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Activity of Anticancer Drugs in Relation to the Tumour
Microenvironment and Potential to Inhibit Repopulation
and Enhance Therapeutic Outcome
by
Jasdeep Kaur Saggar
A thesis submitted in conformity with the requirements for the degree of
Doctor of Philosophy
Graduate Department of Medical Biophysics
University of Toronto
© Copyright by Jasdeep Kaur Saggar 2014
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Activity of anticancer drugs in relation to the tumour microenvironment and
potential to inhibit repopulation and enhance therapeutic outcome
Jasdeep Kaur Saggar
Doctor of Philosophy
Graduate Department of Medical Biophysics
University of Toronto
2014
ABSTRACT
Tumours have a disorganized and convoluted vasculature. Tumour cells that reside close
to patent blood vessels are nourished by oxygen and other nutrients and are rapidly proliferating
while distally located cells slowly proliferating. Anticancer drugs must be delivered in adequate
(toxic) concentrations to all tumour cells to be effective, and it is therefore important to study
their distribution of activity in tumours.
In order to assess the distribution of anticancer agents in tumours using immuno-
histochemistry (IHC) and fluorescence imaging, drugs must be either inherently fluorescent or
have antibodies that recognize their activity. As the vast majority of anticancer drugs fulfill
neither of these criteria, the first objective of this thesis was to develop techniques to assess the
pharmacodynamic activity of anticancer drugs in relation to distance from vasculature and
hypoxic regions using biomarkers that could be recognized by antibodies using IHC. I found that
γH2AX, a biomarker of DNA damage was activated in as little as 10 minutes following
treatment. In addition, changes in biomarkers of apoptosis (cleaved caspase -3 or -6) and cell
proliferation (Ki-67) could be used to determine drug activity at 24 hours following drug
treatment.
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The second objective of this thesis addressed the ability of a novel hypoxia activated pro-
drug, TH-302, used alone or with chemotherapy (doxorubicin or docetaxel) to modify the
distribution of biomarkers in relation to blood vessels and regions of hypoxia. It was found that
TH-302 increased DNA damage and apoptosis not only in hypoxic regions but also in
vascularized areas and this effect was augmented through the addition of chemotherapy. TH-302
increased growth delay due to chemotherapy of human tumour xenografts, consistent with
complementary effects of the drugs in different regions of the tumour microenvironment.
The third objective of this thesis was to study changes in the tumour microenvironment
after chemotherapy. I injected into tumour-bearing mice two markers of hypoxia, EF5 and
pimonidazole, with a variable interval between injections. I studied the effects of chemotherapy
alone on repopulation of hypoxic tumour cells and the ability of TH-302 to inhibit repopulation.
It was found that (i) reoxygenation of previously hypoxic cells and repopulation of the tumour
from them occurs after chemotherapy, and (ii) TH-302 was able to reduce tumour cell
reoxygenation and repopulation. This supports the potential of TH-302 to increase therapeutic
effects of chemotherapy.
The work presented in this thesis highlights the importance of the tumour
microenvironment and its ability to impact the distribution of anticancer drugs to cells.
iv
DEDICATION
I would like to dedicate this thesis to
Biji Kesar Kaur & the late Baba Ramlal Saggar
&
The late Biji Jit Kaur & the late Baba Kartar Singh Kainth
&
my parents and entire family.
v
ACKNOWLEDGEMENTS
I’d like to first and foremost thank God Almighty who has guided me my entire life and to whom
I attribute every blessing and happiness possible in life. With faith in God, all things can be
accomplished.
I’d like to thank my supervisor, Dr. Ian Tannock for his time, guidance and support that has led
to my completion of my doctoral thesis and blossoming into a Scientist. His perfect balance
between informative feedback and encouraging freedom of intellectual development as part of
the scientific process has contributed volumes to my PhD experience. You have been a
wonderful supervisor and an excellent mentor and working in your lab exploring the tumour
microenvironment has been a great privilege. Thanks for your support of all of my
extracurricular activities and involvement in student life initiatives. My PhD experience has been
extremely rewarding ranging from securing a CIHR Banting & Best doctoral fellowship and
presenting award-winning research on 4 continents including: South America: Brazil (Sao
Paolo); Asia: China (Shanghai); Europe: France (Versailles & Berder Island), Greece (Crete &
Corfu) and North America: USA (Orlando, Chicago, Washington & San Diego) would not have
been possible without your support of cross-disciplinary scientific engagement. I was also able to
partake in several initiatives on campus including CEO of the Life Sciences Career Development
Society, science outreach at the Canadian Cancer Society, trainee in CIHR’s Drug Safety and
Effectiveness Cross-Disciplinary Program, Mitacs industry internship, Life Sciences Ontario’s
mentorship program and winning the 3 minute thesis inaugural championship and Gordon Cressy
Student Volunteer award– all of this would not have been possible without your support – thank-
you greatly!
I would like to thank my committee members, Dr. Brad Wouters and Dr. Lothar Lilge; my
committee meetings and thought-provoking scientific exchange sessions have contributed to my
development as a scholar and scientist and would not be possible without your guidance.
I’d like to thank Dr. Dhalla and Ken from the St. Boniface Research Centre for introducing me to
research and providing me with a valuable undergraduate research learning environment that has
helped to lead to this work.
I’d like to thank all members of the Tannock laboratory from my early days until now: Carol,
Krupa, Andrea, Susie, Patricia, Man Yu, Alberto, Qian, Neha and Marina. You have contributed
to the wonderful atmosphere in the lab that I have enjoyed coming in to everyday. Thanks for all
of the in-depth conversations! Best of luck in your future endeavors!
I’d like to thank the entire Pathology Research Program at the Toronto General Hospital, the
Advanced Microscopy Facility (Miria, Judy & James), the Animal Resource Colony and Melania
Pintilie for their technical assistance.
Thank-you to the Canadian Institutes of Health Research for selecting me for a Banting & Best
Doctoral fellowship – it was a true privilege to represent the University of Toronto, Canada and
my research at all the conferences I attended.
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Over the years, I have met amazingly supportive individuals in the form of family and friends
worldwide. I thank each and every one of you for your support. I will never forget your
encouragement, kind words and belief in me. Your thoughtfulness and kindness will not be
forgotten!
I’d like to particularly thank my parents who were always encouraging of my pursuit of a PhD
and of my wishes to make the most out of everything – you have taught me much more than can
be learned in a university alone. I’d also like to thank my entire family Bijis, Rome, Sharon,
Shawn, Matthew, Nick, Neel, Devun, Saiyah and Nancy for their support and well-wishes.
Nancy, thanks for all of the phone calls to check in on how I was doing – it was a saving grace at
times and gave me more support than you can imagine. I also thank my Aunts, Uncles and
cousins for their wonderful support.
Moving to Toronto was a big step in life that had its tough moments. I thank God for sending a
beacon of positivity and happiness into my life in the form of my boyfriend Emanuel. You have
been everything that I have ever asked for and even that which I didn’t know I needed. I look
forward to many more happy moments together and thank you from the bottom of my heart for
your unwavering commitment and support of me as an individual on every level possible. Life as
a PhD student and life in general would be unimaginable without you!
Finally, completion of my PhD has been a long process but I have found strength in the teachings
of Guru Nanak:
“Guraa ik dahi buijaa-ee
Sabhnaa jee-aa kaa ik daataa
So mai visar na jaa-ee”
The Guru has given me one understanding
All souls are gifts of the One Almighty.
May I never forget Him!
- Japji Sahib
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TABLE OF CONTENTS
ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................................ v
DEDICATION……………………………………………………………………………………vi
TABLE OF CONTENTS .............................................................................................................. vii LIST OF TABLES .......................................................................................................................... x LIST OF FIGURES ....................................................................................................................... xi ABBREVIATIONS ..................................................................................................................... xiii
CHAPTER 1 ................................................................................................................................... 1 1.1 OVERVIEW OF SOLID TUMOURS & THEIR MICROENVIRONMENT ..................... 2 1.2 TUMOUR MICROENVIRONMENT .................................................................................. 3
1.3 STROMA IN TUMOURS .................................................................................................... 5 1.4 TUMOUR HYPOXIA .......................................................................................................... 5 1.5 TUMOUR ACIDITY ............................................................................................................ 9
1.6 INTERSTITIAL FLUID PRESSURE IN THE TUMOUR MICROENVIRONMENT .... 10 1.7 CANCER TREATMENT ................................................................................................... 11
1.7.1 Chemotherapy ...................................................................................................................... 12 1.8 CAUSES OF RESISTANCE TO ANTICANCER AGENTS ............................................ 13
1.8.1 Molecular and Cellular Causes of Drug Resistance in Tumours ......................................... 13 1.8.1.1 Impaired Drug Uptake ......................................................................................................... 13
1.8.1.2 Enhanced Drug Efflux .......................................................................................................... 14
1.8.1.3 Increased Drug Inactivation and Decreased Drug Activation ............................................. 14
1.9 FACTORS INFLUENCING DRUG DISTRIBUTION WITHIN SOLID TUMOURS .... 15 1.9.1 Effects of High Cell Density ................................................................................................ 15
1.9.2 Effects of Tumour Hypoxia ................................................................................................. 16 1.10 REPOPULATION ............................................................................................................ 17
1.10.1 Repopulation Following Radiotherapy .............................................................................. 17 1.10.2 Repopulation Following Chemotherapy ............................................................................ 20
1.11 FACTORS INFLUENCING DRUG DISTRIBUTION ................................................... 23
1.12 QUANTIFYING DRUG DISTRIBUTION ...................................................................... 24 1.13 STRATEGIES TO IMPROVE THERAPY BY MODULATING THE TUMOUR
MICROENVIRONMENT ........................................................................................................ 28 1.13.1 Strategies to Reduce Interstitial Fluid Pressure ................................................................. 29
1.13.2 Hypoxia Targeted Agents .................................................................................................. 30 1.13.3 Hypoxia-Activated Pro-Drugs ........................................................................................... 31
1.13.3.1 Aromatic N-oxides ............................................................................................................. 33
1.13.3.2 Aliphatic N-oxides ............................................................................................................. 34
1.13.3.3 Quinones ........................................................................................................................... 34
1.13.3.4 Transition Metal Complexes ............................................................................................. 35
1.13.3.5 Nitroimidazoles ................................................................................................................. 35
1.13.3.5.1 PR-104 ........................................................................................................................ 37
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1.13.3.5.2 TH-302 ........................................................................................................................ 38
1.14 RATIONALE .................................................................................................................... 40
1.15 HYPOTHESES ................................................................................................................. 42 1.16 OBJECTIVES ................................................................................................................... 43 1.17 REFERENCES ................................................................................................................. 44
CHAPTER 2 ................................................................................................................................. 56 2.1 ABSTRACT ........................................................................................................................ 57
2.2 INTRODUCTION .............................................................................................................. 58 2.3 MATERIALS AND METHODS ........................................................................................ 59
2.3.1 Cell lines. ............................................................................................................................. 59 2.3.2 Drugs and reagents. .............................................................................................................. 60 2.3.3 Effect of anti-cancer drugs on biomarkers. .......................................................................... 61
2.3.4 Image Analysis and Quantification. ..................................................................................... 63 2.3.5 Statistical Analysis. .............................................................................................................. 63
2.4 RESULTS ........................................................................................................................... 64
2.4.1 Quantification of Biomarkers in Tumour Sections by IHC: ................................................ 64 2.4.2 Time-dependent distribution of doxorubicin and biomarkers in Tumours: ......................... 64 2.4.3 Distribution of melphalan-induced DNA adducts and biomarkers in Tumours: ................. 65
2.4.4 Effect of docetaxel on biomarker distribution in xenografts: .............................................. 66 2.4.5 γH2AX expression and decay following chemotherapy: .................................................... 66
2.5 DISCUSSION ..................................................................................................................... 67 2.6 TABLES ............................................................................................................................. 73 2.7 FIGURES ............................................................................................................................ 74
2.8 REFERENCES ................................................................................................................... 80
CHAPTER 3 ................................................................................................................................. 84 3.1 NOVELTY .......................................................................................................................... 85 3.2 ABSTRACT ........................................................................................................................ 86
3.3 INTRODUCTION .............................................................................................................. 87 3.4 MATERIALS AND METHODS ........................................................................................ 89
3.4.1 Cell lines .............................................................................................................................. 89 3.4.2 Drugs and reagents ............................................................................................................... 90
3.4.3 Effect of drugs on biomarker distributions .......................................................................... 91 3.4.4 Growth delay ........................................................................................................................ 92 3.4.5 Statistical analysis ................................................................................................................ 93
3.6 RESULTS ........................................................................................................................... 93
3.6.1 Distribution of biomarkers in tumour sections: ................................................................... 93 3.6.2 Effects of doxorubicin, docetaxel and TH-302 on the growth of xenografts: ..................... 95
3.7 DISCUSSION ..................................................................................................................... 96
3.8 TABLES ........................................................................................................................... 101 3.9 FIGURES .......................................................................................................................... 102 3.10 REFERENCES ............................................................................................................... 106
CHAPTER 4 ............................................................................................................................... 108 4.1 ABSTRACT ...................................................................................................................... 109
4.2 INTRODUCTION ............................................................................................................ 110 4.3 MATERIALS AND METHODS ...................................................................................... 111
4.3.1 Cell lines ............................................................................................................................ 111
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4.3.2 Drugs and reagents ............................................................................................................. 112 4.3.3 Effect of drugs on tumour reoxygenation and repopulation .............................................. 113 4.3.4 Statistical analysis .............................................................................................................. 115
4.4 RESULTS ......................................................................................................................... 115
4.4.1 Hypoxia markers in tumour sections ................................................................................. 115 4.4.2 Proliferation in continuously hypoxic cells ....................................................................... 115 4.4.3 Flux through the hypoxic compartment in untreated tumours ........................................... 116 4.4.4 Effects of treatment on reoxygenation and proliferation ................................................... 116 4.4.5 Distribution of Ki-67 in tumour sections ........................................................................... 117
4.5 DISCUSSION ................................................................................................................... 118 4.6 FIGURES .......................................................................................................................... 124 4.7 REFERENCES ................................................................................................................. 129
CHAPTER 5 ............................................................................................................................... 133 5. SUMMARY OF FINDINGS AND IMPLICATIONS FOR FUTURE WORK ................. 134 5.1 Potential sources of error and bias .................................................................................... 135
5.2 Use of molecular biomarkers to quantify the spatial distribution of effects of anticancer
drugs in solid tumours ............................................................................................................. 136
5.2.1 Summary ............................................................................................................................ 136 5.2.2 Implications of the study and future directions .................................................................. 137
5.3 Activity of the hypoxia activated pro-drug TH-302 in hypoxic and perivascular regions of
solid tumours and its potential to enhance therapeutic effects of chemotherapy ................... 140 5.3.1 Summary ............................................................................................................................ 140
5.3.2 Implications of the study .................................................................................................... 141 5.4 Chemotherapy rescues hypoxic tumour cells in tumours and induces reoxygenation and
repopulation - an effect that is inhibited by the hypoxia activated pro-drug TH-302. ........... 143 5.4.1 Summary ............................................................................................................................ 143
5.4.2 Implications of the study .................................................................................................... 144 5.5 LIMITATIONS AND FUTURE DIRECTIONS .............................................................. 146
5.5.1 Tumours, Treatment, Image Collection, Biomarker measurements and Repopulation ..... 146
5.6 CONCLUSIONS............................................................................................................... 150 5.7 REFERENCES ................................................................................................................. 151
x
LIST OF TABLES
Chapter 2: Use of molecular biomarkers to quantify the spatial distribution of effects of
anticancer drugs in solid tumours
Table 2.1 Maximal and minimal levels of doxorubicin fluorescence and biomarkers (γH2AX ,
cleaved caspases, change in Ki-67) in various tumours. . ........................................................... 73
Chapter 3: Activity of the hypoxia activated pro-drug TH-302 in hypoxic and perivascular
regions of solid tumours and its potential to enhance therapeutic effects of chemotherapy
Table 3.1 Drug toxicity. .............................................................................................................. 101
xi
LIST OF FIGURES
Chapter 1: Introduction
Figure 1.1 Schematic representation of the human vascular system. ............................................. 4
Figure 1.2 The tumour microenvironment. . .................................................................................. 6
Figure 1.3 Tumour cell repopulation. ........................................................................................... 19
Figure 1.4 Models of tumour cell repopulation.. .......................................................................... 22
Figure 1.5 Drug penetration into tissues. ...................................................................................... 25
Figure 1.6 In vitro cell culture models used to study drug penetration. ....................................... 27
Figure 1.7 The mechanism of action of hypoxia-activated prodrugs. .......................................... 32
Figure 1.8 Exogenous markers of hypoxia.. ................................................................................. 36
Figure 1.9 Activation of TH-302. ................................................................................................. 39
Chapter 2: Use of molecular biomarkers to quantify the spatial distribution of effects of
anticancer drugs in solid tumours
Figure 2.1 Photomicrographs of biomarkers.. ............................................................................. 75
Figure 2.2 Biomarker distribution following doxorubicin treatment............................................ 76
Figure 2.3 Biomarker distribution following melphalan treatment. ............................................. 77
Figure 2.4 Biomarker distribution following docetaxel treatment. ............................................... 78
Figure 2.5 γH2AX expression in cell lines.. ................................................................................ 79
Chapter 3: Activity of the hypoxia activated pro-drug TH-302 in hypoxic and perivascular
regions of solid tumours and its potential to enhance therapeutic effects of chemotherapy
Figure 3.1 Photomicrographs of biomarkers. ............................................................................. 102
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Figure 3.2 Doxorubicin in combination with TH-302.. .............................................................. 103
Figure 3.3 Docetaxel in combination with TH-302.. .................................................................. 104
Figure 3.4 Growth Delay……………………………………………………………………….103
Chapter 4: Chemotherapy rescues hypoxic tumour cells and induces reoxygenation and
repopulation - an effect that is inhibited by the hypoxia activated pro-drug TH-302
Figure 4.1 MCF-7 tumours treated concurrently with pimonidazole and EF5. .......................... 124
Figure 4.2 Spatial distribution of Ki67 positive cells in relation to formerly hypoxic regions in
MCF-7 tumours treated with pimonidazole. ............................................................................... 124
Figure 4.3 Proliferation in hypoxic tissues. .............................................................................. 1272
Figure 4.4 Flux through the hypoxic compartment of MCF-7 tumours. .................................... 128
Figure 4.5 Flux through the hypoxic compartment of PC-3 tumours…………………………..123
Figure 4.6 Reoxygenation and Repopulation in MCF-7 tumours………………………………124
Figure 4.7 Reoxygenation and Repopulation in PC-3 tumours……………………………...…125
xiii
ABBREVIATIONS
5-FU 5-fluorouricil
γH2AX Phosphorylated histone 2AX
α-MEM Alpha minimal essential medium
ABC ATP binding cassette
AOI Area of interest
ATP Adenosine triphosphate
CAMDR Cell adhesion mediated drug resistance
DiOC7 3,3’-diheptyloxycarbocyanine
DMEM Dulbecco’s modified Eagle’s medium
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
DOC Docetaxel
DOX Doxorubicin
ECM Extracellular matrix
EGF Epidermal growth factor
EGFR Epidermal growth factor receptor
ERK Extracellular signal-related kinase
FBS Fetal bovine serum
FITC Fluorescein isothiocyanate
HER2 Human epidermal growth factor receptor 2
HIF Hypoxia inducible factor
xiv
IHC Immunohistochemistry
IFP Interstitial fluid pressure
IP Intraperitoneal
IV Intravenous
mAb Monoclonal antibody
MAPK Mitogen activated protein kinase
MCC Multilayered cell cultures
MDR Multidrug resistance
MTD Maximum tolerated dose
OCT Optimal cutting temperature
PBS Phosphate-buffered saline
PgP P-glycoprotein
RNA Ribonucleic acid
RPMI Roswell Park memorial institute medium
VEGF Vascular endothelial growth factor
1
CHAPTER 1
INTRODUCTION
2
1.1 OVERVIEW OF SOLID TUMOURS & THEIR MICROENVIRONMENT
Solid tumours are composed of tumour cells, normal cells and stroma (primarily composed of
fibroblasts and immune cells) within a complex extracellular matrix (ECM), and nourished by a
vascular network. Solid tumours can arise in epithelial tissues throughout the body (carcinomas)
or from mesencheymal connective tissues (sarcomas). Solid tumours can be benign, pre-
malignant or malignant. The tumour microenvironment is heterogeneous, containing mixtures of
cells that are exposed to varying concentrations of nutrients and oxygen. The variable levels of
oxygen and nutrients can influence the expression of genes and favour a more aggressive
phenotype within regions of the microenvironment. Patent blood vessels supply nutrients and
oxygen to tumour cells that are necessary for their survival and proliferation. Cells that are
farther away from the blood source are poorly oxygenated and are usually slowly or non-
proliferative. Chemotherapy can kill cells that are immediately adjacent to the vasculature, while
cells that reside farther away can escape treatment because they are i) often not exposed to toxic
amounts of drug and ii) not targeted by cycle-active chemotherapeutic drugs due to their slow
rate of proliferation. These cells can survive and repopulate the tumour following treatment.
This thesis explores mechanisms by which the characteristics of the tumour microenvironment
can be manipulated in order to design more effective therapies that target different populations
within the tumour.
3
1.2 TUMOUR MICROENVIRONMENT
The tumour microenvironment is a crucial regulator of the delivery of nutrients to tumour cells,
and of the removal of products of metabolism, and consequently of tumour growth. Tumour cells
are unlike normal cells in that they lack structured growth. Although tumour cells release factors
that stimulate angiogenesis (such as VEGF), the resultant tumour vasculature lacks structure, and
as tumour cells proliferate, blood vessels may be separated by longer distances than in normal
tissues (figure 1.1) and blood flow becomes irregular 1, 2
. As a result, some regions of the tumour
become deficient in oxygen and other nutrients, while poor clearance of cellular breakdown
products, including lactic and carbonic acid, leads to regions of low pH. The highly disorganized
vascular architecture also causes problems related to drug penetration and drug delivery
throughout the solid tumour mass and thus is an important limitation of chemotherapy 2-5
.
Within nutrient-deprived and hypoxic tumour regions the rate of cell proliferation is relatively
low 6, 7
, and slowly-proliferating cells are resistant to most currently-used anticancer drugs,
including many targeted agents. Finally, because blood flow is irregular within a tumour,
regions distal to blood vessels receive lower amounts of drug than those cells located proximal to
tumour blood vessels. Furthermore, the high interstitial fluid pressure caused by poor lymphatic
drainage from the tumour is a challenge to drug delivery 2.
4
Figure 1.1 Schematic representation of the human vascular system. A) Normal tissue and B)
Solid tumour. Red represents well-oxygenated blood from arteries, blue represents poorly-
oxygenated venous system blood and green represents the lymphatic system.
Modified from Kim and Tannock, Nat Rev Cancer 2005;5(7):516-25.
5
1.3 STROMA IN TUMOURS
The stroma in tumours can be stratified into its basic components of i) non-malignant cells,
including cancer-associated fibroblasts, immune cells and vasculature including endothelial cells
and pericytes 8 and ii) the extracellular matrix which includes the structural proteins collagen and
elastin; and specialized proteins (fibronectin). Compared to normal host tissues, the stroma in
tumours favours a malignant transformation that contains an altered ECM, increased amounts of
tumour associated fibroblasts capable of synthesizing growth factors that support epithelial
growth and a defective vascular network that provides tissues with varying levels of oxygen 9.
1.4 TUMOUR HYPOXIA
Hypoxia is a hallmark of many different tumour types and evidence suggests that upwards of
60% of solid tumours display some form of hypoxia distributed heterogeneously throughout the
tumour 10
. Hypoxia arises due to an imbalance between the oxygen requirements for metabolism
of cells and oxygen availability. The convoluted and irregular vasculature of tumours can
compound this situation due to variable and insufficient oxygen supply to cells through blood
vessels as seen in figure 1.2.
6
Figure 1.2 The tumour microenvironment. Schematic representation of a blood vessel and its
surrounding cells. Cells that are closest to the blood vessel are richly nourished with oxygen- and
nutrient-rich blood and therefore rapidly proliferating, cells that are farther away are slowly
proliferating and develop regions of hypoxia due to an inadequate blood supply.
Modified from Tredan et al, JNCI 2007;99:1441-54.
