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Targeting the tumour microenvironment : characterizing the anti-vascular effects of the hypoxic cytotoxin.. Baker, Jennifer Hazel Elizabeth 2010-04-01

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TARGETING THE TUMOUR MICROENVIRONMENT:  CHARACTERIZING THE ANTI-VASCULAR EFFECTS OF  THE HYPOXIC CYTOTOXIN TIRAPAZAMINE  by  JENNIFER HAZEL ELIZABETH BAKER  B.Sc., Simon Fraser University, 2004      A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF   DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES  (Interdisciplinary Oncology)         THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)    March 2010  © Jennifer Hazel Elizabeth Baker, 2010   ii ABSTRACT  Tirapazamine (TPZ) is a bioreductive prodrug with greater toxicity to hypoxic cells in vitro, and anti-vascular activity in tumours grown in vivo in mice. Considerable inter- and intra- tumour heterogeneity occurs in response to the anti-vascular effects of TPZ. The main hypothesis for the work described in this thesis is that features of the tumour microenvironment confer tumour sensitivity to TPZ-mediated vascular dysfunction. Tumours exhibiting less sensitivity to the anti-vascular effects of TPZ had evidence of greater pre-treatment vascular function, including greater blood flow or permeability measured using DCE-MRI derived biomarkers and tumour mapping data of high molecular weight fluorescent dyes injected intravenously. Modulation of nitric oxide (NO) levels decreased the density of perfused blood vessels and sensitized tumours to the anti-vascular effects of TPZ. Additional vascular phenotype features such as relatively poor vascular maturity were also found to correlate with greater tumour sensitivity to TPZ-mediated vascular dysfunction. Greater toxicity of TPZ to hypoxic cells in vitro led to the hypothesis that blood oxygenation may have an impact on tumour sensitivity to TPZ-mediated vascular dysfunction in vivo. Tumours from mice that had moderate bleeding-induced anemia or that were breathing lowered (7-10%) oxygen were sensitized to the anti-vascular effects of TPZ. In vitro assays showed that human microvascular tube structures are sensitive to damage by TPZ at clinically relevant concentrations and oxygen levels.  TPZ may be reduced by cellular nitric oxide synthase (NOS), and NOS is competitively inhibited by TPZ to result in decreased amounts of NO. Enhanced anti-cancer effects have previously been observed for other vascular disrupting agents (VDAs) and hypoxic cytotoxins when combined with NOS inhibitors. These findings led to studies presented in this thesis, which show that TPZ-mediated vascular dysfunction is enhanced by co-administration with a NOS inhibitor, and this combined activity can lead to reduced cancer growth.   Results from this thesis suggest that features of the tumour microenvironment, including tumour vascular phenotype, blood oxygenation and tumour hypoxia impact tumour sensitivity to TPZ-mediated vascular dysfunction. In addition, inhibiting NOS in combination with TPZ is a therapeutically advantageous strategy that merits further investigation.  TABLE OF CONTENTS  CHAPTER 1 ............................................................................................................................................... 1 IN VIVO CHAPTER 2 ............................................................................................................................................. 31  iv 2.2.6 Scoring vascular dysfunction response.............................................................................. 37 2.2.7      DCE-MRI analysis ............................................................................................................. 40 2.2.8      Radial analysis.................................................................................................................... 40 2.2.9      Statistics .............................................................................................................................. 41 2.3 RESULTS ................................................................................................................................... 42 2.3.1 Histological analysis identifies tumours with an increase in necrosis or a decrease in  perfusion in response to TPZ ............................................................................................. 42 2.3.2 BrdUrd data not included ................................................................................................... 45 2.3.3 DCE-MRI analysis shows decreases in IAUC and Ktrans for TPZ-treated tumours with central  vascular dysfunction .......................................................................................................... 46 2.3.4 Microregional heterogeneity of response to TPZ ............................................................. 48 2.4 DISCUSSION............................................................................................................................. 51 2.4.1 DCE-MRI parameters IAUC and Ktrans ............................................................................. 51 2.4.2 Analysis of DCE-MRI and histological images................................................................ 52 2.4.3 Pre-treatment perfusion as indicator of non-response ...................................................... 