, UK). The gel was immersed in running buffer (5X Tris-
glycine electrophoresis buffer: 25 mM Tris, 250 mM glycine pH 8.3, 0.1%
SDS). 20-75 µg of protein was diluted in 20 µl SDS loading buffer (50 mM
Tris Cl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10%
glycerol) and heated at 70 °C for ten minutes followed by rapid incubation on
ice to denature proteins. 20 µl of sample was loaded into each well.
PageRulerTM Plus Prestained Protein Ladder marker (SM1811, Thermo
Fisher Scientific Inc. Rockford, USA) was loaded into first lane. The marker
76
had a range of size from 10 kDa to 250 kDa. The electrophoresis was run at
20 mA for 90 minutes to separate the proteins.
2.2.3.6.2.3 Western Blot for αV and β3 Integrins
Proteins were transferred from the SDS-polyacrylamide gel to nitrocellulose
membrane (GE Healthcare Life Sciences, Amersham, Buckinghamshire, UK)
using transfer buffer (2.9 g glycine, 5.8 g Tris base, 0.37 g SDS, 200 ml
methanol, 800 ml water). Protein transfer was run at 42 mA for 120 minutes.
After transfer, membranes were immersed in Ponceau S solution to verify
protein transfer, and the SDS-polyacrylamide gel was stained with
Coomassie Brilliant Blue to confirm that all proteins were transferred to the
membrane.
2.2.3.6.2.4 Ponceau S Stain
Protein blotted onto the nitrocellulose membrane was stained with Ponceau
S (1 g Ponceau S plus 50 ml acetic acid, made up to 1 l with distilled water)
for one to three minutes. The membrane was scanned (HP scan jet 2400)
then washed with distilled water three times in order to destain the
membrane. Figure 2.2A shows a representative Ponceau S membrane
where successful transfer has taken place.
2.2.3.6.2.5 Coomassie Brilliant Blue Staining
Complete protein transfer was confirmed by immersing SDS-polyacrylamide
gels in Coomassie Brilliant Blue solution (dissolved in methanol: H2O [1:1 v/v]
with 10% acetic acid) and fixing and staining them for one hour. The gels
77
were then destained in a solution of 40% methanol plus 10% acetic acid and
50% water. Gels were scanned with HP scan jet 2400. Figure 2.2B shows a
representative Coomassie Brilliant Blue stain gel where successful transfer of
most of the proteins has taken place.
A B
Figure 2.2 Confirmation of successful protein transfer

(A) Sample membrane stained with Ponceau S indicating that protein has
been transferred to the membrane from (B) SDS stained with Coomassie
Brilliant Blue and as can be seen most of the protein transferred to the gel.
2.2.3.6.2.6 Immunoblotting of nitrocellulose membrane with anti-β3 and
anti-αV integrin subunits
The nitrocellulose membranes were blocked with either 5% non-fat dried milk
in PBS with 0.05% Brij-35 solution (B4184) or 5% BSA in PBS- with 0.05%
Brij-35 solution for one hour at room temperature, followed by application of
primary antibody. The membrane was then washed with PBS with 0.05%
Brij-35 solution (3 washes for 10 minutes each) followed by application of
secondary antibody as specified in Table 2.1. The ECL plus Western blotting
detection system (NEL104, Perkin Elmer LAS) reagents A and B were mixed
78
in a 1:1 ratio and applied to the membrane for one minute after washing the
membrane with PBS with 0.05% Brij-35 solution (3 minutes for 5 minutes
each). The membrane was exposed to Kodak Scientific imaging film
(Amersham Biosciences) in the dark for five minutes. Films were developed
using IIford Rapid Multigrade developer (1/10 dilution) for three minutes,
rinsed with water and fixed in IIford rapid film and paper fixer solution (1/10
dilution) for two minutes. Expression of the integrin subunits was semi-
quantified by using Molecular Imager FX System (Bio-Rad Laboratories, Inc.,
UK) by dividing the signal intensity of the expression of αV or β3 protein by
the signal intensity of the expression of internal control β-actin.
79
2.3 Results
2.3.1 Detection of the expression of αIIbβ3 and αVβ3 integrin in human
tumour xenograft tissue by IHC
2.3.1.1 Tumour xenograft
Haematoxylin and Eosin (H & E) staining was carried out on M14 human
xenograft tissue sections. Microscopic examination revealed the tumours to
be poorly differentiated due to lack of organisational structure (Figure 2.3, A
and B).
A B
Figure 2.3 Haematoxylin and Eosin stain in M14 human melanoma

xenograft paraffin embedded tissues
Haematoxylin and Eosin stain shows histology of melanoma tissue. The
black arrows indicate tumour cells. Bar length = 200 µm (A) and 60 µm (B).
80
2.3.1.2 Studies on FFPE tissue samples
Detection of the expression of αIIbβ3 and αVβ3 integrin was attempted to
optimise by evaluating several antibodies in FFPE sections, and investigating
the effect of changing the antigen retrieval method, blocking procedure,
antibody incubation time and concentration.
Primary antibodies Q20 (anti-αV), C20 (anti-αIIb), BV4 (anti-β3), B7 (anti-β3)
and LM609 (anti-αVβ3) (Table 2.1) were used to characterise integrin
expression in FFPE tissue. However, assessment of all variables (as
summarised in Figure 2.4) revealed that all methods were unsuccessful for
immunodetection of αV, αIIb, β3 and αVβ3 (as shown in Figure 2.5). All
antibodies except Q20 (anti-αV) showed non-specific immunolabelling in both
sections with and without primary antibody. Q20 (anti-αV) had no
immunolabelling in the section without primary antibody whereas the section
with primary antibody had non-membranous immunolabelling.
81
Studies of FFPE tissue samples such as M14, UACC-62, PC-3, DLD-1 & HCT-116
1-Antigen retrieval
A-Proteinase K B-Pepsin digestion C-Citrate buffer
Damage tissue All primary antibodies: BV4 & B7 (anti-β3), Q20 (anti-αV), C20 (anti-αIIb) &
LM609 (anti-αVβ3) had non-specific immunolabelling in sections without
primary antibody. Q20 had non membranous immunolabelling.
Figure 2.5, A & B Figure 2.5, C & D
2- Blocking
A-Normal serum B-1-5 % BSA

Figure 2.5, E - H Figure 2.5, I - L
No change in the results with all antibodies
3- Primary antibody dilution :1:50 – 1:400
1 hour room temperature Overnight 4 C

Figure 2.5, M & N
4- Secondary antibody dilution 1:200-1:800. Figure 2.5, O & P
All attempts tried gave same result: BV4, B7, C20 &
LM609 gave non specific immunolabelling in
sections without primary antibody. Q20 had non
membranous immunolabelling.
Figure 2.4 Optimisation of IHC to detect αIIbβ3 and αVβ3 integrin

expression in FFPE human xenografts, using different methods
82
Proteinase K No primary antibody Pepsin No primary antibody
A B C D
B7 (anti-β3)
B7 (anti-β3)
Antigen retrieval
Citrate buffer No primary antibody
Citrate buffer No primary antibody
E F G H
Q20 (anti-αV)
B7 (anti-β3)
5% BSA No primary antibody 5% BSA No primary antibody
Blocking
I J K L
Q20 (anti-αV)
B7 (anti-β3)
Ab dilution / incubation
PAb Dilution No primary antibody PAb Dilution No primary antibody
M N O P
Q20 (anti-αV)
B7 (anti-β3)
Figure 2.5 Optimisation of IHC to detect αV and β3 integrin expression in

FFPE M14 human melanoma xenografts using Q20 and B7
M14 tissue (A-H & M-P), blocked with normal serum and immunolabelled
with primary antibodies: B7 (A-F) or Q20 (G&H) at dilution 1:50, overnight
incubation 4 °C. Proteinase K damaged M14 tissue immunolabelled with B7
(A) and without B7 (B). Pepsin digestion damaged M14 tissue with B7 (C)
and without B7 (D). A 30 minute citrate buffer antigen retrieval duration was
effective in sections immunolabelled with B7 (E) but non-specific
immunolabelling developed in sections without B7 (F). A 30 minute citrate
buffer antigen retrieval used in a M14 section immunolabelled with Q20 (G)
showed non-membranous immunolabelling whereas a section without Q20
(H) did not develop any non-specific binding. Under the same conditions with
blocking changed to 5% BSA, M14 immunolabelled with B7 (I) had
membranous immunolabelling but a section without B7 (J) developed non-
specific binding. Under the same conditions, non-membranous
immunolabelling developed in a section with Q20 (K) and a clean section
without Q20 (L). Dilution of B7 to 1:400 and incubation for 1 hour at room
temperature gave the same pattern of sections with (M) and without B7 (N).
Increasing the dilution of primary antibody to 1:400 and the secondary
antibody to 1:800 did not change the result in sections immunolabelled with
Q20 (O) and without Q20 (P). Sections shown are a representative example
of three independent experiments done using different primary antibodies on
different human tumour FFPE tissue. B7 in M14 tissue was chosen as a
representative example with the same results being seen for the other
primary antibodies used. Bar length = 60 µm.
83
2.3.1.3 IHC studies on Frozen Sections.
In order to avoid adverse effects of fixation and processing issues associated
with FFPE tissue, frozen sections were also evaluated. Frozen sections were
used with the same antibodies as used for the paraffin sections. Figure 2.6
summarises the steps of optimisation for different primary antibodies used to
characterise expression of αV, αIIb, β3 and αVβ3 integrins by IHC. The results
were similar to those obtained with FFPE tissues (Figure 2.7).
84
Studies of frozen tissue samples such as M14, UACC-62, SKOV-3, A2780, A549, H460, PANC-1, DLD-1, HCT-116 & PC-3
1- Differentfixatives tried
A-Acetone B-Methanol C-4% PFA
Damaged the tissue Q20 (anti-αV) had diffuse immunlabelling and clean section without primary
antibody .Other antibodies such as BV4 & B7 (anti-β3), C20 (anti-αIIb) & LM609
(anti-αVβ3) had immunoreactivity in both sections,with and withoutPAb.
2- Block endogenous peroxidase with 1%H2O2 for 1, 5 & 20 minutes
3- Block non specific binding of primary antibody
A-Normal serum B-5% BSA
4- Primary antibody at dilution 1:50 -1:400 incubated: 1 hour

at room temperature or at overnight 4 C.
5- Secondary antibody :1:200-1:800, 30 minutes at room

temperature then wash 3 5 minutes with PBS.
6-Avidin biotin complex (ABC) incubation

time decreased from 30 to 15 minutes
7- DAB time decreased from 5 to 2 minutes.
Figure 2.7
Figure 2.6 Optimisation of the protocol used to characterise expression

of αIIbβ3 and αVβ3 integrin in frozen human xenograft mouse tissue by
IHC
85
Acetone No primary antibody 1% H2O2 No primary antibody
A B G H
Block EP
Methanol No primary antibody 5% BSA No primary antibody
Different fixation
C D I J
Block NSB
4% PFA No primary antibody primary antibody No primary antibody
Primary antibody D/I
E F K L
Figure 2.7 IHC of αV integrin expression in human xenograft mouse

frozen tissue using Q20
IHC of M14 with anti αV antibody Q20: non-membranous immunolabelling
developed further to tissue damage in tissue fixed with acetone (A & B),
methanol (C & D) and diffuse immunolabelling in tissue fixed with 4% PFA,
blocked with NGS and incubated with 1:50 Q20 for overnight incubation at 4
°C (E & F). The immunolabelling of M14 tissue blocked with 1% H2O2 for
twenty minutes (G & H) did not change when blocking was changed to 5%
BSA (I & J) or when the Q20 incubation time was reduced to one hour at
room temperature and was diluted to 1:400 (K & L). EP: endogenous
peroxidise, NSB: non-specific binding, D/I: dilution/incubation. Bar length =
60 µm.
One reason why the use of antibodies for αV, αIIb, β3, and αVβ3 integrin was
unsuccessful for immunodetection in FFPE human tumour xenograft and
frozen tissues that is the use of mouse antibodies to detect the expression in
86
mouse tissue led to non-specific binding developing in sections without
primary antibody. Therefore, the next step was to address this issue using a
kit specifically designed to allow the use of mouse antibodies on mouse
tissues.
2.3.2 Immunodetection of β3 and αVβ3 integrin subunits using an
M.O.M. kit.
2.3.2.1 M.O.M. Immunodetection of β3 and αVβ3 integrins in FFPE
sections.
The kit was first used on FFPE sections, where M14 sections were incubated
with B7 (anti-β3), BV4 (anti-β3) or LM609 (anti-αVβ3), (changing variables as
shown in Figure 2.8). Different conditions were tested in order to optimise
final conditions of the above primary antibodies, such as dilution and
incubation: B7 gave concentration-dependent membranous immunoreactivity
at dilutions of 1:50 and 1:200. However, non-specific binding developed in
the blood vessels in the sections incubated with the blocking serum instead
of the B7 primary antibody, whereas sections without primary antibody and
without a secondary antibody did not develop any non-specific binding. This
result indicates that the non-specific binding that developed in the negative
control (without primary antibody) without using the M.O.M. kit was due to the
reaction of mouse secondary antibody on mouse tissue (Figure, 2.9 A - F).
The results of experiments on M14 xenograft sections immunolabelled with
B7 were confirmed by immunolabelling other human xenograft tissue that
87
supposedly have different expressions of β3 integrin; PC-3, DLD-1, HCT-116
and H460. As shown in Figure 2.10, low expression of β3 was found in PC-3
(A and B), weak expression in DLD-1 (C and D) and H460 (G and H), and no
expression was observed in HCT-116 (E and F).
88
Studies of FFPE tissue samples such as M14, using M.O.M kit
Citrate buffer antigen retrieval
3 5 minutes cycle 3 6 minutes cycle
Block endogenous peroxidase
1 % H2O2 3 % H2O2
Primary antibodies B7 and BV4 (anti-β3) & LM609 (anti-αVβ3) used at dilution 1:50
1 hour room temperature Overnight 4 C
Secondary antibody
1:250 for 10 minutes
BV4 and LM609 developed non-specific binding in sections without primary antibody.
B7 developed non-specific binding in section without primary antibody mainly in blood vessels.
B7 used only
1- Section had 30 minutes antigen 2- Same treatment as 3- Same treatment as section 1

retrieval, 3% H2O2 treatment, section 1 except using except using PBS instead of
immunolabelled with B7, 1:50, 1 hour PBS instead of primary primary antibody and secondary
at room temperature. Secondary antibody. antibody.
antibody 1:250 for 10 minutes.
M14 tissue had membranous M14 tissue had non-specific M14 tissue clean no reactivity
expression of β3 integrin. binding in blood vessels only. observed.
Figure 2.8 IHC detection of αVβ3 and β3 integrin in human FFPE tumours
with an M.O.M. kit
89
A B C
D E F
Figure 2.9 IHC detection of β3 integrin expression using B7 with M.O.M.

kit in FFPE M14 melanoma human tumour xenografts in mouse
Membranous expression was observed in M14 sections treated for 30
minutes with citrate buffer as a method of antigen retrieval, blocked with 3%
H2O2, immunolabelled with 1:50 B7 for one hour at room temperature (C) with
clean section without primary antibody (B) just the blood vessels that showed
non-specific binding. The non-specific binding disappeared when the same
tissue (used in B & C) had no primary and no secondary antibody (A). 1:50
dilution of B7 overnight incubation at 4 °C, gave a high background signal (E)
while a section without B7 had non-specific binding in blood vessels (D).
Dilution of B7 to 1:200 and incubation at room temperature (F) changed the
intensity of signal in comparison to Figure C. Bar length = 60 µm.
90
No primary antibody B7: primary antibody
A C E G
B D F H
Figure 2.10 IHC detection of β3 integrin expression in FFPE human

tumour xenografts in mouse using B7 with M.O.M. kit
All tumour tissues were treated with citrate buffer as a method of antigen
retrieval for 30 minutes, blocked with 3% H2O2 and immunolabelled with B7
1:50 for 1 hour at room temperature. PC-3 (A & B), DLD-1 (C & D), HCT-116
(E & F) and H460 (G & H) were used as comparison with the expression of
β3 seen in M14 tissue in Figure 2.9, C. Low expression of β3 was detected in
PC-3 (A), very weak in DLD-1 (C) and H460 (G) and no expression in HCT-
116 (E). Sections without B7 (B, D, F, H) developed non-specific binding in
blood vessels in a similar manner to M14 (Figure 2.9, B). Bar length = 60 µm.
91
2.3.2.2 M.O.M. Immunodetection of β3 and αVβ3 integrins in frozen
sections.
It was hoped that the use of the M.O.M. kit would enable the characterisation
of the β3 expression in FFPE xenograft tissue. However, sections without
primary antibody still showed non-specific binding. The same antibodies used
on FFPE sections were used to characterise the integrin expression in frozen
sections using the M.O.M. kit and optimisation of conditions described in
Figure 2.11. The expression of β3 was detected in PC-3 using BV4 (anti-β3)
(Figure 2.12). B7 (anti-β3) and LM609 (anti-αVβ3) showed non-specific
immunolabelling in both controls. BV4 (anti-β3) was further used to
investigate the expression of β3 in other human tumour xenograft mouse
tissues such as UACC-62, A549, H460, PANC-1, A2780 and SKOV-3, but
non-specific positive immunolabelling resulted. The same non-specific
immunolabelling was obtained with B7 (anti-β3) and LM609 (anti-αVβ3).
92
Studies of frozen tissue samples using M.O.M
Fixation
Ice cooled acetone 4% PFA
Block with 0.1% H2O2 Block with 1% H2O2
BV4 (anti-β3) B7 (anti-β3) LM609 (anti-αVβ3)
Primary antibody 1:50 -1:400, 1 hour room temperature.
Non-specific immunolabelling developed in both controls
Increase blocking time from 1 hour to 2 hours
BV4: high membraneous expression with clean B-7 & LM609: non-specific immunolabelling developed in
section without primary antibody in PC-3. both controls.
Screen other tumours such as A549, H460,

SKOV-3, A2780, PANC-1 & UACC-62.
Not all tumours screened developed the same

membranous expression found in PC-3, further
to non-specific binding developed in section
without primary antibody.
Figure 2.11: IHC optimisation for αVβ3 and β3 integrin expression in

frozen sections using the M.O.M. detection kit.
93
Prostate carcinoma PC-3
BV4(anti-β3) No primary antibody
BV4(anti-β3) No primary antibody BV4(anti-β3) No primary antibody BV4(anti-β3) No primary antibody