HIGH cell proliferation
low cell
proliferation
Decreasing
pH
Decreasing
nutrients
Decreasing
oxygen
Hypoxia
7
This type of hypoxia is known as chronic or diffusion limited hypoxia. Acute hypoxia may also
occur in solid tumours due to intermittent blood flow resulting from changes in the perfusion of
blood in vessels. Cells that have no oxygen available to them are anoxic and typically this results
in immediate cell arrest 10
. Necrosis occurs when cells are severely starved of oxygen and/or
other nutrients and consequently die. Hypoxic tissues exhibit a partial pressure of oxygen
equivalent to 5-10 mm Hg and severely hypoxic tissue has been shown to have a pO2 as low as
<1 mm Hg oxygen 11
. Most non-benign tissues in the body have a pO2 typically >38 mm Hg
oxygen with the exception of the bone marrow, thus making oxygen content (or the lack thereof)
an attractive target for new anticancer therapies.
Cells that reside far away from functional blood vessels may become hypoxic due to the limited
diffusion of oxygen: the distance from blood vessels to hypoxic regions will depend on the rate
of oxygen consumption by the tumour cells, but typically cells residing at a distance greater than
70 µm from functional blood vessels receive inadequate amounts of oxygen 12
. Tumour hypoxia
is particularly problematic as chemotherapy drugs are unlikely to be achieved in toxic
concentrations in these areas. A study by Primeau et al found that the concentration of
doxorubicin falls off exponentially with increasing distance from blood vessels so that at a
distance of 40-50 µm from blood vessels, doxorubicin was at half of the perivascular
concentration; furthermore, hypoxic regions in these tumours were found at a distance of 90-140
µm from blood vessels 13
. Therefore targeting and killing hypoxic cells with traditional
chemotherapy agents is highly improbable because they do not achieve toxic concentrations in
hypoxic cells and furthermore they are inherently resistant to cycle-active chemotherapy due to
their slow rate of proliferation. In addition, chemotherapy agents may have a reduced effect in
8
hypoxic tissues due to their low extracellular pH levels; this is due to the fact that molecules that
are uncharged can pass through the cell membrane easier than those that are ionized 14
.
Hypoxia in tumours is associated with poor clinical outcome as compared to patients with
tumours lacking hypoxia 15-18
. The presence of hypoxia confers resistance to radiotherapy due to
the absence of oxygen, since oxygen acts as a potent radiosensitizer that fixes radiation-
associated DNA damage. Hypoxia can also have an adverse effect on prognosis since its
presence promotes distant metastasis 16
. The presence of hypoxia leads to up-regulation of genes
that promote a more malignant phenotype and favour cell survival. The transcription factor
hypoxia inducible factor 1 (HIF-1) is induced. HIF-1 is a heterodimer consisting of 2 units, HIF-
1α and HIF-1β 19
. HIF-1β is constitutively expressed and HIF-1α is stabilized under hypoxic
conditions. HIF-1 stimulates the synthesis of angiogenesis-relevant proteins (i.e. VEGF),
suppression of apoptosis and enhanced receptor tyrosine kinase signaling 20
. These in turn favour
epithelial to mesenchymal transition (EMT) – a process that is believed to be associated with
tumour invasiveness and metastasis 21
and has been observed in several in vitro studies 22
,
xenograft models 23
and some clinical studies that have reported markers of EMT to be
associated with poor prognosis in breast carcinomas 24
. HIF-1 also induces the expression of
carbonic anhydrase 9 (CA9) which favours the hydration of CO2 leading to the production of
carbonic acid – further contributing to a decrease in extracellular pH 25
.
Tumour hypoxia is linked with loss of the p53 tumour suppressor protein that may result in a loss
of apoptotic ability 26
. Furthermore, hypoxia confers radio-resistance because reactive oxygen
radicals that are produced following radiation under well-oxygenated conditions contribute to
DNA damage 27
. Specifically, the ratio of the effective dose of ionizing radiation in hypoxic cells
verses the dose in oxygenated cells is 2.5-3 28
.
9
Hypoxia may also inhibit the effects of chemotherapy via the same mechanism since in the
presence of oxygen drugs such as doxorubicin can produce reactive oxygen species such as
super-oxides that can further damage DNA beyond the primary mechanism of action i.e.
topoisomerase II inhibition 29
. Hypoxia has also been shown to down-regulate expression of
DNA topoisomerase II, so that drugs such as doxorubicin and etoposide that target this protein
will be inefficient 30
.
Transient hypoxia can stimulate gene amplification, leading to increased expression of genes that
encode proteins that cause drug resistance; these proteins include dihydrofolate reductase, with
associated resistance to methotrexate and the multi-drug resistant transporter p-glycoprotein 31
.
Increased expression of p-glycoprotein results in increased levels of substrate drugs being
pumped out of cells thus resulting in inadequate intracellular levels to cause cytotoxicity 32
.
1.5 TUMOUR ACIDITY
The poor vascular organization and lack of lymphatic drainage of solid tumours contributes to a
build-up of metabolic byproducts such as lactic and carbonic acids leading to a reduced
extracellular pH. The production of lactate arises from glycolysis – a favoured route of energy
production in tumours that uses glucose to produce ATP in the cytosol. Glycolysis typically
takes place under hypoxic conditions, when oxidative phosphorylation is not possible, but in
tumours glycolysis also takes place in oxygenated regions 33
. Tumour acidity influences drug
uptake into tumour cells. When the extracellular tumour environment is acidic, chemotherapeutic
drugs that are basic (such as doxorubicin, mitoxantrone, vincristine and vinblastine) are
protonated; this decreases cellular uptake since charged drugs pass through the cellular
membrane less efficiently than those that are uncharged 34
. In contrast, drugs that are acidic (such
10
as chlorambucil and cyclophosphamide) will tend to concentrate within cells. Even if basic drugs
pass through the cellular membrane, sequestration within acidic organelles such as endosomes
may occur, leaving less drug to attack tumour DNA and produce antitumour effects 35
. It is also
unlikely that toxic concentrations of acidic drugs will pass through the cell membrane in their
uncharged form (due to the low extracellular pH) due to poor penetration from the vasculature.
1.6 INTERSTITIAL FLUID PRESSURE IN THE TUMOUR MICROENVIRONMENT
High interstitial fluid pressure (IFP) within solid tumours is a barrier to the effective delivery of
therapeutic compounds 36
,37, 38
, as it inhibits the penetration of drugs into tumour tissue. This is
particularly true in human pancreatic tumours that are extremely resistant to systemic cancer
therapy 39, 40
. High IFP has been shown to predict overall lower survival following radiation
therapy in cervical cancer patients 36
. Raised IFP is due at least in part, to a dense extracellular
matrix (ECM) leading to stiffness and high cell density that leads to compression of blood
vessels (leaky blood vessels), inadequate lymphatic drainage and contraction of the interstitial
space mediated by fibroblasts 41
. High IFP may have an adverse effect on treatment since it may
cause vascular compression and inadequate drug delivery. Drugs may diffuse from the blood
vessel via diffusion (the movement of molecules from where they are highly concentrated to
regions where they are lowly concentrated) or via convection which depends on gradients of
pressure (both the hydrostatic force imposed by the heart and the osmotic force imposed by the
movement of water from the interstitial space into the blood vessel) 38
. As the IFP is high in
tumours, this works against the effective delivery of drugs from the vasculature to tumour cells
via convection.
11
1.7 CANCER TREATMENT
Cancer therapies can include a combination of surgery, radiation and drug treatment including
molecularly targeted agents, hormonal therapy, biological therapy and chemotherapy.
Molecularly targeted agents such as trastuzumab target specific molecular pathways of cells such
as the HER2 pathway. Hormonal therapy includes the use of drugs that change hormone levels or
block hormone receptors. Biological therapies can work in different ways including to either
prevent cells from dividing, attempt to increase the response of the body’s immune system to
attack cancerous cells rather than attacking tumour cells themselves and seeking out cancer cells
and destroying them. An example of a biological therapy that stimulates the immune system is
the monoclonal antibody rituximab which targets the CD20 antigen on the surface of leukemia
and lymphoma cells which then elicits the immune system to recognize these cells and kill them.
Typically, patients receive a combination of different treatments tailored to treating their specific
cancer; however, therapies – particularly chemotherapy – are quite toxic not only to rapidly
proliferating cancer cells but to normal tissues as well. Due to their narrow therapeutic index,
many anticancer therapies must be administered below levels at which they would have a
maximum effect on cancer cells in order to minimize damage to normal tissues. In addition,
chemotherapy is typically delivered in a three week cycle in order to allow recovery of normal
rapidly proliferating tissues that are damaged (particularly bone marrow). Pharmacokinetic
studies can help profile a drug’s activity within the body following administration and
characterizes a drug’s: 1) absorption into the blood plasma, ii) distribution in the body, iii)
metabolism by different organs and iv) elimination from the body 42
. Pharmacodynamic studies
reveal information regarding the damage induced by drugs in different tissues.
12
1.7.1 Chemotherapy
Chemotherapy can be used as the primary curative modality for some types of cancer including
Hodgkin’s disease and testicular cancer or as palliative therapy for advanced cancers or as
adjuvant therapy to improve local control of the primary tumour. When chemotherapy is used
prior to surgery in order to reduce the size of the tumour it is referred to as neoadjuvant therapy.
Conventional chemotherapeutic agents include those that directly attack DNA and cause cell
death in rapidly proliferating tissues. Generally, most chemotherapeutic drugs are most active at
a given stage of the cell cycle, often the S-phase: these can include topoisomerase II inhibitors
such as doxorubicin, and antimicrotubule agents such as docetaxel. Administration of
chemotherapy is typically on a 3 week cycle to allow for recovery of normal tissues such as the
bone marrow 43
.
Newer anticancer treatments include targeted therapies that do not interfere directly with DNA
replication but instead target different molecular pathways. These include both small molecules
and monoclonal antibodies; monoclonal antibodies include trastuzumab, which targets HER2
and signaling from it, rituximab which enhances the immune response to cause cellular damage
to lymphoma cells and bevacizumab which targets VEGF to inhibit angiogenesis. Small
molecules may inhibit receptor tyrosine kinases such as gefitnib that blocks tyrosine kinase
activity of EGFR or intracellular signaling molecules and include temsirolimus that blocks the
mTOR pathway and thereby inhibits cell proliferation 43
.
Most anticancer drugs have a narrow therapeutic index meaning the dose at which they are
effective against tumour tissues and the dose at which they are toxic to some normal tissues is
quite similar which compromises the treatment’s efficacy.
13
1.8 CAUSES OF RESISTANCE TO ANTICANCER AGENTS
Anticancer agents have limited effectiveness for most solid tumours with the exception of
testicular cancer and some childhood tumours. Lack of efficacy may be due to intrinsic drug
resistance present in a subpopulation of cells in the tumour that have the ability to regenerate the
tumour such that selective pressure favours survival of resistant cell populations. Molecular
mechanisms of drug resistance focus on epigenetic or molecular changes occurring in single cells
whereas causes related to the tumour microenvironment consider the entire population of cells in
the tumour and include such problems as drug distribution.
1.8.1 Molecular and Cellular Causes of Drug Resistance in Tumours
Molecular mechanisms of drug resistance typically focus on genetic or epigenetic modifications
occurring at the cellular level which may lead to impaired drug uptake, enhanced drug efflux,
increased drug inactivation or reduced drug activation.
1.8.1.1 Impaired Drug Uptake
Drugs enter cells via 3 processes: I) simple diffusion by which the drug enters the cell without
interacting with a specific protein or site on the cell membrane, II) facilitated diffusion by which
the drug interacts with a transport carrier in the membrane without the expenditure of energy and
III) active transport by which the drug interacts with a specific transport carrier and requires the
expenditure of energy. Only process III can allow for the transport of molecules against their
concentration gradient 1. Impaired drug influx to cells is a cause of drug resistance. An example
is the nitrogen mustards (e.g. melphalan) that require active transport in order to enter cells;
14
therefore defects in the active transport carrier will inhibit this process thus reducing the amount
of drug that enters the cell.
1.8.1.2 Enhanced Drug Efflux
Following the successful entry of a drug into a cell, drugs can be faced with removal from the
cell via membrane transporter proteins known as drug efflux pumps. Although there are several
ATP-binding cassette (ABC) transporters only 3 are clinically relevant to drug resistance: p-
glycoprotein, multidrug resistance protein 1 and ABCG2. The best characterized drug resistance
pump is p-glycoprotein which is encoded by the multidrug-resistance (MDR1) gene. P-
glycoprotein transports a range of anticancer and non-anticancer drugs and has a broad substrate
specificity, which contributes to its potential to export drugs resulting in less net drug uptake due
to increased drug efflux. Studies in mice that were p-glycoprotein knockouts displayed a marked
increase in sensitivity to several drugs 44
. Furthermore, p-glycoprotein has been found to be over-
expressed in tumours compounding the issue of drug delivery due to enhanced drug efflux.
1.8.1.3 Increased Drug Inactivation and Decreased Drug Activation
Many anticancer drugs are administered in an inactive form and require activation via enzymes.
Therefore reduced activity of drugs can occur when the enzymes responsible for activation are
lacking or if there is an abundance of enzymes that deactivate the activated form of the drug. For
example, 5-fluorouracil requires the enzyme uridine monophosphate for its activation 45
.
Downregulation of uridine monophosphate in normally 5-FU sensitive HCT-8/P colourectal
cancer cells resulted in drug resistance and cell survival 45
. Conversely, 5-FU is catabolised to an
15
inactive form by the enzyme dihydropyrimidine dehydrogenase, rendering the drug ineffective in
patients who overexpress this enzyme 46
.
1.9 FACTORS INFLUENCING DRUG DISTRIBUTION WITHIN SOLID TUMOURS
Anticancer drugs must reach target tumour cells through the vasculature. The penetration of
drugs to tumour cells is reliant upon convection and/or diffusion. Convection depends on
pressure gradients and given that the pressure within tumour blood vessels and the tumour
interstitium are both quite high, there is probably minimal movement of drugs from the
vasculature to the tumour via this mechanism 47
. Diffusion involves the movement of drugs
along a concentration gradient, i.e. from regions where they are concentrated (within the
vasculature) to less concentrated regions (the tumour interstitium). Larger molecules tend to
move more slowly than smaller molecules via diffusion, and tissue penetration will depend on
consumption by the cells 2. Drugs that are water-soluble will diffuse readily through the
extracellular fluid, although the diffusion coefficient will depend on the nature of the ECM.
Drugs with a higher lipid solubility can penetrate into cells easily 42
. Drug half-life is also an
important determinant influencing drug penetration, since drugs with longer half-lives in the
circulation have a better opportunity to establish themselves within tumour tissues as their source
concentration remains higher for a longer period of time 42
.
1.9.1 Effects of High Cell Density
Tumour cells grown in cell culture often display sensitivity to drugs when cell density is low in
dilute culture 48
supporting the concept that cells, which are tightly packed together are more
16
likely to display drug resistance. Drug resistance in highly dense cellular areas may be due to
high levels of cell adhesion proteins such as integrins and is described as cell-adhesion mediated
drug resistance (CAMDR). CAMDR can be related to several factors including decreased drug
uptake into cells due to an acidic microenvironment (particularly of concern for basic drugs as
discussed in section 2.3) and altered apoptosis and cell proliferation 49
.
1.9.2 Effects of Tumour Hypoxia
The presence of hypoxia within a tumour has been associated with a more malignant phenotype
as it has been shown to increase genomic instability. Tumour vasculature is poorly organized and
as a result, does not meet the requirements of providing oxygen and nutrients throughout the
tumour, and many solid tumours develop regions of low oxygen concentration 28
. Cells in
hypoxic regions of tumours are resistant to radiotherapy (because radiation and some drugs are
more active in killing cells in the presence of oxygen, which allows formation of toxic free
radicals) and are probably resistant to chemotherapy because of poor drug access and low
proliferation. The presence of hypoxia in tumours has also been associated with a more
aggressive phenotype and poor clinical outcome through the mechanism of increasing genetic
instability 50
. In addition, hypoxia stimulates the expression of hypoxia-inducible factor 1 (HIF1)
which leads to: (i) upregulation and release of VEGF leading to increased angiogenesis , (ii) loss
of E-Cadherin function – promoting cell motility and invasiveness, (iii) upregulation of
proteolytic proteins such as urokinase-type plasminogen activator (Upa), promoting increased
cancer cell motility. These properties of hypoxic tumour cells may render hypoxia a major
determinant to effectiveness of chemotherapy 10
. As a result, there is considerable potential for
17
therapies, which target hypoxic tumour cells to reduce repopulation and tumour regeneration that
may occur from these sites.
1.10 REPOPULATION
Both radiation therapy and chemotherapy are given on a schedule that allows for periods of rest
so that normal tissues can repair, recover or repopulate. However, repopulation or the
proliferation of surviving tumour cells between daily treatments of radiation therapy can also
occur and is an established cause of treatment failure. Few studies have focused on repopulation
following chemotherapy as it relates to the tumour microenvironment and this topic is addressed
in chapter 3 of this thesis.
1.10.1 Repopulation Following Radiotherapy
Accelerated repopulation following radiotherapy was first described by Malaise and Tubiana by
demonstrating that growth of a mouse fibrosarcoma following irradiation was faster than in non-
irradiated controls 51
. Jung et al studied rhabdomyosarcoma RIH in the rat model and observed
that repopulation of cells increased with increasing radiation dose 52
. In a mouse model of human
squamous cell carcinoma, treatment with fractionated radiotherapy resulted in an initial lag phase
of growth that was followed by rapid repopulation 53
. In another study by Sham and Durand,
mice treated with 50 Gray in 20 fractions over 23 days with a split between 10 fraction-5 day
courses compared to mice treated with 50 Gray in 20 fractions over 10 days had a greater rate of
repopulation than observed with the shorter treatment time frame suggesting that increased time
delay between treatments should be avoided 54
. Withers et al provided evidence of the clinical
18
significance of accelerated repopulation following radiotherapy: their analysis showed that
clonogen repopulation accelerates in squamous cell carcinoma of the head and neck following an
initial lag period 55
. To offset this repopulation, a marked increase of 0.6 Gray daily was needed
to overcome repopulation, consistent with a 4 day clonogen doubling rate. Recently, Pottgen et al
(2013) found that accelerated hyperfractionated (22 days) verses conventional fractionated (31
days) radiation therapy for the treatment of non-small cell lung cancer resulted in pathologic
complete response rates of 37% and 24% respectively, indicating that a shorter treatment time
can contribute to a favourable outcome 56
. The overall response to radiotherapy is based on four
R’s: i) repair of DNA damage, ii) redistribution of cells in the cell cycle, iii) repopulation and iv)
reoxygenation of hypoxic tumour areas 57
. As accelerated fractionated radiotherapy occurs over a
shorter period of time compared to conventional radiotherapy, it is likely that the increased
response rates of accelerated fractionated radiotherapy are due to a reduction in the time allowed
for cells to repopulate the tumour.
Repopulation following radiation therapy has been attributed to reoxygenation of tissues that
occurs from killing oxygenated cells located preferentially closest to blood vessels, leaving more
blood and nutrients for cells that survived therapy located farther away 58, 59
(Figure 1.3a).
Another model of repopulation highlights an increase in tumour stem cells containing clonogenic
potential following radiation therapy thus re-establishing the tumour (Figure 1.3b). In addition,
radiation therapy has been shown to increase levels of EGFR ultimately leading to activation of
the MAP kinase pathways contributing to cellular growth 60
. Methods to reduce the effects of
tumour cell repopulation on therapeutic outcome are under investigation.
19
Figure 1.3 Tumour cell repopulation. A) Schematic of a blood vessel and surrounding cells in
untreated verses treated tumours. Notice that irradiation or chemotherapy preferentially kills cells
located closest to blood vessels thereby improving the nutrient and oxygen content to surviving
cells located farther away which leads to subsequent migration closer to blood vessels. B) In
untreated tumours, stem cells may produce differentiated cells and other stem cells at a constant
rate compared to fractionated radiotherapy that can result in an increased production in tumour
stem cells possessing clonogenic capabilities.
Adapted from Kim and Tannock 2005.
20
1.10.2 Repopulation Following Chemotherapy
Few studies have focused on repopulation following chemotherapy as it relates to the tumour
microenvironment. Studies using tissue culture in which ovarian cells were exposed to lethal
concentrations of cisplatin (relative to doses achievable in humans) resulted in the majority of
cells dying but surviving cells were able to re-establish colonies 61
. However, ovarian cancer
cells that were exposed to the synthetic anti-progestin steroid mifepristone between multiple
rounds of chemotherapy (cisplatin) resulted in markedly reduced clonogenic capacity 62
. Huxham
et al reported that repopulation of HCT-118 colon cancer cells following gemcitabine treatment
commenced sooner in regions distal to blood vessels compared to proximal vessels 4. In another
study, concurrent treatment of gefitnib and paclitaxel in A431 EGFR over-expressing tumours
resulted in increased growth delay over the control and sequential treatments 59
. Another study
evaluating tumour cell repopulation following chemotherapy showed that treatment of MCF-7
xenografts with either 5-FU or paclitaxel led to a greater tumour volume than when the selective
estrogen receptor modulator arzoxifene was added between courses of chemotherapy
treatment 63
. Therefore, there is potential to inhibit chemotherapy-associated repopulation.
Presumably the same reasons pertaining to the tumour microenvironment and the ability of
chemotherapy to kill off rapidly dividing cells as opposed to cells that are slowly proliferating
contribute to tumour cell repopulation.
Repopulation can be attributed to several mechanisms including a sub-optimal level of drug
delivered to tumour cells due to the erratic vascular architecture within solid tumours. Limited
drug distribution even to cells that are relatively close to blood vessels is also a major problem
due to cells that possess multidrug resistance proteins that pump drugs out of cells, the
sequestration of basic anticancer drugs (i.e. doxorubicin) into acidic organelles within cells
21
thereby reducing exposure of the drug target, and high interstitial fluid pressure that inhibits drug
diffusion into the tumour. Our group and others have demonstrated that the concentration of
several drugs including doxorubicin, melphalan and docetaxel decreases rapidly at increasing
distances from blood vessels so that cells that are 40-50 µm away receive about half the
perivascular concentration; this leads to reduced cell death and consequently a surviving
population of cells capable of regenerating the tumour 13, 59, 64, 65
.
Models of tumour cell repopulation have been generated and examples are displayed in Figure 4.
This model assumes that 70% of tumour cells are killed following each round of chemotherapy.
Surviving tumour cells can repopulate the tumour at either a constant rate of repopulation
following chemotherapy treatment as shown in Figure 1.4a or an accelerated rate of repopulating
cells following treatment as seen in Figure 1.4b. Finally an assumption of an initial lag period
following each chemotherapy treatment followed by accelerated repopulation can be built into
the model (Figure 1.4c).
22
Figure 1.4 Models of tumour cell repopulation. These models assume that 70% of tumour
cells are killed after each round of chemotherapy which is given at a 3 week interval. A)
Assuming a constant rate of repopulating tumour cells the doubling time could be either 10 days
or 2 months depending on either a slow or fast rate. B) Assuming accelerated repopulation
following each round of chemotherapy and C) Assuming an initial lag in repopulating tumour
cells followed by accelerated repopulation.
Adapted from Kim and Tannock 2005.
23
Cells with slow proliferation can also evade chemotherapy treatment. There is evidence that
cancer initiating cells (also referred to as cancer stem cells or cancer progenitor cells) have a
slow rate of proliferation and have been implicated in resistance to radiation and chemotherapy
66, 67. Hermann et al showed that pancreatic cancer cells treated with gemcitabine both in vitro
and in vivo led to selection of CD133+ cancer stem cells following treatment 68
. Other cell types
within the tumour microenvironment that divide slowly if at all include hypoxic cells.
Chemotherapy is designed to kill rapidly dividing cells – those that are typically located closest
to blood vessels. Cells that are farther away may not receive sufficient amounts of drug to induce
cell death 1, 13, 59, 65
. The presence of hypoxia has been extensively studied in relation to radiation
treatment; however the effect of hypoxia on repopulation following chemotherapy has not been
studied and chapter 3 of this thesis will investigate the effect of reoxygenated cells on
repopulation.
1.11 FACTORS INFLUENCING DRUG DISTRIBUTION
Several of the characteristics that are present in the tumour microenvironment contribute to the
limited effectiveness of chemotherapy. Drugs reach the tumour through blood vessels: as the
tumour blood supply is irregular, adequate drug distribution to the entire tumour is problematic.