53 2.5 CONCLUSIONS........................................................................................................................ 55 2.6 REFERENCES........................................................................................................................... 56  CHAPTER 3 ............................................................................................................................................. 58 3.1 INTRODUCTION...................................................................................................................... 59 3.1.1 Tirapazamine (TPZ) as a hypoxic cytotoxin with anti-vascular activity ........................ 59 3.1.2 Tumour vasculature as a target for TPZ ............................................................................ 59 3.2 METHODS................................................................................................................................. 61 3.2.1 Mice and tumours ............................................................................................................... 61 3.2.2 Treatments ........................................................................................................................... 61 3.2.3 Immunohistochemistry (IHC) ............................................................................................ 62 3.2.4 Image acquisition and analysis .......................................................................................... 62 3.2.5 Vascular Dysfunction Score (VDS)................................................................................... 63 3.2.6 Endothelial Tube Assay...................................................................................................... 63 3.2.7 Statistics............................................................................................................................... 64 3.3 RESULTS ................................................................................................................................... 64 3.3.1 HT29 colorectal xenograft tumours are resistant to vascular dysfunction effects  of TPZ.................................................................................................................................. 64 3.3.2 Microenvironmental differences between HCT116 and HT29 colorectal xenografts ... 67 3.3.3 Differences in vascular permeability between HCT116 and HT29 colorectal  xenografts ............................................................................................................................ 69 3.3.4 Differences in vascular phenotype between HCT116 and HT29 colorectal  xenografts ............................................................................................................................ 69 3.3.5 10% O2 breathing or induction of anemia enhances the anti-vascular effects of  TPZ in HCT116 colorectal xenografts .............................................................................. 73 3.3.6 7% O2 breathing enhances the anti-vascular effects of TPZ in HT29 colorectal  xenografts ............................................................................................................................ 76 3.3.7 Tirapazamine mediates damage to endothelial tube structures in vitro in a time,  concentration and oxygen-dependent manner................................................................... 76 3.4 DISCUSSION............................................................................................................................. 78 3.4.1 Tumour vasculature and TPZ sensitivity........................................................................... 78 3.4.2 Tumour hypoxia and TPZ sensitivity ................................................................................ 79 3.5 CONCLUSIONS........................................................................................................................ 81 3.6 REFERENCES ........................................................................................................................................82 CHAPTER 4 ............................................................................................................................................. 85 in vivo CHAPTER 5 ........................................................................................................................................... 114  APPENDIX ............................................................................................................................................. 129  vi LIST OF TABLES  CHAPTER 3 Table 3.1 TPZ-mediated vascular dysfunction in HT29 and HCT116  colorectal xenografts.................................................................................................... 67 Table 3.2 Impact of modulation of blood oxygenation on TPZ-mediated vascular  dysfunction in HCT116 and HT29 colorectal xenografts. ............................................. 74   CHAPTER 4 Table 4.1 Impact of modulation of NO on TPZ-mediated vascular dysfunction in  HCT116 colorectal xenografts ..................................................................................... 93 Table 4.2 Impact of NOS inhibition on TPZ-mediated vascular dysfunction in SCCVII  and HT29 tumours. .................................................................................................... 100    vii LIST OF FIGURES  CHAPTER 1  Figure 1.1 Hypoxic cells in the tumour microenvironment. ................................................... 