Pancreatic carcinoma
A2780 - Ovarian carcinoma - SKOV-3

H460 - Lung carcinoma - A 549
PANC
Melanoma
UACC-62
Figure 2.12 Expression of β3 integrin in human tumour xenograft frozen tissues immunolabelled with BV4 (anti-β3) using
M.O.M. detection kit
Tissue was fixed with 4% PFA, treated with 1% H2O2, blocked for 2 hours and immunolabelled with 1:50 BV4 for one hour at room
temperature, PC-3 had a membranous immunolabelling and no non-specific binding developed in section without primary antibody.
Screening a panel of human tumour cell line xenografts in mouse did not give similar results. Non-specific immunolabelling
developed in both sections with and without BV4. Bar length = 60 µm.
94
2.3.3 Detection of the expression of αIIb, αV, and β3 subunits in
homogenised tumour xenograft mouse tissue by immunoblotting
Integrin expression was also evaluated using immunoblotting (IMB) to
characterise the expression of αIIb, αV, and β3 integrin in human xenograft
mouse tissue. IMB was optimised for each antibody, taking into consideration
the type of lysis buffer, centrifugation speed and time, and protein
concentration loaded in the gel. The optimisation steps are summarised in
Figure 2.13. After the conditions for protein extraction had been optimised,
the optimised method was used for detecting expression of αV and β3 integrin.
No expression of αIIb was detected, probably because the cell lines tested
expressed αIIb at negligible levels (in-house data). Once again, non-specific
binding developed in protein from xenograft tissues with B7 (anti-β3) whereas
human tumour cell lines (Chapter 3) did not develop any non-specific binding
under the same conditions (Figure 2.14).
95
Optimise protocol for protein extraction from human xenograft mouse tissue
1- Buffer
PBS Lysis buffer
2- Centrifugation for different speed & time
9000 rcf, 20 minutes 20000 rcf, 20 minutes 100000 rcf, 1 hour
Supernatant collected Pellet resuspended either in lysis buffer or PBS
PBS excluded Lysis buffer selected at 9000 rcf, 20 minutes
Protein concentration
7.5 µg 25 µg 50 µg
It was selected as an optimum extraction of protein using lysis buffer and

centrifuged using 9000 rcf for 20 minutes .
B7 (anti-β3), mouse antibody had non-specific binding with mouse tissue. Confirmed by incubation of the
membrane without primary antibody (B7) and another membrane without primary antibody and without
secondary antibody in presence of human tumour cell line that did not develop any non -specific binding.
Figure 2.13 Optimisation steps for extraction of protein from human

xenograft mouse tissue.
96
B-7 No PAb No PAb, no 2nd Ab
6- M14 Tissue
2- M14 Tissue
4- M14 Tissue
7- M14 Cell
3- M14 Cell
5- M14 Cell
1-Marker
132
Non specific binding 95
72
Figure 2.14 Expression of β3 subunit in M14 human xenograft mouse

tissue using B7 by IMB
β3 expression was detected on the blot at 125 kDa. B7 mouse antibody had
non-specific binding with M14 human xenograft tissue (Lane 2). Non-specific
binding to mouse protein was confirmed by incubation the membrane without
B7 (Lane 4) and another membrane without B7 and without secondary
antibody (Lane 6). Protein extracted from M14 cells grown in vitro did not
demonstrate any non-specific binding (Lane 3, 5 and 7).
97
2.3.3.1 Expression of αV and β3 integrin in homogenised human
xenograft mouse tissue compared to negative control tissue
and human tumour cells
Once blotting conditions were optimised for each antibody, the expression
level of αV and β3 integrin subunits was evaluated in a panel of human
tumours. Figure 2.15 shows a representative blot and analysis using the
Molecular Imager FX System.
98
Cell Tissue Cell Tissue
Mouse liver
Mouse liver
UACC-62
UACC-62
HCT-116
HCT-116
Marker
DLD-1
DLD-1
HT-29
HT-29
PC-3
PC-3
M14
M14
M14
M14
αV
132
92 A B
132
β3
92
72 C D
β-actin 1
3
Expression of αv and β3 integrin
αV
αV
related to β-actin control
2.5 β3
β3
2
106
1.5
0.5
C: cell, T: tissue
Figure 2.15 Expression of αV and β3 integrin in homogenised tissue

In all membranes, 20 µg of M14 and HT-29 cell protein were loaded and 50 µg
of homogenised tissue. A: Expression of αV detected using Q20 antibody, B:
membrane treated same as A, but with PBS instead of Q20. C: Expression of
β3 detected using B7 antibody as bordered with red rectangle, D: membrane
treated with PBS instead of B7; anti-mouse secondary antibody shows non-
specific binding of mouse antibody on mouse tissue. Films were
semiquantitatively analysed by Bio-Rad, Molecular Imager FX.System. While
αV was similarly expressed in all human xenograft tissues, β3 showed a range
of expression with M14 and UACC-62 showed the highest expression followed
by PC-3 and weaker expression was seen in DLD-1 and HCT-116 cells.
Normal mouse liver tissue was negative for human integrins and used as a
control here since it had non-specific binding with B7. Human tumour cell lines
M14 and HT-29 used as a positive and negative control for β3 respectively.
Results in the graph calculated from three independent experiments.
99
2.4 Discussion
The overall aim of this part of the study was to characterise αIIbβ3 and αVβ3
integrin expression in human tumour xenograft tissue in mice. This is
because the ultimate goals of the drug discovery project of which this thesis
is a part require in vivo models that can be used to test the efficacy of novel
integrin antagonists as drug targets in cancer therapy, and also to guide
selection of cell lines for in vitro studies.
The expression of αIIb and αVβ3 integrin in tissue can be evaluated by
immunohistochemistry and immunoblotting (only αIIb subunit was investigated
because it hetrodimer with β3 subunit only so expression of αIIb subunit and
β3 indicated the expression of αIIbβ3). However, immunohistochemistry has
been the more widely used of the two techniques reported in the literature
because it gives information about specific location of integrin in tissue. In a
study on clinical human tumours, archival FFPE tissue was utilized and
immunohistochemistry was able to characterise the expression of αVβ3
integrin in glioma tissue, where it was associated with poor prognosis
(Schittenhelm et al. 2013). Bisanz et al. used immunohistochemistry to detect
the expression of αV subunit. The study treated PC-3 cells with anti-αV siRNA
inhibitor and indicated role of αV subunit mediated interaction of prostate
cancer cells with bone stroma when the tumour cells were implanted in
tumour as xenografts (Bisanz et al. 2005).
The expression of αIIbβ3 and αVβ3 integrin was initially investigated in FFPE
human xenograft tissues using immunohistochemistry. However,

100
immunohistochemistry was unsuccessful due to non-specific immunolabelling
that developed in the controls.
The problems experienced with immunohistochemistry may have been due
to masking antigenic epitopes; thus, different antigen retrieval methods were
tried. The use of citrate buffer as a method for antigen retrieval in human
xenograft tissue was unsuccessful. As shown in the results section, the use
of different primary antibodies did not reveal any that worked with the citrate
buffer microwave method of antigen retrieval. Therefore, pepsin and
proteinase K digestion were also tried.
Initially, LM609 was used to immunolabel different human xenograft mouse
tissue treated with pepsin, but pepsin digestion was unsuccessful with LM609
and also with other primary antibodies tried, such as B7 and BV4 (anti-β3),
C20 (anti-αIIb), and Q20 (anti-αv). Proteinase K antigen retrieval methods led
to tissue damage. All three different methods of antigen retrieval were
ineffective, due to the non-specific immunolabelling that developed in both
controls. The problem with antigen retrieval seen in this study has been
reported previously, where the embedding of tumour tissue in paraffin after
fixation can block integrin detection, requiring the use of an antigen retrieval
method to expose the integrin receptor to facilitate immunolabelling of the
integrin receptor with the primary antibody (Liapis & Hutton 1997). The failure
of the citrate buffer method in the present study was similar to that reported
in another study where the citrate buffer antigen retrieval method was
101
unsuccessful for integrin detection in formalin-fixed tissue (Cattoretti et al.
1993).
The non-specific binding observed in this study likely resulted from cross-
reactivity between the secondary antibody and endogenous mouse
immunoglobulins. Both sections (with primary antibody and without primary
antibody) developed immunoreactivity. These findings were confirmed by
using different fresh frozen human xenograft tumour tissue, which also
developed the same non-specific binding.
The detection of αVβ3 integrin in FFPE tissue remains a challenge. Goodman
et al. have summarised the problems with currently available antibodies, the
non specific staining developed in formalin fixed paraffin embedded tissue,
and have developed new rabbit monoclonal antibodies for characterisation of
integrin expression in archival FFPE human tumour tissue. Goodman’s
results reflected the problem faced in the present study with currently
available antibodies; LM609, BV4, B7 (mouse monoclonal antibodies) which
failed to detect specific expression of αVβ3 and β3 integrin in FFPE tissues
(Boger et al. 2013).
The use of the M.O.M. immunodetection kit has been proposed as a solution
for these issues (Jin et al. 2007). Using a mouse primary antibody in a mouse
xenograft tissue produced background labelling. The M.O.M. kit is specifically
designed to localize mouse primary antibodies in mouse tissues and to
eliminate background immunolabelling. The M.O.M. kit has been used
successfully with LM609 to detect the expression of α Vβ3 integrin in the

102
frozen tissue of human IGROV-1 ovarian carcinoma nodules disseminated in
the peritoneal cavity of a mouse (Garanger et al. 2005). Here, β3 expression
was detected using BV4 (anti-β3) in cryo-sections, but not in paraffin
sections, probably because the integrin epitopes are affected by fixation. BV4
(anti-β3) showed β3 subunit membranous immunolabelling, which was
observed in a PC-3 frozen section with a M.O.M. kit, but the resulting
expression was not reproducible. Furthermore, no expression was detected
in other fresh frozen human tumour xenografts using the same protocol,
although they were expected to express β3; the heavy background obscured
the results further to non-specific immunolabelling developed in sections
without BV4 (anti-β3) as a primary antibody.
The search for the β3 subunit integrin in tissue was continued using another
antibody B7 (anti-β3), aiming to result in membranous immunolabelling with a
clean negative control with a lack of non-specific binding. B7 gave a
membranous immunolabelling, but without a clean negative control. Although
a M.O.M. kit was used, the outcome was not changed: the same results were
obtained with the use of mouse monoclonal antibodies BV4, B7, and LM609.
This indicated that either non-specific immunolabelling developed in both
controls was due to the use of mouse antibody on mouse tissue, or it was
due to the inability of the primary antibodies used here to detect the
expression of αIIbβ3/αVβ3 in FFPE and frozen tissue using
immunohistochemistry.
103
The results obtained in the present study indicate an inability to detect the
expression of αIIbβ3 and αVβ3 integrin in xenograft tissue using
immunohistochemistry. Liapis and Hutton used a panel of primary antibodies
to detect the expression of α2β1, αVβ3, α3, α4, α5, α6, β1, and β5 integrin in
FFPE human ovarian carcinoma, and reported that not all antibodies work
with the microwave citrate buffer method of antigen retrieval, although they
used clinical tissues (Laurens et al. 2009; Liapis & Hutton 1997; Singh et al.
2000).
The second reason for the failure of antibodies used in this study may be due
to difficulties in characterising the expression of αIIbβ3 and αVβ3 integrin in
tumours grown in mouse tissue using a mouse antibody. The observation
that indirect immunohistochemical detection with a primary antibody
homologous with the test tissues usually results in non-specific background,
has been mentioned in the literature; for example, Lu et al. reported that
binding of a Fc fragment of secondary antibody to a mouse tissue component
was the cause of background immunolabelling (Lu & Partridge 1998).
Due to the issues of non-specificity seen in the immunohistochemistry
experiments, immunoblotting was also utilised to detect the expression of
human αVβ3 integrin, in the presence of normal mouse tissue as a negative
control. Expression of αV and β3 subunits was successfully detected using
the immunoblotting technique in a panel of human tumour xenograft tissue;
the xenograft tissues had varying levels of expression of β3 whereas αV was
expressed in all human tumour xenograft tissues tested. Expression of β 3
104
was highest in melanoma xenograft tissue (UACC-62 and M14), followed by
prostate adenocarcinoma (PC-3), which had moderate expression, with very
weak expression detected in colonic carcinoma (DLD-1 and HCT-116).
These results were confirmed by comparing them with normal mouse liver
tissue that has no expression of human αV and β3 integrin subunits (Taylor et
al. 2012).
The B7 antibody was able to reveal the expression of β3 but gave an
additional band just below the expected position of a band for β3 expression,
indicating non-specific binding. In order to confirm the non-specific binding
developed with B7, several variables were tried such as incubating the
membrane without B7 as a primary antibody and incubating another
membrane without a primary antibody and without a secondary antibody. It
was obvious that normal mouse liver tissue and human xenograft mouse
tissue developed non-specific binding in a membrane incubated with anti-
mouse secondary antibody only, due to the reaction of anti-mouse on mouse
tissue.
Further confirmation came from using human tumour cell lines M14 and HT-
29, which did not develop any non-specific binding in the blot membrane
without B7 as a primary antibody. The lack of non-specific binding in cell lines
indicates it is a problem with proteins present in mouse tissue, as shown in
Figure 2.15.
In addition, rabbit antibody Q20 (anti-αV) did not show any non-specific
binding in membrane without Q20 as a primary antibody, either in normal

105
mouse liver tissue or in human xenograft mouse tissue. This further confirms
the results obtained with B7 (anti-β3).
The results convincingly revealed that M14, UACC-62, PC-3, HCT-116 and
DLD-1 expressed αV. The expression of the αV integrin subunit has been
detected by immunoblotting in other types of human tumour cells however,
homogenised human tissue is rarely utilised to detect the expression of
integrin using immunoblotting. A study done by Schnell et al. detected the αV
and β3 integrin subunits in homogenised glioma tumour tissue using
immunoblotting (Schnell et al. 2008). The non-specific binding observed in
this study was also noted by Gouon et al. when immunoblotting was utilised
to detect the expression of β3 integrin in HT-144 melanoma homogenised
human xenograft mouse tissues (Gouon et al. 1996).
The expression of β3 varies among the different tissues, with the highest
levels seen in melanoma cells such as UACC-62 and M14. Studies found
strong β3 integrin expression was associated with the presence of tumour
necrosis, increased tumour thickness, tumour ulceration, vascular invasion,
tumour cell proliferation and high level of cytoplasmic p-cadherin. Significant
association of both αV and β3 integrin with tumour necrosis was also
associated with reduced overall survival in nodular melanoma (Bachmann et
al. 2008; Hieken et al. 1999; Si & Hersey 1994). The expression of β3 is
significant in malignancy, and αVβ3 integrin expression could be an important
step with in situ melanoma development and invasion.
106
The expression of αV, β3 subunits and αIIbβ3 integrin had previously been
detected by other groups in human melanoma tissue embedded in paraffin
(Trikha et al. 1997). Goodman has confirmed earlier work that found no β3
integrin expression in colon carcinoma tissue (Goodman et al. 2012). This
supports this study result where very weak expression of β3 integrin in colon
carcinoma xenograft tissue DLD-1 and HCT-116, was observed using
immunoblotting, whilst the αV subunit was detected in the same tissue
through immunohistochemistry labelling. In addition, this previous study
supports the finding of the present study where the αV subunit was detected
in all human xenograft tissues. Since no expression of β3 integrin was
observed, the αV subunit must be present as a heterodimer with another β
subunit such as β1, β5 and β6 (Agrez et al. 1996).
2.5 Conclusion
In conclusion, αIIb, αV, β3, and αVβ3 integrin expression could not be detected
in human xenograft tissue grown in mice using immunohistochemistry.
However, using the immunoblotting technique, αV and β3 integrin expression
could be successfully characterised in the panel of xenograft mouse tissues.
Differential β3 expression was seen over the panel of tumours, while αV levels
were similar across the panel whereas αIIb could not be detected due to in
ability of the primary antibody to work with IMB.
107
a panel of human tumour cell lines
108
Chapter 3
3.1 Introduction
Evaluation of any novel targeted therapy requires careful selection of
appropriate models. In this thesis, the target is the β3 integrins. A panel of cell
lines with a range of expression levels of the different β3 integrins is therefore
required for the evaluation of novel integrin antagonists.
The expression of the β3 integrins is widely reported in the literature in
various human tumour cell lines and tissues, as reviewed in section 1.2.5.
Whilst the findings of the previous chapter may give some guidance for the
cell lines worthy of further investigation, the expression is not likely to be the
same in vitro as in vivo for all cell lines evaluated (Taylor et al. 2012). Thus,
this part of the study focused on characterisation of the expression in human
tumour cell lines grown in vitro.
Ideally, a panel for novel therapeutic evaluation should include: 1) cell lines
that express both αVβ3 and αIIbβ3 integrin; 2) cell lines that are negative for
both αVβ3 and αIIbβ3 integrin; 3) cell lines that express αVβ3 but are negative
for αIIbβ3; 4) cell lines that are negative for αVβ3 but express αIIbβ3 integrin.
The cell lines selected as models should ideally grow as a monolayer in cell
culture flasks whilst maintaining the specific integrin expression. They must
also have a reproducible expression of αIIbβ3 and αVβ3 integrins.
Optimisation of the protocols for the antibodies to be used in this part of the
study first requires a cell line known to express αVβ3 and αIIbβ3 integrin as a
109
positive control. A number of researchers have investigated the expression of
αVβ3 and αIIbβ3 in PC-3 cells, using different techniques for protein detection,
such as flow cytometry and immunoblotting (Cooper et al. 2002; Trikha et al.
1998; Wang et al. 2005). αVβ3 integrin plays an important role in prostate
cancer development and progression (Cooper et al. 2002; McCabe et al.
2007) and the expression of αVβ3 integrin has been detected in PC-3 cells
using the LM609 antibody (Arosio et al. 2009; Chatterjee et al. 2001). In
addition, Trikha et al. detected the expression of αIIb in PC-3 using the MAB
1990 antibody (Trikha et al. 1998).
The principle aim of this chapter will be to assemble a panel of cell lines that
can be used for screening novel antagonists. This will be done through
characterising integrin expression in human tumour cell lines.
The aims will be addressed using the following objectives:
1- Characterisation of the growth patterns of selected cell lines in order to
optimise seeding densities for further experiments.
2- Optimisation of the immunoblotting protocols for the antibodies used to
characterise the expression of αV, αIIb and β3 integrins.
3- Optimisation of the immunocytochemistry protocols for the antibodies
used to characterise the expression of αV, αIIb, β3, and αVβ3 integrins.
110
4- Evaluation of the expression of αV, αIIb, β3, integrin subunits in a panel
of cell lines using the immunoblotting and immunocytochemistry
protocols optimised in 2 and 3.
111
3.2 Materials and Methods
3.2.1 Materials
All general chemicals, media and media supplements were from Sigma-
Aldrich (Poole, UK) unless otherwise specified. Primary antibodies and
related secondary antibodies were used in this part of the study to detect
expression of αV, αIIb, β3 and αVβ3 as summarised in Table 3.1. The blocking
reagents (BSA, NRS, and NHS), and Vectashield hard-set mounting medium
with DAPI were all from Vector Laboratories Ltd (Peterborough, UK). All cell
lines used as summarised in Table 3.2 were stored in liquid nitrogen prior to
use.
3.2.2 Methods
3.2.2.1 Cell maintenance
The cell lines were selected to represent a range of tumour types (Table 3.2).
Cell culture procedures were performed in a sterile Micro-flow class II cabinet
(NUAIRE, biological safety cabinet, Plymouth, UK).
Cells were routinely maintained as monolayer cultures in 75 cm 2 cell culture
treated flasks (T75 flask) (Corning, Amsterdam, Netherlands) in 10 ml
complete Roswell Park Memorial Institute (RPMI) 1640 medium
supplemented with 10% foetal bovine serum (FBS) 200 mM L-glutamine and
100 mM sodium pyruvate.
112
immunocytochemistry (ICC) and IMB techniques.
PAb R Type Source 2nd Ab Source 2nd Ab Source
(IMB) (ICC)
Anti-β3 MMAb BV4 ab 7167, PRAM, P0260, Dako PRAM R0270,
Abcam, Ig HRP Cytomation, Igs, Dako
BV4
Cambridge, Glostrup, TRITC Cytomation,