Drugs penetrate into tissues via diffusion and convection, but given the high interstitial fluid
pressure in tumours movement via convection is minimal. Pharmacokinetics also plays a role
including distribution of the drug in the plasma and its half-life. Whereas monoclonal antibodies
may have longer half-lives that can help increase drug distribution (e.g. cetuximab) due to a
longer circulation time in the plasma and corresponding longer time to penetrate into tissues;
other drugs such as doxorubicin possess a short half-life and consequently tissue penetration
24
occurs in a shorter period of time 5. Water soluble drugs can move about the ECM quite readily
whereas lipid soluble drugs can penetrate into cell membranes easily depending on the density of
the ECM. High extracellular tumour acidity also makes it difficult for basic drugs such as
doxorubicin and mitoxantrone to cross the cell membrane as they exist in their protonated or
charged forms at low pH. Despite this, basic drugs that cross the cell membrane may be
sequestered into acidic endosomes, thus compromising the amount of drug available to attack
DNA. This non-specific sequestration contributes to the drug’s consumption as does the target-
specific binding (i.e. tumour DNA or growth factor receptor). A schematic summarizing the
factors that influence drug distribution are presented in Figure 1.5. Other physiochemical
properties of drugs including their size and molecular weight, shape, charge and solubility
(aqueous and lipid) play a role in drug distribution.
1.12 QUANTIFYING DRUG DISTRIBUTION
Quantification of drug distribution is important in order to determine a drug’s ability to penetrate
tissue within solid tumours. Both in vitro and in vivo techniques have been used for quantifying
drug distribution. A common in vitro technique uses tumour spheroids, and adherent tumour
cells can grow spheroids up to 3 mm in diameter 69
. Spheroids develop hypoxic areas as well as
central necrosis once they have reached ~500 μm in diameter 70
.
25
Figure 1.5 Drug penetration into tissues. A) As a drug moves from the blood vessel into the
tumour the amount of drug administered as well as its plasma half-life will be limiting factors as
to how much drug the tumour actually receives. B) A drug’s movement in the ECM is
dependent upon its size, charge and solubility. Small, neutrally-charged molecules that are lipid
soluble will have an easier time penetrating cell membranes compared to large, basic drugs. C)
Drug consumption relates to metabolism of the drug through binding to target receptors as well
as sequestration into non-specific areas.
Adapted from Minchinton and Tannock 2006.
26
Drug distribution in spheroids (Figure 1.6a) can be studied for fluorescent drugs, or by using
autoradiography to determine the distribution of labelled drugs 71, 72
. An alternative is to generate
multicellular layers (MCL) on collagen-coated micro-porous membranes: the rate of drug
penetration can then be evaluated by adding a drug on one side of the MCL and measuring its
concentration on the other as a function of time, as shown in Figure 1.6b 21
. Spheroids and MCLs
have been used to study the distribution of a wide range of drugs 73
, and most drugs show rather
poor distribution in tumour tissue.
Drug distribution can also be studied in tumours grown in animals. Growth of tumours in
window and ear chambers allows for direct observation of tumour microcirculation, but a
disadvantage is that tumours are relatively small with limited areas of hypoxia and/or necrosis 74
.
Tissue sections can be obtained after drug treatment of animals bearing transplanted tumours or
human tumour xenografts and used for immunohistochemical analysis. This analysis will allow
for the quantification of fluorescent drugs in relation to blood vessels or regions of hypoxia, and
the technique can be applied to human biopsies 13, 59, 64
. These studies have revealed decreasing
concentration of fluorescent doxorubicin, mitoxantrone or topotecan with increasing distances
from blood vessels (4). Distribution of other drugs such as cetuximab, trastuzumab 5 and
melphalan 65
within tumour sections can be quantified with the use of anti-IgG specific (for the
former two) or melphalan DNA adduct specific (for the latter) monoclonal antibodies that
recognize the drug’s activity.
Most anticancer drugs are non-fluorescent in the UV-visible range so their distribution within
tumour tissues is difficult to assess.
27
Figure 1.6 In vitro cell culture models used to study drug penetration. A) Tumour spheroids
containing several layers of cells can be generated and stained with cell cycle markers to observe
changes due to drugs. B) A multicellular culture can be used to study drug penetration where
drug is administered in the top compartment (blue) and its diffusion rate into the bottom
compartment can then be assessed.
Adapted from The Basic Science of Oncology (2013)
A B
28
An alternative is to evaluate molecular markers of drug effect, using antibodies that recognize
cell proliferation (Ki67, cyclin D1 or bromodeoxyuridine incorporation into DNA), antibodies
that mark cell death or apoptosis (e.g. activated caspase -3 or -6), and markers of DNA damage
such as γH2AX . We recently used antibodies to γH2AX , caspase-3 or -6 and Ki-67, and a
computer-based algorithm, to quantify the distribution of (non-fluorescent) docetaxel 65
, this
technique is elaborated upon in chapter 2 of this thesis.
Given the limited penetration of many chemotherapeutic agents, cells that are distal from blood
vessels do not receive adequate amounts of drug to cause cell death. Thus, tumour cell
repopulation arising from areas where cells are not killed and previously under-nourished (e.g.
hypoxic regions) is probable, and indeed we have recently shown that previously hypoxic cells
may reoxygenate and repopulate after treatment of human tumour xenografts with doxorubicin or
docetaxel, this study is expanded upon in chapter 3 of this thesis.
1.13 STRATEGIES TO IMPROVE THERAPY BY MODULATING THE TUMOUR
MICROENVIRONMENT
Given the importance of the tumour microenvironment, methods to target its components are
under investigation. A variety of strategies to improve drug distribution are being researched and
include: strategies to reduce interstitial fluid pressure, increasing the delivery of oxygen to
tissues and the use of hypoxia-targeted agents such as hypoxia-activated pro-drugs.
29
1.13.1 Strategies to Reduce Interstitial Fluid Pressure
The interstitial fluid pressure (IFP) within solid tumours is often high 37, 38
, and this can inhibit
the penetration of drugs into tumour tissue. This is particularly true in human pancreatic tumours
that are extremely resistant to systemic cancer therapy 39, 40
. Raised IFP is due, at least in part, to
a dense ECM and high cell density that leads to compression of blood vessels, and to inadequate
lymphatic drainage 41
. High IFP may have an adverse effect on treatment since it may cause
vascular compression and inadequate drug delivery. Strategies to reduce IFP are being
investigated and include the use of angiogenesis (VEGF) inhibitors that have been shown to
increase the uptake of chemotherapy (CPT-11) 75
. Platelet-derived growth factor (PDGF)
contributes to raising IFP by controlling the extracellular matrix through increased production of
collagen76
. Inhibition of PDGF receptor kinases with agents such as imatinib (Glivec) has been
shown to reduce IFP in immunocompromised mice bearing thyroid carcinoma and in rats with
colon carcinoma 77
.
A recent study by Provenzano et al 40
showed that there is an abundance of hyaluronic acid (HA)
in the ECM of pancreatic tumours. HA is a large glycosaminoglycan that is associated with
elevated IFP, and treatment with a HA-targeting enzyme (PEGPH20) was able to diminish HA
levels and result in patent blood vessels and a corresponding increase in doxorubicin penetration
40. Other methods of improving vascular perfusion have also been investigated: Olive et al.
reported that reduction in levels of tumour-associated stromal fibroblasts through disruption of
Hedgehog signaling resulted in increased angiogenesis and greater penetration of gemcitabine
into pancreatic tumours 39
. The use of HA-targeting enzymes (PEGPH20) and Hedgehog
signaling disruptors (GDC-0449 & LDE225) are being investigated in clinical trials. Despite the
strategies to reduce IFP in order to increase drug distribution, it is likely that most drugs will be
30
unable to penetrate the needed distance of up to 200 µm in order to be effective against
pancreatic tumours.
1.13.2 Hypoxia Targeted Agents
Mechanisms for targeting hypoxia have been varied. These include placing patients in hyperbaric
oxygen chambers in order to decrease the levels of hypoxic tissues present in their bodies.
Hyperbaric oxygen chambers are designed to deliver 100% pure oxygen to patients under
increased atmospheric pressure so that more oxygen is dissolved in the plasma and then
delivered to tissues.
Carbogen breathing has also been used as a means of increasing oxygen levels in tissues.
Carbogen is a mixture of two gases: carbon dioxide and oxygen. Once carbon dioxide is detected
by the brain it is tricked into believing that oxygen availability has decreased (which would
normally be the case, however levels of oxygen in carbogen are as high at 70-95%). The brain
reacts by increasing the rate of breathing, elevating the heart rate and causing cells to release
buffering agents to remove carbonic acid from the blood. This results in an increase in dissolved
oxygen in the plasma.
Hypoxic cell radiosensitizers mimic the radiosensitizing abilities of oxygen. These compounds
are electrophillic and the nitromidazoles are the best known; of these misonidazole is the most
extensively studied. These agents are reduced under hypoxic conditions and then bind to DNA;
although they have no effect as single agents, they are able to chemically fix the damage caused
by ionizing radiation. Clinical trials involving the use of misonidazole have been inconclusive
but their limited therapeutic effects may be attributed to the toxicity of this agent (neuropathy) in
31
normal tissues. However, the less toxic nimorazole has been reported to produce superior loco-
regional control in conjunction with radiotherapy in a phase III trial 78
.
1.13.3 Hypoxia-Activated Pro-Drugs
Since hypoxic cells may survive after systemic drug treatment, and since tumour hypoxia confers
a particularly metastatic and aggressive tumour phenotype, it is a logical target for new
approaches to therapy. Hypoxia-activated pro-drugs (HAPS) have been developed, such that a
pro-drug is administered in an inactive form, and is activated via a reduction reaction in hypoxic
regions to a toxic moiety that damages DNA 79
. Since the pro-drug does not bind to DNA in
oxygenated cells, it should diffuse readily to hypoxic tumour regions. Several HAPs have been
investigated including tirapazamine, AQ4N, PR-104 and TH-302. The efficacy of a HAP
depends on i) selective activation in areas >100 µm from blood vessels, i.e. extremely hypoxic
areas (<0.5% Oxygen), ii) resistance to non-selective activation, i.e. anything other than extreme
hypoxia, iii) resistance to inactivation via non-selective enzymes, iv) maintenance of low toxicity
in normal tissues and v) ability to diffuse to tumour cells that are not themselves hypoxic
following activation, i.e. bystander effect. All HAPs function on the ability of the original non-
toxic compound to undergo an electron reduction thereby forming a radical that may be back
oxidized by oxygen to produce superoxide and the original pro-drug. In the absence of oxygen
the radical can undergo further reaction to form a toxic compound that is capable of attacking
tumour DNA. The basic mechanism of a hypoxia-activated pro-drug is demonstrated in Figure
1.7. The most common feature of bioreductive pro-drugs is to damage the DNA replication fork
21. There are 5 different chemical moieties with the ability to be metabolized by enzymatic
reduction under hypoxic conditions.
32
Figure 1.7 The mechanism of action of hypoxia-activated pro-drugs. A) Under normoxic
conditions, hypoxia activated pro-drugs may become reduced by reductases and undergo back
oxidation by oxygen that is present in the cell. B) In hypoxic cells, hypoxia-activated pro-drugs
become reduced and in the absence of oxygen their toxic moiety is free to attack DNA in
hypoxic tumour cells.
Adapted from Brown and Wilson; exploiting tumour hypoxia in cancer therapy. Nature reviews
cancer 2004.
33
These include: aromatic N-oxides, Aliphatic N-oxides, quinones, transition metal complexes and
nitroimidazoles 80
.
1.13.3.1 Aromatic N-oxides
Tirapazamine is the most widely studied aromatic N-oxide. Tirapazamine was first reported in
1986 to have 300 times more selective cytotoxicity in anoxic cells than oxygenated cells in vitro
81. Tirapazamine is an aromatic N-oxide that undergoes reduction via the cytochrome P450
oxioreductase thus generating a free radical, this tirapazamine free radical then gives rise to
another radical capable of attacking DNA 82, 83
. Several preclinical in vivo studies in which
tirapazamine was combined with either radiotherapy or chemotherapy were promising 84
;
however results of clinical trials results have been disappointing. While phase II results appeared
satisfactory 85, 86
, tirapazamine failed to provide superior tumour control in patients with
squamous cell carcinoma of the oral cavity, oropharynx, hypopharynx or larynx over the
standard of care (radiotherapy and cisplatin) in phase III trials 87
. Due to limited clinical benefit
(perhaps because of poor distribution in tumour tissue of both the pro-drug, and the activated
drug), further clinical investigation was halted 88
. There are several problems with the design of
the drug that include i) the requirement of only moderate levels of hypoxia in order to activate
the drug that may lead to greater toxicity of normal tissues (i.e. bone marrow), ii) irreversible
inactivation by an oxygen insensitive reductase and iii) an extremely limited bystander effect. All
of these factors may have attributed to the poor performance of this drug in larger phase III
clinical trials. A new analogue of tirapazamine – SN30000 has been synthesized to possess a
greater selectivity for hypoxia and is scheduled to undergo phase I clinical trials.
34
1.13.3.2 Aliphatic N-oxides
AQ4N is an aliphatic N-oxide that is metabolized to AQ4 in hypoxic regions following a 2-
electron reduction by members of the cytochrome P450 family. AQ4 then functions as a
topoisomerase II inhibitor and DNA alkylator that will attack hypoxic cells. However AQ4N is
activated in moderately hypoxic regions and has relatively no bystander effect 89, 90
. Imaging
studies showed evidence of the fluorescent AQ4N/AQ4 to distribute well to hypoxic regions 91
.
Preclinical studies combining AQ4N with radiation or chemotherapy in vivo reported significant
activity and provided the basis for the first clinical trials 92, 93
. Specifically, murine tumours with
induced hypoxia via hydrazaline breathing showed a 13 times greater response rate than control
tumours 93
. Despite these promising results, there has been no data reported to date regarding the
outcome of clinical trials in humans. Development of this drug has been discontinued.
1.13.3.3 Quinones
The quinone prototype is mitomycin C. Mitomycin C requires activation via reductases and
possesses greater activation in hypoxic verses oxygenated regions 94
. Unfortunately mitomycin C
is readily inactivated by a non-reversible reduction 95
. This led to the development of other
quinones with greater hypoxic selectivity for activation such as apaziquone. Apaziquone
undergoes a 1-electron reduction reaction to produce a cytotoxic species that damages DNA
causing DNA double strand breaks. Unfortunately pharmacokinetic studies have revealed
apaziquone as having a very short half-life contributing to inadequate tissue distribution and
rapid clearance from plasma 96
. Overall quinones have not been the most promising hypoxic
cytotoxic agents.
35
1.13.3.4 Transition Metal Complexes
Transition metals have been reported to have the potential to form free radicals by accepting
electrons and then attacking DNA. However, the transition metals undergo these reactions non-
selectively, that is they occur in both oxygenated and hypoxic tissues so their use as hypoxic
selective cytotoxic agents is not promising 97
. The platinum compounds: cisplatin, oxaliplatin
and carboplatin have all demonstrated increased control of tumours following radiation therapy
perhaps due to inhibition of DNA repair or the increased production of free radicals able to
attack DNA, but these effects are not selective to hypoxic regions and as a result possess
significant toxicity to normal tissues 97
.
1.13.3.5 Nitroimidazoles
Nitroaromatic compounds vary in their hypoxic selective cytotoxicity. Nitro compounds were
first used for their radiosensitizing abilities. They are able to mimic oxygen in that they can fix
DNA damage caused by radiation. The nitroimidazoles: pimonidazole and EF5 (as seen in Figure
1.8) are reduced by the NADPH:cytochrome P450 oxioreductase enzymes following a 1-
electron reduction reaction in the absence of oxygen and are fixed onto thiol groups in hypoxic
cells with less than 10 mm Hg Oxygen. Direct visualization of these hypoxic tracers is possible
with immunohistochemistry. There have been no reported adverse events upon the
administration of pimonidazole 98
or EF5 at doses required for visualization of hypoxic tissues
and have been safely injected into humans in clinical studies 99, 100
.
36
Figure 1.8 Exogenous markers of hypoxia. Chemical structures of A) Pimonidazole and B)
EF5. These nitroimadazoles are frequently employed in the study of hypoxia as they are
exogenous markers of hypoxia.
A B
37
In addition, neither pimonidazole nor EF5 are reduced in dead cells, so only viable hypoxic cells
will generate signals 101
. EF5 can identify hypoxic tissue about 2 hours following injection in
mice and pimonidazole at 30-60 minutes. Once bound to hypoxic tissues, EF5 adducts remain
visible for up to 24 hours while pimonidazole adducts are stable for 5 days depending on the rate
of hypoxic cell turnover. Following tumour excision, these adducts can be detected with the
appropriate antibodies and were utilized in my studies (chapter 3 of this thesis) whereby I
evaluated the change in hypoxia (reoxygenation) over time following drug treatment using both
pimonidazole and EF5. In the clinic, the most widely studied hypoxic imaging maker is 18
F-
MISO and is detected with PET. This technique has been employed to assess tumour
responsiveness following treatment.
1.13.3.5.1 PR-104
PR-104 is a water soluble pro-drug that undergoes hydrolysis of its phosphate group via plasma
phosphotases to generate the lipophilic intermediate PR-104A. In hypoxic cells, PR-104A
undergoes enzymatic reduction to PR-104H and PR-104M, which are much more toxic then the
parent drug. Unfortunately, PR-104A can also be activated by aldo-keto reductases under aerobic
conditions 102
. Results from a phase I clinical trial of PR-104 with sorafenib in hepatocellular
carcinoma showed that treatment was not well tolerated and the trial was discontinued 103
.
38
1.13.3.5.2 TH-302
TH-302 is a 2-nitroimidazole containing a toxic alkylating moiety that is released only when its
attached nitro-heterocyclic trigger molecule fragments in hypoxic cells to release the DNA
crosslinking agent bromo-isophosphoramide mustard that binds to DNA 104
. TH-302 is reduced
by the NADPH: cytochrome P450 family of reductases and requires severe hypoxia (~0.1%
Oxygen) for maximal activation 105
. Following reduction, the TH-302 radical anion fragments
releasing the toxic bromo-isophosphoramide moiety capable of attacking DNA and causes
crosslinkage to occur as seen in Figure 1.9.
Preclinical studies evaluated TH-302 across 32 human cancer cell lines using a clonogenic assay
and reported cytotoxicity results following TH-302 treatment for 2 hours under hypoxic or
normoxic (air) conditions followed by 3 days incubation in normoxic (air) conditions 105
. TH-302
cytotoxicity was modest under normoxic conditions, however an aerobic to hypoxic cell
cytotoxicity ratio of up to 550 was found 105
. TH-302 has been shown to decrease the hypoxic
fraction and increase necrosis following treatment of many different tumours including small cell
lung cancer and pancreatic cancer in animals 106
. Anti-tumour efficacy was related to the overall
dose of TH-302 given regardless of regimen where animals bearing H460 tumours given a total
dose of 500 mg/kg over 15 days with dosing twice weekly compared to smaller daily increments
equaling 500 mg/kg over 2 weeks showed equivalent tumour growth inhibition of 89% compared
to controls 106
. In studies evaluating the effects of TH-302 in combination with chemotherapy,
tumour growth delay and tumour growth inhibition were favoured when TH-302 was delivered 4
hours prior to chemotherapy as tested in non-small cell lung cancer (with cisplatin, docetaxel,
gemcitabine), fibrosarcoma (doxorubicin) and prostate (docetaxel) cancer models 107
.
39
Figure 1.9 Activation of TH-302. TH-302 enters cells in an inactive form and becomes reduced
by reductases to generate a radical intermediate. This intermediate can undergo back-oxidization
to generate the initial pro-drug or exert a bystander effect by diffusing to surrounding tissues
prior to fragmentation to release the toxic warhead bromo-isophosphorimide drug that attacks
hypoxic cell DNA.
Adapted from William and Hay; Targeting hypoxia in cancer therapy. Nature reviews 2011.
40
Delivery of TH-302 prior to chemotherapy would allow for TH-302 to attack the tumour’s
hypoxic fraction, which may be reduced by chemotherapy through the induction of
reoxygenation of formerly hypoxic cells – this hypothesis is investigated in chapter 3 of this
thesis. Co-administration of TH-302 with chemotherapy also resulted in toxicity, with loss of
body weight in animals, and hematology studies revealed that the combination of TH-302 with
doxorubicin or gemcitabine resulted in lowered white blood cell counts 107
. TH-302 has a plasma
half-life of 8-10 minutes in mice, thus by 4 hours the majority of TH-302 would be cleared from
the plasma making a drug-drug interaction improbable 108
. Favourable in vivo study results have
paved the way for the investigation of TH-302 in clinical trials. A phase 1 study of the safety and
efficacy of TH-302 as monotherapy established a maximum tolerated dose of 575 mg/m2
weekly
with no clinically significant myelosuppresion; dose limiting toxicities were reported to be due to
skin and mucosal irritations 109
. In a phase 1 study evaluating the combination of TH-302 with
doxorubicin in 16 patients with advanced soft tissue sarcomas, the maximum tolerated dose of
TH-302 was 300 mg/m2
when combined with doxorubicin 110
.
In a randomized phase II clinical trial of gemcitabine and TH-302 in pancreatic cancer,
combined therapy increased progression-free survival from 3.6 to 5.6 months 111
and a phase III
trial is in progress. Thus, TH-302 appears to be a promising addition to traditional chemotherapy,
and recent studies in our laboratory suggest that it can inhibit the repopulation and reoxygenation
of formerly hypoxic cells following treatment of human tumour xenografts with chemotherapy.
1.14 RATIONALE
Drug distribution is an important cause of drug resistance since many cells within tumours do not
receive adequate amounts of drug needed to kill cells. The distribution of the frequently used
41
fluorescent drug doxorubicin in solid tumour xenografts has already been studied and has been
shown to decrease with increasing distances from blood vessels. As many tumour cells do not
receive enough drug to cause cell death, there is potential for chemotherapy administration to
selectively target susceptible populations of cells thus leaving more resistant cells within the
tumour to proliferate. Repopulation of tumour cells following radiotherapy is well-established,
and although repopulation of tumour cells following chemotherapy has not been studied
comprehensively, it is believed to contribute to treatment failure. Most anti-cancer drugs are not
fluorescent and their distribution in tumour sections is difficult to assess; however techniques
evaluating the pharamcodynamic distribution of biomarkers that reflect a drug’s activity may be
used. By using biomarkers of drug effect, the ability of drugs to penetrate deeply into the tumour
with respect to the vasculature and hypoxia can be measured. This tool can then be used in the
testing of novel therapeutics and the ability of different drug combinations to target certain cell
populations within the tumour microenvironment.
Tumour hypoxia has been associated with metastatic disease and poor treatment outcome. As
most anticancer drugs target either rapidly proliferating cells (cytotoxic agents) that are closest to
the vasculature, or certain cells that express growth factor cell receptors (cytostatic agents) or
attempt to block angiogenesis (VEGF inhibitors), there is a need to develop drugs that target
hypoxic cells. Hypoxia activated pro-drugs have been evaluated in the clinic but to date there is
no HAP that is approved for use in conjunction with standard chemotherapy. The HAP, TH-302
is the farthest advanced in clinical trials, however since it is non-fluorescent, its direct
visualization in tumour tissue is not possible. Therefore, evaluating the distribution of
biomarkers of TH-302-based activity in relation to the vasculature and hypoxia, alone and in
combination with chemotherapy, is important in supporting the mechanism of action of this drug.
42
Comparing the biomarker changes in different regions of the tumour will allow for greater
understanding of the use and scheduling of combination therapy.
Since chemotherapy targets rapidly dividing oxygenated tissue and spares hypoxic cells, it is
possible that following chemotherapy, hypoxic tissues that may have died in the absence of
treatment, now reoxygenate and start proliferating. This hypothesis is tested in chapter 3 of this
thesis using two sequentially administered markers of hypoxia: pimonidazole and EF5 that bind
to hypoxic tissues. Isolating tissues that are hypoxic prior to treatment and are no longer hypoxic
(i.e. have reoxygenated) can elucidate changes in the transiently hypoxic tumour fraction.
In theory, HAPs can inhibit the process of proliferation of hypoxic tissues by binding to DNA
and killing such cells. The effects of treatment with a HAP (TH-302) in hypoxic tissues and
throughout the tumour can then be determined by measuring the fraction of cells that are
proliferating using Ki-67 as a marker. The efficacy of TH-302 to inhibit tumour cell proliferation
in formerly hypoxic tissues can thus be determined, providing valuable information about the
drug and its efficacy. Studying the effects of chemotherapy on tumour cell repopulation and the
potential of TH-302 to inhibit this process will contribute to the optimal scheduling and
combination of these agents.