3 Figure 1.2 Model for TPZ metabolism. .................................................................................4 Figure 1.3 Vascular dysfunction visualized using tumour mapping. ......................................8 Figure 1.4 NO synthesized by nitric oxide synthase (NOS). ................................................ 16  CHAPTER 2  Figure 2.1 Experiment overview. ........................................................................................ 34 Figure 2.2 Fiducial markers................................................................................................. 35 Figure 2.3 Rationale for combined Vascular Dysfunction Score (VDS)............................... 39 Figure 2.4 Compartmental modeling of DCE-MRI contrast agent ....................................... 41 Figure 2.5 DCE-MRI and histological parameter maps of HCT116 tumours. ...................... 43 Figure 2.6 DCE-MRI and histological parameter maps of HCT116 tumours. ...................... 44 Figure 2.7 Tirapazamine-mediated changes in vascular function measured using  histological analysis. .................................................................................................... 45 Figure 2.8 Tirapazamine-mediated changes in vascular function measured using  DCE-MRI parameters IAUC and Ktrans......................................................................... 47 Figure 2.9 Tumour mapping microregional heterogeneity in response to TPZ. .................... 48 Figure 2.10 MRI microregional heterogeneity in response to tirapazamine.......................... 49 Figure 2.11 Correlation of MRI and histology features........................................................ 50  CHAPTER 3  Figure 3.1 HT29 and HCT116 colorectal xenograft sensitivity to anti-vascular effects  of TPZ. ........................................................................................................................ 65 Figure 3.2 HT29 tumour maps. ........................................................................................... 66 Figure 3.3 Tumour microenvironment of HCT116 and HT29 colorectal xenografts. ........... 68 Figure 3.4 Vascular function in HCT116 and HT29 colorectal xenografts. .......................... 70 Figure 3.5 CD31 and  SMA staining in HCT116 and HT29 colorectal xenografts.............. 71 Figure 3.6 CD31 and Collagen Type IV (CIV) staining in HCT116 and HT29 colorectal xenografts. ................................................................................................................... 72 Figure 3.7 Modulation of tumour hypoxia in HCT116 and HT29 colorectal xenografts. ...... 74 Figure 3.8 Hypoxia enhances anti-vascular effects of TPZ in HCT116 and HT29  xenografts. ................................................................................................................... 75 Figure 3.9 TPZ mediates damage to endothelial tube structures in a concentration, time  and oxygen dependent manner. .................................................................................... 77   viii CHAPTER 4  Figure 4.1 NOS inhibition enhances anti-vascular effects of TPZ in HCT116 tumours; quantitative data........................................................................................................... 93 Figure 4.2 NOS inhibition enhances anti-vascular effects of TPZ in HCT116 tumours; tumour maps. ............................................................................................................... 94 Figure 4.3 Excess NO enhances anti-vascular effects of TPZ in HCT116 tumours. ............. 96 Figure 4.4 Decrease in S-phase cells when TPZ combined with NO modulation. ................ 97 Figure 4.5 Inhibition of NOS by L-NNA enhances the growth inhibition effects of TPZ in  HCT116 tumours. ........................................................................................................ 98 Figure 4.6 NOS inhibition enhances the anti-vascular effects of TPZ in SCCVII tumours. 101 Figure 4.7 NOS inhibition does not enhance anti-vascular effects of TPZ in HT29  tumours...................................................................................................................... 102 Figure 4.8 Modulation of NO effects on hypoxia in HCT116 and HT29 tumours. ............. 103 Figure 4.9 NOS expression variation between HCT116 and HT29 tumours. ...................... 105      ix LIST OF ABBREVIATIONS   SMA   alpha smooth muscle actin  BrdUrd  5-bromo-2-deoxy-uridine  BTZ   benzotriazine  Ca2+   calcium  CA-4-P  combretastatin-A-4-phosphate  CFU   colony forming units  CIV   collagen type IV  CO2   carbon dioxide  DAB   diamino benzidine  DCE-MRI  dynamic contrast enhanced magnetic resonance imaging  DMSO   di-methyl sulfoxide  DMXAA  5,6-dimethylxanthenone-4-acetic acid  DNA   deoxy-ribonucleic acid  ECM   extra-cellular matrix  EF5  2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-    pentafluoropropyl)acetamide  EGF   epidermal growth factor  eNOS   endothelial nitric oxide synthase  ETC   electron transport chain  FBS   foetal bovine serum  FITC   fluorescein isothiocyanate  FLASH  fast low-angle shot sequence    xii SOD   superoxide dismutase  TPZ   tirapazamine  TR   repetition time  uNOS   universal nitric oxide synthase  VDA   vascular disrupting agent  VDS   vascular dysfunction score  VF   viable fraction  VEGF   vascular endothelial growth factor  VTA   vascular targeting agent     xiii ACKNOWLEDGEMENTS  I would like to thank my supervisor Dr. Andrew Minchinton for providing me with the opportunity to pursue graduate school and for creating a fantastic laboratory and working environment. It is truly a pleasure coming to the lab every day, and I am so grateful.  