UK Denmark Glostrup,
Denmark
Anti-β3 MMAb Clone B7, SC- PRAM, P0260, Dako PRAM R0270,
46655, Ig HRP Cytomation, Igs, Dako
Santa Cruz Glostrup, TRITC Cytomation
B7
Biotechnology Denmark Glostrup,

Santa Cruz, Denmark,
USA
Anti- PGAb SC-6602, Goat P0449, Dako PRAG F0250,
αIIb Santa Cruz Igs Cytomation, Igs, Dako
C20
Biotechnology Glostrup, FITC Cytomation,

Santa Cruz, Denmark Glostrup,
USA Denmark
Anti-αV MMAb SC-9969, PRAM, P0260, Dako PRAM R0270,
Biotechnology, Ig HRP Cytomation, Igs, Dako
P2W7
Santa Cruz Glostrup, TRITC Cytomation,

Biotechnology Denmark Glostrup,
Santa Cruz, Denmark
USA
Anti-αV PRAb SC-6617-R, PGAR, P0448, Dako GAR, A-11010,
Santa Cruz Ig HRP Cytomation, Alexa Invitrogen,
Q20
Biotechnology Glostrup, fluor California,

Santa Cruz, Denmark 546 CA, USA
USA
Anti- MMAb Clone LM609, - - PRAM R0270,
αV β 3
LM609
MAB 1976, Igs, Dako

Chemicon- TRITC Cytomation,
Millipore, Glostrup,
Watford, UK Denmark
Anti β- MMAb Sigma-Aldrich, PRAM, P0260, -
actin Poole, UK Ig HRP DAKO
A1978
Cytomation,
Glostrup,
Denmark
nd
Key: PAb: primary antibody, R: receptor, 2 Ab: secondary antibody, MMAb: monoclonal
mouse antibody, PGAb: polyclonal goat antibody, PRAb: polyclonal rabbit antibody, PRAM:
polyclonal rabbit anti mouse, Ig. Immunoglobulin, PGAR: polyclonal goat anti rabbit, PRAG:
polyclonal rabbit anti goat. TRITC: tetramethylrhodamine isothiocyanate, FITC: fluorescein
isothiocyanate. GAR: goat anti rabbit.
113
The cells were maintained at 37 °C in humidified conditions and a 5% CO2
atmosphere. When the cells attained 75 to 80% confluence, they were
passaged by discarding the medium and rinsing the contents of the flask
twice with 10 ml of Hank’s Balanced Salt Solution (HBSS), prior to
trypsinisation using 3 ml of a 0.25% trypsin-EDTA solution, and incubating at
37 ˚C for three to five minutes. When cells became detached from the flask,
10 ml of medium was added to deactivate the action of trypsin/EDTA. The
trypsinised cells were then centrifuged for five minutes at 1000 rcf at room
temperature (Heraeus Megafuge 1.0 centrifuge, DJB Labcare Ltd. UK). The
resulting supernatant was discarded and cell pellet was resuspended in 10
ml of fresh complete culture medium. Cells were then counted with 10 µl of
cell suspension placed in each chamber of a Neubauer Haemocytometer
(7201004, VWR International Ltd. Poole, UK). An inverted microscope
(Olympus, CK2, x-20 objective lens magnification) was used to count cells in
ten 1 mm2 areas of the haemocytometer and the mean count was calculated.
Cell counts were then expressed as (mean cell count) × 104 cells/ml medium,
and the required amount of cell suspension was seeded into new flasks
containing the appropriate amount of complete medium.
114
Table 3.2 Cell lines
Cell line Origin Identification code
A549 Human non small cell lung carcinoma ATCC CCL-185
(NSCLC)
DLD-1 Human colorectal adenocarcinoma ATCC CCL-221
DU-145 Prostate adenocarcinoma ATCC HTB-81
H460 Human NSCLC ATCC HTB-177
HCT-116 Human colorectal carcinoma ATCC CCL-247
HT-29 Human colorectal adenocarcinoma ATCC HTB-38
M14 Human melanoma NCI-0507466
MCF-7 Human breast adenocarcinoma cell line ATCC HTB-22
from metastatic site: pleural effusion
PC-3 Human prostate adenocarcinoma derived ATCC CRL-1435
from grade IV cancer metastasised to bone
SK-MEL-2 Human malignant melanoma ATCC HTB-68
UACC-62 Human malignant melanoma NCI-0507381
Key: ATCC: American Type Culture Collection. NCI: National Cancer Institute
3.2.2.2 Tumour cell line growth kinetics
The growth kinetics of all the tumour cell lines used in this study were
characterised to determine when cells would be in a log phase and hence be
functionally active. Cells were seeded at 1 × 104 cells/ml in 5 ml fresh
complete medium in T25 flasks, and were maintained at 37 °C, 5% CO2 as
described above. Cell counts were performed over 7 days and growth curves
plotted. Growth curves were carried out in this study using cells from different
passages. Readings are reported as the average (± SD) recorded from three
different flasks per day.
3.2.2.3 Immunoblotting
Immunoblotting (IMB) was carried out using whole cell lysates in order to
characterise the expression of αV, αIIb, and β3 integrin in a panel of human
tumour cell lines.

115
3.2.2.3.1 Cell Lysis
Once the cells lines reached 70 to 80 % confluence, cells were scraped from
the flask surface after a gentle wash with HBSS. Cells were centrifuged
(Eppendorf centrifuge, G417R, Natheler-Hizz, Hamburg, Germany) for five
minutes at 1000 rcf, the cell pellet was washed three times with PBS for four
minutes with centrifuging (4600 rcf) after each wash. Lysis buffer (2X lysis
buffer [100 mM Tris pH 8.8, 5 mM EDTA, 300 mM NaCl, 2% Triton X-100],
protease inhibitor (Complete mini EDTA-free (Roche, Mannheim, Germany)
and water) was applied for one hour on ice, after which sonication (Philip
Harries Scientific Sonicator, Scientific Laboratory) was carried out for two
pulses of five seconds each. Sonicated cells were centrifuged at 9000 rcf for
ten minutes at -20 °C, after which the cytosolic supernatant of the lysed cells
was collected and the cellular pellet resuspended in fresh lysis buffer. Since
functional integrin receptors span the cell membrane, but can also be
endocytosed or present in a storage pool within the cell both the plasma
membrane (cell pellet) and cytosolic content were collected here for further
confirmation of their integrin expression.
3.2.2.3.2 IMB of nitrocellulose membrane with different anti αV, αIIb and
β3 integrin antibodies
The Bradford assay, SDS-polyacrylamide gel electrophoresis and
immunoblotting were carried out using the same conditions as described in
Chapter 2. In this part of the study, the primary antibodies were optimised to
characterise expression of αIIb, αV and β3 integrin in human tumour cell lines.

116
With the initial optimisation protocol for each antibody, the nitrocellulose
membranes were blocked with either 5% non-fat dried milk in PBS-buffered
saline (with 0.05% Brij, 35 solution), or 5% BSA/PBS, for one hour at room
temperature. Then, the primary antibody as summarised in Table 3.1 was
applied at different dilutions ranging from 1:200 to 1:5000 for either one hour
at room temperature or overnight at 4 °C; each antibody had its own optimum
dilution and incubation condition (Table 3.4). The membrane was washed
with PBS (0.05% Brij) (3 washes of 10 minutes each). The recommended
secondary antibody (Table 3.1) was applied in the dilution range from 1:1000
to 1:5000 for one hour at room temperature followed by washing with PBS
(0.05% Brij) (3 washes for 5 minutes). The ECL Plus Western blotting
detection system (NEL104, Perkin Elmer LAS) reagents A and B were mixed
in 1:1 ratio and applied to membrane for one minute. The membrane was
exposed to Kodak Scientific Imaging film in the dark for five to thirty minutes.
Films were developed using IIford Rapid Multigrade developer (1/10 dilution)
for three minutes, rinsing with water and fixing in IIford Rapid Film and Paper
fixer solution (1/10 dilution) for two minutes. The immunoblotting was
repeated three times to confirm the reproducibility of the optimised protocol.
117
3.2.2.4 Immunocytochemistry (ICC)
3.2.2.4.1 Detection of αV, αIIb and β3 subunits, and αVβ3 integrin
expression in different tumour cell lines by ICC
3.2.2.4.1.1 Materials
All materials used here were the same as described in section 3.2.1 with the
addition to the primary antibody panel of LM609, anti-αVβ3 integrin.
3.2.2.4.1.2 Cell preparation
In order to detect the expression of αV, αIIb and β3 subunits and αVβ3 integrin,
1 × 104 to 1 × 105 cells/ml were seeded on autoclaved 22mm × 22mm cover
slips in a six-well plate and incubated at 5% CO2, 37 ˚C until cells were seen
adhering to the cover slip when viewed under the microscope no more than
12 hours. The medium was removed and the cells washed for two washes of
two minutes with 1 ml HBSS.
Cells were fixed with either ice pre-cooled methanol for twenty minutes at -20
°C, 1% PFA or 4% PFA for ten minutes at room temperature, or four to five
dips with ice pre-cooled acetone. The fixative was removed and the cells
allowed to air dry. Fixed cells were either used for immunolabelling directly or
stored at -20 °C. Following rehydration, blocking was followed by direct
application of primary antibody (Table 3.1) at different dilutions and
incubation conditions followed by washing for 3 washes for 5 minutes each
(Table 3.3). The secondary antibody (Table 3.1) was then applied at different
incubations times after labelling with secondary antibody, and cells washed
118
for 3 washes for 5 minutes each as shown in Table 3.3. Cells were mounted
with Vectashield with DAPI and kept at 4 °C until dry and examined by
fluorescence microscope (Leica DMRB, Leica Microsystems, Wetzlar,
Germany).
3.2.2.4.1.3 Confocal microscopy
Confocal microscopy was used in order to confirm the expression observed
by fluorescent microscope. Cells were prepared as in section 3.2.2.4.1.2 and
were analysed and images captured with a Zeiss LSM510 confocal system
attached to an Axiovert 200 M inverted microscope using LSM510 software
(all from Zwiss, Welwyn Garden City, UK).
3.2.2.4.1.4 ICC analysis
Semi-quantitative analysis of integrin expression in the tumour cells was
done by counting cells in 10 fields at 40 magnification, and giving a score to
the labelling of each cell dependent upon its intensity. The expression
intensity scoring scheme was confirmed by an independent observer. The
high expression was designated as +++, moderate ++, low + and weak ±.
The mean intensity was calculated for each sample.
119
Table 3.3 Conditions investigated while optimising detection of expression of αV, αIIb, β3 and αVβ3 integrin in human tumour
cell lines using ICC.
PAb Anti-β3 subunit, BV4 Anti-β3 subunit, B7 Anti-αV subunit, Q20 Anti-αIIb subunit, C20 Anti- αVβ3 integrin, LM609
Rehydration PBS 5 minutes, R.T. PBS 5 minutes, R.T. PBS 5 minutes, R.T. PBS 5 minutes, R.T. PBS 5 minutes, R.T. or
PBS + 0.1% Triton 5
minutes, R.T. or 4 °C
Blocking 2 & 15% NRS/PBS, 2% NRS/PBS, 10 2% NGS/PBS, 10 2% NRS/PBS, 10 2% NRS/PBS, 10 minutes,
10 minutes, R.T. minutes, R.T., or 5% minutes, R.T. or 5% minutes, R.T. or 1- 5% R.T. 5% BSA/PBS, 1 hour
BSA/PBS, 1 hour. R.T. BSA/PBS, 1 hour. BSA/PBS, 1 hour R.T. R.T.
R.T.
Primary 1:50 - 1:800 1:20 - 1:400 1:25 - 1:400 1:20 - 600 1:50 – 1:400
antibody dilution
Primary 30 minutes & 1hour at 30 minutes & 1hour at 30 minutes at R.T., or 30 minutes & 1 hour at 30 minutes & 1 hour at R.T.,
antibody incubation R.T., or 1 hour at 37° R.T., or 1 hour at 37 1 hour at 37° C, or R.T., or 1 hour at 37 or 1 hour at 37 °C, or
C, or overnight at 4 °C °C, or overnight at 4 °C overnight at 4 °C °C, or overnight at 4 °C overnight at 4 °C
Secondary antibody PRAM, TRITC PRAM, TRITC GAR IgG, Alexa fluor PRAG, FITC, 1:50 in PRAM, TRITC
dilution 1:50/PBS 1:50/PBS 546, 1:50 in PBS, or PBS, or1:50 in 5% 1:50 in PBS, or 1:50 in 5%
or 1:50/5%BSA/PBS
1:50 in 5% BSA/PBS BSA/PBS, or 1:50 in BSA/PBS
1% BSA/PBS
Secondary antibody 30 minutes to 1 hour in 30 minutes to 1 hour in 30 minutes to 1 hour 30 minutes to 1 hour in 30 minutes to 1 hour in dark
Incubation dark dark in dark dark
Washing PBS (3 × 5 minutes) PBS or 5% BSA/PBS PBS or 5% BSA/PBS PBS or 5% BSA/PBS PBS or 5% BSA/PBS
after primary and (3 × 5 minutes) (3 × 5 minutes) or 1% BSA/PBS (3 × 5 minutes)
secondary antibody (3 × 5 minutes)
Key: PAb: Primary antibody, PBS: Phosphate buffered saline, R.T: room temperature, BSA: bovine serum albumin, NGS: normal goat serum. NRS: normal
rabbit serum, PRAM: polyclonal rabbit anti mouse, GAR: goat anti rabbit, PRAG: polyclonal rabbit anti goat.
120
3.3 Results
3.3.1 Characterisation of cellular growth kinetics
Growth rates varied between tumour cell lines as demonstrated by
differences in their growth curves and the duration of lag, log and plateau
phases.
Most of the cell lines had a lag phase, except DU-145 and DLD-1, which
were started in log phase and showed exponential growth from the first day.
Cell lines were in plateau phase once the cells became confluent except
DLD-1 and PC-3 which continued in log phase until day 7. Figure 3.1 shows
the growth curves obtained for the cell panel.
121
104/ml
Number of cell × 104/ml

Number of cell × 104/ ml
H460 HT-29

100.00 A549 100.00 100.00 MCF-7 100.00
10.00 10.00 10.00 10.00
Number of cell
1.00 1.00 1.00 1.00
0.10 0.10 0.10 0.10

0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6
Time (Days) Time (Days) Time (Days) Time (Days)
PC-3
Number of cell 104/ ml
104/ml
DLD-1

100.00 100.00 HCT-116 100.00 100.00 DU-145
10.00 10.00
Number of cell
10.00 10.00
1.00 1.00 1.00 1.00
0.10 0.10 0.10 0.10

0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6
Time (Days) Time (Days) Time (Days) Time (Days)
M14

100.00 UACC-62 100.00 SK-MEL-2
Number of cell × 104/ ml
100.00
10.00 10.00 10.00
1.00 1.00 1.00
0.10 0.10 0.10

0 2 4 6 0 2 4 6 0 2 4 6
Time (Days) Time (Days) Time (Days)
Figure 3.1 Growth parameters of human tumour cell lines

Growth parameters were obtained as described in section 3.3.1.
Values correspond to the mean ± SD of 3 independent experiments.
122
3.3.2 Detection of αV, αIIb, and β3 integrins using IMB
3.3.2.1 Optimisation of IMB
In order to optimise the IMB protocols for each antibody used here, different
variables were tried. These variables as shown in the flowchart in Figure 3.2
included: protein concentrations, blocking the non-specific binding of primary
antibody, primary antibody dilution and incubation time, and film exposure
time. All primary antibodies detected expression of integrin at a protein
concentration of 20 µg except C20 (anti-αIIb). C20 did not detect expression
even when using a protein concentration of 100 µg. B7 (anti-β3) and Q20
(anti-αV) were able to detect expression in membrane blocked with 5%
BSA/PBS whereas the rest of the antibodies worked only with 5% non-fat
milk. The different dilutions and incubations tried for primary antibodies
clearly showed that 1:2000 dilution, overnight incubation at 4 °C was
optimum for B7 (anti-β3) and Q20 (anti-αV) whereas 1:500 at room
temperature for two hours was optimum for the rest of the antibodies. All
antibodies detected expression after five minutes exposure time except C20
(anti-αIIb) which did not detect clear expression even if exposed for thirty
minutes. Thus no further work was carried out for this antibody and hence
attempts at detecting αIIb. Figure 3.2 shows an example of a representative
blot (three independent experiments were done for each antibody using
optimum conditions for each antibody as summarised in Table 3.4). From the
above results B7 (anti-β3) and Q20 (anti-αV) were identified as specific
antibodies that can be used to screen the expression of α V and β3 integrin in
123
a panel of different tumour cell lines using the optimised protocol as
summarised in Table 3.4.
IMB optimisation of primary antibodies used for detection αV, αIIb, and β3
1- Loading PC-3 different protein concentrations: 5, 10, 15, 20, 75 & 100 µg
Q20, P2W7, B7 & BV4 cannot detect expression < 20 µg C20 did not detect expression even at 100 µg
2- Blocked with 5% non fat milk/PBS or 5% BSA/PBS1 hour R.T
BV4, P2W75%
and non fat with
C20 work milk/PBS
5% non fat milk/PBS B7 and Q20 work with 5% BSA/PBS
3- primary antibody dilution / incubation
1:500 1:1000 1:2000 1:3000
2 hours R.T. Overnight 4 C
BV4, P2W7 & C20 at 1:500. B7 & Q20 at 1:2000

.
4- Secondary antibody (2nd Ab) dilution, 1:2000 to 1:5000 1 hour R.T
5- Film exposing time: 5, 10 15 & 30 minutes
BV4: double band at 2nd P2W7: multiple band, 2nd Ab C20: no clear band, 2nd Ab B7 & Q20: single band, 2nd
Ab1:5000 , 5 minutes exposing 1:5000, 5 minutes exposing 1:3000, 30 minutes exposing Ab,1:3000, 5 minutes exposing
αIIb αV β3
C20 P2W7 Q20 BV4 B7
132
95
72
No No Yes No Yes
Figure 3.2 Optimisation of IMB protocol.