1.15 HYPOTHESES
A) Pharmacodynamic markers of drug effect can be used to predict the distribution of anticancer
drugs in tumour tissue as a function of distance from vasculature and hypoxic regions.
B) Tumour cells in regions distant from blood vessels (i.e. hypoxic regions) are a major source of
repopulation after chemotherapy.
43
C) The hypoxia activated pro-drug (TH-302) will inhibit repopulation of cancer cells stimulated
to proliferate between cycles of conventional chemotherapy and thereby reduce the probability of
tumour relapse.
1.16 OBJECTIVES
The aims of this thesis are to: i) develop techniques to assess drug penetration in relation to
blood vessels and regions of hypoxia using pharmacodynamic markers of drug effect, ii)
determine the ability of chemotherapy to rescue previously hypoxic cells that may have died in
the absence of treatment and thus repopulate or regrow the tumour and iii) evaluate the ability of
the hypoxia activated pro-drug TH-302 to inhibit this process. These objectives will be
completed by:
1. Evaluating the distribution of the fluorescent anti-cancer drug doxorubicin and comparing its
fluorescence pattern to the distribution of pharmacodynamic markers of drug effect through its
effects on apoptosis, DNA damage and/or change in cellular proliferation. These techniques will
then be extended to other non-fluorescent drugs (melphalan, docetaxel and TH-302). Melphalan
will serve as further validation for the use of these techniques as monoclonal antibodies that
recognize the unique DNA adducts formed by this drug will be used to compare to the pattern of
distribution observed of the biomarkers.
2. Determining the degree to which hypoxic cells (at time of chemotherapy treatment) are able to
redistribute in relation to blood vessels and contribute to repopulation and the ability of TH-302
to distribute to hypoxic tissues and inhibit repopulation between cycles of chemotherapy.
44
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56
CHAPTER 2
Use of molecular biomarkers to quantify the spatial distribution of
effects of anticancer drugs in solid tumours
Jasdeep K. Saggar1, Andrea S. Fung
1, Krupa J. Patel
1 and Ian F. Tannock
1,2
1Department of Medical Biophysics, University of Toronto, Toronto ON, Canada and
2Division of Medical Oncology and Hematology, Princess Margaret Hospital, Toronto ON,
Canada
This data chapter has been published in Molecular Cancer Therapeutics:
Saggar JK, Fung AS, Patel KJ, Tannock IF. Use of molecular biomarkers to quantify the
spatial distribution of effects of anticancer drugs in solid tumours. Molecular Cancer
Therapeutics. 2013 Apr;12(4):542-52.
57
2.1 ABSTRACT
Poor distribution of anticancer drugs within solid tumours may limit their effectiveness. Here we
characterize the distribution within solid tumours of biomarkers of drug effect. γH2AX, cleaved-
caspase -3 or -6 and Ki-67 were quantified in tumour sections in relation to blood vessels
(recognized by CD31) using monoclonal antibodies and immunohistochemistry. To validate
their use we compared their time-dependent distribution with that of (i) fluorescent doxorubicin
and (ii) a monoclonal antibody that detects melphalan-induced DNA adducts. The biomarkers
were then used to quantify the distribution of docetaxel in relation to tumour blood vessels.
Activation of γH2AX was evaluated following in vitro exposure of tumour cells to multiple
drugs. Distributions of doxorubicin in MDA-MB-231 and MCF-7 xenografts and of melphalan-
induced DNA adducts in MCF-7 & EMT-6 tumours decreased with distance from blood vessels,
similar to the distributions of (i) γH2AX at 10 minutes, (ii) cleaved caspase-3 or -6 and (iii)
change in Ki-67 at 24 hours following treatment. The distribution of these biomarkers following
treatment with docetaxel also decreased with increasing distance from tumour blood vessels.
Activation of γH2AX occurred within 1 hour after exposure to several drugs in culture. Multiple
anticancer drugs show a decrease in activity with increasing distance from tumour blood vessels;
poor drug distribution is an important cause of drug resistance. The above biomarkers may be
employed in designing strategies to overcome therapeutic resistance by modifying or
complementing the limited spatial distribution of drug activity in solid tumours.
58
2.2 INTRODUCTION
Solid tumours develop an imperfect vasculature. Although tumour cells release factors
that stimulate angiogenesis (such as VEGF), the resultant tumour vasculature lacks structure, and
as tumour cells proliferate, blood vessels may be separated by longer distances than in normal
tissues and blood flow becomes irregular 1,2
. As a result, some regions of the tumour become
deficient in oxygen and other nutrients, while poor clearance of cellular breakdown products,
including lactic and carbonic acid, leads to regions of low pH 3,4
. The disorganized vascular
architecture also causes problems of limited drug penetration and drug delivery throughout the
solid tumour. Tumour regions distal to patent blood vessels may receive lower amounts of drug
than those cells located proximal to tumour blood vessels, while changes in blood flow may lead
to intermittent delivery of drugs to some tumour regions. Within nutrient-deprived and hypoxic
tumour regions the rate of cell proliferation is relatively low, and slowly-proliferating cells are
resistant to most currently-used anticancer drugs, including many targeted agents 5-7
. As a result
of these factors, tumour cells within poorly-nourished regions of solid tumours are likely to
survive drug therapy, regardless of their intrinsic sensitivity to the drugs that are used. This
important mechanism of drug resistance has been rather neglected in comparison to molecular
causes of resistance operative at the level of the single cell, which dominate when cells are
exposed to drugs under optimal conditions in tissue culture.
The spatial and temporal distribution of clinically-used drugs in tumour tissue are
important factors in determining tumour response to therapy, while modifying or complementing
such distributions with other agents represent potential strategies to improve therapeutic index.
In previous studies our group has demonstrated limited distribution from tumour blood vessels of
doxorubicin and mitoxantrone using immuno-histochemistry (IHC) to quantify these fluorescent
59
drugs in tumour sections 8,9
. However, most anti-cancer drugs are not fluorescent and antibodies
that recognize them in tissue are not generally available. While autoradiography has been used
to demonstrate limited distribution of other drugs such as paclitaxel, 10,11
this technique is
cumbersome. Moreover, it would be preferable to quantify the distribution not only of the native
drugs, but of the pharmacodynamic effects that they and their metabolites have on tumour cells.
The aim of the present study was to characterize the spatial distribution within solid tumours of
biomarkers of drug effect: changes in the phosphorylated histone γH2AX (a marker of DNA
damage), cleaved caspase-3 or -6 (a marker of apoptosis) and Ki-67 (a marker of cell
proliferation), in relation to blood vessels (recognized by an antibody to CD31). We elected to
first study immunohistochemical (IHC) methods for quantifying markers of effect for
doxorubicin, which is fluorescent, and for melphalan, where adducts with DNA can be
recognized by an antibody; this strategy enables a comparison between drug concentration and
changes in the pharmacodynamic markers of drug effect in relation to tumour blood vessels. We
then extended these techniques to study a widely-used non-fluorescent agent (docetaxel) and to
evaluate expression of γH2AX after treatment of cultured cells with a range of anticancer drugs.
2.3 MATERIALS AND METHODS
2.3.1 Cell lines. Studies were conducted using the following cell lines: human breast carcinomas
MDA-MB-231 and MCF-7, mouse mammary EMT-6, human vulvar epidermoid carcinoma A-
431 and human prostate cancer PC-3. EMT-6 cells were originally provided by Dr. Peter
Twentyman (University of Cambridge; Cambridge, UK) and all other cell lines were purchased
from the American Type Culture Collection (Manassas, VA) in 2011. MCF-7, MDA-MB-231,
60
A-431 and EMT-6 cells have been maintained in our laboratory and were grown in α-MEM
supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT). PC-3 cells were grown
in Ham's F-12K medium (Life Technologies Inc.) supplemented with 10% FBS. All cells were
grown in a humidified atmosphere of 95% air/5% CO2 at 37ºC. Routine tests to exclude
mycoplasma in all cell lines were performed several times each year. Short tandem repeat
analysis was performed to ensure cells (PC-3, MCF-7, MDA-MB-231, A-431) were of human or
(EMT-6) murine origin in August, 2012. These cell lines were employed to test the efficacy of
drugs on tumour models relevant to their clinical use (e.g. the use of doxorubicin for human
(MCF-7, MDA-MB-231) and murine (EMT-6) breast tumours and docetaxel for human prostate
(PC-3) and cervical (A-431) tumours.
To generate tumours, 4-6 week old male athymic nude mice (Jackson, Bar Harbor,
Maine, USA) were injected subcutaneously in both flanks with 2x106 PC-3 cells, and 4-6 week
old Female athymic nude mice (Harlan Sprague-Dawley, Madison, WI) with implanted 17β
estradiol tablets (60 day release; Innovative Research of America, Sarasota, FL) were injected
subcutaneously with 5x106 MCF-7 cells per side; non-estradiol implanted female athymic nude
mice were injected with 5x106 MDA-MB-231 cells or 1x10
6 A-431 cells. Female balb/c mice
were injected with 1x106 EMT-6 cells. There were six mice per treatment group (~10-12
tumours) and each experiment was repeated twice.
2.3.2 Drugs and reagents. Doxorubicin (Pharmacia, Mississauga, Ontario, Canada), melphalan
(Glaxo Wellcome Inc., Mississauga, Ontario, Canada), docetaxel (Sanofi-aventis, Laval, Quebec,
Canada), 5-FU, cabazitaxel (Sanofi-aventis, Laval, Quebec, Canada), gemcitabine (Eli Lilly,
Toronto, Ontario, Canada), methotrexate (Pfizer, Kirkland, Quebec, Canada), topotecan
61
(GlaxoSmithKline, Toronto, Ontario, Canada) paclitaxel (Bristol Myers Squibb, Montreal,
Canada) and vinblastine (Mayne Pharma, Montreal, Quebec, Canada) were purchased from the
Princess Margaret Hospital pharmacy; they were provided as solutions with concentrations of 2
mg/mL, 5 mg/mL or 40 mg/mL.
Purified rat anti-mouse CD31 (platelet/endothelial cell adhesion molecule 1) monoclonal
antibody was purchased from BD PharMingen (Mississauga, Ontario, Canada) and the Cy3-
conjugated goat anti-rat IgG secondary antibody was purchased from Jackson ImmunoResearch
Laboratories, Inc. (West Grove, PA). The MP5 monoclonal antibody that recognizes
melphalan/DNA adducts 12
was generously provided by Dr. M.J.Tilby (U.K.) as a hybridoma
culture supernatant, dilution 1:100. γH2AX was stained with a rabbit anti-human γH2AX
antibody (Cell Signaling; HRP chromogen), cleaved caspase-3 with rabbit anti-human cleaved-
caspase 3 antibody (Cell Signaling; HRP chromogen), cleaved caspase-6 with rabbit anti-human
cleaved caspase-6 antibody (Novus Biologicals, HRP chromogen) and Ki-67 with rabbit anti-
human Ki-67 antibody (Novus; HRP chromogen).
2.3.3 Effect of anti-cancer drugs on biomarkers.
In vitro studies were performed in chamber slides with one chamber per slide (Lab-Tek II, Nunc,
Roskilde, Denmark) using a protocol described elsewhere 12
. Briefly, PC-3 cells were grown in
chamber slides and then treated with 50nM docetaxel for 0, 10, 30, 60 and 90 minutes at 37C.
Following the removal of culture media, cells were washed with phosphate-buffered saline
(PBS) and then fixed for 10 minutes at room temperature in 0.3% H2O2 solution supplemented
with acetone in order to block endogenous peroxidase. Cells were subsequently air-dried for 10
62
minutes and stored at -20ºC while awaiting immunohistochemical staining with γH2AX
antibody.
To determine the distribution of drugs and/or pharmacodynamic biomarkers in vivo, mice
bearing tumours of mean cross-sectional area 0.7-0.8 cm2 were given a single intravenous
injection of doxorubicin (25 mg/kg) or intraperitoneal injection of melphalan (6 mg/kg) or
docetaxel (15 mg/kg). These doses were selected as the maximum tolerated doses that caused
minimal weight loss in mice. Animals were killed and tumours were excised from control
(untreated) mice and from treated animals at varying times after drug injection. Samples were
embedded immediately in OCT compound and flash frozen in liquid nitrogen and stored at
−70°C prior to tissue sectioning and IHC staining. Single cryostat sections (10μm thick) were
then cut from each tumour. Whole tumour sections were analyzed and artifacts and regions of
necrosis were omitted; a minimum of 10 tumours were analyzed per treatment group.
Tumour sections were stained for blood vessels using the rat anti-CD31 primary antibody
and Cy3-conjugated goat anti-rat IgG secondary antibody; they were stained for γH2AX, cleaved
caspase-3 (or -6) and Ki-67 with the appropriate antibodies. Since MCF-7 cells contain a
deletion in the exon 3 that encodes the caspase-3 gene, they do not express caspase-3 13
. A study
by Inoue et al found that the effector caspase-6 processes caspases-8 and -10 leading to
apoptosis, and we therefore chose to study the expression of cleaved caspase-6 14
. Tumour
sections were imaged for CD31 using the Cy3 (530-560 nm excitation/573-647 nm emission)
filter set. γH2AX, cleaved caspase-3 (or -6) and Ki-67 were imaged using transmitted light.
Photomicrographs of these biomarkers in relation to blood vessels are illustrated for control and
docetaxel-treated A-431 xenografts in Figure 2.1.
63
2.3.4 Image Analysis and Quantification.
Image analysis and quantification were performed using Media Cybernetics Image Pro PLUS
software. In order to minimize noise due to tumour autofluorescence, a minimal threshold for
detection (below the level of detection of doxorubicin) was determined for each tumour.
Doxorubicin was quantified according to the method described by Primeau et al 8.
A novel protocol developed by Fung et al 15
was developed to analyze composite images
based on the method described by Primeau et al. 8. Briefly, binarized CD31 images were created
and then used to create a distance map such that each pixel is represented by its distance to the
nearest functional blood vessel in the section. Distributions of the pharmacodynamic biomarkers
γH2AX, cleaved caspase-3 (or -6) and Ki-67 were determined by creating binary masks (black
and white images). The biomarker mask was then combined with the blood vessel distance map
to form a composite image with distance measurements that corresponded only to the biomarker
(γH2AX, cleaved caspase-3, Ki-67) positive pixels. The data are represented graphically as the
percent of pixels that are biomarker positive at any given distance from the nearest blood vessel
in the section; a cut-off of 60µm was used in order to minimize interference from neighboring
blood vessels that are out of the plane of the section. For in vitro studies γH2AX positive pixels
were counted and expressed as a percentage of the total number of pixels.
2.3.5 Statistical Analysis.
A one-way ANOVA, followed by Tukey’s post-hoc test, determined statistical differences
between treatment groups. P<0.05 was used to indicate statistical significance; all tests were 2-
sided and no corrections were applied for multiple significance testing. Drug and biomarker
distributions are represented as mean values +/- SEM.
64
2.4 RESULTS
2.4.1 Quantification of Biomarkers in Tumour Sections by IHC:
The biomarkers γH2AX, cleaved caspase-3 or -6 and Ki-67 could be recognized and
quantified by applying appropriate antibodies as shown in Figure 2.1, panels A-C. γH2AX and
the apoptotic markers cleaved caspase-3 or -6 increased after drug treatment (see below for time
course) while proliferation as indicated by Ki-67 decreased.
2.4.2 Time-dependent distribution of doxorubicin and biomarkers in Tumours:
In order to determine the time-dependent distribution of the biomarkers, we studied
previously frozen, doxorubicin-treated MDA-MB-231 tumours. These tumours were excised at
10 minutes, 3, 6, 24 and 48 hours after doxorubicin treatment or control (untreated). The
fluorescent distribution of doxorubicin at all time points displayed decreased levels of drug at
greater distances from blood vessels (Figure 2.2A). As seen in Figure 2.2B, γH2AX was
maximal at 10 minutes after treatment and then decreased to undetectable levels. Cleaved
caspase-3 levels increased with time up to 24 hours and decayed by 48 hours (Figure 2.2C). The
Ki-67 distribution remained suppressed from 3 to 48 hours (Figure 2.2D). We chose to evaluate
subsequently γH2AX distribution at 10 minutes, and cleaved caspase-3 or change in Ki-67
distributions at 24 hours, after drug treatment.
Results for MCF-7 tumours were qualitatively similar to the above with highest
doxorubicin concentration adjacent to blood vessels (Figure 2.2E). γH2AX expression was
65
increased compared to control at 10 minutes and maximal in regions closet to blood vessels
(Figure 2.2F). There was increased apoptosis in regions close to blood vessels at 24 hours
compared to control (Figure 2.2G). Ki-67 staining was highest in regions closest to blood
vessels with a marked reduction 24 hours after doxorubicin treatment (Figure 2.2H).
Maximum (close to blood vessels) and minimum levels (at 60μm, largest distance from
blood vessels evaluated) of doxorubicin fluorescence, and of the biomarkers (in relation to values
in control tumours) are shown in Table 2.1, Reductions in γH2AX at 10 minutes and cleaved
caspase-3 or -6 at 24 hours reflect the distribution of doxorubicin fluorescence. Interpretation of
change in Ki-67 is more complex because of two effects: drug-induced decrease in proliferation
proximal to blood vessels and low proliferation in distant regions even in control tumours.
2.4.3 Distribution of melphalan-induced DNA adducts and biomarkers in Tumours:
Melphalan-induced DNA adducts were assessed in MCF-7 xenografts and murine EMT-6
tumours by staining with the MP5 antibody, which recognizes adducts formed as a result of
melphalan binding to N7 of guanine 12
. Such adducts were evident at 10 minutes after injection
and decayed only slightly at 24 hours (Figures 2.3A and 2.3E). The frequency of adducts
decreased with increasing distance from tumour blood vessels.
At 10 minutes after administration of melphalan, there was a marked increase in γH2AX
compared to control and γH2AX decreased with increasing distance from blood vessels; this
distribution reflected that of melphalan-induced DNA adducts in both tumour types (Figures
2.3B and 2.3F). There were few apoptotic cells in untreated tumours but at 24 hours after
treatment there was increased staining for cleaved caspase-3 or -6 and this was most pronounced
in regions proximal to blood vessels (Figures 2.3C and 2.3G). Control tumours showed
66
decreased proliferation with increasing distance from blood vessels in both tumours. There was a
reduction in Ki-67 levels at 24 hours following treatment with melphalan, more pronounced in
regions close to blood vessels (Figures 2.3D and 2.3H). As shown in Table 2.1, the fall in levels
of γH2AX at 10 minutes and of cleaved caspase-3 at 24 hours reflect quite closely the
distribution of melphalan-DNA adducts.
2.4.4 Effect of docetaxel on biomarker distribution in xenografts:
Staining of the biomarkers in relation to blood vessels in control and docetaxel-treated A-
431 tumours is shown in Figure 2.1. At 10 minutes after docetaxel treatment there was marked
activation of γH2AX in PC-3 and A-431 xenografts and γH2AX decreased with increasing
distance from blood vessels (Figures 2.4A and 2.4D). There was a corresponding increase of
cleaved caspase-3 over control levels at 24 hours after treatment, again with more marked
apoptosis in proximal as compared to distal regions (Figuress 2.4B and 2.4E). There was a fall in
Ki-67 at 24 hours after docetaxel treatment, especially in regions close to blood vessels (Figures
2.4C and 2.4F). The distribution of biomarkers after docetaxel treatment is quite similar to that
after treatment with doxorubicin and melphalan (Table 2.1).
2.4.5 γH2AX expression and decay following chemotherapy:
To evaluate the expression of γH2AX following a range of anticancer drugs with
different mechanisms of action, we treated PC-3 cells for 1 hour with: 5-FU (10 uM), cabazitaxel
(100 nM), gemcitabine (100 nM), methotrexate (400 nM), topotecan (50 nM), paclitaxel (50 nM)
67
or vinblastine (50 uM). As seen in Figure 2.5A, all chemotherapeutic agents induced expression
of γH2AX.
PC-3 cells were treated with 50 nM of docetaxel and samples were taken at different time
points. As seen in Figure 2.5B, γH2AX expression is highest at 10 minutes following treatment
and then decays to near basal levels by 90 minutes.
2.5 DISCUSSION
Our group and others have shown that the distribution of doxorubicin 8,16
, mitoxantrone
and topotecan 9,17
decrease as a function of distance from the nearest blood vessel in solid
tumours. However, most anticancer agents are not fluorescent, and their visualization and
quantification in solid tumours is difficult. Moreover, the activity of many drugs depends on
metabolites as well as the parent drug, so that evaluation of the distribution in tumours of cellular
damage caused by drugs is more relevant than the distribution of the drugs themselves. We have
therefore evaluated the use of biomarkers of drug effect, which can be recognized by fluorescent-
or chromogen-tagged monoclonal antibodies, in order to determine the distribution of activity of
anticancer drugs. Using fluorescent doxorubicin (25 mg/kg) to initiate our studies, we have
shown that not only does the distribution of doxorubicin decrease at increasing distances from
the vasculature, but also markers of drug effect mimic drug distribution in different solid
tumours. We chose a high dose of doxorubicin in order to visualize the autofluorescence of the
drug in tumour tissue; however previous studies from our lab have shown that even a single
smaller dose (8 mg/kg) of doxorubicin resulted in delayed growth delay of various tumour types
including EMT-6 and MCF-7 8, 16
.
68
We chose to evaluate γH2AX, cleaved caspase-3 (or -6) and Ki-67. The three drugs that
we evaluated are all approved for use in patients and proven to have a therapeutic effect. Each
treatment led to stimulation of cleaved caspase-3 or -6 and reduction of Ki-67 at 24 hours after
treatment, and these could be used as biomarkers of drug distribution and activity. Huxham et
al18
evaluated the distribution of cell proliferation (using bromodeoxyuridine) following
treatment of human colon cancer xenografts with gemcitabine (240mg/kg) and found complete
inhibition of uptake of the S-phase marker at 24 hours following treatment. Gemcitabine is a
nucleoside analog that was administered at a very high dose in these experiments and has a
different mechanism of action from that of the drugs that we studied in vivo. Although we
observed a reduction in Ki-67 positive cells after treatment with the three drugs evaluated, we
did not find complete inhibition of proliferation. Also this assay is complicated because low
proliferation in regions of control tumours distal from blood vessels makes it difficult to assess
drug activity in those regions.
Biomarkers of drug activity reflect accumulated drug activity and not drug distribution at
a fixed time. Changes in drug distribution, loss of damaged cells, and changes in the spatial
distribution of surviving or damaged but intact cells can occur during the 24 hour period that is
required to evaluate the distribution of cleaved caspase, or changes in Ki-67. γH2AX is formed
rapidly in response to induction of double-strand DNA breaks following ionizing radiation or
treatment with some drugs, due to phosphorylation of serine 139 of histone 2aX 19
. γH2AX
expression has been detected as soon as 3 minutes following ionizing radiation 20
and then
gradually decreases (presumably due to repair of DNA double strand breaks or lysis of lethally
damaged cells) and returns to basal levels 21
. Other studies have suggested that γH2AX may be a
more general sensor of DNA damage following treatment with agents that are not known to
69
cause double strand DNA breaks 21,22
; this is consistent with our results, which indicate that
γH2AX is formed within 10 minutes following treatment with a variety of anticancer drugs in
vitro. In single cells, γH2AX levels fell to near basal levels by 90 minutes after treatment with
docetaxel, while in solid tumours treated with doxorubicin, melphalan or docetaxel it diminished
to pre-treatment control levels at 3 hours. The distribution of γH2AX as a biomarker of cellular
damage at 10-60 minutes after drug administration has the advantage that the time interval is
sufficiently short to minimize any changes in the spatial distribution of the tumour cells between
drug administration and the assay.
Doxorubicin, a DNA topoisomerase II inhibitor can intercalate with DNA which leads to
DNA damage and production of γH2AX 23
. Our results show that the distribution of γH2AX
decreased with increasing distance from the nearest blood vessel following a single treatment of
doxorubicin in two human xenografts (MDA-MB-231 and MCF-7). The pattern of distribution
of induced γH2AX was similar to that observed for drug distribution. Furthermore, subsequent
changes in cleaved caspase-3 or -6 showed similar effects, despite possible kinetic changes
within 24 hours. These results demonstrate that not only is there poor distribution of doxorubicin
in solid tumours, but also poor distribution of drug activity.