Thank you to Dr. Alastair Kyle for building so many useful things and for always having insightful, cheerful advice. Thank you to Dr. Lynsey Huxham for guidance, making tirapazamine and for doing great work with tirapazamine prior to this project. Alastair and Lynsey have been wonderful mentors and I greatly appreciate their impact on my research and experience as a graduate student. Many thanks to the undergraduate co-op students who have participated on this project or who have been involved in the Minchinton laboratory: Kirsten Bartels, Erin Flanagan, Stephen Methot, Firas Moosvi, Andrew Balbirnie, Melissa Woodward, Erin Gabriel and Jordan Cran.  Thanks also to Kirstin Lindquist, a fellow graduate student at aimLab. Thank you to Dr. Stefan Reinsberg and Lauren Bains who provided expertise and contributed much of the work with DCE-MRI. Thank you to my supervisory committee Drs. Marcel Bally, Aly Karsan and Sylvia Ng for helpful comments, advice and encouragement. Special thanks to Dr. Ng for her thorough reading of this thesis. Thank you also to Dr. Don Yapp for useful imaging advice and collaborations. Thank you to Wil Cottingham for always being so helpful and supportive, to the department of Medical Biophysics for general support and encouragement, and to the many other BC Cancer graduate students who have become friends. Finally, I owe a huge thank you to my wonderful family and great friends who have been so important in my motivation and ability to do this work. You have been so patient, supportive and above all enthusiastic - I am so very grateful! Special mention to my loving parents Brian & Janette, my wonderful husband Mark, my awesome siblings Melissa & Simon, and to Courtney, Hugh, et al.  Financial support for my graduate work was provided by a senior graduate studentship from the Michael Smith Foundation for Health Research. I have also been the grateful recipient of travel awards from the American Association of Cancer Research, the Tumour Microenvironment Society, the Interdisciplinary Oncology Program, and the European School of Hematology.   xiv  CO-AUTHORSHIP STATEMENT  CHAPTER 2 A version of this chapter has been published: Bains* LJ, Baker* JHE, Kyle AH, Minchinon AI and Reinsberg SA. (2009). International Journal of Radiation Oncology Biology Physics 74 (3) pp. 957-965.  (*Bains and Baker are co-first authors of this publication). Author contributions: Baker JHE designed and carried out experiments and wrote 50% of the manuscript including designing and creating figures. Bains LJ designed and carried out experiments and wrote 20% of the manuscript; parts of this work contributed to the MSc work of LBains. Kyle AH designed and built the robotic microscope and image analysis software. Minchinton AI is the principal investigator and supervisor of JBaker and AKyle, and contributed in experiment design, data interpretation and editing of the manuscript. Reinsberg SA is the principal investigator and supervisor of LBains, and contributed by designing and carrying out experiments and writing 30% of the manuscript.  CHAPTER 3 A version of this chapter has been submitted for publication and is under review. Author list: Baker JHE, Kyle AH, Flanagan E, Methot S, Bartels K and Minchinton AI.  Author contributions: Baker JHE designed and carried out all the experiments, performed all data analysis and interpretation of results, and authored 100% of the manuscript including design and creation of figures. Kyle AH designed and built the robotic microscope and image analysis software. Flanagan E, Methot S and Bartels K were all undergraduate student assistants who helped carry out mouse handling and histological processing for experiments. Minchinton AI is the principal investigator and supervisor of all the authors in this manuscript.    xv CHAPTER 4 A version of this chapter will be submitted for publication. Author list: Baker JHE, Kyle AH, Balbirnie A, Gabriel E, Cran J and Minchinton AI.  Author contributions: Baker JHE designed and carried out all the experiments in this paper, performed all data analysis and interpretation of results, and authored 100% of the manuscript including design and creation of figures. Kyle AH designed and built the robotic microscope and image analysis software. Balbirnie A, Gabriel E and Cran J were all undergraduate assistants who helped carry out mouse handling and histological processing for experiments. Minchinton AI is the principal investigator and supervisor of all other authors in this manuscript.    Please see Appendix A for a complete list of publications by Jennifer H.E. Baker.  1 TABLE OFCFF  NSHPR1.2H3RSF            2 1.1 Introduction  The treatment of cancer relies on three principal strategies: surgery, radiotherapy and chemotherapy. Localized tumours may often be effectively treated with one or a combination of these strategies, but eradication of occult metastases must be achieved by systemically administered chemotherapies. Most anti-cancer chemotherapies work by killing cells that are rapidly dividing, and are described as cytotoxic anti-proliferative drugs. Other, normal tissues of the body such as the bone marrow, hair follicles and epithelial cells lining the gastrointestinal tract also have rapidly proliferating cells, therefore cytotoxic anti- proliferatives can lead to dose-limiting side effects (Tannock 1998). Considerable drug development efforts have therefore shifted from broad-spectrum, poorly selective cytotoxic anti-proliferative drugs towards more cancer-specific, targeted treatments that should have an improved therapeutic index.  