Expression of αV, αIIb and β3 were detected in PC-3 cells using different
antibodies. Q20 and P2W7 were anti-αV, C20 anti- αIIb, and BV4 and B7 were
anti-β3. Protein concentrations, blocking conditions, PAb dilutions and
incubations and film exposing time were optimised for each antibody.
Key: Nc: no clear band. Mb: multiple bands. Db: double bands. Sb: single band.
124
Table 3.4 The optimised protocol for all antibodies used to detect the expression of αV, αIIb, and β3 subunit and β-actin
using IMB technique.
Immunolabelling BV4 anti-β3 B7 anti-β3 P2W7 anti-αV Q20 anti-αV C20 anti-αIIb β-Actin
conditions
Protein 20 µg 20 µg 20 µg 20 µg 100 µg 20 µg
concentration
Blocking 5% non-fat milk, 5% BSA/PBS, 5% non-fat milk, 5% BSA/PBS , 5% non-fat milk, 5% BSA/PBS ,
1 hour R.T. 1 hour R.T. 1 hour R.T. 1 hour R.T. 1hour R.T. 1 hour R.T.
Primary antibody 1:500 in 5% non- 1:2000 in 1:500 in 5% non- 1:2000 1:500 in 5% 1:5000
Dilution/Incubation fat milk, 5%BSA/PBS, fat milk, in 5% BSA/PBS non-fat milk, in 5% BSA/PBS
2 hour R.T. overnight 4 °C 2 hour R.T. overnight 4 °C 2 hour R.T. overnight 4 °C
Washing PBS PBS (0.05 % Brij), PBS (0.05 % Brij) PBS (0.05 % Brij) PBS (0.05 % Brij) PBS (0.05 % Brij)
(0.05 % Brij), (3 × 10 minutes) (3 × 5 minutes) (3 × 10 minutes) (3 × 5 minutes) (3 × 10 minutes)
(3 × 5 minutes)
Secondary PRAM 1:5000 in PRAM 1:3000 in PRAM 1:5000 in PGAR 1:3000 Goat Igs 1:3000 PRAM 1:5000
antibody non-fat milk, non-fat milk, non-fat milk, in non-fat milk, in non-fat milk, in non-fat milk,
Dilution/Incubation 1 hour R.T. 1 hour R.T. 1 hour R.T. 1 hour R.T. 1 hour R.T. 1 hour R.T.
Washing PBS (0.05% Brij), PBS (0.05% Brij), PBS (0.05% Brij) PBS (0.05% Brij) PBS (0.05% Brij) PBS (0.05% Brij)
(3 × 5 minutes) (3 × 5 minutes) (3 × 5 minutes) (3 × 5 minutes) (3 × 5 minutes) (3 × 5 minutes)
Exposing 5 minutes 5 minutes 5 minutes 5 minutes 30 minutes 2 minutes
Result Double bands Single band size Multiple bands Single band size No detectable Single band size
125 KDa 132 KDa band 42 KDa
Key: PGAb: polyclonal goat antibody. PRAM: polyclonal rabbit anti mouse. R.T: room temperature.
125
3.3.2.2 Screening different tumour cell lines for αV and β3 integrin
expression by IMB technique using the optimised conditions
for anti-αV, Q20, and anti-β3, B7, antibodies
In the preceding section, two antibodies were identified as giving reliable and
specific detection of integrin subunits, Q20, anti-αV and B7, anti-β3. These
were therefore used to screen the extended cell line panel. The expression of
αV and β3 was detected in the supernatant (Figure 3.3), as well as in the
pellet resuspended in lysis buffer (Figure 3.4) indicating expression of the
integrin subunits in both the cell membrane and cytosol. αV was expressed in
all cell lines with HCT-116 demonstrating a notably lower expression,
whereas β3 expression levels showed more variation between cell lines. The
melanoma cell lines UACC-62, M14 and SK-MEL-2 had the highest
expression of β3 subunit. Moderate expression of β3 was seen for the
prostate lines PC-3 and DU-145, whilst weak expression was seen in the
NSCLC lines, A549 and H460 followed by the colon lines DLD-1 and HCT-
116 and very weak in breast line MCF-7. As would be expected from earlier
studies (section 2.3.3.1), no expression was seen in the HT-29 colon line
which used here as a negative control.
126
A
SK-MEL-2
UACC-62
HCT-116
DU1-45
DLD-1
MCF-7
HT-29
H460
A549
PC-3
M14
M
M
132
92 αV αV
132
92
β3 β3
72
β-actin
B 3
β-6
to10
to β actin control
2.5
V and β3
αv and β3 xof10α6 related
2
control
1.5 αv
αV
Expression
related
1 β3
β3
actin
0.5
Expression of
Figure 3.3 Expression of αV and β3 integrin in a panel of human tumour

cell lines from a supernatant
A. Expression of αV and β3 integrin subunits in a panel of human tumour cell
lines. All cell lines expressed the αV integrin subunit. Expression of β3 integrin
was highest in M14 followed by UACC-62 and SK-MEL-2. Moderate
expression of β3 integrin was seen in the PC-3 and DU-145. A549, H460,
DLD-1 and HCT-116 and MCF-7 showed weak expression of β3 integrin. HT-
29 was negative with no clear band observed. αV integrin was expressed at
molecular weight 132 kDa and β3 at 125 kDa. Data shown are representative
of at least three independent experiments. Lane 1 is Page Ruler Plus
Prestained Protein Ladder marker range from 10 to 250 kDa but because of
a short exposure time of five minutes it is not clearly seen on the film. B.
Results are presented as mean ± SD of three independent experiments.
Films analysed using Bio Rad Molecular Imager FX. The expression of α V
and β3 band density and β-actin band density were subtracted from the film
background density. αV and β3 signal intensity were divided by β-actin signal
intensity to obtain relative expression levels.
127
A
SK-MEL-2
UACC-62
HCT-116
DU-145
MCF-7
DLD-1
HT-29
H460
A549
PC-3
M14
M
M
132
92 αV αV
132
92
β3 β3
72
β-actin
B 3
Expression of αV and β3 106
2.5
related to β actin control
1.5 αv
αV
1 β3
β3
0.5
Figure 3.4 Expression of αV and β3 integrin protein in a panel of human

tumour cell lines from a pellet resuspended in lysis buffer
A. Expression of αV and β3 integrin subunit in the panel of human tumour cell
lines pellets resuspended in lysis buffer. Expression in the cell pellet showed
that αV and β3 subunit levels correlated with the expression found in cell
supernatant (Figure 3.3) except UACC-62 and PC-3 cell lines where the
expression was higher in their supernatant. B. Results are presented as
mean ± SD of three independent experiments. Films were analysed as
described in Figure 3.3.
128
3.3.3 Detection of αV, αIIb, β3 and αVβ3 integrins using ICC
3.3.3.1 Optimisation of ICC
Whilst IMB identifies the expression of a specific integrin subunit within a cell
line, it does not give any information as to the localisation of the integrin
expression within the cell and further cannot detect intact heterodimers.
Therefore, the ICC technique was also used to evaluate the cell panel. In
order to optimise the detection of expression of αV, αIIb, β3 and αVβ3 by ICC,
several variables were evaluated as shown in Figure 3.5. The optimised
protocol for each antibody is summarised in Table 3.5.
129
Optimise protocol for PAbs to characterise expression of αVβ3 and αIIbβ3 in human tumour cell lines by
Different fixatives tried
ICM ICA 4% PFA 1% PFA
Block non-specific binding of PAb
15% NS 2 % NS 5 % BSA 1 % BSA
All PAbs had non- BV4: +VE All other LM609 +VE Q20 weak All other C20 weak All other PAbs
specific binding in with all PAbs -VE with 4% PFA with 4% PFA PAbs -VE with 1% PFA -VE
section without PAb fixatives
PAbs dilution 1:50 , incubated 1 hour R.T, 1 hour. 37 C and 4 C overnight
BV4: +VE at R.T & 37 C Q20, B7, P2W7 & LM609: +VE 4 C only with 4%PFA C20 +VE at 4 C with all fixatives
Dilution of 1:50 -1:800
BV4: can be detected B7 & C20: can be detected Q20, P2W7 & LM609 can
untill 1:600 at 1:50 & 1:100 only be detected untill 1:400
Q20 (anti-αV) P2W7 (anti-αV) C20 (anti-αIIb)
BV4 (anti-β3) B7 (anti-β3) LM609 (anti-αVβ3)
Figure 3.5 Detection of αV, αIIb, β3 and αVβ3 integrin in PC-3 using ICC.
Different antibodies: Q20 and P2W7 anti-αV, BV-4, and B7 anti-β3, C20, anti-
αIIb, and LM609, anti-αVβ3 integrin were used to immunolabel PC-3 cells
using the optimum conditions for each antibody as summarised in Table 3.5.
Bar length = 60 µm.
KEY: ICM: ice-cooled methanol, ICA: ice-cooled acetone, PFA: paraformaldehyde.
130
Table 3.5 Optimised protocols for all antibodies used to detect the expression of αV, αIIb, β3 subunits, and αVβ3 integrin in
cell membrane using ICC
Labelling P2W7 (anti-αV) Q20 (anti-αV) C20 (anti-αIIb) BV4 (anti-β3) B7 (anti-β3) LM609 (anti-αVβ3)
Conditions
Fixation 4% PFA, 4% PFA, 1 or 4% PFA, ICM, 20 minutes 4% PFA, 4% PFA,
10 minutes R.T. 10 minutes R.T. 10 minutes R.T. or at -20 °C 10 minutes at R.T. 10 minutes at R.T.
ICM 20 minutes
at -20 °C
Blocking 5% BSA/PBS, 5% BSA/PBS, 1% BSA/PBS, 2% NRS/PBS, 5% BSA/PBS, 5% BSA/PBS,

1 hour R.T. 1 hour R.T. 1 hour R.T. 10 minutes R.T. 1 hour R.T. 1 hour R.T.
PAb D/I 1:50 in 5% 1:50 in 5% 1:50 1:50 in 2% 1:50 in 5% 1:50 in 5%
BSA/PBS, BSA/PBS, in 1% BSA/PBS, NRS/PBS, BSA/PBS, BSA/PBS,
overnight 4 °C overnight 4 °C overnight 4 °C 30 minutes R.T. overnight 4 °C overnight 4 °C
2nd Ab D/I TRITC, TRITC, 1:50 TRITC, 1:50 TRITC,1:50 TRITC, 1:50 TRITC, 1:50 in 5%
1:50 in PBS, in 5% BSA/PBS, in 1% BSA/PBS, in 5%BSA/PBS, in 5%BSA/PBS, BSA/PBS,
30 minutes in dark 1 hour in dark 1 hour in dark 1 hour in dark 1 hour in dark 1 hour in dark
Washing PBS 5% BSA/PBS 1% BSA/PBS 5% BSA/PBS 5% BSA/PBS 5% BSA/PBS
(3 × 5 minutes) (3 × 5 minutes) (3 × 5 minutes) (3 × 5 minutes) (3 × 5 minutes) (3 × 5 minutes)
nd
Key: ICM: ice pre-cooled methanol, PAb: primary antibody, 2 Ab: secondary antibody, D/I: dilution/ incubation.
131
3.3.3.2 Evaluation of the effect of method of cell harvesting on integrin
expression
Trypsinisation of cells can affect integrin expression (Brown et al. 2007).
Since cells were harvested by scraping for IMB and trypsinisation for ICC,
any effect on the integrin expression was investigated to ensure correlation
between expression of integrins by both techniques. Both scraped and
trypsinised PC-3 cells were immunolabelled with LM609 (anti-αVβ3) following
the optimum ICC protocol (Table 3.5). As shown in Figure 3.6, there was no
obvious difference in αVβ3 integrin expression between scraped and
trypsinised PC-3 cells using ICC.
A B
Figure 3.6 Comparison of cell harvesting techniques

No difference was seen in αVβ3 integrin expression on the membrane of PC-3
cells which had been scraped (A) and trypsinised (B). Cells were
immunolabelled with LM609 using ICC. Bar length = 60 µm.
132
3.3.3.3 Screening different cell lines for expression of αV, β3, αIIb and
αVβ3 integrins by ICC technique.
A panel of tumour cell lines was tested for the expression of αV, β3, αIIb
subunits and αVβ3 integrin immunoreactivity according to the optimised
protocols (Table 3.5). Following optimisation of the antibodies for ICC, the
panel of cell lines was screened for expression of αV, β3, αIIb subunits and
αVβ3 integrin (Figure 3.7 and 3.8).
133
Q20, anti-αV C20, anti-αIIb B7, anti-β3 LM609, anti-αVβ3
A549
H460
DU-145
PC-3
M14
SK-MEL-2
Figure 3.7 Screening of the first part of the cell line panel with anti-αV
(Q20), anti-αIIb (C20), anti-β3 (B7), and anti-αVβ3 (LM609) using ICC.
The antibodies were used for immunodetection of α V, αIIb, β3 subunits and
αVβ3 integrin in the panel of human tumour cell lines (A549, H460, DU-145,
PC-3, M14 and SK-MEL-2) according to protocols optimised above. Apart
from C20, all samples were counterlabelled with DAPI (blue) to visualize cell
nuclei. Bar length = 60 µm
134
Q20, anti-αV C20, anti-αIIb B7, anti-β3 LM609, anti-αVβ3
UACC-62
MCF-7
DLD-1
HCT-116
HT-29
Figure 3.8 Screening of the second part of the cell line panel with anti-
αV (Q20), anti-αIIb (C20), anti-β3 (B7), and anti-αVβ3 (LM609) using ICC.
The antibodies were used for immunodetection of α V, αIIb, β3 subunits and
αVβ3 integrin in the panel of human tumour cell lines (UACC-62, MCF-7,
DLD-1, HCT-116 and HT-29) according to protocols optimised above. Apart
from C20, all samples were counterlabelled with DAPI (blue) to visualize cell
nuclei. Bar length = 60 µm.
135
3.3.3.4 Analysis of the expression of αV, αIIb, β3 and αVβ3 integrin in
different human tumour cell lines detected by ICC.
After screening a panel of human tumour cell lines for the expression of α V,
αIIb, β3 and αVβ3 integrin using ICC technique, the expression was as either:
high expression, moderate expression, low expression, weak expression or
no expression. Table 3.6 summarises the expression seen in all cell lines
based on three independent experiments for each cell line.
Table 3.6 Screening different tumour cell lines for the membranous
expression of αV, β3, αIIb and αVβ3 by ICC
Cell lines BV4 B7 Q20 LM609 C20
anti-β3 anti-β3 anti-αV anti-αVβ3 anti-αIIb
Lung cancer cell lines
A549 + ± +++ ± ±
H460 ± ± + ± ±
Colon cancer cell lines
DLD-1 ++ ± +++ ++ +
HCT-116 ++ ± + ± +
HT-29 ± - ++ - ++
Prostate adenocarcinoma
cell lines
DU-145 ++ ++ ++ ++ ++
PC-3 +++ ++ ++ +++ +
Melanoma cell lines
M14 + +++ ++ ++ ±
SK-MEL-2 +++ ++ +++ ++ ±
UACC-62 ++ +++ +++ ++ +
Breast carcinoma cell line
MCF-7 + ± +++ ± +
Key: +++: High cell membrane expression, ++: Moderate cell membrane expression, +: Low
cell membrane expression, ±: Weak cell membrane expression.
136
3.3.3.5 Confirmation of sub-cellular immunolocalisation of αIIb, β3 and
αVβ3 integrin using confocal microscopy.
The sub-cellular localisation of expression of αIIb, β3 and αVβ3 integrins were
confirmed using ICC by observing the expression using confocal microscopy.
As shown in Figure 3.9, membranous expression of αVβ3 integrin and the β3
subunit were observed in human tumour cells with high, moderate, and weak
expression of β3, αVβ3, and αIIb integrin according to IMB and ICC result for
M14, PC-3, and MCF-7.
137
No primary antibody B7 No primary antibody LM609 No primary antibody C20
M14
PC-3
MCF-7
Figure 3.9 The expression of αIIb, β3, and αVβ3 integrin in human tumour cell lines by confocal microscopy.
The human melanoma tumour cell line, M14, the human prostate carcinoma cell line, PC-3, and the human breast carcinoma, MCF-
7, were selected as examples of high, moderate, and weak expression of β3 and αVβ3 respectively, and were examined using
confocal microscopy. β3 and αVβ3 integrin immunoreactivity was detected in the cell membrane using B7 and LM609, respectively.
Weak expression of αIIb was detected in M14, and low expression was detected in PC-3 and MCF-7 cell membrane using C20
under the optimum conditions summarised in Table 3.5. The white arrow indicates the different expression of β 3 and αVβ3 integrin in
human tumour cell line and weak and low expression of αIIb. Bar length for C20 images = 30 µm and for B7 and LM609 = 150 µm.
138
3.3.4 Comparison of result of IMB and ICC
The inability to detect αIIb by IMB and the possibility that expression obtained
by ICC might represent false positive expression (since no expression was
detected by IMB), meant that this was excluded from comparison of integrin
expression between the two detection techniques, as shown in Table 3.7.
The expression of αV and β3 integrin was similar in all the human tumour cell
lines included the panel.
Table 3.7 Comparison of integrin expression in different tumour cell