Melphalan is a bifunctional alkylating agent that forms adducts with DNA leading to
DNA damage 24
. Melphalan-DNA adducts expressed a pattern of damage such that tumour cells
located closest to blood vessels were affected preferentially. The spatial distribution of γH2AX
expression at 10 minutes mirrored this distribution and increases in cleaved caspase-3 or -6
expression levels at 24 hours also reflected the spatial distribution of melphalan activity.
Finally we studied changes in biomarker distribution following treatment with docetaxel,
a drug that is used widely in the clinic and is non-fluorescent. We chose a dose of 15 mg/kg that
70
our lab has previously shown to cause delayed growth delay of PC-3 tumours 25
. Docetaxel binds
to microtubules, preventing their disassembly and turnover, leading to accumulation of cells in
the G2/M phase of the cell cycle and to cell death 26
. Kolfschoten et al 27
have reported that p53
expression levels increase in ovarian cells expressing wild-type p53 following treatment with
docetaxel. Histone 2aX (H2aX) and p53 are both substrates for phosphorylation by the ATM
kinase which is involved in repairing DNA damage 28
and we found higher levels of γH2AX
following docetaxel treatment. Zhang et al 29
also observed increased levels of γH2AX assessed
by flow cytometry in non-small cell lung cancer (A549) cells following docetaxel treatment,
supporting the hypothesis that γH2AX as a general marker of DNA damage.
γH2AX expression at 10 minutes, increased apoptosis and reduced proliferation at 24
hours following docetaxel treatment was highest proximal to blood vessels and decreased at
greater distances. Thus, the activity of docetaxel also decreases substantially at increasing
distance from tumour blood vessels. We are unaware of direct assessment of the distribution of
the drug itself, but studies of radiolabelled paclitaxel by autoradiography have shown a similar
distribution in hypopharyngeal tumour xenografts 10
.
To evaluate γH2AX as a general marker of activity of anticancer drugs, we evaluated its
expression at 1 hour after treating cultured cells with a range of chemotherapeutic agents with
differing mechanisms of action: anti-microtubule activity (vinblastine, paclitaxel, cabazitaxel,
docetaxel), nucleoside analog (gemcitabine), anti-folate activity (methotrexate) and
topoisomerase-I inhibition (topotecan). Increased γH2AX expression after drug treatment of PC-
3 cells supports the use of γH2AX to study the distribution of activity of many anticancer agents
in solid tumours.
71
Our study has limitations. We assessed distributions of biomarkers and drugs in two-
dimensional tumour sections, whereas solid tumours are three-dimensional. We limited our
studies to a distance of 60μm from blood vessels because of unknown effects from blood vessels
out of the plane of the tumour section, and cells at greater distances, some of which are likely to
be hypoxic, are likely to have even lower levels of activity than those documented here. In
addition, we did not use a flow marker to indicate patent blood vessels.
In summary, we have used the induction of biomarkers γH2AX and cleaved caspase-3,
and inhibition of Ki-67, to show that the effects of three quite different anticancer drugs decrease
with increasing distance from blood vessels of a variety of solid tumours. These results suggest
that limited delivery of drugs to regions distal to blood vessels, and/or limited response of cells
within them because of their low proliferative rate or microenvironment, is a general problem
that is likely to be an important cause of clinical drug resistance. We observed that γH2AX
serves as an early marker of DNA damage that is induced within 10 minutes following treatment
with different types of drugs. This early induction is an advantage since this interval is too short
to be influenced by changes in drug distribution and by the kinetics of the tumour cells that may
occur at later times after drug administration. We are evaluating the distribution of biomarkers in
biopsies from patients; our techniques require 10 micron sections which are easily cut from
biopsies. Our results suggest that induction of γH2AX might be used more generally to predict
the spatial distribution of drug activity within solid tumours. This will be essential information in
designing strategies to overcome therapeutic resistance by modifying or complementing the
limited spatial distribution of drug activity in solid tumours.
72
Acknowledgements
Supported by grants from the Canadian Institutes of Health Research (Tannock laboratory
funding) and Canadian Institutes of Health Research Banting & Best doctoral student funding
(Ms. Jasdeep K. Saggar).
We thank all members of the Pathology Research Program (PRP), and the Advanced Optical
Microscopy Facility (AOMF).
73
2.6 TABLES
Table 2.1 Maximal and minimal levels of doxorubicin fluorescence and biomarkers
(γH2AX, cleaved caspases, change in Ki-67) in various tumours. Values represent percent of
positive pixels at a given distance from blood vessels. Maximum values are at or very close to
the blood vessel at 0μm and minimal values are at 60μm.
Drug Tumour type
MDA-MB-231 MCF-7
Doxorubicin Max Min MinValue
(% Max.)
Max Min Min Value
(% Max.)
Doxorubicin – 10 mins 7.7 5.8 75 7.8 5.5 70
Doxorubicin – 24 hrs 4.1 3.7 92 4.6 3.3 72
γH2AX – 10 mins 9.3 6.7 71 3.7 1.6 44
Cleaved caspase-3 (or-
6) 24 hrs
1.8 1.2 63 3.5 2.4 67
Reduction in difference
of Ki-67- 24 hrs
6
0.02
0.3
4
0.08
2
MCF-7 EMT-6
Melphalan Max Min MinValue
(% Max.)
Max Min Min Value
(% Max.)
Melphalan-DNA
adducts – 10 mins
0.80 0.26 33 0.39 0.24 62
Melphalan- DNA
adducts – 24 hrs
0.49 0.20 41 0.38 0.23 61
γH2AX – 10 mins 1.37 0.63 46 5.0 2.6 52
Cleaved caspase-3 (or-
6) 24 hrs
0.77 0.25 33 1.1 0.6 54
Reduction in difference
of Ki-67- 24 hrs
3.7
0.1
2.7
3.6
0.3
8.3
PC-3 A-431
Docetaxel Max Min MinValue
(% Max.)
Max Min Min Value
(% Max.)
γH2AX – 10 mins 5.4 2.6 48 7.5 5.0 66
Cleaved caspase-3 - 24
hrs
4.7 2.2 48 7.4 3.3 45
Reduction in difference
of Ki-67- 24 hrs
7.8
0.5
6.4
9.6
1.5
15
74
2.7 FIGURES
75
Control 24 hrs
Figure 2.1 Photomicrographs of biomarkers. (A) γH2AX (cyan) (B) Cleaved caspase-3
(yellow) and (C) Ki-67 (magenta) in untreated (control) or docetaxel treated A-431 tumour
xenografts in relation to CD31 labelled blood vessels (red).
A
B
C
10 mins Control
Control 24 hrs
Control 24 hrs
76
Doxoru
bic
in F
luore
scen
t
Inte
nsi
ty
Ki-
67
Per
cen
t of
posi
tive
pix
els
(%)
γH
2aX
C
leaved
-Casp
ase
3 o
r 6
MDA-MB-231 MCF-7
Figure 2.2 Biomarker distribution following doxorubicin treatment. MDA-MB-231 (panels A-D) and
MCF-7 (panels E-H) tumours were treated with doxorubicin (25 mg/kg) or untreated controls. Panels A
and E show doxorubicin fluorescence in relation to tumour blood vessels at different times after injection.
Corresponding changes in biomarkers of drug effect: γH2AX (panels B and F), cleaved caspase-3 (MDA-
MB-231, panel C) or 6 (MCF-7, panel G), and Ki67 (panels D and H). The figures represent percent of
positive pixels as a function of distance from the nearest blood vessel. Points indicate average for 10 mice
per group; bars, SE. Legend: Control, 10 minutes, 3 hrs, × 6 hrs, 24 hrs, 48 hrs.
C
Distance from the nearest blood vessel (μm)
A
B
D
E
F
G
H
77
Cle
aved
-Casp
ase
3 o
r 6
Per
cen
t of
posi
tive
pix
els
(%)
Ki-
67
γH
2aX
M
elp
ha
lan
DN
A A
dd
uct
s
MCF-7 EMT-6
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0
2
4
6
8
10
0 10 20 30 40 50 60
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
1.2
0
2
4
6
0
2
4
6
8
10
12
14
0 10 20 30 40 50 60
0
0.2
0.4
Figure 2.3 Biomarker distribution following melphalan treatment. MCF-7 (panels A-D) and EMT-6
tumours (panels E-H) treated with melphalan (6 mg/kg) or untreated controls. Panels A and E show
changes in melphalan-DNA adducts in relation to tumour blood vessels at 10 minutes and 24 hours after
injection. Corresponding changes in biomarkers of drug effect γH2AX: (panels B and F), cleaved
caspase-6 (MCF-7, panel C) or 3 (EMT6, panel G), and Ki67 (panels D and H). The figures represent
percent of positive pixels as a function of distance from the nearest blood vessel. Points indicate average
for 10 mice per group; bars, SE. Legend: Control, 10 mins, 24 hrs.
Distance from the nearest blood vessel (μm)
C
B
A
D
E
F
G
H
78
Cle
aved
-Casp
ase
3
γH
2aX
K
i-67
Per
cen
t of
posi
tive
pix
els
(%)
PC-3 A-431
0
2
4
6
0
2
4
6
8
10
12
14
0 10 20 30 40 50 60
0
2
4
6
0
2
4
6
8
0
2
4
6
8
0
2
4
6
8
10
12
14
0 10 20 30 40 50 60
Figure 2.4 Biomarker distribution following docetaxel treatment. PC-3 (panels A-C) & A431
tumours (panels D-F) treated with docetaxel (15 mg/kg) or untreated controls. Changes in
biomarkers of drug effect are shown at 10 minutes for γH2AX: (panels A and D) and 24 hours
for cleaved caspase-3 (panels B and E) and Ki67 (panels C and F) compared to untreated
controls. The figures represent percent of positive pixels as a function of distance from the
nearest blood vessel. Points indicate average for 10 mice per group; bars, SE. Legend: Control, 10 mins, 24 hrs.
F
Distance from the nearest blood vessel (μm)
A
B
C
D
E
Distance from the nearest blood vessel (μm)
79
Figure 2.5 γH2AX expression in cell lines. (A) γH2AX expression in PC-3 cells after treatment
for 1 hour with different chemotherapeutic agents: 5-FU (10 uM), cabazitaxel (100 nM),
gemcitabine (100 nM), methotrexate (400 nM), topotecan (50 nM), paclitaxel (50 nM) and
vinblastine (50 uM). (B) Time course of γH2AX expression in PC-3 cells treated with docetaxel
(50 nM).
A B
80
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12. Rothbarth J, Koevoets C, Tollenaar RA, Tilby MJ, van de Velde CJ, Mulder GJ, et al.
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human colourectal liver tumours in vivo. Biochem Pharmacol. 2004;67:1771-8.
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distribution of Ki-67 and other biomarkers in tumour sections and use of the method to study
repopulation in xenografts after treatment with paclitaxel. Neoplasia. 2012;14:324-34.
16. Lankelma J, Dekker H, Luque FR, Luykx S, Hoekman K, van der Valk P, et al.
Doxorubicin gradients in human breast cancer. Clin Cancer Res. 1999;5:1703-7.
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the distribution of doxorubicin in breast tumours. BMC Cancer. 2009;9:356.
18. Huxham LA, Kyle AH, Baker JH, Nykilchuk LK, Minchinton AI. Microregional effects
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19. Paull TT, Rogakou EP, Yamazaki V, Kirchgessner CU, Gellert M, Bonner WM. A
critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage.
Curr Biol. 2000;10:886-95.
20. Cucinotta FA, Pluth JM, Anderson JA, Harper JV, O'Neill P. Biochemical kinetics model
of DSB repair and induction of gamma-H2AX foci by non-homologous end joining. Radiat Res.
2008;169:214-22.
21. Lobrich M, Shibata A, Beucher A, Fisher A, Ensminger M, Goodarzi AA, et al.
gammaH2AX foci analysis for monitoring DNA double-strand break repair: strengths,
limitations and optimization. Cell Cycle. 2010;9:662-9.
22. Cleaver JE, Feeney L, Revet I. Phosphorylated H2Ax is not an unambiguous marker for
DNA double-strand breaks. Cell Cycle. 2011;10:3223-4.
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phosphorylation of multiple downstream targets in part through the generation of reactive
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CHAPTER 3
Activity of the hypoxia-activated pro-drug TH-302 in hypoxic and
perivascular regions of solid tumours and its potential to enhance therapeutic
effects of chemotherapy
Jasdeep K. Saggar and Ian F. Tannock
This data chapter has been published in The International Journal of Cancer:
Saggar JK & Tannock IF. Activity of the hypoxia-activated pro-drug TH-302 in hypoxic
and perivascular regions of solid tumours and its potential to enhance therapeutic effects of
chemotherapy. International Journal of Cancer. 2013 Nov 8 (E-publication ahead of print).
85
3.1 NOVELTY
Hypoxia in tumours is associated with poor therapeutic outcome. Chemotherapy targets rapidly-
proliferating cells close to tumour blood vessels and spares hypoxic cells. The hypoxia-activated
pro-drug TH-302 is in clinical trials, but its spatial distribution of activity within tumours is
unknown. Here we study the spatial distribution of biomarkers of drug effect to show that TH-
302 increases DNA damage and apoptosis in hypoxic regions, but also has effects in perivascular
regions, and enhances the effectiveness of chemotherapy.
86
3.2 ABSTRACT
Many chemotherapy drugs have poor therapeutic activity in regions distant from tumour blood
vessels because of poor tissue penetration and low cytotoxic activity against slowly-proliferating
cells. The hypoxia activated pro-drug TH-302 may have selective toxicity for hypoxic and
neighboring cells in tumours. Here we characterize the spatial distribution and ability of TH-302
to selectively target hypoxic regions and complement the effect of doxorubicin and docetaxel by
modifying biomarker distribution. Athymic nude mice bearing human breast MCF-7 or prostate
PC-3 tumours were treated with doxorubicin or docetaxel respectively and TH-302 alone or in
combination. Biomarkers of drug effect including γH2AX (a marker of DNA damage), cleaved
caspase-3 or -6 (markers of apoptosis) and reduction in Ki-67 (a marker of cell proliferation)
were quantified in tumour sections in relation to functional blood vessels (recognized by DiOC7)
and hypoxia (recognized by EF5) using immunohistochemistry. γH2AX expression at 10
minutes and cleaved caspase-3 or -6 at 24 hours after doxorubicin or docetaxel alone decreased
with increasing distance from tumour blood vessels, with minimal expression in hypoxic regions;
Ki67 levels were reduced the greatest in regions closest to blood vessels at 24 hours. TH-302
induced maximal cell damage in hypoxic and neighboring regions, but was also active in tumour
regions closer to blood vessels. TH-302 given 4h prior to chemotherapy increased DNA damage
and apoptosis throughout the tumour compared to chemotherapy alone. When given with
doxorubicin or docetaxel, TH-302 complements and enhances anticancer effects in both
perivascular and hypoxic regions but also increases toxicity.
87
3.3 INTRODUCTION
Most solid tumours contain hypoxic regions, and hypoxic cells may be important in
limiting the effects of both radiotherapy and chemotherapy. The presence of oxygen increases
the effects of radiation via increased fixation of free radicals on DNA strands and hypoxic cells
are relatively radio-resistant 1,2
. Hypoxic cells in tumours are deficient in other nutrients and are
slowly or non-proliferating: they may be spared by chemotherapy because of poor penetration of
drugs to hypoxic tumour regions3, and because most anti-cancer drugs are more effective against
rapidly-proliferating cells. In addition to their radio-resistance, the presence of hypoxia leads to
a more aggressive phenotype due to the expression of pro-survival genes (including mutated
p53), increased levels of proteins favouring an epithelial-to-mesenchymal transition, increased
metastasis, angiogenesis and inhibition of apoptosis 4. For these reasons, hypoxic cells may be
an important cause of treatment failure in solid tumours and may repopulate the tumour.
Therapies that target hypoxic tumour cells therefore have potential to augment the effects of
radiotherapy and chemotherapy1. Also, hypoxic cells occur rarely in normal tissues, so that
targeting them might offer tumour selectivity. Hypoxia-targeted therapies such as gene therapy
(using a promoter highly responsive to HIF-1), recombinant anaerobic bacteria (whose spores
activate in necrotic centers of tumours and kill tumour tissue) and hypoxia-activated pro-drugs
(HAPs) are all under investigation 1,5
.
Hypoxia-activated pro-drugs are administered in an inactive form and are reduced to their
active metabolites within hypoxic regions of tumours1. Once activated they may diffuse to act
against cells in neighboring tumour regions (“the bystander effect”). The first HAP to undergo
clinical testing was tirapazamine; and although activity against hypoxic cells in human tumours
could be shown 6, large randomized trials evaluating tirapazamine in combination with
88
radiotherapy or chemotherapy did not establish therapeutic benefit7-9
. Tirapazamine had several
properties that limited its effectiveness including poor tissue penetration10
and toxicity unrelated
to its hypoxic activation, which led to declining interest in this strategy.
Newer HAPs such as TH-302 have been designed with more favourable properties than
tirapazamine. TH-302 is a 2-nitroimidazole containing a toxic alkylating moiety that is released
only when its attached nitro-heterocyclic trigger molecule fragments in hypoxic cells to release
the DNA crosslinking agent bromo-isophosphoramide mustard11
. Traditional chemotherapy
combined with TH-302 can lead to greater cell kill and growth delay of various rodent tumours
and human xenografts12
, and promising results have been reported from early clinical trials
evaluating TH-302 combined with gemcitabine for treatment of pancreatic carcinoma13
.
However, there have been few studies evaluating the microenvironmental distribution of activity
of TH-302 within solid tumours and the mechanisms which lead to antitumour effects remain
uncertain.
Previous studies from our laboratory using immunohistochemistry (IHC) have shown that
the combination of mitoxantrone with another HAP, AQ4N, resulted in effective drug exposure
throughout solid tumours and improvement in therapeutic index when used to treat human breast
tumour xenografts14
. These studies were possible because both mitoxantrone and AQ4N are
fluorescent. It is important for the development of TH-302 to characterize its distribution in
tumour tissue, including its ability to diffuse to hypoxic regions, where it is activated, and for its
active metabolite(s) to diffuse to neighboring better-nourished and oxygenated tumour regions
(i.e. the bystander effect). We have validated techniques to analyze the pharmacodynamic
changes that occur after treatment with non-fluorescent drugs by using the biomarkers γH2AX (a
marker of DNA damage), cleaved caspase-3 or -6 (markers of apoptosis) and reduction in Ki-67
89
(a marker of cell proliferation)15
. Here we quantify the distribution of these biomarkers
following treatment of two human xenografts with TH-302 alone or with doxorubicin or
docetaxel.
3.4 MATERIALS AND METHODS
3.4.1 Cell lines
Experiments were conducted using human breast carcinoma (MCF-7) and human prostate cancer
(PC-3) cell lines. Cell lines were purchased from the American Type Culture Collection
(Manassas, VA). MCF-7 cells were cultured in α-MEM supplemented with 10% fetal bovine
serum (FBS; Hyclone, Logan, UT). PC-3 cells were cultured in Ham's F-12K medium (Life
Technologies Inc.) supplemented with 10% FBS. Cells were incubated in a humidified
atmosphere of 95% air/5% CO2 at 37ºC. Routine tests to exclude mycoplasma were performed.
Short tandem repeat analysis was performed to characterize the origin of these cells. MCF-7
cells were chosen to represent estrogen receptor positive breast cancer and PC-3 cells to
represent prostate cancer.
To generate MCF-7 tumours, 4-6 week old female athymic nude (nu/nu) mice (Harlan
Sprague-Dawley, Madison, WI) were implanted with 17β-estradiol tablets (60-day release;
Innovative Research of America, Sarasota, FL) and then injected subcutaneously with 5x106
MCF-7 cells per side. To generate PC-3 tumours, 4-6 week old male athymic nude (nu/nu) mice
(Jackson, Bar Harbor, Maine, USA) were injected subcutaneously in both flanks with 2x106 PC-
3 cells. There were five mice per treatment group (8-10 tumours) in each experiment and each
experiment was repeated.
90
3.4.2 Drugs and reagents
Doxorubicin (Pharmacia, Mississauga, Ontario, Canada) and docetaxel (Sanofi-Aventis,
Laval, Quebec, Canada) were purchased from the Princess Margaret Cancer Centre pharmacy;
they were provided as solutions with concentration of 2 mg/mL and 40 mg/mL respectively. TH-
302 was provided by Threshold pharmaceuticals (San Francisco, USA) as a powder and then
dissolved in 0.9% saline prior to use.
EF5 was provided by the National Cancer Institute as a powder and then dissolved in
distilled water supplemented with 2.4% ethanol and 5% dextrose to make a 10-mM stock
solution that was stored at room temperature. Cy5-conjugated mouse anti-EF5 antibody was
purchased from Dr. Cameron Koch, University of Pennsylvania, PA. DiOC7 was purchased from
AnaSpec Inc. (San Jose, CA) and a stock solution (2.5 mg/ml) was made by dissolving in
dimethyl sulfoxide; this stock was diluted 1:10 in phosphate-buffered saline and 10% Solutol HS
15 (Sigma-Aldrich, Oakville, ON). γH2AX was recognized with a rabbit anti-human γH2AX
primary antibody (Cell Signaling, Danvers, MA). Cleaved caspase-3 was recognized with
primary rabbit anti-human cleavedcaspase-3 antibody (Cell Signaling, Danvers, MA) and
cleaved caspase-6 with rabbit anti-human cleaved caspase-6 antibody (Novus Biologicals,
Oakville, ON). Ki-67 was identified with primary rabbit anti-human Ki-67 antibody
(NovusBiologicals, Oakville, ON). Application of all primary antibodies was followed by Cy3-
conjugated goat anti-rabbit IgG secondary antibody and visualized using the Olympus
fluorescent upright microscope.
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3.4.3 Effect of drugs on biomarker distributions
Animals bearing tumours with a mean cross-sectional area 0.7-0.8 cm2 were given a
single intravenous injection of doxorubicin (8 mg/kg) or intraperitoneal injection of docetaxel
(15 mg/kg) and/or TH-302 (150 mg/kg) or saline (controls); these doses were used based on the
maximum tolerated dose established and used to provide therapeutic benefit in previous
studies11,16-18
. In most experiments evaluating drug combinations, TH-302 was administered 4
hours prior to doxorubicin or docetaxel, but we also evaluated γH2AX expression using this
schedule as compared to concurrent delivery of TH-302 and doxorubicin in MCF-7 tumours; this
was done to test observations of a previous study that TH-302 administration 4 hours prior to
chemotherapy was superior to concurrent administration19
. Mice were killed and tumours excised
from saline (control) and treated animals at 10 minutes and 24 hours following drug injection;
EF5 and DiOC7 were administered 2 hours and 1 minute respectively, prior to animal death.
Samples were embedded immediately in OCT compound, flash frozen in liquid nitrogen
and stored at −70°C prior to tissue sectioning and IHC staining. Cryostat sections (10μm) were
cut from each tumour. Whole tumour sections were imaged and analyzed with artifacts and
regions of necrosis omitted. At least six tumours were analyzed per treatment group.
Functional blood vessels were identified in tumours by injection of DiOC7 – a
fluorescent carbocyanine dye – that stains cells immediately adjacent to functional vasculature20
.
Hypoxic regions were identified by binding of EF5.
Separate tissue sections were stained for γH2AX, cleaved caspase-3 (or -6) and Ki-67
with the appropriate antibodies. We evaluated cleaved caspase-6 expression in MCF-7 tumours
because MCF-7 cells do not express caspase-3 due to a deletion in exon 3 that encodes the
caspase-3 gene21
; the effector caspase-6 processes caspases-8 and -10 leading to apoptosis22
.
92
Tumour sections were imaged for biomarkers using the Cy3 filter set (530-560 nm
excitation/573-647nm emission).
Media Cybernetics Image Pro PLUS software was used for image analysis and
quantification. A minimal threshold for detection (below the level of detection of drug or
biomarker) was determined for each tumour in order to minimize noise due to auto-fluorescence,
Biomarkers were analyzed using a protocol described previously15,23
. Briefly, binarized
DiOC7 or EF5 images were created and then used to create distance maps. Biomarker images
(γH2AX, cleaved caspase-3 or -6 and Ki-67) were used to create binary masks which were then
combined with the distance map to form a composite image with distance measurements that
corresponded only to the biomarker. The data are represented graphically as the percent of pixels
positive for any given biomarker at a given distance from the nearest functional blood vessel or
hypoxic region in the section; a cut-off distance of 60µm was used in order to minimize
interference from neighboring blood vessels or hypoxic regions that are out of the plane of the
section.
3.4.4 Growth delay
MCF-7 or PC-3 tumour-bearing mice were assigned randomly to treatment groups of 5-6
mice. Treatments with saline (control), chemotherapy (doxorubicin, 8 mg/kg or docetaxel, 15
mg/kg), TH-302 (150 mg/kg) or TH-302 in combination of chemotherapy were administered
weekly for three weeks: doses were selected based on previous experiments12,16,17
in order to
minimize animal weight loss. The longest and perpendicular tumour diameters were estimated
using calipers three times per week until tumours reached a maximum diameter of 1 cm or
tumours became necrotic, when animals were killed humanely. Tumour volume was estimated
93
by the formula: (longest tumour diameter) x (smallest tumour diameter)2/2. Body weight of the
mice was also measured.
3.4.5 Statistical analysis
Biomarker distributions & growth delay: A one-way ANOVA was used to determine the
statistical differences between treatment groups. Tests were 2-sided and no corrections were
applied for multiple significance testing. A p-value of less than 0.05 was used to indicate
statistical significance. Tumour volumes and biomarker distributions are represented as mean
values +/- SEM.
3.6 RESULTS
3.6.1 Distribution of biomarkers in tumour sections:
Expression of the biomarkers γH2AX, cleaved caspase-3 or -6 and Ki-67 were
recognized with specific antibodies as shown in Figure 3.1A-C in control, docetaxel, TH-302 or
combination- treated PC-3 tumours. Expression of γH2AX was evaluated at 10 min after
treatment with TH-302 alone or after chemotherapy (given alone or 4h after TH-302), while
increase in the apoptotic markers and decrease in Ki-67 was evaluated at 24 hours after these
treatments; these time points have been shown to give optimal expression of the biomarkers after
drug treatment 15
.
The distribution of biomarkers after treatment of MCF-7 tumours with doxorubicin, TH-
302 and the combination in relation to the nearest patent functional vessel (left panels) and the
nearest region of hypoxia (right panels) is shown in Figure 3.2. In control tumours expression of
94
γH2AX and cleaved caspase-6 was low and Ki67 expression decreased with increasing distance
from blood vessels (Figure 3.2, left panel) to low levels near hypoxic regions (Figure 3.2, right
panel). Following treatment with doxorubicin, expression of γH2AX and cleaved caspase-6 was
increased significantly (p < 0.05) above control levels with maximal expression in regions
closest to blood vessels (Figure 3.2A and B, left panel); expression of these markers declined in
regions distal to blood vessels with levels similar to control in hypoxic regions (Figure 3.2A and
B, right panel). Cell proliferation following treatment with doxorubicin was reduced in regions
close to blood vessels (Figure 3.2C, left panel) but was near (already low) control levels in
regions close to hypoxia (Figure 3.2C, right panel). TH-302 treatment alone caused an increase
in γH2AX at 10 minutes and this effect was augmented at 4 hours following treatment. TH-302
treatment alone also increased expression of cleaved caspase-6and while these effects were
maximal adjacent to hypoxic regions they were observed in all tumour regions. Ki-67 was
suppressed throughout the tumour. Combination therapy with doxorubicin and TH-302 led to
the greatest increase in expression of γH2AX and cleaved caspase-6 in regions both close to
blood vessels (Figure 3.2A and B, left panel) and hypoxia (Figure 3.2A and B, right panel), with
greater increase in γH2AX levels seen when TH-302 was administered 4 hours prior to
doxorubicin as compared to concurrent treatment. Ki-67 expression was suppressed throughout
the tumour following combined treatment.
Results for PC-3 tumours treated with docetaxel and/or TH-302 were qualitatively similar
to those obtained in MCF-7 tumours and are shown in Figure 3.3. There was low expression of
γH2AX and caspase-3 in control tumours, and the distribution of cell proliferation (Ki67)
decreased with distance from the nearest blood vessel (Figure 3.3 A-C, left panel) with slow
proliferation near to hypoxic regions (Figure 3.3 A-C, right panel). Following treatment with
95
docetaxel alone, the expression of γH2AX and of caspase-3 was increased significantly (p <
0.05) above that in the control, but it decreased with increasing distance from a functional blood
vessel, and was low in hypoxic regions. Suppression of Ki67 followed a similar pattern. As
expected treatment with TH-302 alone increased expression of biomarkers further from blood
vessels and near to hypoxic regions, but also led to significantly increased expression of the
biomarkers and suppression of cell proliferation, in all regions compared to controls (p < 0.05).
The combination of docetaxel and TH-302 produced the most profound effects on the
biomarkers and on suppression of cell proliferation (p < 0.001), with increased evidence of
activity both in regions proximal to and distal from blood vessels (including hypoxic regions).
3.6.2 Effects of doxorubicin, docetaxel and TH-302 on the growth of xenografts:
Doxorubicin had minimal effects to inhibit growth of MCF-7 xenografts as compared to
controls and TH-302 treatment alone also had limited effect. The combination of TH-302 with
doxorubicin led to the greatest delay in growth (Figure 3.4A). Docetaxel treatment of PC-3
xenografts resulted in growth delay that was increased when combined with TH-302 (Figure
3.4B).
Animal body weights were also measured. Chemotherapy treatment alone led to a
significant 4% (doxorubicin) and 1.5% (docetaxel) decrease in body weight compared to control
mice (p<0.05) but TH-302 alone had no significant effect (table 3.1). Combined treatment led to
a significant 11% (MCF-7 tumours) and 8.4% (PC-3 tumours) decrease in body weight
compared to controls (p<0.0001). Combination treatment also led to a statistically significant
6.8% (MCF-7 tumours) and 7.0% (PC-3 tumours) decrease in body weight compared to
chemotherapy alone treated animals (p<0.05) (table 3.1).
96
3.7 DISCUSSION
Causes of resistance to chemotherapy include not only molecular changes in cancer cells
but also mechanisms related to the tumour microenvironment. We have shown previously that
γH2AX, cleaved caspase-3 (or -6) and Ki-67 are useful biomarkers to quantify the distribution of
drug activity within solid tumours15
. By using these markers and other methods, our group and
others have shown limited distribution of activity of multiple anticancer drugs from blood
vessels14,15,17,18,24
. Thus, anticancer drugs are often effective against cells adjacent to vasculature
but penetrate poorly through tumour tissue so that they are not delivered in effective
concentrations to tumour cells in regions distal to functional blood vessels; moreover such cells
have low rates of cell proliferation and are resistant to cycle-active chemotherapy. Therefore the
development of drugs that can kill cells located distant from vasculature, including hypoxic cells,
is of clinical importance. Here, we have evaluated the ability of the hypoxia-activated pro-drug
TH-302 to enhance the effects of doxorubicin and docetaxel against human tumour xenografts,
and have studied the distribution of its activity within the tumour microenvironment.
Consistent with previous results from our group and others, docetaxel and doxorubicin
alone induced γH2AX, cleaved caspase-3 (or -6) and reduced Ki-67 expression and this effect
occurred predominantly in regions close to functional blood vessels; there were minimal effects
in and near to hypoxic regions15,23
.
TH-302’s is a pro-drug that is activated specifically in hypoxic cells, and has
demonstrated promising results both in preclinical studies and in early clinical trials11-13,25,26
.
However, since TH-302 is non-fluorescent in the UV-visible spectrum, its direct visualization in
tumour tissue is not possible. Therefore, we extended our techniques of assessing biomarker
97
distribution following drug treatment to evaluate the effects of TH-302 in hypoxic and vascular
regions of solid tumours. As expected we found that TH-302 induced expression of γH2AX ,
cleaved caspase-3 (or -6) and reduced Ki-67 expression immediately adjacent to hypoxic
regions, supporting the mechanism of action that TH-302 is activated in hypoxic areas and
diffuses to nearby tissues (i.e. consistent with its bystander effect). More surprisingly, our data
show that TH-302 also induced these biomarkers in all regions of the tumours, including those
close to functional blood vessels, and that it augmented the effects of chemotherapy in all tumour
regions. The ability of TH-302 to induce expression of γH2AX has also been reported by Sun et
al11
, who administered TH-302 to mice as well as pimonidazole (to detect hypoxia) given an
hour prior to animal death. These authors showed that induction of γH2AX was located
preferentially near pimonidazole positive cells. Our results support the findings of Sun et al11
with respect to γH2AX expression near hypoxic regions (here identified by EF5); however, our
evaluation of γH2AX and other biomarkers (cleaved caspase -3 or -6 and reduction of Ki-67) in
regions close to functional blood vessels identified by DiOC7 revealed that TH-302 can also
change the expression of these biomarkers in well-oxygenated regions of tumours. We have
evaluated previously biomarker distributions following treatment with another nitrogen mustard
melphalan, and found increased γH2AX, cleaved caspase and reduced Ki-67 expression close to
blood vessels. Although both melphalan and TH-302 are nitrogen mustards, differences in their
distributions may be related to their activation: TH-302 is a prodrug and is designed to become
activated in hypoxic cells and is able to penetrate into these areas as it is not activated in oxic
cells whereas melphalan is not a prodrug and readily attacks DNA in cells proximal to the
vasculature, thus reducing the amount of drug available to diffuse to distantly located hypoxic
regions.
98
It is possible that the combination of TH-302 with doxorubicin or docetaxel led to greater
DNA damage and cell death due to activation of TH-302 in hypoxic cells and its resulting
bystander effect – i.e. the ability of the activated drug to diffuse to surrounding non-hypoxic
tissues27
. The 2-nitroimidazole portion of TH-302 undergoes electron rearrangement forming a
radical anion in regions with less than about 0.5% oxygen27
. The radical anion can then fragment
to the toxic bromo-isophosphoramide mustard which can diffuse to surrounding tissues.
Therefore, the diffusible toxic bromo-isophosphoramide coupled with doxorubicin or docetaxel
may have a heightened effect on the expression of biomarkers γH2AX, cleaved caspase-3 (or -6)
and reduction in Ki-67.
Sun et al have reported that TH-302 delivery 4 hours prior to chemotherapy results in
greater tumour growth inhibition than does concurrent administration of these agents19
. Our
findings support this conclusion in that we found greater γH2AX expression in both perivascular
and hypoxic regions when TH-302 was given 4 hours prior to doxorubicin than following
concurrent treatment, thus providing further evidence for the use of this schedule.
Interestingly, TH-302 treatment alone resulted in an increase in γH2AX, cleaved caspase-
3 (or -6) and reduction in Ki-67 in regions close to blood vessels. Under aerobic conditions, TH-
302 is fragmented to its radical anion and then back oxidized to its pro-drug form27
. A byproduct
of this reaction is the generation of superoxide via the reduction of oxygen. Superoxides can
react with free iron generating hydroxyl radicals; these radicals in turn can act as oxidants and
damage DNA28
. It is possible that the production of excess hydroxyl radicals leads to the
relatively uniform expression of γH2AX as a function of distance from blood vessel that is
observed following TH-302 treatment alone. While this effect might further augment the anti-
tumour effects of chemotherapy, it is less likely to be tumour-specific since it might also occur in
99
well-oxygenated normal tissues. TH-302 enhances the antitumour effects of chemotherapy, but
also increases toxicity in normal tissues. It is also possible that TH-302 is activated and diffuses
to surrounding oxygenated tumour tissue within seconds, and that the relatively uniform
expression of γH2AX at 10 minutes after injection of TH-302 is due to the bystander effect.
Weiss et al26
reported the presence of both TH-302 and its toxic metabolite bromo-
isophosphoramide in the plasma following drug administration in patients; it is possible that this
toxic moiety that was activated during circulation (i.e. via hepatic activation) then caused
activation of the biomarkers in perivascular regions in our study. TH-302 has also exhibited
activity as monotherapy in a phase I clinical trial26
; our results including elevated levels of
apoptosis in tumours provide further validation for its activity when used alone.
A phase I clinical trial that combined TH-302 with doxorubicin in soft tissue sarcomas
suggested added benefit in the form of a response rate of 33% compared to the projected 12-23%
in patients treated with doxorubicin alone29
. The enhanced effects on DNA damage, apoptosis
and reduction in cell proliferation that we found when TH-302 was combined with doxorubicin
or docetaxel in the present study may provide some explanation for the enhanced effect found in
the clinic.
Our study has limitations. Assessment of biomarker distributions was undertaken in two-
dimensional tumour sections, whereas solid tumours are three-dimensional. We limited our
analysis to a distance of 60μm from blood vessels or hypoxic regions to decrease the
confounding effects of undetected blood vessels or hypoxic regions outside of the tumour
section, but a three-dimensional analysis would be preferred. Also, although the biomarkers
evaluated show consistent changes, none of them provide direct evaluation of loss of
reproductive activity of tumour cells, the most important endpoint of cell death. However, the
100
elevated biomarkers after combined treatment correspond to significant increases in tumour
growth delay at the cost of added toxicity.
In summary, our evaluation of biomarkers of drug effect following chemotherapy with
doxorubicin or docetaxel applied to two human xenografts showed greatest drug activity
proximal to functional blood vessels, and decreased activity within or close to regions of
hypoxia. The distribution of cellular damage was modulated by the addition of TH-302 4 hours
prior to doxorubicin or docetaxel and resulted in greater activation of biomarkers in hypoxic
regions, but also in regions closer to blood vessels. The activity of both doxorubicin and
docetaxel can be complemented and enhanced by the activity of the hypoxia-activated pro-drug
TH-302, resulting in better distribution of drug activity throughout the tumour but also increased
toxicity as well. Our results support the hypothesis that use of hypoxia-activated pro-drugs with
cycle-active chemotherapy might reduce drug resistance and treatment failure in solid tumours.
Acknowledgements
Supported by grants from the Canadian Institutes of Health Research (CIHR) and Threshold
Pharmaceuticals to Dr. Tannock and a CIHR Banting & Best doctoral studentship to Ms. Saggar.
We thank all members of the Pathology Research Program (PRP), and the Advanced Optical
Microscopy Facility (AOMF). We thank Threshold pharmaceuticals for providing us with TH-
302 and for previous grant support.
101
3.8 TABLES
Table 3.1 Drug toxicity. Mean loss in body weight (%) of MCF-7 or PC-3 tumour bearing
animals treated with either: saline (control), chemotherapy (doxorubicin [DOX] or docetaxel
[DOC]), TH-302 or combination (TH-302 4 hours prior to DOX or DOC). P values compared to
control are also displayed. *Combination treatment group is also statistically significant
compared to DOX or DOC alone: -6.8% (MCF-7) & -7% (PC-3) loss in body weight (p=<0.05).
Treatment MCF-7
tumours (%)
p-value PC-3
tumours
(%)
p-value
Control - - - -
DOX or DOC -4 p = <0.05 -1.5 p = <0.05
TH-302 +0.8 p = >0.05 -0.7 p = >0.05
TH-302 + DOX or
DOC*
-11 p = <0.0001 -8.4 p = <0.0001
102
3.9 FIGURES
Figure 3.1 Photomicrographs of biomarkers. (A) γH2AX (red) (B) Cleaved caspase-3 (blue)
and (C) Ki-67 (magenta) in PC-3 tumour xenografts following treatments: saline (control),
docetaxel, TH-302, or docetaxel preceded 4hrs earlier by TH-302 (combination). γH2AX was
evaluated at 10 minutes post-treatment and cleaved caspase-3 & Ki-67 24 hours post-treatment;
co-localization with hypoxia (green) produces yellow. Scale bar = 100 μm.
A
B
C
103
Ki-
67
Cle
aved
-Casp
ase
6
γH
2aX
Per
cen
t of
posi
tive
pix
els
(%)
Figure 3.2 Doxorubicin in combination with TH-302. MCF-7 tumours treated with: saline control ( ),
doxorubicin (8 mg/kg; - ), TH-302 (150 mg/kg at 10 mins or 4 hrs ) or TH-302 + doxorubicin
combination (concurrent administration or TH-302 4 hours prior to DOX ). The distributions of
biomarkers of drug effect in relation to the nearest functional blood vessel (panels A-C) or region of
hypoxia (panels D-F) are shown. Distribution of γH2AX at 10 minutes & 4 hours after treatment (panels
A and D); cleaved caspase-3 (panels B and E) and Ki67 at 24 hours after doxorubicin treatment (panels C
and F) are compared to untreated controls. Points indicate mean for 5-6 mice per group; bars, SE.
Treatments in panels A-F are statistically significantly different from one another (p<0.05).
D
B
C
E
F
Distance from the nearest functional blood vessel (μm) Distance from the nearest hypoxic region (μm)
A
104
Cle
aved
-Casp
ase
3
γH
2aX
K
i-67
Per
cen
t of
posi
tive
pix
els
(%)
Figure 3.3 Docetaxel in combination with TH-302. PC-3 tumours treated with: saline control ( ),
docetaxel (15 mg/kg; -), TH-302 (150 mg/kg; ) or TH-302 4 hours prior to docetaxel ( ). The
distribution of biomarkers of drug effect in relation to the nearest functional blood vessel (panels A-C) or
region of hypoxia (panels D-F) are shown. Distribution of γH2AX at 10 minutes after treatment (panels A
and D); cleaved caspase-3 (panels B and E) and Ki67 at 24 hours after docetaxel treatment (panels C and
F) are compared to untreated controls. Points indicate mean for 5-6 mice per group; bars, SE. Treatments
in panels A-F are statistically significantly different from one another (p<0.05).
F
Distance from the nearest functional blood vessel (μm)
A D
B
C
E
Distance from the nearest hypoxic region (μm)
105
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
1 3 6 9 12 15 18 21 24 27 30 33
Me
an
Tu
mo
r V
olu
me (
cm
3)
Time (days)
CONTROL
DOXORUBICIN
TH-302
DOXORUBICIN+TH-302
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35
Me
an
Tu
mo
r V
olu
me
(c
m3)
Time (days)
CONTROL
DOCETAXEL
TH-302
DOCETAXEL+TH-302
Figure 3.4 (A) Growth delay of MCF-7 xenografts in nude mice treated with: saline control,
doxorubicin (8 mg/kg i.v.), TH-302 (150 mg/kg) or the combination of doxorubicin preceded 4h
earlier by TH-302 (treatments were given once weekly for three weeks). (B) Growth delay of
PC-3 xenografts in nude mice treated with: saline control, docetaxel (15 mg/kg i.p.), TH-302
(150 mg/kg) or combination docetaxel preceded 4h earlier by TH-302 (treatments were given
once weekly for three weeks). Points indicate mean for 5 mice per group; bars, SE. * and **
signify statistical significance (p<0.05). Legend: Control, - Doxorubicin or Docetaxel, TH-
302, TH-302 4 hrs + Doxorubicin or Docetaxel.
A
B
*
* *
**
*
*
**
*
106
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108
CHAPTER 4
Chemotherapy rescues hypoxic tumour cells and induces
reoxygenation and repopulation - an effect that is inhibited by the
hypoxia-activated pro-drug TH-302
Jasdeep K. Saggar1 and Ian F. Tannock
1,2
1Department of Medical Biophysics, University of Toronto, Toronto ON, Canada and
2Division of Medical Oncology and Hematology, Princess Margaret Hospital, Toronto ON,
Canada
This chapter has been prepared as a future submission to Cancer Research.
109
4.1 ABSTRACT
Introduction: Chemotherapy targets rapidly proliferating cells and may spare hypoxic cells
because of their slow proliferation and poor drug distribution to them. Intervals between
chemotherapy administrations allow normal tissues to recover but reoxygenation and increased
proliferation of formerly hypoxic cells might also occur. Hypoxia activated pro-drugs (e.g. TH-
302) may kill hypoxic cells. Here we evaluate repopulation and reoxygenation following
chemotherapy and the effect of TH-302 to inhibit these processes. Methods: Mice bearing
human breast (MCF-7) or prostate (PC-3) tumours were treated with chemotherapy using
doxorubicin or docetaxel, with or without TH-302. Two specific markers of hypoxia
(pimonidazole [pimo] and EF5) were used to recognize hypoxic cells: pimo was given at time of
chemotherapy and EF5 after a variable interval of 24-120 hours. Changes in proliferation and
oxygen status of formerly hypoxic (pimo+ve) cells were quantified by their Ki-67 status and
uptake of EF5 as a function of time, using immunohistochemistry. Results: Chronically hypoxic
cells had very limited proliferation in control tumours and this decreased further over time. There
was flux through the hypoxic compartment as previously hypoxic cells died and tumour cells
became hypoxic in control tumours: pimo+ve cells were reduced by half of their initial staining
by 96 hours in both tumour types. At all times after chemotherapy, there was reoxygenation and
increased proliferation of previously hypoxic cells, and these processes were inhibited by
administration of TH-302. Conclusions: Chemotherapy rescues hypoxic cells from dying, and
leads to the reoxygenation and proliferation of previously hypoxic cells in solid tumours at all
times following treatment. Repopulation from hypoxic cells that were likely to die in the
absence of treatment contributes to treatment failure, and can be inhibited by TH-302, thereby
increasing the response to chemotherapy.
110
4.2 INTRODUCTION
Hypoxia in tumours can occur due to limited perfusion of blood vessels (acute hypoxia) or
because of limited oxygen diffusion and its consumption by tumour cells (chronic hypoxia) that
leads to hypoxic regions at distances of ~100-200 microns from functional blood vessels1.
Hypoxic cells in tumours have a limited lifespan, and chronically hypoxic regions in untreated
tumours appear to be in a state of dynamic equilibrium, where new hypoxic cells produced by
proliferation and migration of cells from regions closer to blood vessels is matched by cell death
from the hypoxic compartment2. Hypoxic cells are resistant to radiotherapy because its toxic
effects are mediated in part by oxygen-dependent free radicals1. Most types of chemotherapy are
designed to target rapidly proliferating cells and there is poor penetration of many drugs to
tumour cells that are located distal to functional blood vessels, including tumour cells in hypoxic
regions3-7
; hence hypoxic cells are also likely to be resistant to chemotherapy.
Both chemotherapy and radiotherapy are delivered in multiple doses, with chemotherapy often
scheduled in 3-week cycles to allow for the repopulation or recovery of critical normal tissues
such as bone marrow. During the intervals between treatments repopulation of surviving tumour
cells can occur, and is an established mechanism that reduces the effectiveness of fractionated
radiotherapy8-10
. Repopulation in the longer intervals between cycles of chemotherapy has also
been implicated to limit the effectiveness of treatment11,12
, but few studies have focused on
repopulation following chemotherapy as it relates to the tumour microenvironment. Because
hypoxic cells are likely to be resistant to chemotherapy, they might reoxygenate and proliferate,
thereby contributing to treatment failure. As hypoxia is associated with poor clinical outcome
and treatment failure, there is a need for the development of hypoxia-targeted agents capable of
killing these cells.
111
Hypoxia activated pro-drugs (HAPS) are administered in an inactive form, and are activated via
a reduction reaction in hypoxic regions to a toxic moiety that damages DNA13
. HAPs are back-
oxidized to their native form in oxygenated cells and can diffuse to distantly-located hypoxic
regions. The HAP TH-302 is in clinical trials and consists of a nitroimidazole conjugated to the
cytotoxic bromo-isophosphoramide mustard that is released in hypoxic tissues14
, so that TH-302
targets hypoxic cells selectively15,16
. The combination of chemotherapy to target the oxygenated
compartment of solid tumours and TH-302 to target the hypoxic compartment might inhibit
survival and/or repopulation of hypoxic cells and augment the effects of chemotherapy. In the
present study, we evaluate the hypotheses that i) cells in hypoxic regions of solid tumours are
destined to die in the absence of chemotherapy, ii) reoxygenation and proliferation of formerly
hypoxic regions occurs following chemotherapy and iii) survival and repopulation of hypoxic
cells can be inhibited by treatment with TH-302 in combination with chemotherapy.
4.3 MATERIALS AND METHODS
4.3.1 Cell lines
The human breast carcinoma (MCF-7) and prostate cancer (PC-3) cell lines were purchased from
the American Type Culture Collection (Manassas, VA). Cells were cultured in α-MEM (MCF-7
cells) or Ham's F-12K (PC-3) media (Life Technologies Inc., Carlsbad, CA) supplemented with
10% fetal bovine serum (FBS; Hyclone, Logan, UT) and incubated in a humidified atmosphere
of 95% air/5% CO2 at 37ºC. Routine tests to exclude mycoplasma and characterize the origin of
the cells (short tandem repeat analysis) were performed.