lines using specific antibodies for αV and β3 integrin subunits using ICC
and IMB.
Human tumour cell lines Q20 (anti-αV) B7 (anti-β3)
IMB ICC IMB ICC
A549 +++ +++ ± ±
H460 ++ + ± ±
DLD-1 ++ +++ ± ±
HCT-116 + + ± ±
HT-29 ++ ++ - -
DU-145 ++ ++ + ++
PC-3 ++ ++ + ++
M14 ++ ++ +++ +++
SK-MEL-2 +++ +++ ++ ++
UACC-62 ++ +++ +++ +++
MCF-7 +++ +++ ± ±
Key for IMB and ICC: +++: High expression, ++: Moderate expression, +: Low expression,
±: Weak expression, -: no expression.
The expression of αV and β3 integrin subunits was quantified visually by giving the highest
cell expression +++ and correlated to the rest of the cell lines expression.
139
3.4 Discussion
The main aim of this chapter was to characterise αIIb and αVβ3 integrin
expression in a panel of human tumour cell lines, in order to develop a set of
models to be use in screening novel β3 integrin antagonists.
Initially, growth curves were recorded to identify the log phase where cells
functionally active in the days where cells reach 70 to 80% confluency. Cells
were collected in this period of growth to detect expression of αIIbβ3 and αVβ3
integrin because, according to previous reports, integrin expression is
reduced when cells are highly confluent (Haywood-Reid et al. 1997). In the
present study the expression was detected only in the log phase of the
tumour cells, to know how the expression of αIIbβ3 and αVβ3 integrin changed
during different growth phase, further study needs to be done.
The expression of αIIbβ3 and αVβ3 integrin was investigated by
immunoblotting, thereby confirming integrin protein expression in human
tumour cell lines and the specificity of the primary antibodies used.
Immunocytochemistry was then used to localise the site of expression as
either membranous or intracellular, using the same antibodies.
The expression of αV and β3 integrin subunits in tumour cells lines was
successfully detected using the immunoblotting technique but αIIb expression
was not. Different antibodies were used comprising anti-β3 BV4 and B7 and
anti-αV P2W7 and Q20 and anti-αIIb C20. Looking at this range of antibodies,
some seemed to be non-specific; for example, BV4 (anti-β3) and P2W7 (anti-
140
αV) showed double and multiple bands in immunoblotting. A specific antibody
should give a single band on the exposed film and any non-specificity that
develops is due to binding of the primary antibody to other non-target
proteins. C20 (anti-αIIb) did not reveal any obvious expression, which is most
likely due to the presence of the target protein at negligible levels in the cell
lines investigated. Further investigations involving this antibody should use
known expressors (e.g. human platelet lysates) (Lau et al. 2004) or inducible
samples (PMA treatment of K562) (Sevinsky et al. 2004) as a positive
controls to optimise the antibody reaction. For these reasons, P2W7, C20
and BV4 were excluded from further study. Q20 and B7 successfully
detected the expression of integrin subunits in tumour cell lines showing a
single band in the immunoblotting indicating the specificity in detecting the
expression. B7 binds to amino acids 635-730, mapping near the C-terminus
of the β3 integrin and Q20 recognises a peptide mapping to the C-terminus of
integrin αV indicating the chance of αV and β3 subunits to form αVβ3 integrin
heterodimer. Q20 and B7 were then used to screen a panel of cell lines for
the expression of αV and β3 integrin using immunoblotting. Q20 had been
used previously for immunoblotting in human 1321N1 astrocytoma (Liao et
al. 2007).
After optimising the protocol for each antibody, specific antibodies were
utilised to screen a panel of human tumour cell lines. All the human tumour
cell lines expressed αV integrin, whereas the expression of β3 integrin differed
between different tumour cell lines. High expression of β3 integrin was
141
observed in melanoma human tumour cell lines which agrees with the
literature (Li et al. 2001; Voura et al. 2001).
In order to support the expression seen by immunoblotting,
immunocytochemistry was utilised using the same antibodies in addition to
an anti-αVβ3 integrin antibody LM609. This antibody binds to a conformational
epitope resulting from the post-translational association of the αV and β3
subunits and detects the αVβ3 complex, so it could not be used with the
immunoblotting technique because cell lysis causes dissociation of the dimer.
LM609 has been used in several studies to detect the expression of α Vβ3
integrin in different cell membranes (Arosio et al. 2009; Hofmann et al. 2000;
Seftor et al. 1992). The protocol of Arosio et al. (Arosio et al. 2009) was
followed for immunodetection of αVβ3 by immunocytochemistry using LM609.
Optimised protocols for other antibodies were developed by altering variables
until optimisation was reached, indicating that each antibody had different
protocol requirements.
Moderate expression of αV and β3 and αVβ3 integrin was observed in PC-3
using Q20, B7, and LM609, respectively, by immunocytochemistry and
immunoblotting. The human prostate carcinoma cell line PC-3 was selected
to optimise the methodologies because earlier studies have documented high
expression of αV, αIIb, β3 and αVβ3 in this prostate adenocarcinoma cell line
(Arosio et al. 2009; Trikha et al. 1998; Trikha et al. 1996; Zheng et al. 1999).
Zheng et al. detected high expression of αVβ3 integrin in a PC-3 cell line
using flow cytometry analysis and immunoblotting techniques, and αV was
142
shown to complex with β3 using an immunoprecipitation technique (Zheng et
al. 1999). Trikha et al. detected intracellular expression of αIIbβ3 in PC-3 and
DU-145 cells, and showed that αIIbβ3 can translocate to the cell surface
following the activation of PKC (Trikha et al. 1996). They had previously
detected membranous expression of αIIbβ3 in DU-145, using
immunocytochemistry using with the anti-αIIbβ3 integrin antibody, MAB 1990
(Trikha et al. 1998). However, very little expression was reported by some
others; for example, Arosio et al. found little expression of αVβ3 integrin in
PC-3 using LM609 by immunofluorescence technique (Arosio et al. 2009).
Chatterjee et al. also reported little expression of αVβ3 integrin in PC-3 in
comparison to LNCaP (Chatterjee et al. 2001), and Goodman et al. found no
expression of αVβ3 and αIIbβ3 integrin in PC-3 (Goodman, Grote & Wilm
2012). This variation in αVβ3 expression in the PC-3 cell line may be due to
different culture conditions, as proposed by Nemeth et al. who suggested that
expression of αVβ3 integrin in PC-3 can be lost after a long period of culturing
(Nemeth et al. 2003). Here in the present study, the cells were not used after
passage 10 to avoid loss of the expression which can occur if late-passage
cells are used. Variation in the expression reported between laboratories may
be due to different culture conditions, and the use of fetal bovine serum from
different batches (Haywood-Reid, Zipf & Springer 1997).
After successful detection of integrin expression in PC-3 cells, a panel of
tumour cell lines was screened for the expression of αV, αIIb, β3 and αVβ3
integrins. To address the objectives of the immunodetection of αIIb, αV and β3
subunits and αVβ3 integrins in melanoma, three cell lines were used; M14,
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UACC-62 and SK-MEL-2. The αV, β3 subunits and αVβ3 integrin complex
were expressed in all cell line membranes whereas αIIb subunit expression
was weak. Melanoma cell lines had the highest expression of α V, β3 subunits
and αVβ3 integrin. This is the first study to investigate the expression of αV, β3
and αVβ3 integrin in the UACC-62 melanoma cell line using immunoblotting
and immunocytochemistry techniques.
Expression of αIIbβ3 and αVβ3 and their respective subunits have been
reported in melanoma cell lines. For example, Seftor et al. found that αVβ3
integrin was expressed in the melanoma cell line A375M using LM609 with
immunofluorescence (Seftor et al. 1992; Trikha et al. 1997). Petitclerc et al.
found the expression of αVβ3 integrin in the M21 melanoma cell line and used
it as a model to explore αVβ3 integrin expression on melanoma growth in vivo
(Petitclerc et al. 1999). Del Bufalo et al. found that M14 expresses αV and β3
integrin subunits but did not show any expression of αIIb indicating that M14
expressed αVβ3 integrin as the only β3 containing receptor complex (Del
Bufalo et al. 1998). Seoane et al. reported the expression of αV and β3
integrin and no expression of αIIb in another melanoma cell line (SK-MEL-28)
using PCR and flow cytometry techniques (Seoane et al. 2010). Trikha et al.
reported that the expression of αIIbβ3 integrin was not limited to only one
single cell line; all melanoma cell lines expressed αIIbβ3 integrin, furthermore,
translocation of αIIbβ3 integrin from the intracellular pool to the cell surface
can be mediated under the effect of an agonist (Trikha et al. 1997).
144
Both positive and negative control cell lines needed to be investigated for
each integrin subunit. The presence of positive and negative controls in the
study confirms the expression detected in other cell lines is true expression
rather than a false negative/positive.
In this part of the study, MCF-7 was utilised and as indicated by
immunoblotting very weak expression of β3 integrin. Previous studies have
detected the expression of αV, αIIb, β3 subunits and αVβ3 integrin in breast
carcinoma cell lines, such as MCF-7, using different techniques such as
immunoblotting and immunocytochemistry (Beauvais et al. 2004). The weak
β3 subunit expression in MCF-7 was also seen by Wang et al. who found that
MCF-7 had a lower expression than another breast cell line, MDA-MB-231
(Wang et al. 20100.
The A549 human NSCLC cell line was used as a possible negative control
for use in the functional assay for αVβ3. Goodman reported no expression of
αVβ3 integrin in A549 (Goodman, Grote & Wilm 2012) however, in the present
study α Vβ 3 expression was detected, by immunoblotting and
immunocytochemistry, in human NSCLC cell lines such as A549 and H460,
with both cell lines showing weak expression of the β 3 subunit and αVβ3. This
difference may be due to different culture conditions between labs.
The expression of αV and β3 subunits and αVβ3 integrin was also detected in
the colon carcinoma cell lines, DLD-1 and HCT-116, by immunoblotting and
immunocytochemistry, but no expression of β3 was noted in HT-29. The
immunodetection of β3 integrin by immunocytochemistry indicated that colon

145
carcinoma cell lines DLD-1 and HCT-116 had weak expression of the β3
subunit; the expression of the αVβ3 integrin was lower than the β3 subunit.
Few reports describe the expression of αV, β3 and αIIb subunit and αVβ3
integrin in colon cancer. Most of the previous reports concentrate on β1
integrins rather than β3 (Gong et al. 1997; O'Brien et al. 1996).
Immunocytochemistry and immunoprecipitation detected different integrins
expression in HT-29, i.e. α2, α3, α6, β1 and β5 but weak expression of αV and
no expression of β3 integrin (Haier et al. 1999). Burbridge et al. detected the
expression of αVβ3 integrin in HCT-116 and no expression in HT-29 by
immunoblotting and immunocytochemistry (Burbridge et al. 2003). This result
agrees with Trikha et al. who used LM609 and reported that HT-29 was
negative for αVβ3 integrin (Trikha et al. 2002), and other studies which also
found no expression of αVβ3 integrin in HT-29 cell (Caltabiano et al. 1999)
using the immunofluorescence technique (Liu et al. 2011).
αV was expressed at a high level in all screened cell lines, which is to be
expected as αV does not bind exclusively to β3 but can also bind to β1, β5, β6,
and β8. The expression obtained from immunoblotting and
immunocytochemistry in this chapter was similar to previous reports (Trikha
et al. 1998; Trikha et al. 2002; Zheng et al. 1999; Wang et al. 2005). β3
integrin expression differed between cell lines; β3 expression was highest in
the melanoma cell lines M14, SK-MEL-2 and UACC-62, followed by the
prostate carcinoma cell lines: PC-3 and DU-145. Lung carcinoma cell lines
A549 and H460, colon carcinoma cell line DLD-1 and HCT-116 cells and
146
breast carcinoma cell line MCF-7 showed weak expression of β3 integrin. The
HT-29 colon carcinoma cell line did not express β3 integrin.
A weak expression of the αIIb subunit was noted using immunocytochemistry
technique. However, although there was evidence among a panel of tumour
cell lines not all of them had an expression, it was not convincing. The result
obtained by immunocytochemistry did not agree with immunoblotting
technique which suggests the expression of αIIb observed by
immunocytochemistry is may be false positive result or the conditions in
western blot did not optimised properly. In addition the serum added to the
media may also play an important role in the expression because the growth
factors and the serum can be differes between labs
αVβ3 is believed to be present over the entire surface of the cell (Wang et al.
2005). Confocal microscopy was used to localise expression as intracellular
or on the cell surface. αVβ3 integrin was found to be membranous in all cell
lines tested. Immunolabelling was done on cells adhering to a coverslip, so
reducing the chance for cell to cell contact resulting in increasing
membranous expression, since the expression of αVβ3 was known to be
more prominent when cells start to adhere and migrate (Carreiras et al.
1999).
147
3.5 Conclusion
Immunoblotting and immunocytochemistry were used to characterise the
expression of αV and β3 integrins in a panel of cell lines, whose growth
characteristics have been determined. Expression of αVβ3 was successfully
detected and found to be highest in M14, UACC-62 and SK-MEL-2,
moderate in PC-3 and DU-145, weak in A549, H460, DLD-1, HCT-116 and
MCF-7, negative in HT-29. The αIIb subunit was expressed in the human
tumour cell lines by ICC but not with IMB. The αV subunit was expressed in
all human tumour cell lines while expression levels of the β3 subunit were
more variable.
148
4 Chapter 4: Investigation of the effect of αVβ3 integrin inhibition on
tumour cell migration
149
Chapter 4
4.1 Introduction
There is strong evidence that αVβ3 integrin plays a role in tumour
dissemination, and therefore developing a therapeutic strategy to antagonise
this integrin should be of value in reducing dissemination.
One of the key stages of the tumour dissemination process is cell migration.
Tumour cell migration is initiated by cell polarization and the formation of
membrane protrusions at the leading edge of the cell (Wang et al. 2005;
Brakebusch et al. 2002). There are two types of tumour cell migration: single
cell migration and collective cell migration. When a single cell migrates, it
needs to polarize and protrude at the leading edge, then attach the leading
edge to the substratum over which it migrates, followed by proteolytic
degradation of the tissue component, actomyosin contraction, and finally,
forward sliding of the cell rear. Collective cell migration occurs when cells
maintain cell-cell junctions at the leading edge and lateral regions inside the
moving cell group via cadherins (Vicente-Manzanares et al. 2005).
As previously discussed, research supports the view that the β3 integrins,
particularly αVβ3, play an important role in various stages of tumour
metastasis including migration (Seftor et al. 1992; McCabe et al. 2007;
Takayama et al. 2005). Voura et al. indicated that the strongest expression of
the αVβ3 integrin was seen in the protrusions and pseudopods of migrating
cells, and also in cells spreading on matrigel. This indicates a role for αVβ3
150
integrin in extravasation and in cell-extracellular matrix interactions (Voura et
al. 2001). Blocking β3 integrin can inhibit interaction of tumour cells with the
ECM and block tumour cell migration.
Several experimental assays (summarised in Table 4.1) are available for
measuring tumour cell migration in vitro, such as the Boyden chamber assay,
ring assay and scratch assay (Kramer et al. 2013). The scratch assay
involves scratching a confluent monolayer of the cells and then evaluating
the extent of tumour cell migration into the scratch. This assay has several
advantages that make it an attractive assay; it is simple, inexpensive and can
record the cell movement in real time (Liang et al. 2007). Furthermore, the
scratch assay can be used to study cell migration by cell-cell interaction and
cell interaction with ECM when cells are seeded on coated plate as a
monolayer, rather than held in suspension, which disrupts cell-to-cell and
cell-to-ECM interactions (Goetsch & Niesler 2011; Liang, Park & Guan 2007).
The scratch assay has been used to investigate the effect of anti-integrin
antagonists in inhibition of tumour cell migration in vitro and to study the
functional activity of tumour cells. For example, it has been used to evaluate
the anticancer effects of the disintegrin contortrostatin on the PC-3 prostate
cancer cell line (Lin et al. 2010), and to investigate the role of receptor
glycosylation on αVβ3 function (Kremser et al. 2008).
In order to utilise this assay in the evaluation of novel β 3 antagonists, the
assay must be initially validated using known anti-αVβ3 integrin antagonists.
Cyclic RGD peptides such as (cRGDfV) (section 1.2.5.3.2) (Manzoni et al.

151
2009) and the LM609 antibody are known αVβ3 integrin antagonists (section
1.2.5.3.1). Whilst LM609 is specific to the αVβ3 integrin (Trikha et al. 2002;
Voura et al. 2001; Zheng et al. 1999), cRGDfV may also antagonise the αVβ5
integrin (Manzoni et al. 2009).
The overall aim of the work described in this chapter is to develop and
validate a functional assay of cell migration, the scratch assay, for use in the
evaluation of β3 integrin antagonists.
This aim was achieved by addressing the following objectives:
1- Setting up the scratch assay using the cell line models characterised
in Chapter 3.
2- Determination of a safe dose of the known αVβ3 integrin antagonists,
cRGDfV and LM609.
3- Validation of the assay using cRGDfV and LM609.
152
Table 4.1 Comparison of cell migration assays (Kramer et al. 2013)
Trans-well Wound Cell Fence assay Micro- Spheroid Capillary Capillary tube Leukocyte Colloidal Time-lapse
migration healing assay exclusion carrier bead migration chamber assays migration particle cell tracking
assay (Scratch zone assay assay assay assays agarose assay
assay) technique
assay
Dimensionality 2D 2D 2D 2D 2D (3D) 2D (3D) 2D 2D 2D 2D 2D
Chemotaxis + - - - - - + + + - Assay
dependent
Stable - - - - - - + + + - Assay
chemokine dependent
gradient
Measurement Cell count Migration area Migration area Migration area Migration Migration Cell count Migration Migration Cell Cell
fluorescence area area migration distance distance migration migration
area area path path
Live imaging - + + + + + + + + + +
IF, IHC - + + + + + + - n.d. - +
Substrate PC, PET Plastic, glass, Plastic, glass, Plastic, glass, Plastic, Plastic, Glass, Glass Plastic, Plastic, Plastic,
membrane or coated or coated or coated or coated or coated or coated glass, glass, glass,
or coated or coated or coated or coated
Direction of Horizontal Horizontal Horizontal Horizontal Vertical then Mixed then Horizontal Horizontal Horizontal Horizontal Horizontal
movement then vertical horizontal horizontal
HTS + + + ± ± ± - - - + +
Type of analysis End point Kinetic kinetic Kinetic kinetic kinetic kinetic Endpoint Endpoint kinetic kinetic
(kinetic) (kinetic)
Recapitulated in Single cell Collective Collective Collective Attachment Migration Single cell Leukocyte Leukocyte Single cell Assay
vivo migration migration migration of migration of migration of and from migration, migration, migration migration dependent
mode chemotaxis epithelial epithelial epithelial migration cell cluster, Chemotaxis single cell
sheets, EMT sheets, EMT sheets, EMT established migration
cell-cell
interaction
Key: 3D: Three dimensional, - : Not suitable, +: Suitable, PC: Polycarbonate, PET: Polyethylene terphthalate, BME: Basal membrane extract, IF:
Immunofluorescence, IHC: Immunohistochemistry, HTS: High throughput screening, n.d.: Not done. EMT: Epithelial mesenchymal transmission
153
4.2.1 Materials
Human tumour cell lines (Table 3.2) were maintained as described in section
3.2.2.1. All general chemicals, media and media supplements were
purchased from Sigma-Aldrich (Poole, UK), unless otherwise specified.
cRGDfV (ENZO Life Sciences, Farmingdale, NY) 4.3 Mm was dissolved in
PBS, aliquoted and stored at -20 °C, and LM609 (see Table 2.1) was diluted
in RPMI medium and used fresh. Ki-67 rabbit polyclonal primary antibody
(AB9260, Chemicon Millipore Watford, UK), LM609 (see Table 2.1) and the
Alexa fluor 546, anti rabbit, IgG (A11010, Invitrogen, USA) were used for
immunocytochemical studies.
4.2.2 Methods
4.2.2.1 Wound healing migration assay
Cells were seeded into six-well plates (culture area: 9.6 cm2) at different
concentrations in 2 ml of RPMI 1640 medium and then incubated at 37 C in
5% CO2 humidified atmosphere for three different durations (24, 48 and 72
hours) to determine the concentration and time at which a confluent
monolayer was formed. Once a confluent monolayer of cells had formed, the
cell monolayer was scratched in a straight line with a sterile P200 pipette tip
to create a gap (approximately 2 cm in length and 650 µm in width) in the
centre of the well. After scratching, the medium was slowly aspirated and
discarded, and the cells were washed with 1 ml of HBSS to remove the
154
debris and to smooth the edge of the scratch. After washing, fresh medium
was added to the cells, and the plate was incubated for 3, 6, 12, 24, 48 or 72
hours in order to investigate the time of wound healing for each cell line
(Figure 4.1). During incubation, the plates were periodically monitored using
phase-contrast microscopy (Olympus, Model: CK2). The real-time tracking of
the scratch perimeter was done by measuring the average between the
leading cells at five points along the scratch field using an eyepiece graticule.
After determining the time of wound healing for each cell line, the cells were
washed twice with HBSS and fixed with ice pre-cooled methanol for thirty
minutes at -20 °C. After fixation, the cells were hydrated by two washes in
PBS and counterstained with Harris’s Haematoxylin solution for two minutes.
The cells were then washed in tap water for one minute and left to dry at
room temperature.
The resulting plates were observed using an inverted microscope and
images were taken (Nikon camera, model: C-D 55230, Japan) at ten
positions throughout the scratch area. Scratch width was calculated using the
grid and percentage migration was calculated as follows:
Migration at time x (%) = (average scratch width at Tx /average scratch width
at T0 ) × 100.
The data were expressed as means ± SD of three independent experiments.
155
Figure 4.1 Wound healing assay steps (Hulkower 2011)
Cells were seeded as a confluent monolayer in six-well plates (A). The
monolayer was scratched using a sterile P200 pipette tip (B). The initial
perimeter was washed twice with HBSS and incubated with 1 ml of RPMI
media for 24 hours, the initial scratch measured at five points as indicated by
blue arrows (C). Cells that migrated toward the wound area were indicated by
narrowing of the scratch area after cell migration as indicated by blue arrows
(D).
4.2.2.1.1 Characterisation of the phenotype of the migrated cells in
terms of proliferation and integrin expression
ICC was carried out with LM609 to confirm that cells migrating into the wound
area expressed αVβ3 integrin, and with Ki-67, a marker for proliferation
(Gasparini et al. 1998), to confirm that wound healing was due to cell
migration rather than proliferation. M14 cells were seeded on autoclaved
coverslips in six-well plates using the optimum seeding concentration and the
required time for each cell line to grow to give a confluent monolayer. The
confluent monolayer was scratched and left to heal for 24 hours. Both healed
and initial perimeter scratches were immunolabelled by ICC technique as
previously described in Chapter 3. For Ki-67 immunolabelling, M14 cells were
fixed with 4% PFA, and blocked with 5% BSA/PBS for one hour at room
temperature. Cells were immunolabelled with Ki-67, 1:50 at 4 °C overnight
156
then the secondary antibody (Alexa fluor 546, anti rabbit) was applied, 1:50
for one hour in the dark at room temperature (for optimisation see Figure
3.5).
4.2.2.2 Evaluation of the cytotoxicity of cRGDfV and LM609 using the
MTT assay
The cytotoxicity of cRGDfV and LM609 was measured in M14 and HT-29
human tumour cell lines using the MTT assay (Mosmann 1983). The assay is
based on the ability of viable cells to reduce a yellow water-soluble
tetrazolium salt (MTT: 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium
bromide) to a purple water-insoluble formazan product by mitochondrial
succinate dehydrogenase, with the amount of formazan produced being
proportional to the number of viable cells present. The formazan products are
dissolved in DMSO and the absorbance of the resulting solution is
determined spectrophotometrically. In the first lane (8 wells per lane) of a 96-
well plate, 200 µl of RPMI medium was added to serve as a blank. To
subsequent lanes, 1×104 /ml tumour cells were plated in a final volume of 180
µl of medium and incubated for 24 hours. 20 µl of the test compounds: 0.01,
0.05, 0.5, 5.0 and 50 µM cRGDfV and 2.5, 5.0, 7.5 and 10 µg/ml LM609 were
then added to all wells except the RPMI blank control and untreated control
for 96 hours of continuous drug exposure at 37 °C in a humidified 5% CO 2
atmosphere.
Following incubation, the medium was removed and 200 µl of MTT solution
(5.0 mg/ml) was added per well. The plate was incubated for four hours in the
157
dark at 37 °C, 5% CO2 humidified atmosphere. The plate was centrifuged at
1000 rcf for five minutes, the supernatant was removed and 150 µl/well
DMSO was added with vigorous mixing in the pipettor to dissolve the
formazan crystals. The absorbance of the resulting solution was determined
at 550 nm using a Lab system Multiscan Plus spectrophotometer (Lab-
systems Group, UK). The adjusted mean absorbance for each compound
concentration was calculated by subtracting the mean background
absorbance (average of the blank well) from the mean absorbance of the test
wells. A dose response curve of adjusted mean absorbance against
concentration was constructed, and the IC50 values for each compound
against each cell line were calculated using least squares method in
Microsoft Excel.
4.2.2.3 Validation of the scratch assay using the integrin antagonists
cRGDfV and LM609
The scratch assay was carried out as described in section 4.2.2.1. cRGDfV
used on A549, DLD-1, DU-145, M14, PC-3 and UACC-62. LM609 used on
A549, DLD-1, M14 and PC-3. cRGDfV (0.05, 0.5, 5.0 and 50 µM) and LM609
(0.5 and 2.5 µg/ml) were diluted in a final volume of 1 ml RPMI 1640
medium, added to the scratched monolayer after the washing step, and
incubated for 24 hours. In each six-well plate, two wells were used as
controls, one for the initial perimeter, and the other for 24 hours of wound
healing. The specific positions were photographed as described in section
4.2.2.1 and analysed using a superimposed grid (Figure 4.2, A & B).
158
Migration compared to untreated control (%) was calculated as % migration =
100 × (ST0-ST24)/ (SU0-SU24) × 100.
Where ST0 is scratch at initial perimeter, SU24 is untreated scratch after 24
hours and ST24 is treated scratch after 24 hours. The data were expressed as
the means ± SD of three independent experiments.
Scratch at time 0 (ST0) Untreated scratch after 24 Treated scratch after 24