112
Female athymic nude mice (4-6 weeks old; Harlan Sprague-Dawley, Madison, WI) were
implanted with 17β-estradiol tablets (60-day release; Innovative Research of America, Sarasota,
FL) and then injected subcutaneously with 5x106 MCF-7 cells per flank. Male athymic nude
mice (4-6 weeks old; Jackson, Bar Harbor, Maine, USA) were injected subcutaneously in both
flanks with 2x106 PC-3 cells. There were five mice per treatment group (8-10 tumours) in each
experiment and each experiment was repeated.
4.3.2 Drugs and reagents
Doxorubicin (Pharmacia, Mississauga, Ontario, Canada) and docetaxel (Sanofi-Aventis,
Laval, Quebec, Canada) were purchased from the Princess Margaret Cancer Centre pharmacy
and provided as solutions with concentrations of 2 mg/mL and 40 mg/mL respectively. TH-302
was provided by Threshold pharmaceuticals (San Francisco, USA) as a powder and then
dissolved in 0.9% saline prior to use.
Pimonidazole (pimo) and a FITC-conjugated mouse IgG1 monoclonal antibody that
recognizes pimo adducts were purchased from Hypoxyprobe (Burlington, MA). The National
Cancer Institute provided EF5 as a powder, which was prepared by dissolving it in distilled water
supplemented with 2.4% ethanol and 5% dextrose to make a 10-mM stock solution that was
stored at room temperature. Cy5-conjugated mouse anti-EF5 antibody was purchased from Dr.
Cameron Koch, University of Pennsylvania, PA. Ki-67 was identified with primary rabbit anti-
human Ki-67 antibody (NovusBiologicals, Oakville, ON) followed by Cy3-conjugated goat anti-
rabbit IgG secondary antibody and visualized using the Olympus fluorescent upright microscope.
113
4.3.3 Effect of drugs on tumour reoxygenation and repopulation
Tumour-bearing mice (mean cross-sectional area 0.7-0.8 cm2) were given a single
intraperitoneal injection of pimo (60 mg/kg) and chemotherapy: either an intravenous injection
of doxorubicin (MCF-7, 8 mg/kg) or intraperitoneal injections of docetaxel (PC-3, 15 mg/kg)
and/or TH-302 (150 mg/kg) or saline (controls). Previous studies have established these doses as
the maximum tolerated5,6,16,17
. In groups treated with chemotherapy and TH-302, TH-302 was
administered 4 hours prior to chemotherapy; this was based on data from previous studies
establishing greater tumour cell death, DNA damage7 and increased growth delay
18 in tumours
treated with TH-302 4 hours prior to chemotherapy as compared to concurrent scheduling. Mice
were injected with a second marker of tumour hypoxia, EF5, to mark hypoxic regions at various
times following initial treatment (24, 48, 72, 96 and 120 hours) and animals were killed 2 hours
later. Tumours were excised and embedded immediately in OCT compound and then flash
frozen in liquid nitrogen and stored at −70°C prior to tissue sectioning and immunohistochemical
staining. 10μm thick cryostat sections were cut from each tumour. Whole tumour sections were
imaged and analyzed; artifacts and regions of necrosis were omitted. At least six tumours were
analyzed per treatment group.
Originally hypoxic cells were identified by binding of pimo and visualized using FITC;
hypoxia occurring in tumours at subsequent times was recognized using EF5 and visualized with
a Cy-5 far-red filter set. Changes in hypoxia were elucidated by identifying cells that were
pimo+/EF5- (formerly hypoxic cells) as well as cells that were pimo+/EF5+ (chronically hypoxic
cells). Ki-67 was detected using the Cy-3 filter set.
114
Media Cybernetics Image Pro PLUS software was used for image analysis and
quantification. A minimal threshold for detection (below the level of detection of drug or
hypoxia) was determined for each tumour in order to minimize noise due to auto-fluorescence.
Regions of hypoxia were analyzed as described previously3,7
. Binarized pimo and EF5 images
were used to make a composite displaying regions of only pimo staining (pimo+/EF5-) or EF5
staining (EF5+/pimo-): these composites represented the reoxygenated or newly hypoxic regions
and were reported as a percent of the initially hypoxic [pimo+/EF5-]/[pimo+] or newly hypoxic
[EF5+/pimo-]/[EF5+] regions, respectively. Chronically hypoxic regions were identified with
dual pimo+ and EF5+ labeling. The Ki-67 biomarker image was then used to identify
proliferating cells within the reoxygenated region by using the image processing overlay feature.
The Ki-67 positive area was quantified and reported as a percent of that in the total formerly
hypoxic region (i.e. to represent change in proliferation of the reoxygenated cells) or chronically
hypoxic region. For the assessment of Ki-67 distribution in relation to current hypoxia in
tumours, a binarized EF5+image (black and white image with white corresponding to only
biomarker positive pixels) was created, and used to create a distance map such that each pixel
was represented by its distance to the nearest hypoxic region (EF5+) in the section. The data are
represented graphically as the percent of pixels positive for Ki-67 at a given distance from the
nearest currently hypoxic region in the section; a cut-off distance of 60µm was used in order to
minimize interference from neighboring hypoxic regions that are out of the plane of the section.
115
4.3.4 Statistical analysis
A one-way ANOVA was used to determine the statistical differences amongst treatment groups.
Tests were 2-sided and no corrections were applied for multiple significance testing. A p-value
of less than 0.05 was used to indicate statistical significance.
4.4 RESULTS
4.4.1 Hypoxia markers in tumour sections
Photomicrographs displaying the overlap of concurrent administration of pimo and EF5 and of
the spatial distribution of Ki-67 positive cells following treatment are illustrated for MCF-7
xenografts in Figures 4.1 and 4.2 respectively.
4.4.2 Proliferation in continuously hypoxic cells
The proportion of Ki-67 positive cells in the tumour region that remains hypoxic in control
tumours (i.e. pimo+/EF5+) is depicted in Figure 4.3 A (MCF-7) and B (PC-3). As can be seen
there is low proliferation in chronically hypoxic regions of both tumour types starting at 1.5%
and 1.2% at 24 hours, which decreases continuously over time to 0.98% and 0.88% at 120 hours
in MCF-7 and PC-3 tumours respectively. Thus the (low) rate of cell proliferation decreases with
increasing duration of exposure to hypoxia.
116
4.4.3 Flux through the hypoxic compartment in untreated tumours
The proportion of viable tumour that was originally hypoxic in control samples (i.e. pimo+) and
the generation of new hypoxic (i.e. EF5+/pimo-) regions is illustrated in Figure 4.4A (MCF-7
tumours) and Figure 4.5A (PC-3 tumours). The fraction of originally hypoxic pimo-labelled cells
decreased over time from 24 to 120 hours in both MCF-7 and PC-3 tumours, consistent with
death of cells from the hypoxic compartment. In MCF-7 tumours, greater than half of the
originally (1.7%) pimo labelled cells were lost (0.7%) by the 96 hour time point and were further
reduced by 120 hours (0.58%) (Figure 4.4A). The accumulation of new hypoxic cells
(EF5+/pimo-) increased over time, such that by 24 hours, half of the currently hypoxic cells were
due to the flux of previously oxygenated cells into hypoxia and this increased to 80% by 120
hours (Figure 4.4B). PC-3 xenografts had a similar response, the pimo labelled fraction of cells
at 24 hours (1.5%) dropped by about half at 96 hours (0.8%) and further reduced to 0.3% at 120
hours (Figure 4.5A). Flux of previously oxygenated cells into hypoxia as determined by uniquely
EF5+ labelling, increased over time to nearly 100% by 120 hours (Figure 4.5B).
4.4.4 Effects of treatment on reoxygenation and proliferation
Reoxygenation of hypoxic tissues occurred in untreated MCF-7 control tumours
(consistent with some fluctuating hypoxia) at all times observed (Figure 4.6, panels A-E)
however, proliferation as assessed by the presence of Ki-67 was low in these reoxygenated cells
(Figure 4.6, panels F-J). Reoxygenation of formerly hypoxic cells (pimo+/EF5 -) in
chemotherapy and/or TH-302 treated MCF-7 tumours is shown in Figure 4.6 panels A-E.
Although all treatments resulted in a significant increase in reoxygenation as compared to that in
117
untreated control tumours (p<0.05), treatment with doxorubicin led consistently to the highest
rate of reoxygenation. Furthermore, proliferation in the reoxygenated compartment was
increased following treatment with doxorubicin (Figure 4.6, panels F-J) at all tested time points.
Treatment with TH-302 alone increased reoxygenation compared to controls, but resulted in
reduced cell proliferation in the formerly hypoxic region, and this effect was enhanced when
combined with chemotherapy. Combined treatment resulted in the greatest inhibition of
proliferation (p<0.05, compared with all other groups).
Reoxygenation in PC-3 treated tumours is similar to that observed in MCF-7 tumours
(Figure 4.7, panels A-E). All treatments resulted in greater reoxygenation compared to untreated
control tumours with maximum reoxygenation in tumours treated with docetaxel alone (p<0.05).
Docetaxel treatment alone resulted in increased proliferation of reoxygenated cells above that
seen in the control treatment at each time observed (p<0.05) and TH-302 reduced cell
proliferation in the reoxygenated region and this effect was maximal upon combination with
docetaxel (Figure 4.7, panels F-J).
4.4.5 Distribution of Ki-67 in tumour sections
Ki-67 expression as a function of distance from the nearest hypoxic region in the tumours
was evaluated at 24, 48, 72, 96 and 120 hours in controls and following chemotherapy, TH-302
or the combination (Figures 4.6 and 4.7, panels K-O). Ki-67 expression was significantly
(p<0.05) increased above that found in the control tumours at every time point (24, 48, 72, 96
and 120 hours) following treatment of MCF-7 tumours treated with doxorubicin and PC-3
tumours treated with docetaxel. Treatment with TH-302 alone reduced Ki-67 proliferating cells
to a level below that of the control (p<0.05) and this effect was amplified when combined with
118
chemotherapy so that the combination treatment had the lowest levels of proliferating cells near
hypoxic regions (p<0.05).
4.5 DISCUSSION
Chemotherapy is delivered frequently in 3-week cycles where intervals between cycles allow for
the repopulation of cells in the peripheral blood through proliferation and maturation of
precursors in the bone marrow. Unfortunately, bone marrow cells are not the only ones to
proliferate as surviving tumour cells can also proliferate. Repopulation of tumour cells following
radiotherapy is well documented but repopulation following chemotherapy has not been studied
extensively. Limited studies have found that the rate of proliferation of surviving tumour cells
may increase following chemotherapy19-22
. The objectives of the present study were to evaluate
the changes in hypoxia that occur in two solid tumour xenografts in the absence of treatment the
reoxygenation and repopulation that occur in tumour cells that were formerly hypoxic following
chemotherapy, and the potential of TH-302 to inhibit these processes.
We addressed the above questions by using two sequentially administered markers of hypoxia:
this method allowed identification of cells that were hypoxic at time of treatment and then
underwent reoxygenation following treatment with two widely-used chemotherapeutic agents:
doxorubicin and docetaxel. In control tumours, the originally hypoxic fraction in the viable
tumour (as labelled by pimonidazole) decreased over time, consistent with loss of pimonidazole
labelled cells due to cell death or possibly to some dilution of the pimonidazole-labelled adducts.
We are able also to determine rates of reoxygenation by identifying the population of
pimo+/EF5- cells at each time point. We report the flux of cells into hypoxia by determining the
population of EF5+/pimo- cells in control tumours and this steadily increases over time,
119
suggesting that in untreated tumours, cells continuously enter the hypoxic compartment, and
eventually undergo cell death.
We used Ki67 to investigate changes in proliferation amongst the chronically hypoxic and
reoxygenated cells. Proliferation rates were very low in chronically hypoxic cells and decreased
further over time, indicating that cells that remain in hypoxia for extended periods of time have
markedly reduced proliferative ability. Proliferation of cells within newly reoxygenated tissues
was contingent upon drug treatment. In both MCF-7 and PC-3 tumours, chemotherapy alone
resulted in the greatest proliferation of reoxygenated tissue at all times following treatment
compared to untreated controls. It is probable that following chemotherapy-induced cell death,
surviving tumour cells benefit from an enhanced supply of nutrients (including oxygen) resulting
from interrupted metabolism and/or clearance of dead cells that were located closer to blood
vessels, thus leaving more nutrients for cells that survive therapy and undergo proliferation3,23
.
Durand and Aquino-Parsons tracked SiHa cells that were pimonidazole labelled under transient
hypoxic conditions in spheroids over 9 days using flow cytometry24
. Upon the return of the
spheroids to normoxic (air) conditions, it was found that the outermost layer of cells in the
spheroids which was better oxygenated had a greater turnover rate of pimonidazole compared to
the innermost (and presumably most hypoxic) layer24
; these results were attributed to rapid cell
turnover in the outermost layers presumably due to reoxygenation and rapid cell proliferation.
Durand and Aquino-Parsons also investigated biopsies taken from patients treated with pimo
prior to chemo-radiotherapy and found that prior to treatment, pimo+ cells were primarily in
unproliferative Go/G1 phases of the cell cycle as assessed using flow cytometry; following
treatment, the pimo-label disappeared as cells entered proliferation24
.
120
Our study enhances the current understanding of how chemotherapy contributes to treatment
failure by identifying hypoxic cells that have been rescued (i.e. they may have died in the
absence of treatment) and contribute to repopulation. Administration of other treatments can
inhibit repopulation of tumour cells between courses of chemotherapy but it is important that
such treatments be relatively specific for tumours. For example our group found that
administration of the selective estrogen receptor modulator arzoxifene to estrogen receptor
positive MCF-7 xenografts between courses of 5FU or paclitaxel chemotherapy resulted in
substantial tumour inhibition effects compared to chemotherapy alone25
. Due to our observations
of reoxygenation and proliferation of formerly hypoxic cells following chemotherapy, we chose
to evaluate the potential of TH-302 to inhibit these processes and thereby increase therapeutic
efficacy by reducing the proliferation of reoxygenated, formerly hypoxic, cells.
TH-302 is a hypoxia activated pro-drug that contains an oxygen sensing 2-nitroimidazole group
conjugated to a bromo-isophosphoramide mustard which undergoes fragmentation releasing the
toxic mustard moiety that binds to DNA and causes cross-linkage26
. TH-302 is reduced by the
NADPH: cytochrome P450 family of reductases and requires severe hypoxia (~0.1% Oxygen)
for maximal activation15
. We hypothesized that TH-302 would be able to inhibit proliferation of
formerly hypoxic cells. Indeed, we found that although treating tumours with TH-302 did not
appear to inhibit the process of reoxygenation of hypoxic tissue, it reduced proliferation of the
reoxygenated cells. It is possible that many cells in the reoxygenated region might have been
lethally damaged by TH-302. In a previous study7, we evaluated the distribution of TH-302 in
relation to vascular and hypoxic regions and reported maximum DNA damage (as measured by
γH2AX ), cell death (as measured by cleaved caspases -3 or -6) and reduced cell proliferation in
regions closest to hypoxia in MCF-7 and PC-3 xenografts, consistent with this hypothesis.
121
Furthermore, we noted that the combination of TH-302 with either doxorubicin or docetaxel
resulted in the greatest cell death and reduced proliferation compared to either treatment alone in
both perivascular and hypoxic regions. In the present study, we found that combined treatment
inhibited proliferation in the reoxygenated compartment – an important finding as it provides
support for the activation of TH-302 in hypoxic tissues and its ability to reduce repopulation
from this region. Huxham et al reported that following gemcitabine treatment of HCT-116
tumours, the first cells to proliferate where those located at the border of necrotic (presumably
hypoxic) regions19
. Fung et al also reported that cell proliferation (repopulation) occurred in
regions close to hypoxia following chemotherapy3. In the present study, we observed that TH-
302 treatment alone was able to suppress Ki-67 proliferation in areas closest to current hypoxia
as well, an effect that was increased with the addition of chemotherapy. Thus in our present
study, the additive anticancer effects of combined TH-302 and chemotherapy may be attributed
to the complementary roles of these agents. For example, chemotherapy will target rapidly
dividing cells in perivascular regions while TH-302 predominantly attacks hypoxic tissues,
although the combination therapy did not abolish all Ki-67 proliferating cells. This might be due
to the multifactorial nature of repopulation which can be attributed to several reasons aside from
reoxygenation of hypoxic tissues, including loss of p53 activity leading to reduced apoptosis 27
or a need to increase the dosage and frequency of TH-302. We chose to give a single high dose
of TH-302 alone and 4 hours prior to chemotherapy that yielded superior antitumour activity
based on our 7 previous study as well as data from others
16,18.
The combination of TH-302 and chemotherapy in two xenografts resulted in the greatest
inhibition of proliferating cells. In our previous study we reported that the combination therapy
resulted in the greatest growth delay and increased DNA damage not only in hypoxic regions but
122
also in vascularized regions. The current observation of the greatest reduction in Ki-67
proliferation in the formerly hypoxic area as well as the greatest inhibition in proliferation of
cells in regions closest to hypoxia in the combination group may be due to several factors. TH-
302 produces free radicals as it is back oxidized to its prodrug form in the presence of oxygen as
it diffuses through the tumour. Furthermore, TH-302 is activated in extremely hypoxic areas
(<0.1% oxygen) and releases its cytotoxic moiety that is capable of diffusing to surrounding
tissue that may have a higher oxygen content and kill these cells, thus having a bystander effect -
an ability that is crucial in maximizing the effects of a hypoxia targeted agent15
.
Our study has limitations including our inability to assess changes in cell proliferation as it
relates to the formerly hypoxic compartment beyond 5 days due to the limited stability of
pimonidazole28
. Also, we assessed cell proliferation and hypoxia using a 2 dimensional
approach although tumours are 3 dimensional. Furthermore, it was difficult to assess actual cell
death at the time points we measured as we’ve previously demonstrated that cleaved caspase -3
signal – a marker of apoptosis – is most robustly measured at 24 hours following drug treatment;
given our time course, we were impeded in this measurement.
Overall, our data demonstrates the ability of chemotherapeutic agents (doxorubicin and
docetaxel) to induce repopulation by rescuing previously hypoxic tissue and allowing their
reoxygenation and proliferation. This effect can be inhibited with the use of TH-302, which
thereby has potential to improve therapeutic outcome.
123
Acknowledgements
Supported by grants from the Canadian Institutes of Health Research (CIHR) and Threshold
Pharmaceuticals to Dr. Tannock and a CIHR Banting & Best doctoral studentship to Ms. Saggar.
We thank all members of the Pathology Research Program (PRP), and the Advanced Optical
Microscopy Facility (AOMF). We thank Threshold pharmaceuticals for providing us with TH-
302 and for previous grant support.
124
4.6 FIGURES
Figure 4.1 MCF-7 tumours treated concurrently with pimonidazole and EF5. Images
display staining of hypoxia as labelled with (A) pimonidazole adducts (green), (B) EF5 adducts
(red) and (C) overlay of pimonidazole with EF5 (yellow). Notice the colocalization of
pimonidazole with EF5 to produce yellow.
Figure 4.2 Spatial distribution of Ki67 positive cells in relation to formerly hypoxic regions
in MCF-7 tumours 24 hours following treatment with pimonidazole &: (A) saline control
(B) doxorubicin (C) TH-302 or (D) TH-302+doxorubicin combination. Green represents
formerly hypoxic region and red represents Ki67 positive cells; overlapping regions of formerly
hypoxic cells with current Ki-67 positive cells have produced a yellow colour.
A B C
B A C D
125
Figure 4.3 Proliferation in hypoxic tissues. Ki-67 proliferation (%) in chronically hypoxic
(pimo+/EF5+) portions of MCF-7 (A) & PC-3 (B) control tumours over time
Figure 4.4 Flux through the hypoxic compartment of MCF-7 tumours. (A) Percent of entire
viable tumour that was originally hypoxic (pimo+) in MCF-7 control (untreated) tumours over
time & (B) Percent of newly hypoxic cells (EF5+/pimo-) in relation to total hypoxic cells (EF5+)
over time in control tumours.
A B
A B
A B
126
Figure 4.5 Flux through the hypoxic compartment of PC-3 tumours. (A) Percent of entire
viable tumour that was originally hypoxic (pimo+) in PC-3 control (untreated) tumours over time
& (B) Percent of newly hypoxic cells (EF5+/pimo-) in relation to total hypoxic cells (EF5+)
over time in control tumours.
.
B A
127
72 H
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Figure 4.6 Reoxygenation and Repopulation in MCF-7 tumours treated with pimonidazole &: saline
(control; ), doxorubicin (8 mg/kg; ), TH- 302 (150 mg/kg; ) or combination ( ). Panels A- E show
reoxygenation (percent of cells that were pimo+ and are now EF5-) of tumour tissue that has occurred
following treatment over time (24-120 hours). Panels F-J depict proliferation (Ki67 positive staining) in
reoxygenated tissues over time following treatment & panels K-O show the spatial distribution of KI67 in
relation to the nearest currently hypoxic area. Points indicate average of 6 mice per group; bars, SE.
A
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128
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Ki67 SPATIAL DISTRIBUTION Ki67 PROLIFERATION REOXYGENATION
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Figure 4.7 Reoxygenation and Repopulation in PC-3 tumours treated with pimonidazole and: saline
(control; ), docetaxel (15 mg/kg; ), TH-302 (150 mg/kg; ) or combination ( ). Panels A- E show the
reoxygenation (percent of cells that were pimo+ and are now EF5-) of tumour tissue that has occurred
following treatment over time (24-120 hours). Panels F-J depict proliferation (Ki67 positive staining) in
the reoxygenated tissues over time following treatment & panels K-O show the spatial distribution of
KI67 in relation to the nearest currently hypoxic area. Points indicate average for 6 mice per group; bars,
SE.
A
B
C
D
E
F
G
H
I
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Distance to hypoxic area
129
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133
CHAPTER 5
CONCLUSIONS AND FUTURE DIRECTIONS
134
5. SUMMARY OF FINDINGS AND IMPLICATIONS FOR FUTURE WORK
The effectiveness of chemotherapy is limited by intrinsic and acquired drug resistance. While
many studies are devoted to the molecular causes of drug resistance, the tumour
microenvironment also plays a role. Specifically, limited drug distribution within the tumour
microenvironment is a cause of drug resistance. Repopulation of surviving tumour cells between
courses of chemotherapy also contributes to treatment failure. Since the majority of anticancer
drugs are non-fluorescent, their visualization and distribution within tumour tissue is difficult to
assess. By focusing our research on validating methods to assess non-fluorescent drug
distribution as it relates to the tumour microenvironment, we can evaluate drug distribution in
tumours and assess methods of modifying or complementing it. This thesis is focused on
developing techniques to assess the distribution of biomarkers of drug effect that reflect drug
distribution in vivo. My research also evaluated the hypothesis that chemotherapy leads to the
survival and increased proliferation of tumour cells that were previously hypoxic and may have
died in the absence of treatment. Finally, this thesis assessed whether tumour cell repopulation
between courses of chemotherapy treatment was a modifiable phenomenon by using a hypoxia-
activated pro-drug, TH-302, to target hypoxic cells in tumours. TH-302 was found to result in
increased cell death throughout the tumour and was able to inhibit tumour cell repopulation when
combined with chemotherapy.
135
5.1 Potential sources of error and bias
In my experiments, tumour sections are stained with different antibodies and are then imaged on
the fluorescent upright microscope. During the image acquisition process, there are sources of
natural variability that we did not account for such as the change in the intensity of the lamp used
for imaging, the loss of fluorophore signal intensity due to photobleaching or changes that occur
due to the specimen processing (e.g. tumour sections that are stained at different times with
antibodies).
The scanned tumour sections are then used to identify pixels in the image that have stained
positively for the biomarker in question; the number of biomarker positive pixels is reported as a
percent of the total number of pixels at any given distance from a blood vessel or hypoxic region.