hours (SU24) hours (ST24)
Figure 4.2 Scratch assay analysis
Figure A shows a black line drawn at the back of each well to separate it into
two to ensure the images were captured at the same position each time.
After image capture, the original image was analysed by superimposing a
grid over the image, as shown in B.
159
4.2.2.4 Statistical analysis
The student’s t-test and Kruskall wallis test were used for statistical analysis
of the scratch assays, with results considered statistically significant and
highly significant for p < 0.05 and p < 0.01 respectively. Data are presented
as mean ± standard deviation, with each experiment repeated at least three
times.
160
4.3 Results
4.3.1 Development of the wound healing migration assay
A scratch wound healing assay was performed using cell lines that showed
high, low, weak or no expression of αVβ3 integrin. Developing this assay
required: optimisation of seeding density required for each cell line to form a
confluent monolayer, working on the scratching technique to produce a
consistent scratch width, determination of the wound healing times for the
different cell lines, and confirmation that cells occupying the wound area had
migrated there and were not just the result of proliferation by cells at the
wound edges.
4.3.1.1 Seeding density
As the scratch assay method is dependent on the cell density per well
leading to a confluent monolayer before scratching, the different tumour cell
lines were seeded into six-well plates at different concentrations for 24, 48
and 72 hours in order to determine the optimum seeding concentration for
each cell line.
Initially different concentrations of cells were seeded into six-well plates,
ranging from 1 × 103 to 1 × 106 cells /ml and incubated at 37 °C, 5% CO2 for
24 hours. After 24 hours, the appearance of the monolayer was analysed
visually, and if a confluent monolayer was reproducibly formed, then the
concentration was deemed suitable for seeding. Cell lines that did not form a
confluent monolayer after 24 hours were incubated for longer times. Different
161
cell lines required different seeding concentrations and incubation times to
form a confluent monolayer (Figure 4.3).
A549 (2 105, 48 hrs) H460 (2 105, 48 hrs) MCF-7 (1 106, 48 hrs)
DLD-1 (2 105, 48 hrs) HT-29 (5 105, 72 hrs) HCT-116 (1 106, 24 hrs)
M14 (8 105, 48 hrs) UACC-62 (2 105, 48 hrs) SK-MEL-2 (1 106, 48 hrs)
PC-3 (2 105 , 48 hrs) DU-145 (3 105, 24 hrs)
Figure 4.3 Formation of confluent monolayers for the cell line panel
(cell density per ml and time in hours)
Bar length = 200 µm.
162
4.3.1.2 Healing time
The time required for wound closure in each cell line was evaluated. After 24
hours, only M14 cells showed a high percentage (85%) of wound closure.
PC-3, DLD-1, UACC-62, DU-145 and A549 showed a moderate wound
closure in comparison to the initial perimeter after 24 hours; PC-3, DLD-1 and
UACC-62 monolayers were respectively 94%, 90%, and 78% healed after 48
hours and DU-145 100% healed after 72 hours. H460, HT-29, SK-MEL-2,
MCF-7 and HCT-116 had slow wound closure and scratch was not healed
after 72 hours (Figure 4.4).
163
A
T0 T 24 hours T 48 hours T 72 hours
A549
H460
MCF-7
DLD-1
HT-29
HCT-116
M14
UACC-62
SK-MEL-2
PC-3
DU-145
B 100
** ** **
**
** **
** ** **
**
80
** **
** **
60 **
** **
T 24 hours
40 ** T 48 hours.
Migration (%)
T 72 hours
* * * * * *
20 * * *
* * * * *
*
Figure 4.4 Migration of different tumour cell lines as assessed by the

scratch assay
A: A confluent monolayer was scratched and incubated with complete
medium and wound closure was measured using the grid. Images are
representative of three independent experiments. Bar length = 200 µm.
B: Results were analysed and the mean percentage of wound closure shown
±SD based on three independent experiments. ** indicated p < 0.01 and *
indicated p <0.05.
164
As a part of assay development, the phenotype of the cells seen in the
wound area was characterised using ICC. The αVβ3 integrin expression of
M14 cells migrating into the wound area was evaluated by labelling cells with
LM609. As shown in Figure 4.5, all cells found in the wound area expressed
αVβ3 integrin (Figure 4.5 A).
M14 cells were immunolabelled with Ki-67 to determine whether cells moving
into the wound area had migrated and not proliferated. Nuclear labelling was
obtained in cells which had moved toward the wound area, however there did
not seem to be a higher rate of proliferation in the cells found in the wound
area (Figure 4.5 B).
165
A TRITC DAPI Overlay X10 Overlay X40
Initial perimeter
24 hours wound healing
B
TRITC DAPI Overlay x40 Overlay x10
24 hours wound healing
Figure 4.5 Labelling M14 cells at initial wound perimeter (T=0) and after
24 hours of wound healing with LM609 and Ki-67 using ICC
A. Expression of αVβ3 integrin at the initial wound perimeter and after 24
hours of wound healing using LM609, anti-αVβ3 integrin antibody. All M14
cells were labelled with LM609 at the initial wound perimeter and after 24
hours. Cells migrating into the wound area showed αVβ3 integrin expression
as indicated by arrow. B. The immunolabelling with Ki-67 confirmed that M14
cells moving into the wound area had migrated. Examining M14 cells under
higher magnification showed that Ki-67 is a nuclear immunolabelling and it
did not seem that M14 had a high rate of proliferation. Bar length (A) = 60 µm
and (B) = 200 µm.
166
4.3.2 Evaluation of the cytotoxicity of cRGDfV and LM609 using the
MTT assay
The MTT assay was used to assess the cytotoxicity of cRGDfV against M14
and HT-29 cells and LM609 against M14 cells over a range of different
concentrations for 96 continuous hours of drug exposure. As shown in Figure
4.6, neither antagonist demonstrated cytotoxicity in the cells at the
concentration tested, and was it not possible to obtain an IC50 for either
compound. Thus, both antagonists are non-toxic within this concentration
range for these cell lines and could be used in the scratch assay.
100 * * * *
* 100
* *
80 80
Cell survival (%)
Cell survival (%)
60 60
40 40
M14
20 Ht29
HT-29 20 M14
0 0
0.01 0.05 0.5 5 50 2.5 5 7.5 10
cRGDfV (µM) LM609 (µg)
Figure 4.6 Evaluation of cRGDfV and LM609 cytotoxicity against human

tumour cell lines M14 and HT-29
Cells were exposed to the antagonists for 96 hours. There was a negligible
effect on cell survival by either compound. The results was statistically
significant p< 0.05.
167
4.3.3 The effect of cRGDfV on the migration of human tumour cell lines
Cell lines with varying αVβ3 integrin expression that demonstrated definite
wound healing above were used to validate the wound healing assay
because HT-29 (no αVβ3 integrin expression) can not be used as a model in
scratch assay due to slow wound healing. Initially the M14 cell line was used
due to its high expression of αVβ3 integrin in addition to its good growth and
rapid wound healing. All the concentrations of cRGDfV tested led to inhibition
of M14 migration. UACC-62 also showed high expression of αVβ3 integrin,
and again all cRGDfV concentrations led to inhibition of UACC-62 migration,
although with less of an effect than on M14 except at 0.05 µM, which showed
the same percentage of inhibition in both cell lines with other concentrations
tried. 50 µM cRGDfV was not toxic to M14 and UACC-62 cells. The effect of
all cRGDfV concentrations was statistically significant p < 0.01 expect with
DLD-1 the significant reached at 50 and 5 µM only.
50 µM cRGDfV was toxic to PC-3 and A549 cells (Figure 4.7). Thus, the
effect of cRGDfV on the migration of PC-3 and A549 cells was tested at 5.0,
0.5 and 0.05 µM and it was statistically significant p < 0.01 except for A549 at
0.05 µM, p < 0.05 (Figure 4.7).
DLD-1, used as tumour model of weak expression of αVβ3 integrin, did not
show a dose dependent response to cRGDfV. The inhibition of DLD-1
migration seen with high concentrations of cRGDfV may be due to inhibition
of αVβ5 integrin in DLD-1 (Appendix 1). The effect of 50 and 5 µM was
168
statistically significant with p <0.01. There was no significant effect of
cRGDfV at 0.5 and 0.05 µM on DLD-1 cell migration (Figure 4.7).
cRGDfV was effective in inhibition of all tumour cell migration at 5.0, 0.5, and
0.05 µM except DLD-1 which had an effect at 50 and 5.0 µM only. It was vital
to know why cell lines with moderate and weak expression of αVβ3 integrin
were affected by cRGDfV. These cell lines may also express αVβ5 integrin
(Appendix 1) which can also be antagonised by cRGDfV. To investigate this
possibility, LM609, which is reported to be specific to αVβ3 integrin, was also
used in the scratch assay.
169
A T0 Untreated 0.05 µM 0.5 µM 5.0 µM 50 µM
DLD-1
UACC-62
M14
PC-3
A549
DU-145
B PC-3 A549
100 *
** **
** ** **
**
80
** **
**
** **
**
Untreated
60 **
0.05 µM
Migration (%)
**
** ** 0.5 µM
** **
40
5.0
5 µMµM
50 µM
20 **
T T
0
DLD-1 UACC-62
UACC62 M14 PC-3 A549 DU-145
Figure 4.7 Effects of cRGDfV on tumour cell migration in the cell panel
using the scratch assay
A: Migration of different human tumour cell lines toward wound area was
inhibited using different concentrations of cRGDfV. Images shown as are
representative of three independent experiments. Bar length = 200 µm.
B: Toxicity was observed with PC-3 and A549 at 50 µM as indicated by (T) in
the chart. ** indicated p < 0.01 and * indicated p <0.05.
170
4.3.4 The effect of LM609 on human tumour cell migration
LM609 was used first with M14 cell line at two different concentrations, 0.5
and 2.5 µg/ml. LM609 at both of concentrations inhibited M14 cell migration,
but had little effect on migration of the PC-3 and DLD-1 cells and a negligible
effect on the migration of A549 cells, which showed cytotoxicity with cRGDfV
(Figure 4.8). The effect of LM609 on tumour cells migration was statistically
significant with p < 0.01. M14, PC-3, DLD-1 and A549 (as one cell line from
each type of tumour) were selected as a model of high, moderate and weak
expression to validate the scratch assay.
171
A T0 Untreated 0.5 µg/ml 2.5 µg/ml
M14
PC-3
A549
DLD-1
100
**
**
80
**
**
**
60
Untreated
cell migration
**
(%)
0.5 µg/ml LM609

40 2.5 µg/ml LM609
% TumourMigration
20
0
M14 PC-3 A549 DLD-1
Figure 4.8 The effect of LM609 on tumour cell migration

A: Migration of tumour cells toward the scratch area was inhibited by LM609.
Bar length = 200 µm. B: Images were analysed and aveage result shown as
representative of three independent experiments. ** indicated P < 0.01.
172
4.4 Discussion
The main aim of the work described in this part of the study was to develop a
scratch assay that can be used to measure one of functional aspects of
antagonising integrin expression, migration, for assessing novel integrin
antagonists.
The scratch assay has some key advantages that led to its use in this study.
The assay is easy to set up and does not require any specific equipment, and
all the required materials are available in any cell culture laboratory (Liang,
Park & Guan 2007). Nevertheless, the in vitro scratch assay presented some
technical challenges: the first was the difficulty of creating scratched areas of
equal sizes. This variation in wound size between wells of the same plate
was resolved by practicing to achieve scratches of the same width (Appendix
2). Furthermore, there was difficulty achieving a wound without any cells.
This problem was overcome by tilting the plate and washing the wound from
the corner of the plate, which resulted in the removal of all unwanted cells
from the wound area.
The intention was to use cell lines with different levels of αVβ3 integrin
expression to investigate the effect of αVβ3 antagonism. Thus, tumour cell
lines with different αVβ3 integrin expression which demonstrated clear wound
healing were identified for use. The screening of a panel of tumour cell lines
for wound healing (Figure 4.4) offered a chance to select the best model for
testing β3 antagonists. Not all cell lines showed rapid wound healing due to
the differing properties of the various cells in growing cultures. Some cells
173
grow in clumps rather than in flat spreading way. Cells growing in clumps are
associated with difficulty in defining the starting point of cell migration which
complicates image analysis.
Melanoma cell lines that exhibited high expression of αVβ3 integrin were
screened for wound healing in order to select the best model. The only cell
line that showed rapid wound healing (scratch nearly closed after 24 hours)
was M14, followed by UACC-62, which exhibited moderate wound healing
whereas SK-MEL-2 showed slow wound healing that did not close even after
72 hours. SK-MEL-2 has previously been shown to migrate very slowly, and
to lack a response to TGF-β1, which caused a change in phenotype and
signalling promoting cell migration in other melanoma cell lines (Janji et al.
1999).
PC-3 and DU-145 cell lines exhibited moderate wound healing after 24
hours. PC-3 has been reported to migrate rapidly, with a scratch being
completely healed after 12 hours (Lin et al. 2010). The slower wound healing
of PC-3 seen here may be due to different culture conditions or due to the
original study seeding PC-3 cells on matrigel coated plates which promote
integrin-mediated adhesion, whereas in the present study the PC-3 cells
were seeded on uncoated plates which may have led to a slower migration of
PC-3. Wang et al. found that wound healing was faster in PC-3 than in DU-
145 (Wang et al. 2005), which supported the present study result where both
cell types showed moderate wound healing after 24 hours, but PC-3 had
wound closure after 48 hours, whereas DU-145 did not.
174
Colon carcinoma cell lines were screened for wound healing to be used as a
αVβ3 weak or negative control models. However, only DLD-1 had moderate
wound healing after 24 hours whereas HCT-116 and HT-29 did not show
wound closure even after 72 hours. Thus, both HCT-116 and HT-29 were
excluded from the rest of the scratch assay study, and DLD-1 was selected
for further scratch assays as a model of weak expression of αVβ3 integrin.
Other cell lines with low αVβ3 integrin expression were also investigated.
MCF-7 (weak αVβ3) did not achieve wound closure after 72 hours. Lung
carcinoma cell lines had weak αVβ3 integrin expression; moderate wound
healing was observed for A549 while H460 did not have wound closure even
after 72 hours. Cell lines that showed very slow wound healing (HCT-116,
HT-29, H460 and MCF-7) in the scratch migration assay were excluded from
the rest of the study.
The scratch assay provides valuable results regarding the expression of the
αVβ3 integrin in tumour cell migration. Immunolabelling with LM609 of M14
cells in the scratch assay has never been done before, although this
technique has been demonstrated by Carreiras et al. who showed that
IGROV-1 ovarian carcinoma cells expressing αVβ3 integrin migrated toward a
scratch area by immunolabelling migrating cells with LM609 (Carreiras et al.
1999).
The assay was validated before utilising it to evaluate novel anti-β3 integrin
antagonists by using well-characterised commercially available anti-αVβ3
integrin antagonists as positive controls. Before using the antagonists on

175
tumour cells, the cytotoxicity of cRGDfV and LM609 was tested using the
MTT assay, which showed that cRGDfV had no toxic effects on the M14 and
HT-29 human tumour cell lines. LM609 also did not show any toxicity to M14
cells. The only toxicity observed to other cell lines (did not tested by MTT
assay) was with 50 µM cRGDfV to PC-3 and A549 only.
An anti-migratory effect was obtained with cRGDfV on cells with high,
moderate and low αVβ3 integrin expression (Figure 4.7). PC-3 cells showed
moderate expression of the αVβ3 integrin with high response to cRGDfV at
5.0, 0.5 and 0.05 µM. This may be due to expression of αVβ5, which is also
antagonised by cRGDfV. Bisanz et al. have previously shown that the
expression of β3 was lower than for αVβ1 and αVβ5 in PC-3 cells (Bisanz et al.
2005).
The LM609 antibody has been used to detect the expression of the αVβ3
integrin and as an inhibitor (Leavesley et al. 1992; Seftor et al. 1992; Trikha
et al. 2002). A previous study used 20 µg/ml LM609 to study endothelial cell
migration (Li et al. 2006). However, use of 20 µg/ml on M14 cells led to loss
of M14 adhesion to the plate. Thus, lower concentrations (2.5 and 0.5 µg/ml)
were used to study the effect on migration.
LM609 is specific for αVβ3, and does not antagonise related integrins such as
αVβ5. The observed inhibition of cell migration suggests that αVβ3 plays a
significant role in the migration of M14, and PC-3 cells and may also be
involved in migration of DLD-1. αVβ3 integrin has been shown to play an
important role in migration of other cell lines; for example, a study done by
176
Leavesley et al. reported that LM609 inhibited migration of pancreatic FG
cells transfected with αVβ3 integrin in the Boyden chamber migration assay
(Leavesley et al. 1992).
4.5 Conclusion
This work has developed and validated an assay for evaluating integrin-
mediated cell migration, which can be used to evaluate novel αVβ3
antagonists in the following chapter.
177
5 Chapter 5: Investigation of the cytotoxicity of potential novel β3
integrin antagonists and their effect on tumour cell migration
178
Chapter 5
5.1 Introduction
The previous chapter described the development and validation of the
scratch assay to evaluate novel β3 integrin antagonists. M14 cells were
chosen as the optimal cell line to use in the scratch assay due to their high
αVβ3 integrin expression and rapid wound healing. αVβ3 integrin is required
for melanoma cell motility and it has been reported that blocking αVβ3 integrin
leads to blocking of melanoma cell migration (Li et al. 2001).
In this chapter, the optimised scratch assay was utilised to evaluate potential
novel β3 integrin antagonists synthesized at the Institute of Cancer
Therapeutics, using M14 as the model for testing for inhibition of tumour cell
migration due to its high αVβ3 integrin expression.
In the present study the cytotoxic effect of potential novel β3 integrin
antagonists will be tested on tumour cells before they are evaluated in the
functional scratch assay, in order to know that the effect of the potential novel
β3 integrin antagonists was due to inhibition β 3 integrin function in mediating
tumour cell migration rather than cytotoxicity. Dayam et al. identified highly
selective αVβ3 integrin antagonists through screening a panel of potential
αVβ3 integrin antagonists they had synthesized. Six compounds were tested
in breast tumour cells, MDA-MB-435 and MCF-7 as high and low αVβ3
models respectively. They found one of anti αVβ3 small molecules was toxic
whereas the others showed no toxicity. This study indicated the importance
179
of testing the toxicity of small molecules before evaluating their effects on
cancer cell migration and attachment (Dayam et al. 2006).
The overall aim of this chapter was to evaluate potential novel β3 integrin
antagonists in terms of their cytotoxicity and effect on tumour cell migration.
This aim was achieved through the following objectives:
1- Evaluation of the cytotoxicity of potential novel β3 integrin antagonists
using the MTT assay for cytotoxicity.
2- Determination of the effects of potential novel β3 integrin antagonists
on M14 cell migration using the scratch assay.
180
5.2.1 Materials
Human tumour cell lines M14, MCF-7, and HT-29 (see Table 3.2 for details)
were used in this chapter and maintained as described in section 3.2.2.1. All
general chemicals, media and media supplements were purchased from
Sigma-Aldrich (Poole, UK), unless otherwise specified. cRGDfV, was
sourced as described in section 4.2.1. A series of potential novel β3 integrin
antagonists was synthesized at the Institute of Cancer Therapeutics (see
Table 5.1 for details). All antagonists dissolved readily in 0.1% DMSO and a
stock of 100 mM was prepared. For compound structures see attached file.
LM609 details are summarised in Table 2.1.
Table 5.1 Compounds used

No Compound Molecular weight
1 ICT9003 320
2 ICT9018 531
3 ICT9019 503
4 ICT9020 517
5 ICT9053 538
6 ICT9055 542
7 ICT9057 500
8 ICT9062 542
9 ICT9064 528
10 ICT9068 421
11 ICT9072 474
12 ICT9073 516
181
5.2.2 Methods
5.2.2.1 Evaluation of the cytotoxicity of potential novel β3 integrin
antagonists using the MTT assay
The cytotoxicity of the potential novel β3 integrin antagonists was evaluated
in M14 (high expression of αVβ3 integrin), MCF-7 (weak expression of αVβ3
integrin), and HT-29 (no expression of αVβ3 integrin) human tumour cell lines
using the MTT assay as described in section 4.2.2. The β3 integrin
antagonists were used at the following concentrations 0.01, 0.1, 1.0, 10 and
100 µM.
5.2.2.2 Wound healing migration assay
The anti-migratory effect of the potential novel β3 integrin antagonists on M14
cell migration was evaluated using the scratch assay as described in section
4.2.3. Briefly, wounded cultures of cells were exposed to 0.01, 0.1, 1.0, 10
and 100 µM of the potential novel β3 integrin antagonists for 24 hours, and
then the cultures were processed as described in Chapter 4. cRGDfV at 0.5
and 5.0 µM was included as a positive control.
5.2.2.3 Statistical analysis
The student’s t-test (parametric) and kruskall wallis (non parametric) test
were used for statistical analysis of the scratch assays, with results
considered statistically significant and highly significant for p < 0.05 and p <
0.01 respectively. Data are presented as mean ± standard deviation, with
each experiment repeated at least three times.

182
5.3 Results
5.3.1 Evaluation of the cytotoxicity of potential novel β3 integrin
antagonists
The MTT assay was used to assess the cytotoxicity of potential novel β3
integrin antagonists against M14, HT-29 and MCF-7 cells over a range of
different concentrations for 96 continuous hours of drug exposure. As shown
in Figure 5.1 and Table 5.2, the majority of compounds demonstrated
minimal toxicity, with it not being possible to obtain IC50 at the highest dose
assayed, 100 µM.
As can be seen from Table 5.2, ICT9062, ICT9072, and ICT9073 were toxic
to all cell lines with IC50 calculated from three independent experiments.
183
100 100 100 100
80 80 80 80
Cell survival (%)
Cell survival (%)
Cell survival (%)

Cell survival (%)
60 60 60 60
40 40 40 40
M14 M14 M14 M14
20 MCF-7 20 MCF-7 20 MCF-7 20 MCF-7
HT-29 HT-29 HT-29 HT-29
0 0 0 0
0.01 0.1 1 10 100 0.01 0.1 1 10 100 0.01 0.1 1 10 100 0.01 0.1 1 10 100
ICT9003 (µM) ICT9018 (µM) ICT9019 (µM) ICT9020 (µM)
100 100 100 100
80 80 80 80
Cell survival (%)
Cell survival (%)

Cell survival (%)
Cell survival (%)

60 60 60 60
40 40 40 40
M14 M14 M14 M14
MCF-7 MCF-7 MCF-7 MCF-7
20 20 20 20
HT-29 HT-29 HT-29
HT-29
0 0 0 0
0.01 0.1 1 10 100 0.01 0.1 1 10 100 0.01 0.1 1 10 100 0.01 0.1 1 10 100
100
100 100 100
80
80 80 80
Cell survival (%)

Cell survival (%)
Cell survival (%)
Cell survival (%)

60
60 60 60
40 40 40 40
M14 M14 M14 M14
MCF-7 MCF-7 20 MCF-7 MCF-7
20 20 20
HT-29 HT-29 HT-29 HT-29
0 0 0 0
0.01 0.1 1 10 100 0.01 0.1 1 10 100 0.01 0.1 1 10 100 0.01 0.1 1 10 100
Figure 5.1 Evaluation of the cytotoxicity of the potential novel β3 integrin antagonists in the tumour cell line panel
184
Table 5.2 IC50 values for the potential novel β3 integrin antagonists in
the tumour cell line panel
Compound M14 MCF-7 HT-29
IC50 ± SD (µM) IC50 ± SD (µM) IC50 ± SD (µM)
ICT9003 > 100 >100 >100
ICT9018 > 100 >100 >100
ICT9019 > 100 >100 >100
ICT9020 36 ± 18.6 >100 80 ± 5.8
ICT9053 72.7 ± 9.8 >100 >100
ICT9055 > 100 >100 25 ± 5.8
ICT9057 > 100 >100 >100
ICT9062 3.3 ± 0.8 1.4 ± 0.5 21.2 ± 30
ICT9064 > 100 >100 >100
ICT9068 > 100 9.5 ± 9 >100
ICT9072 50 ± 47 14 ± 8.5 0.8 ± 0.8
ICT9073 61 ± 32.9 6.3 ± 6.9 36.7 ± 42.2
5.3.2 Characterisation of the anti-migratory effect of potential novel β3
integrin antagonists using the scratch assay
The potential novel β3 integrin antagonists were tested in two batches; with
information obtained from the first batch informing the concentrations used in
testing the second batch. In the first batch, ICT9003, ICT9055, ICT9057 and
ICT9064 were screened at four concentrations 0.1, 1.0, 10 and 100 µM. At
the lowest concentration, ICT9055 showed the highest effect of inhibiting
M14 cell migration; whilst the other potential novel β3 integrin antagonists had
lower effects at the same concentrations (Figure 5.2). The results were seen
to be highly statistically significant p < 0.01 for all novel β3 integrin
antagonists. IC50 values were calculated as 4.8 ± 0.2 µM for ICT9003, 1.0 ±
0.09 µM for ICT9057 and 0.2 ± 0.06 µM for ICT9064 (Table 5.3).
185
A T0 T24 hours 0.5 µM cRGDfV 5.0 µM cRGDfV
0.1 µM 1.0 µM 10 µM 100 µM

ICT9003
CB57
ICT9055
ICT9057
ICT9064
B
100
80
** ** **
0.1 µM
** **
60 ** 1.0
1 µMµM
Migration (%)
**
** ** ** 10 µM
** 100 µM
40 **
**
** 0.5 µM
**
20 **
55.0
µMµM
** **
0
ICT9003 ICT9055 ICT9057 ICT9064 cRGDfV
Figure 5.2 Effect of potential β3 integrin antagonists on M14 cell

migration
A: M14 cells were wounded and then treated with different concentrations of
different potential novel β3 integrin antagonists for 24 hours. Bar length = 200
µm. B: The migrated cells were counted and related to 100% untreated
cells. Results were statistically significant for all antagonists with p <0.01.
186
A concentration of 10 µM was selected as an initial screening concentration
for the second batch of compounds, which were ICT9018, ICT9019,
ICT9020, ICT9053, ICT9062, ICT9068, ICT9072 and ICT9073. As seen in
the cytotoxicity assay, some of the antagonists (ICT9062, ICT9072 and
ICT9073) were observed to be toxic. In the MTT assay cells were treated for
96 hours, whereas in the scratch assay the M14 cells were treated for only
24 hours, thus as the exposure time was shorter it was assumed that these
concentrations would not have a cytotoxic effect, however this was not the
case and the toxicity was still observed (Figure 5.3). Therefore, the β3
integrin anatgonists were tried at lower concentrations which were 1.0, 0.1
and 0.01 µM.
187
T0 T24 hours 0.5 µM cRGDfV 5.0 µM cRGDfV ICT9018 ICT9019
ICT9020 ICT9053 ICT9062 ICT9068 ICT9072 ICT9073

Figure 5.3 Effect of 10 µM β3 antagonists on the inhibition of M14 cell migration by the scratch assay.
A confluent monolayer of M14 cells was scratched, and then treated with 10 µM of the second batch of novel anti-β3 integrin
antagonists for 24 hours. Bar length = 200 µm.
188
Further screening was carried out for the β3 integrin antagonists that showed
an inhibitory effect on M14 cell migration using a more extensive
concentration range, from 0.01 µM up to 10 µM (Figure 5.4). The compounds
that were toxic at 10 µM were also evaluated, and these demonstrated a high
percentage of inhibition of M14 cell migration at lower concentrations, with
ICT9073 having the highest percentage of inhibition at 0.01 µM. IC50s were
9.5 ± 0.9 µM for ICT9019, 0.15 ± 0.03 µM for ICT9062 and 0.15 ± 0.04 µM
for ICT9072 (Table 5.3).
189
A 0.01 µM 0.1 µM 1.0 µM 10 µM
ICT9019
ICT9062
ICT9068
ICT9072
ICT9073
100
**
** **
**
**
80 **
** ** **
**
**
**
60 **
** 0.01 µM
**
Migration (%)
** 0.1 µM
40 1 µM
**
10 µM
**
20 0.5 µM
**
5 µM
T T T
0
ICT 9019 ICT 9062 ICT 9068 ICT 9072 ICT 9073 cRGDfV
Figure 5.4 Effect of potential β3 integrin antagonists in inhibition M14

cell
migration by scratch assay
A: M14 cells were wounded then treated with different concentrations of
different potential novel β3 integrin antagonists for 24 hours. Bar length = 200
µm. B: The migrated cells were counted and related to 100% untreated
cells. Result was statistically significant p <0.01. ICT9062, ICT9072 and
ICT9073 were toxic at 10 µM as indicated by T.
190
Table 5.3 A summary of effect of the potential novel β3 integrin
antagonists in inhibition of M14 cell migration after 24 hours treatment
with the scratch assay
Compound IC50
ICT9003 4.8 ± 0.2 µM
ICT9018 -
ICT9019 9.5 ± 0.9 µM
ICT9020 -
ICT9053 -
ICT9055 < 0.1 µM
ICT9057 1.0 ± 0.09 µM
ICT9062 0.15 ± 0.03 µM
ICT9064 0.2 ± 0.06 µM
ICT9068 > 50 µM
ICT9072 0.15 ± 0.04 µM
ICT9073 < 0.01 µM
191
5.4 Discussion
In the previous chapters characterised cell lines were selected for their
integrin expression patterns and then used to set up and validate a functional
assay of integrin antagonism, the scratch assay. In this chapter the validated
assay was used to evaluate novel antagonists in inhibition of migration of a
cell line expressing β3 integrin, M14.
Initially β3 integrin antagonists were evaluated for their relative cytotoxicity.
As shown in Table 5.2, different anti β3 integrin antagonists had different
cytotoxic effects. ICT9003, ICT9018, ICT9019 and ICT9057 were not toxic to
M14, MCF-7 and HT-29 cells. ICT9053 had a cytotoxic effect on M14 only,
and ICT9055 was cytotoxic to HT-29 only. ICT9064, the free acid of ICT9055
showed no obvious cytotoxicity with all tested cell lines. ICT9062 showed a
cytotoxic effect with all tested cell lines, which is surprising since it differs
from ICT9055 only in the stereochemical arrangement of bonds around the
central linker. ICT9068 showed cytotoxic effect with MCF-7 only, and
ICT9020 was cytotoxic with both M14 and HT-29.
The cytotoxic effect was evaluated using the MTT assay, to ensure that the
results observed were due to blockade of integrin function (reduction in cell
migration in the scratch assay), rather than general toxicity.
There was no obvious pattern in cytotoxicity across compounds and cell
lines. Since compounds that were shown to be active in the scratch assay
(and other work in-house) such as ICT9055, are not toxic in the high β 3
192
expressing M14 cells, the toxicities observed are unlikely to be β 3-mediated.
However, cell line specific cytotoxicity may be due to effects mediated by
other integrins: for example, αVβ6 is known to be important in colon cancer
survival, so the toxicity observed with HT-29 treated with ICT9055 may result
from antagonism of β6 (Peng et al. 2009). Future work on these compounds
should involve assessment of their selectivity for similar integrins.
ICT9072 and ICT9073 had a cytotoxic effect with all tested cell lines, which
may be due to the presence of the amino pyrimidine group at the start of the
carbon chain, which has previously been demonstrated to cause toxicity
(Gorneva 2005). Cilengitide induced cytotoxicity in glioma cells U-251MG
and U-87MG (Lomonaco et al. 2011) whereas cRGDfV did not show any
cytotoxic effect to same cells (Chatterjee et al. 2000).
The effect of the potential novel β3 integrin antagonists was investigated by
the scratch assay. The results indicated that different antagonists had
different effects on inhibition of M14 cell migration. ICT9003 had the lowest
effect, which may be due to the absence of both tetrahydronaphthyridine
(THN) at the beginning of the carbon chain and the NHSO 2Mes exosite
binding group at the end of the carbon chain.
As shown in Figure 5.3, ICT9018, ICT9019, ICT9020 and ICT9003
(pyrimidine) (Figure 5.4) at 10 µM were not effective in inhibiting M14 cell
migration. In contrast, ICT9055, ICT9057 and ICT9064 which all contain the
THN group, were all effective inhibitors of M14 cell migration. This consistent
effect of ICT9057, ICT9062 and ICT9064 indicated that the presence of the
193
exosite binding NHSO2Mes group is also required for activity. The ICT9062
had the same structure as ICT9055, but with different stereochemistry which
led to it being cytotoxic to all tested cell lines suggesting that stereochemistry
is a major consideration in antagonist design. ICT9055 was thus the most
effective functional antagonist with no cytotoxic effect on M14 cells, among
the compounds screened.
5.5 Conclusion
A total of 12 potential β3 integrin antagonists were screened in the scratch
assay, with some of the compounds demonstrating significant antagonistic
effects. ICT9055 was the most effective of the compounds screened and
would warrant further investigation.
194
6 Chapter 6: Discussion and future work
195
6.1 General discussion and future perspective
The current strategy for cancer therapy is still inadequate; chemotherapy
continues to be associated with long-term toxicities and drug resistance,
while surgery and radiotherapy are not suitable for treating the whole body
after cancer metastasis. Thus, research has begun to focus on new
strategies for cancer therapy based on targeted therapy. The development of
targeted therapies improves differentiation between normal and neoplastic
cells.
Integrins have become an attractive receptor target for cancer therapy
(Goodman & Picard 2012; Sheldrake & Patterson 2009). However, among
the panel of integrin antagonists summarised in Table 1.3, none of them
specifically targets tumour cell integrins and their interaction with platelet and
endothelial cells to inhibit angiogenesis and tumour metastasis. Furthermore,
a large number of the available antagonists available are either antibodies or
peptides and small molecules are preferred as antagonists as they are less
costly to manufacture than antibodies, have better stability and
pharmacokinetic/pharmacodynamic (PKPD) properties, and can be
administered orally (Alig et al. 1992; Millard, Odde & Neamati 2011).
Blockade of αIIbβ3 and αVβ3 integrins has a critical role in preventing cancer
metastasis and angiogenesis (Liu et al. 2009). Blockade of both tumour cell
αVβ3 and host cell β3 integrins (endothelial αVβ3 and platelets αIIbβ3) can
enhance inhibition of tumour growth in vivo (Trikha et al. 2002). At the
Institute of Cancer Therapeutics we hypothesise that dual αIIbβ3 and αVβ3