As we are reporting biomarker-positive pixels, these must be distinguished from directly
reporting cells. Our images were acquired at 10x magnification, and the size of one pixel is 0.4
µm2; due to variations in the size of cells, we are unable to correlate an exact pixel count to the
size of any given cell. However, since our program accounts for the total number of pixels in the
tumour image this will relate to the overall size of the tumour, i.e. larger tumours possess a
greater number of pixels (and hence cells) than do smaller tumours, so the area of the entire
tumour is taken into account in our reporting metrics. Theoretically, the pixels could be
attributed to different cellular locations for example γH2AX is found in the nucleus whereas
cleaved caspase -3 or -6 and Ki-67 are mainly in the cytoplasm, but we did not attempt to
correlate the biomarker positive pixels with one another amongst different sections, e.g. how
much γH2AX signal is required for cleaved caspase-3 or -6 levels to increase. We also did not
report the number of pixels per tumour section or quantify the size of tumour blood vessels to see
if these correlated with intensities of biomarker expressions.
136
Our statistical analysis of biomarker distributions, growth delay, reoxygenation and
reproliferation involved a one-way ANOVA. All tests were performed with an alpha level of
0.05. No multiple comparison adjustment was performed. The Bonferroni adjustment is
considered conservative especially when the markers are correlated, as in this study. The
intention was to have a relaxed type I error to minimize the false negative results. Given that our
degree of independence is unknown, in cases where the p value = < 0.01 this is not likely to
cause any change, but where our p values were very close to 0.05 the results should be regarded
as hypothesis-generating and approached with caution.
5.2 Use of molecular biomarkers to quantify the spatial distribution of effects of anticancer
drugs in solid tumours
5.2.1 Summary
In chapter 2, I evaluated the use of the molecular biomarkers: γH2AX, cleaved caspase -3 or -6
and Ki-67 to characterize the distribution of effects of different chemotherapeutic drugs in the
tumour microenvironment. In order to validate the use of these biomarkers, I first assessed the
distribution of the auto-fluorescent drug doxorubicin as a function of distance from the
vasculature in MDA-MB-231 and MCF-7 xenografts at different times after treatment: 10 mins
and 3, 6, 24 and 48 hours. My data show that the concentration of doxorubicin in tumour cells at
10 minutes following treatment decreased with increasing distance from the vasculature and this
effect was sustained at the other time points. I next used the above biomarkers to elicit the
distribution of their activity in tumour tissue and found that they were similar to the distribution
of doxorubicin fluorescence. In particular, γH2AX at 10 minutes following treatment displayed a
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similar profile to doxorubicin distribution indicating its usefulness as an early marker of drug
effect. Induction of cleaved caspase -3 or -6 and reduction in Ki-67 were maximal at 24 hours
following drug treatment. Again, the greatest increase in cleaved caspase and reduction in Ki-67
was observed closest to the blood vessel – so that the distribution was similar to that found with
doxorubicin fluorescence. To further validate the use of these biomarkers, I studied their
distributions following treatment of MCF-7 and EMT-6 tumours with melphalan. Melphalan was
chosen because we had available a monoclonal antibody that specifically recognizes melphalan-
induced DNA adducts. I observed that the density of melphalan-induced DNA adducts decreased
rapidly with increasing distances from the vasculature and this was comparable to the
distributions of γH2AX at 10 minutes and cleaved caspase -3 and -6 and reduction in Ki-67 at
24 hours. The biomarkers were then used to evaluate the distribution of activity of the non-
fluorescent and frequently used chemotherapeutic drug docetaxel. This drug also had decreasing
activity with increasing distance from tumour blood vessels, indicating that this is a property of
many anticancer drugs in clinical use.
5.2.2 Implications of the study and future directions
Poor distribution of anticancer drugs within solid tumours will limit their effectiveness. I have
validated the use of biomarkers to assess the distribution of activity of non-fluorescent anticancer
drugs using doxorubicin and melphalan and extended these techniques to docetaxel and TH-302.
Previously, analysis of non-fluorescent drugs in tumour tissue was limited to the use of radio-
labelled compounds, my technique facilitates the study of a variety of drugs. These techniques
are now being used by others in our laboratory and several external collaborators. The
biomarkers γH2AX, cleaved caspase -3 or -6 and Ki-67 allow for assessment of the distribution
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of a wide variety of anticancer drugs within the tumour microenvironment. Indirectly, our results
demonstrate that poor penetration of the anticancer drugs from blood vessels results in reduced
tumour cell death at increasing distances from the vasculature. While both cleaved caspase -3 or
-6 and Ki-67 are reflective of the accumulation of drug effect at later time points, the early
appearance of γH2AX at 10 minutes following treatment reflects drug distribution and is a
predictor of the later pattern of activity of the other biomarkers, thus providing a powerful
predictor of drug efficacy. I was able to use this technique to validate treatment protocols in
chapter 3, for example. It was reported previously that TH-302 administration 4 hours prior to
chemotherapy results in increased growth delay versus concurrent treatment protocols. Applying
the biomarker techniques, I was able to investigate differences in the distribution of γH2AX with
TH-302 administered with chemotherapy either sequentially or concurrently; I found that
γH2AX response was highest with concurrent treatment. This technique is rapid and cost
effective and was able to provide evidence that the reason for increased growth delay in the
sequential treatment pattern was in part attributed to increased DNA damage.
This technique can also be applied to test the ability of different delivery vehicles to increase
drug penetration or that attempt to target the tumour exclusively, for example the use of
nanocarriers conjugated to drugs that are then released uniquely in the tumour, microbubbles that
are burst using ultrasound in order to increase drug diffusion and antibody-drug conjugates that
lead to specific targeting of certain tumour cells Furthermore, our techniques can be used to
evaluate the ability of different drug combinations and scheduling protocols to increase cell
death – this was done in chapter 3 of this thesis in order to determine the differences with respect
to biomarker distributions following sequential and concurrent treatment protocols. Another
application of our technique is the ability to evaluate the ability of a proton pump inhibitor (e.g..
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pantaprazole) to increase drug distribution by raising endosomal pH thereby decreasing the
amount of drug that is sequestered into endosomes and allowing more drug to attack tumour
DNA. The focus of chapters 3 and 4 of this thesis, investigates the ability of a hypoxia-activated
prodrug to complement drug distribution as well. Furthermore, the results of my studies
demonstrated that limited drug distribution into tissues is not solely a problem observed with the
auto-fluorescent drugs doxorubicin and mitoxantrone 1, but applies to a variety of anticancer
drugs and contributes to drug resistance. Drug resistance as it relates to the tumour
microenvironment is a neglected but important cause of treatment failure. Drug distribution
within the tumour microenvironment should be taken into consideration when evaluating new
therapies alone or in combination with existing drugs. The above biomarkers can be employed in
designing strategies to overcome therapeutic resistance by modifying or complementing the
limited spatial distribution of drug activity in solid tumours.
It is important to note that the evaluation of biomarkers of drug effect reflect an accumulation of
drug activity and are not necessarily representative of actual drug distribution at any fixed time.
Doxorubicin fluorescence patterns were reflective of observed biomarker activity and drug
activity can be correlated with poor drug distribution to tumour tissues. Likewise, our use of the
antibody that recognizes DNA adducts formed by melphalan suggests that melphalan – a drug
that is not specific to cells in S-phase – also demonstrates poor penetration into tumour tissues.
Docetaxel is not fluorescent and antibodies that recognize its distribution are unavailable; our
observations suggest that docetaxel is causing DNA damage and cell death in regions closest to
blood vessels – it is possible that this is a result of this S-phase specific drug damaging cells that
are closest to the vasculature because of their more rapid proliferation (as indicated by the Ki-67
staining reported in our control tumours). However, given our combined results with doxorubicin
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and melphalan which demonstrate poor penetration of drugs to tumours and results showing poor
penetration of radio-labelled paclitaxel in tumours 2,3
and in MCLs 4
it appears likely that our
results for docetaxel are due, at least in part, to poor drug penetration from tumour blood vessels.
5.3 Activity of the hypoxia activated pro-drug TH-302 in hypoxic and perivascular regions
of solid tumours and its potential to enhance therapeutic effects of chemotherapy
5.3.1 Summary
In chapter 3 I extended the biomarker techniques that I validated in chapter 2 to determine the
distribution of the hypoxia-activated pro-drug TH-302. Secondly, I evaluated the ability of TH-
302 to modify/complement the distribution of the anticancer drugs doxorubicin and docetaxel in
the tumour microenvironment. Athymic nude mice bearing human breast MCF-7 or prostate PC-
3 tumours were treated with doxorubicin or docetaxel respectively and TH-302 alone or in
combination. Biomarkers of drug effect including γH2AX, cleaved caspase-3 or -6 and
reduction in Ki-67 were quantified in tumour sections in relation to both functional blood vessels
(recognized by DiOC7) and hypoxia (recognized by EF5) using immunohistochemistry. I first
evaluated the distribution γH2AX at 10 minutes following two different treatments, comparing
the effects of concurrent administration of TH-302+doxorubicin versus TH-302 followed by
doxorubicin 4 hours later, a schedule reported to be more effective in a prior study5. I confirmed
that pre-administration of TH-302 4 hours prior to chemotherapy verses concurrent
administration resulted in greater activation of γH2AX. Similar to chapter 2, both doxorubicin
and docetaxel alone caused increases in γH2AX and cleaved caspase -3 or -6 and reduction in
Ki-67 in regions closest to blood vessels but failed to have much effect in hypoxic regions. TH-
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302 treatment alone resulted in a robust increase in γH2AX and cleaved caspase -3 or -6 activity
in hypoxic regions and surprisingly resulted in an increase in these biomarkers in regions close to
vasculature as well. This may have been due to the generation of free radicals as TH-302 back-
oxidizes into its initial pro-drug form in the presence of oxygen. Combination treatment (with
TH-302 administered 4 hours prior to chemotherapy) resulted in the greatest increase in γH2AX
and cleaved caspase -3 or -6 and reduction of Ki-67; it also resulted in the greatest inhibition of
growth delay amongst all treatments.
5.3.2 Implications of the study
This study demonstrated that the intra-tumour distribution of toxic effects of anticancer drugs is
modifiable and that the hypoxia-activated pro-drug TH-302 actively targets hypoxic and
neighboring regions. Hypoxia is an indicator of poor therapeutic outcome 6,7
, and targeting
hypoxic cells may lead to improved treatment outcome. The results of chapter 3 provide
evidence for the spatial distribution and ability of TH-302 to selectively target hypoxic tumour
regions and cause cell death, but also to augment toxic effects in other regions of the tumour.
Furthermore, TH-302 can complement the effect of chemotherapy and resulted in increased
tumour growth delay, although combined treatment also resulted in increased toxicity. These
studies support the ability of TH-302 to diffuse to and become activated in hypoxic regions. Its
ability to increase biomarker distributions can be attributed to its bystander effect 8. Other HAPs
such as tirapazamine had a limited bystander effect and were activated at higher concentration of
oxygen. Recent data from a phase II clinical trial in which TH-302 was combined with
gemcitabine for the treatment of pancreatic cancer resulted in a 27% vs 12% (gemcitabine alone)
response rate 9. Our study evaluated the use of a single high-dose of TH-302 (150 mg/kg) in
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tumour tissues, it would be interesting to observe the change in biomarker distributions following
different treatment protocols, e.g. once a week for 3 weeks with animals killed after each
treatment in order to track the change in the distribution of biomarkers that may occur following
each drug treatment. Also, we evaluate changes in biomarkers on separate tissue sections cut
from the same tumour after any given treatment; ideally all biomarkers should be quantified on
the same tumour section, but given the issue of increasing competitive binding between different
antibodies we opted to use different tumour sections.
Other methods to attack hypoxic cells include the use of proton pump inhibitors such as
pantaprozole. Autophagy is known as a process of self-eating and is believed to aid cells by i)
degrading damaged cellular components in order to avoid mutational accumulation, ii) function
as an alternative pathway to apoptosis and iii) serve as an alternative energy source by recycling
proteins during periods of metabolic stress such as during hypoxia. Autophagy is associated with
a poor clinical outcome 10,11
, thus strategies targeting autophagy such as the use of pantoprazole
are currently being investigated by our lab. The administration of pantoprazole results in
increased endosome pH; this is due to pantoprazole’s ability to block the fusion of
autophagisomes with endosomes - a critical step in autophagy. Our lab is currently evaluating the
distribution of biomarkers in xenografts following treatment with different combinations of
pantoprazole with chemotherapy as a method to target tumour hypoxia.
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5.4 Chemotherapy rescues hypoxic tumour cells in tumours and induces reoxygenation and
repopulation - an effect that is inhibited by the hypoxia activated pro-drug TH-302.
5.4.1 Summary
In experiments described in chapter 4, I evaluated the hypothesis that chemotherapy contributes
paradoxically to treatment failure by rescuing previously hypoxic cells in tumours from cell
death and causes them to reoxygenate and proliferate. I studied these effects by sequentially
administering two markers of hypoxia: pimonidazole (pimo) and EF5 separated by a varying
time interval. These markers could be recognized in tumours by IHC using different antibodies.
This allowed me to follow the fate of initial (pimo+) hypoxic cells and of subsequent (EF5+)
hypoxic cells at different time points in control tumours and following treatment with
chemotherapy. I also studied the ability of TH-302 to inhibit the repopulation of previously
hypoxic cells.
Changes in proliferation and oxygen status of formerly hypoxic (pimo+ve) cells were quantified
by their Ki-67 status and uptake of EF5 as a function of time following treatments. The flux of
cells out of and into hypoxia can be traced in untreated control tumours and used as a baseline to
compare to other treatments. A small amount of reoxygenation was confirmed in control
tumours, consistent with fluctuating hypoxia, likely due to changes in blood flow. Loss of
pimonidazole labeling was attributed to cell death such that by 96 hours, half of the originally
labelled pimo compartment disappeared in both tumour types. Flux into hypoxia (cells labelled
uniquely with EF5) continued to increase to nearly 80% (MCF-7 tumours) and 100% (PC-3
tumours) of the currently hypoxic region, suggesting that as pimo-labelled cells die, other tumour
cells become hypoxic and may also undergo eventual cell death. Both doxorubicin and docetaxel
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induced reoxygenation and increased proliferation of previously hypoxic cells above that of the
control, suggesting that chemotherapy rescues previously hypoxic cells that may have died in the
absence of treatment. TH-302 was able to reduce proliferation of the reoxygenated cells below
that found in the control and this effect was increased upon combination with chemotherapy.
5.4.2 Implications of the study
Few studies have evaluated tumour cell repopulation following chemotherapy. We elucidated the
changes in hypoxia that occur following treatment using two different markers of hypoxia and
found that chemotherapy contributes to increased proliferation of previously hypoxic cells. This
may be due to inadequate drug available for diffusion to these regions and increase in availability
of oxygen and nutrients resulting from the clearance of cells closest to the vasculature.
Chronically hypoxic cells in control tumours have low proliferation rates and this decreases
further over time, suggesting that hypoxic cells lose proliferative capacity. There are
reoxygenated cells in control tumours, and there is a steady rate of proliferation in these regions
(5%). Chemotherapy alone was observed to result in an increase in reoxygenation and
proliferation (within the formerly hypoxic compartment) at all times following treatment
compared to the control. This provided evidence that chemotherapy paradoxically induces
reoxygenation and proliferation from previously hypoxic cell populations that may be destined to
die in the absence of treatment. Proliferating cells within the reoxygenated region were reduced
upon the addition of TH-302 and the anti-proliferative effect was maximized upon combination
with chemotherapy. These results suggest that the addition of a hypoxia-activated pro-drug that
targets hypoxia can complement the effects of chemotherapy that attacks predominantly the well-
oxygenated compartment. By targeting the hypoxic compartment with hypoxia-targeted agents,
145
therapeutic outcome can be increased. Furthermore, our techniques can be used to study the
optimal scheduling of chemotherapy with hypoxia-targeted agents to inhibit repopulation. The
presence of hypoxia is associated with poor therapeutic outcome and interferes with the effects
of radiotherapy 12,13
. Oxygen functions as a radiosensitizer and is also involved in formation of
free radicals which may damage DNA following chemotherapy treatment; its absence (i.e.
hypoxia) confers resistance to radiotherapy and drugs. A study by Huxham et al demonstrated
that cells of a colon cancer xenograft that were located distal from blood vessels commenced
cycling sooner than proximal cells following gemcitabine treatment indicating that previously
hypoxic cells cycle sooner than normoxic cells 14
, consistent with results presented here.
Our current experiments only determined cell proliferation following a single treatment with a
chemotherapeutic drug and TH-302 and while the combined therapy decreased cell proliferation
to the greatest extent, the levels of proliferating cells tended to increase over time. In future
studies, it would be interesting to determine if cell proliferation levels could be controlled with
more frequent administration of TH-302. We also opted to deliver TH-302 4 hours prior to
chemotherapy based on previously reported data showing that this schedule led to maximal
tumour growth delay 15
. We also determined through our biomarker analysis that pretreatment
with TH-302 results in greatest DNA damage levels and apoptosis. Future studies should also
evaluate the impact of consecutive treatments on biomarker distribution profiles to determine
optimal scheduling of agents and different drug combinations. In addition, agents that may lead
to an increase in the size of the hypoxic compartment such as VEGF targeted agents that can
inhibit angiogenesis may thereby increase hypoxia in tumours. The pretreatment of tumours with
a VEGF inhibitor followed by use of TH-302 would be a worthwhile investigation in an attempt
to maximize TH-302’s hypoxia-targeting ability.
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5.5 LIMITATIONS AND FUTURE DIRECTIONS
5.5.1 Tumours, Treatment, Image Collection, Biomarker measurements and Repopulation
Experimental subcutaneous tumours were generated in athymic nude mice for our studies.
Although the tumours we excise are 3-dimensional, our analysis is limited to 2 dimensions. In
using a 2-dimensional approach, we are overestimating the distance to the nearest blood vessel or
region of hypoxia since out-of-plane blood vessels and regions of hypoxia exist. Due to this
uncertainty, we use a cut-off of 60µm when plotting distance from blood vessels or regions of
hypoxia. However, incorporating only the 2-dimensional readings in our results can lead to an
underestimation of the slopes (gradients) of biomarker distributions. It would be interesting to
compare our results with those that use 3-dimensional analysis.
Our analysis was performed using subcutaneous tumours and their vasculature is different from
that found in spontaneous human tumours or in other tumour models such as orthotopic tumours
or in mice that are engineered to spontaneously generate tumours16
. It would be ideal to compare
drug distribution using all 3 models. Our lab has previously evaluated the distribution of
doxorubicin in orthotopic MCF-7 tumours and found a similar distribution profile of the drug as
compared to subcutaneous tumours (Fung et al, unpublished findings). It would also be of
interest to elucidate the distribution of drugs and biomarkers in tumour biopsies taken from
patients at different time points following treatment. Preliminary analysis assessing doxorubicin
fluorescence and cleaved caspase -3 distributions in 2 biopsies of nasopharyngeal tumours from
a patient revealed a similar pattern of distribution comparable to our xenograft studies; i.e.
doxorubicin fluorescence and cleaved caspase -3 distribution decreases as you move further
147
away from a blood vessel. It would be interesting to evaluate drug distribution in other tumour
biopsies from patients that had received recent drug treatment.
The tumour microenvironment can change over time, and it would be desirable to determine the
changes that occur in vasculature, hypoxic regions, and drug and biomarker concentration over
time. Previous studies have employed a window-chamber model, but the tumours generated are
quite small (5-10 mms in diameter) 17
and there are few regions of hypoxia. Doppler Optical
Coherence Tomography has been used to detect changes in the velocity of blood within vessels
with a resolution of <10µms in tumours generated using the window-chamber model but again
the disadvantage is that generated tumours are quite small 18
. Bioluminescence imaging might be
used to assess changes in the tumour environment such as fluctuations in hypoxia using dual
markers, but it is not sensitive enough to detect changes at the cellular level (micrometer range)
so evaluating the spatial distribution of biomarkers over time is not possible using this method.
In all of our studies, we used single dose treatments and then proceeded to evaluate drug
distribution profiles or repopulation of cells. However, patients are treated with multiple rounds
of chemotherapy. It would be of importance to track drug and biomarker distributions following
several rounds of chemotherapy. In chapter 3 it was reported that the pre-administration of TH-
302 4 hours prior to doxorubicin and docetaxel resulted in the greatest increase in γH2AX ,
cleaved caspase -3 or -6 and reduction in Ki-67. Therefore it is possible that other dosing
schedules could produce a different biomarker profile. It would be interesting to track the
distribution of biomarkers in tumours following each round of chemotherapy in patients.
Previously, our lab was restricted to evaluating the distribution of fluorescent drugs with the use
of a quantification program. In order to evaluate non-fluorescent drugs and their effects on
biomarkers, we have developed a new technique and this has several advantages. The previous
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technique was limited to quantifying fluorescent stains and as such was susceptible to artifact
from background auto-fluorescence despite attempts to set thresholds for this. The new technique
is not subject to artifact from background autofluorescence of the tissue as chromogenic stains
are used in lieu of fluorescent stains and the new program reports the number of positive pixels
for any binarized biomarker mask instead of reporting the fluorescence of pixels. Another source
of error with the previous method was its inability to distinguish between artifact-generated and
drug-generated fluorescence - although notably the program was mainly correct in its reporting
overall. Our evaluation of biomarkers to assess drug activity following different treatments
produces a percent value that represents the number of pixels that are positive for any given
biomarker over the total number of pixels at any given distance. During our analysis, each
biomarker image is collected on a separate slide, and in order to obtain a specific signal we were
limited to one biomarker and blood vessel label (e.g. CD31) per slide. Ideally one would quantify
each biomarker (γH2AX, cleaved caspase -3 or -6 and Ki-67) in a single tumour section, so this
does pose a limitation. Future studies ideally should attempt to correlate the levels of γH2AX
with cleaved caspase -3 or -6 in individual cells in an attempt to determine how much γH2AX
activation is required to initiate apoptosis. Ideally this would first be determined using
doxorubicin whose autofluorescence could be used as a guide in determining i) the intensity of
doxorubicin fluorescence (localized in the cell’s nucleus) with ii) the γH2AX activation in those
nuclei and finally iii) the levels of cleaved caspase -3 or -6 in the entire cell. Our current method
of analysis allows us to evaluate the entire tumour section as opposed to the previous
quantification program that was limited to evaluating several regions of interest that may have
been selected in a biased manner.
149
In chapter 4, I describe my repopulation studies. These studies were performed using two
sequentially administered exogenous markers of hypoxia (Pimo and EF5) to elucidate changes in
hypoxia that occur following treatment over time. This study was limited to evaluating single
treatments with drugs. It would be of interest to analyze the rate at which Ki-67 positive cells
proliferate following multiple rounds of treatment (and what the effects are on normal tissue
repopulation as well). I was also limited to using different tumours for studying effects of drug
treatment and controls at each time point, and there is considerable inter-tumour variation in
proportion of hypoxic cells and other parameters. It would be ideal to track changes in hypoxia
and in tumour cell repopulation in the same control and treated tumours over time, preferably
using a 3-dimensional approach. Also it would be interesting to identify cancer stem cells since if
the stem cell model is valid, it is changes in reoxygenation and repopulation of stem cells that are
important in determining long-term outcomes of cancer treatment. Current studies in our lab are
focused on identifying cancer stem cells in the tumour microenvironment with the use of
appropriate cancer stem cell markers and IHC. Ideally, the cancer stem cell markers would be
used to identify such cells that may exist in the hypoxic compartment; changes that may occur
following chemotherapy could then be evaluated and the ability of a stem cell to create more
stem cell progeny could then be evaluated. The effect of TH-302 on such a population of cells
should also be investigated.
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5.6 CONCLUSIONS
In summary, research completed in this thesis has contributed to the field of cancer biology by:
i) Developing techniques to assess the distribution of non-fluorescent anticancer drugs in
tumours,
ii) Demonstrating that drug distribution can be modified with the administration of a
hypoxia-activated pro-drug,
iii) Studying reoxygenation and repopulation and showing that chemotherapy may rescue
previously hypoxic cells from cell death.
iv) Demonstrating that chemotherapy-induced tumour cell repopulation can be inhibited by
TH-302.
This thesis illustrates the complexity of the tumour microenvironment and its role in drug
resistance. The efficient penetration of anticancer drugs from the vasculature into the tumour
remains a challenge for several agents, but this appears to be modifiable. Chemotherapy alone
can induce tumour cell repopulation and paradoxically can contribute to treatment failure even as
it causes shrinkage of tumours – but this has the potential to be modified with the use of hypoxia-
targeted agents. Although it is difficult to predict the ease with which preclinical studies can be
translated into effective clinical outcome, this thesis has broadened our understanding of the role
of the tumour microenvironment in influencing the outcome of systemic cancer treatment and the
potential to improve therapeutic outcome with the use of a hypoxia-activated prodrug.
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