196
antagonism will have a direct effect on β3-expressing tumour cells and it will
also through targeting tumour cell interaction with endothelial cells and
platelets lead to inhibition of angiogenesis and metastasis.
In this thesis, the aim was to evaluate the effect of potential novel β3 integrin
antagonists in inhibition of human tumour cell migration using cell based
assays. The main objectives of the work were to detect αIIbβ3 and αVβ3
integrin expression in human tumour cell lines and human xenograft tissue to
develop models that can be used for both in vitro and in vivo evaluation of
novel β3 integrin antagonists.
After characterisation of αIIbβ3/αVβ3 integrin expression in a panel of human
tumour cell lines, M14 was selected as a suitable cell line to be used as a
model in the scratch assay. This cell line allows the study of migration
mediated by αVβ3 integrin due to negligible expression of αIIbβ3 integrin in the
cell line. Using this cell line the potential novel β3 integrin antagonists
ICT9055 and ICT9073 were identified as providing blockade of cell migration
at non-toxic concentrations, with IC50 < 0.1 and < 0.01 µM respectively.
However, the other antagonists that had cytotoxic effect need to be
confirmed that it non toxic to control cells in other functional assay.
Initially, the expression of αIIbβ3 and αVβ3 was investigated in human
xenograft mouse tissue using immunohistochemistry in order to obtain
information that would guide the selection of cell lines to be used when the
potential novel β3 integrin antagonists progress to in vivo screening.
Detection of integrin expression in xenograft mouse tissue was really

197
important in preparing an in vivo model since it is known that integrin
expression varies in response to extracellular environment (Zhao et al. 2007).
After different methodologies were followed, all the primary antibodies
evaluated in these studies gave non-specific immunolabelling in sections
both with and without primary antibodies, except B7 (anti-β3) using the
M.O.M. kit which gave a clean negative control with non-specific
immunolabelling of blood vessels only. Furthermore, non-membranous
immunolabelling developed with Q20 (anti-αV). These results were confirmed
by immunoblotting which showed that mouse antibody (B7) on mouse tissue
developed non-specific binding while rabbit antibody Q20 did not develop any
non-specific binding.
According to tissue homogenisation and protein extraction in this study, it can
be said that the human xenograft tissues used in this study expressed αV and
β3 integrin subunits. The problem faced with immunohistochemistry was due
to the inability of the mouse primary antibodies used to selectively detect the
expression of human integrin subunits in the presence of mouse tissue. In
normal mouse tissue there was non-specific binding whereas there was no
non-specific binding in samples from human tumour cell lines grown in vitro
when the membrane was incubated without primary antibody. In order to
investigate integrin expression in an in vivo model using either
immunohistochemistry or immunoblotting it seems to be vital to avoid using
mouse antibodies on mouse tissue. For example anti-αV (Q20) did not
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develop any non-specific binding with immunohistochemistry or with
immunoblotting.
The results from immunoblotting encouraged evaluation of the expression of
αVβ3 and αIIbβ3 integrin in other human tumour cell lines. After optimisation of
a panel of primary antibodies, the expression of αV, β3 and αVβ3 integrin was
detected on the surface of a panel of tumour cell lines using
immunocytochemistry, and also the expression of αV and β3 integrin subunits
was detected in different cell lines using immunoblotting. However, the
overall aim of this study can be best investigated in vitro using a model
expressing both αIIbβ3 and αVβ3 integrin and I did not detect expression of αIIb
integrin in this study. Thus, to achieve a model of α IIbβ3/αVβ3 integrin dual
expression, it will be vital to transfect the cells chosen for the study with the
αIIb integrin subunit (O'Toole et al. 1989). The transfection of αIIbβ3 integrin
has been achieved previously; for example, αIIbβ3 integrin was transfected
into the melanoma cell line WM 983B which lead to increased tumour cell
adhesion, spreading and invasion in vitro and decreased apoptotic rate in
vivo (Trikha et al. 2002).
Here, αV protein was expressed in all tumour cell lines evaluated, and was
observed to be localised to the cell surface using immunocytochemistry. This
result was expected since αV can bind to other β subunits besides β3.
Goodman et al. have also shown that αV is expressed in all human tumour
cell lines (Goodman, Grote & Wilm 2012). αV has been shown to play an
important role in prostate cancer angiogenesis and bone metastasis through
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validation of αV as a target by knockdown, and also pharmacological
inhibition by GLPG0187 (Table 1.3), using in vivo and in vitro models.
GLPG0187 was effective in inhibiting angiogenesis and bone metastasis
through its action on prostate cancer stem cells (van der Host et al. 2011). In
addition, αV blocking with GLPG0187 in PC-3 cells prevented the growth of
the tumour in vivo (van der Host et al. 2010). The anti-αV antibody CNTO95
(Table 1.3) inhibits HUVECs and melanoma cell (A375.S2) adhesion,
migration, and invasion in vitro. CNTO95 also leads to inhibition of tumour
proliferation and angiogenesis in vivo (Trikha et al. 2004). Bisanz et al.
treated PC-3 cells with αV si-RNA which lead to decreased growth of PC-3
xenografts in bone, suggesting that αV mediated communication between
tumour cell and bone microenvironment is required for bone metastasis
development (Bisanz et al. 2005). The above indicate that αV has an
important role in cancer. In the present study, it was not possible to use a
model with no αVβ3 integrin because the only line explored that had no αVβ3
integrin expression, HT-29, could not be used in the scratch assay due to its
low rate of migration/wound closure.
The present study developed a semi-quantitative method for the detection of
αVβ3 integrin expression in human tumour cell lines and xenografts. A more
quantitative measure would be to use flow cytometry, it was the advantage
over the immunocytochemistry is being able to detect even weak expression
(Goodman, Grote & Wilm 2012). LM609 has been used previously to
characterise the expression of αVβ3 integrin in human tumour cell lines using
flow cytometry (Landen et al. 2008). However, since flow cytometry does not
200
assess morphological expression, it must still be run in parallel with
immunohistochemistry or immunocytochemistry.
The overall aims of this study were to characterise αVβ3/αIIbβ3 integrin
expression in tumour cell lines and tissues, and to evaluate the
pharmacological effect of novel small molecule, anti αVβ3 and/or αIIbβ3
antagonists. Using different tumour cell lines with different α Vβ3 integrin
expression, a range of wound-healing responses was seen in the scratch
assay for migration. LM609 and cRGDfV were used as positive controls to
demonstrate that M14 migration was mediated by αVβ3 integrin. Albelda et al.
showed increased expression of αVβ3 integrin correlates with increased
melanoma cell migration (Albelda et al. 1990). Although both inhibitors were
effective in inhibition of M14 cell migration, cRGDfV (target αVβ3/αVβ5 integrin)
and LM609 (target αVβ3 integrin) this indicated that αVβ5 integrin may also had
a role in M14 cell migration.
After optimising the scratch assay conditions, the potential novel β3 integrin
antagonists were screened. The effect of potential novel β3 integrin
antagonists in inhibition of M14 cell migration was evaluated. From screening
a panel of 12 novel β3 integrin antagonists, ICT9055 had the highest effect in
inhibition of M14 cell migration. This small molecule did not exert any
cytotoxic effect even at high concentrations on M14 cells but was toxic to HT-
29. ICT9053 was toxic to M14 cells only, ICT9020 was toxic to M14 and HT-
29 but not to MCF-7 and ICT9068 was toxic to MCF-7 not to M14 and HT-29.
Different human tumour cell lines may have different survival pathways and
201
this can be explained by the variation in cytotoxic effect of some potential
novel β3 integrin antagonists on some tumour cells and not others. For
example, while cRGDfV was non-toxic to LNCaP prostate carcinoma cells
(Chatterjee et al. 2001), and U-87MG and U-373MG glioma cells (Chatterjee
et al. 2000), Cilengitide was toxic to glioma cells U-251MG and U-87MG
plated onto vitronectin coated dishes. It has been shown Cilengitide can
induce autophagy leading to cell death (Lomonaco et al. 2011) whilst
cRGDfV can induce apoptosis through blocking FAK which mediates the
signal transduction pathway necessary for cell survival (Chatterjee et al.
2001).
ICT9055 was toxic to HT-29 and this cytotoxicity may be due to expression of
β6 integrin (in house data, Andrew Gordon) which has been shown to have a
role in colon cancer cell survival through ERK signalling inducing tumour cell
proliferation (Peng et al. 2009). 10D5 (IgG2a, monoclonal antibody target
αVβ6) induced apoptosis of HT-29 cells through increase in caspase-3 and
caspase-9 expression and release of cytochrome c from the mitochondria to
the cytoplasm (Zhao-Yang et al. 2008). The above result suggests that the
effects of the novel β3 antagonists tested in the present study on HT-29
maybe were via antagonism of αVβ6 integrin, and further work needs to be
done to prove this by using anti αVβ6 integrin inhibitor.
As shown in the scratch assay done here, not all cell lines were adherent or
had rapid cell migration toward wound area, therefore another assay should
be evaluated that may work with a broader range of cell type. Another assay
202
that could be used to investigate integrin antagonism is the Boyden chamber
transwell migration assay. The transwell migration assay has been used to
test the effect of compounds on the inhibition of cell migration in vitro, by
using a transwell migration assay chamber coated with ligands such as
laminin, vitronectin or fibronectin (Trusolino et al. 2000). Using the transwell
migration assay coated with substrates (Byzova et al. 2000) can give an idea
about the effect of potential novel β3 integrin antagonists in inhibition of
tumour cell migration in vivo. Further, the use of the transwell assay will give
a chance to detect the effect of integrin antagonists on adherent and non
adherent cells. The Boyden chamber assay permits migration of cells toward
the ECM protein. Cell contact allows cells to exchange information that can
be used to control the cell migration process (Carreiras et al. 1999).
Here, the wound healing assay was done using uncoated plates. Coating
plates with substrate would provide a more physiologically relevant model to
help to identify integrin antagonists. The choice of extracellular matrix ligand
can be used to determine the selectivity of the antagonists or the relative
contribution of related integrin to cell migration. PC-3 cells which have no
expression of αVβ3 integrin can adhere and migrate on vitronectin mediated
by αVβ1 and αVβ5 integrin (Bisanz et al. 2005). PC-3 cell expressing αVβ5 and
αVβ1 and α5β1 integrin have been shown to migrate on fibronectin and
vitronectin, since these cells had little expression of αVβ3 a selective αVβ3
antagonist did not have an effect on PC-3 plated on vitronectin (Lin et al.
2010). Thus to fully understand our scratch assay results, the expression of
other integrins must be detected; for example, other studies have reported
203
expression of α5β1 integrin in melanoma cell lines (Leavesley et al. 1992;
Seftor et al. 1992). Eventually, targeting multiple integrins in tumour cells and
the microenvironment may be investigated as a therapeutic strategy (Mark
Sutherland 2012).
It will be vital to test the effects of novel integrin antagonists on the ability of
tumour cell lines to adhere to ECM protein such as vitronectin or fibronectin.
The adhesion assay can be used to identify novel integrin antagonists and to
characterise their selectivity, using antibody controls and cell lines with fully
characterised integrin expression (Caltabiano et al. 1999; Del Bufalo et al.
1998). Gentilucci, et al. used an adhesion assay to test the selectivity of
cyclotetrapeptides as αVβ3 and/or α5β1 antagonists. Selectivity was
determined by studying the effects of the peptides on the inhibition of
adhesion of K562 (human erythroleukemic cells) expressing α5β1 integrin, or
SK-MEL-24 (human melanoma cells) expressing αVβ3 integrin to fibronectin,
and HUVECs expressing αVβ3 integrin to vitronectin (Gentilucci et al. 2010).
Wang et al. investigated the role of β3 integrins in transendothelial migration
in vitro by evaluating the migratory capacity of PC-3 cells through a
monolayer of HUVECs. The membranous expression of β3 integrin in PC-3
was associated with active motility from its location at the edges of
pseudopodial extensions, and the study concluded that PC-3 played an
important role in TEM through its interaction with the matrix underneath the
endothelium. Blocking β3 integrin led to inhibition of the interaction of PC-3
cell with the ECM (Wang et al. 2005). Voura et al. found that cRGDfV (5 µM)
204
was able to inhibit transmigration of melanoma cell WM 239 through
HUVECs by 70% and LM609 (40 µg/ml) led to 40-50% inhibition indicating
that transmigration was partly mediated by αVβ5. These assays will be useful
in identifying compounds that would be effective against early stages of
metastasis (Voura et al. 2001).
The effect of potential novel β3 integrin antagonists on downstream cell
signalling needs to be tested since as has been described earlier, integrins
function through inside-out and out-sidein signalling. The
RAS/RAF/MEK/ERK signalling pathway is commonly activated in melanoma
(Meier et al. 2005). Ligation of αVβ3 integrin with ECM has been shown to
lead to focal contact formation and autophosphorylation of FAK at Y397
which provides a recognition site for c-src SH2 domain, and recruitment of
Src that lead to phosphorylation of FAK at Y925 and provides an additional
site to SH2 (adaptor protein) of Grb-2 that recruits SOS to activate Ras then
Raf then MEK then ERK (Chen et al. 2007). For example, cRGDfV blocking
FAK activation led to cleavage of FAK and reduced expression of Akt/PKB
further to activation of caspase 3 and caspase 9 which induced programmed
cell death in LNCaP, prostate carcinoma cells (Chatterjee et al. 2001).
Cilengitide has been used with HUVECs and glioma cells where it induced
cell detachment and apoptosis in cells grown on uncoated plates. The effect
of Cilengitide was associated with reduction in FAK and Src phosphorylation
in both endothelial and glioma cells. Akt phosphorylation was reduced in
glioma cells, but there was no effect on ERK in HUVECs (Oliveira-Ferrer et
al. 2008).
205
After successfully identifying potential novel β3 integrin antagonists that
inhibit tumour cell adhesion, migration and invasion using the functional
assays in vitro, the effect of novel antagonists can be evaluated in term of
inhibition of tumour cell angiogenesis, migration and invasion in vivo.
The results presented in this thesis indicated a very good start for the
development of potential novel β3 integrin antagonists. However, much work
still needs to be done to evaluate the selectivity of novel integrin antagonists.
This will be a big challenge since the cell line model prepared to use with the
scratch assay may not work with the other assays such as adhesion assay
and invasion assays. Therefore, other models will need to be investigated.
6.2 Conclusion
In conclusion, tumour dissemination is a major issue with the treatment of
cancer, and there is a pressing need for therapies which can control this
process. As has been discussed here, the integrins represent a good
potential target for therapeutic intervention to control tumour dissemination.
Using characterised cell lines in a validated functional assay for migration,
promising potential novel β3 integrin antagonists have been identified. Further
work is required to confirm their activity as anti-cancer agents.
206
7 Chapter 7: References and Appendixes
207
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7.2 Appendices
7.2.1 Appendix 1
The expression of integrins subunits in tumour cell lines: PC-3, DLD-1, DU-
145 and HT-29 by PCR.
1.5 PC3 DLD-1

1.5
Relative Intensity
Relative Intensity
1 1
0.5 0.5
0 0
GAPDH αIIb α5 αv β1 β3 β5 β6 GAPDH αIIb α5 αv β1 β3 β5 β6
1.5 DU-145 HT-29

0.3
Relative Intensity
Relative Intensity
0.25
1 0.2
0.15
0.5 0.1
0.05
0 0
a2 a2b a5 aV b1 b3 b5 aV b6 b8
280
7.2.2 Appendix 2
Analysis of scratch width for each cell line in the panel.
A549 H460 MCF-7 DLD-1 HCT-116 HT-29
PC-3 DU-145 SK-MEL-2 UACC62 M14
180
160
140
Width of initial scratch
120
100
80 Width of
60 wound at To
40
20
0
Tumour cell lines
281
282

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University of Bradford eThesis

OF β3 INTEGRIN ANTAGONISM AS A THERAPEUTIC STRATEGY IN

OF β3 INTEGRIN ANTAGONISM AS A THERAPEUTIC STRATEGY IN

Characterisation, development, and utilisation of preclinical cancer

models to investigate novel β3 integrin anatgonists

Submitted for the degree of Doctor of Philosophy

Institute of Cancer Therapeutics

List of Figures ........................................................................................... viii

List of Tables ............................................................................................... xi

Acknowledgement ..................................................................................... xii

Abbreviations ............................................................................................ xiii

1 Chapter 1: Introduction ........................................................................ 1

1.1 Cancer background........................................................................... 1

1.2 The Integrins.................................................................................... 15

1.3 Aims and objectives........................................................................ 57

2 Chapter 2: Characterisation of integrin αIIbβ3 and αVβ3 expression in

2.1 Introduction ..................................................................................... 60

2.2 Materials and methods ................................................................... 64

A. Citrate buffer antigen retrieval ............................................................. 69

B. Pre-warmed pepsin digestion method antigen retrieval .................... 70

C. Proteinase K method antigen retrieval ................................................ 70

2.3 Results ............................................................................................. 80

2.4 Discussion ..................................................................................... 100

2.5 Conclusion..................................................................................... 107

3 Chapter 3: Characterisation of integrin αIIbβ3 and αVβ3 expression in

3.1 Introduction ................................................................................... 109

3.2 Materials and Methods.................................................................. 112

3.3 Results ........................................................................................... 121

3.4 Discussion ..................................................................................... 140

3.5 Conclusion..................................................................................... 148

4 Chapter 4: Investigation of the effect of αVβ3 integrin inhibition on

4.1 Introduction ................................................................................... 150

4.2 Materials and methods ................................................................. 154

4.3 Results ........................................................................................... 161

4.4 Discussion ..................................................................................... 173

4.5 Conclusion..................................................................................... 177

5 Chapter 5: Investigation of the cytotoxicity of potential novel β3

5.1 Introduction ................................................................................... 179

5.2 Materials and methods ................................................................. 181

Table 5.1 Compounds used .................................................................... 181

5.3 Results ........................................................................................... 183

5.4 Discussion ..................................................................................... 192

5.5 Conclusion..................................................................................... 194

6.1 General discussion and future perspective ................................ 196

6.2 Conclusion..................................................................................... 206

7 Chapter 7: References and Appendixes ......................................... 207

7.1 References ..................................................................................... 208

7.2 Appendices .................................................................................... 280

Firstly, I would like to express my profound gratitude to my supervisors Dr.

providing me with an exciting and challenging research project. I would like to

throughout my PhD studies.

I am extremely thankful to the staff of Institute of Cancer Therapeutics who

had helped in this research either by their support or encouragement. I would

Andrew Gordon (who provided PCR data shown in the appendix).

Training (PAAET) for their funding.

My special thanks must also go to my dear parents (Khairyah Al Shammari

and Owayed Al Shammari), my husband Abdul Mohsen Al Shammari and for

my four daughters Sarah, Nourah, Noof and Shaikha.

ABC: Avidin biotin complex

ADMIDAS: Adjacent to metal-ion-dependent adhesion site

ADP: Adenosine diphosphate

Akt/PKB: Protein kinase B

ARMD: Age-related macular degeneration