While targeted treatment strategies continue to be developed for cancer-specific proteins at the molecular and cellular level, there are also tissue level or pathophysiological, cancer-specific features that may be targeted for therapy (Brown and Giaccia 1998). In addition to cancer cells, tumours may contain blood, immune cells, fibroblasts and cells that make up the tumour vasculature, all of which are collectively described as the tumour stroma. A solid tumour therefore contains a mix of cancer cells, stromal cells, non-cellular material such as ECM, and soluble signaling molecules. These individual components may be abnormal relative to healthy tissues, and their organization is highly heterogeneous. Microregional variations in tumour components result in subpopulations of cells that may be described based on their location within the tumour microenvironment. For example, tumour microenvironments may have a varying availability of nutrients, oxygen and signaling molecules, as well as a distinct local pH, interstitial fluid pressure or reducing environments (Vaupel et al. 1989; Brown and Giaccia 1998; Dewhirst 2003). Figure 1.1 shows tissue from a human colorectal carcinoma xenograft where the inter-vessel distances cause cells distal to the vasculature to become hypoxic (shown in green).  The purpose of work presented in this thesis is to investigate the mechanism for activity of an experimental targeted anti-cancer agent, tirapazamine (TPZ), which has greater toxicity in conditions of low oxygen and is therefore described as a hypoxic cytotoxin. TPZ is   3 in clinical trials for the treatment of various cancers including head and neck, lung and cervical cancers alone or in combination with radiotherapy or chemotherapy (Marcu and Olver 2006; Reddy and Williamson 2009). While the principal mechanism for tissue-level targeted toxicity of TPZ to tumours is thought to be its selective activation in hypoxic cells, TPZ has also been observed to damage tumour blood vessels in vivo (Huxham et al. 2006; Huxham et al. 2008). The mechanism for TPZ-mediated vascular dysfunction in solid tumours is the subject of this thesis, where features of the tumour microenvironment such as hypoxia and blood vessels are investigated for their impact on tumour sensitivity to the anti- vascular effects of TPZ.    hypoxia (pimonidazole) proliferation (BrdUrd) tissue background (hematoxylin) vasculature (CD31) perfusion (carbocyanine) necrosis   Figure 1.1 Hypoxic cells in the tumour microenvironment.  In this HCT116 human colorectal xenograft model the tumour cells are arranged around blood vessels in a corded structure. Staining illustrates vasculature (blue) labeled with perfusion marker (cyan) surrounded by tumour cells stained lightly with hematoxylin (grey), some of which are labeled for incorporated BrdUrd as an indicator of S-phase (black). At distances far from vasculature cells label positively for pimonidazole (green), a bioreductive marker of hypoxia, before becoming necrotic at distances greater than the diffusion limit of oxygen. Scale bar 150 µm.   LIS T OFGUFGFRECHAP1F1E2H2HAP3FF in vitro et al. et al. N N N O O NH2 - + + - N N N O NH2 - + N N N OH O NH2 - + N N N O NH - + O2 TPZ TPZ BTZ SR 4317 1 electron reductase H+ protonation - H2O O2 - OH+ Figure 1.2 Model for TPZ metabolism.  et al. et al.  5  The in vivo efficacy of TPZ has typically been assessed using growth delay or clonogenic survival assays. Growth delay studies measure tumour volumes over time and assess if a treatment reduces the volume or rate of growth of tumours. Clonogenic survival assays involve harvesting tumours following treatment and dissociating the tissues to single cell suspensions that are then plated in culture. The number of detectable colonies after several days of growth in culture is a reflection of the number of cells recovered from the tumours that are capable of proliferating through several generations. Data from clonogenic survival studies may be described as the number of colonies per gram of tissue or per number of viable cells at time of plating. While both of these assays are capable of demonstrating anti-cancer activity, neither specifically confirm the microregional location or microenvironmental status of cells that were killed. Therefore, neither the growth delay nor the clonogenic assay are able to specifically confirm selective cytotoxicity of TPZ directly to hypoxic tumour cells as the mechanism for its anti-cancer activity in vivo. Growth delay and clonogenic assays have been used to demonstrate that TPZ has anti-cancer effects in vivo. TPZ significantly enhances cell kill of both radiotherapy and cisplatin (Zeman et al. 1986; Brown and Lemmon 1990; Dorie and Brown 1993). Radiotherapy is effective at killing oxygenated cells, while hypoxic cells are more resistant by a factor of three and often survive to repopulate irradiated tumours (Gray, Conger et al. 1953). The synergistic activity of TPZ in combination with radiotherapy, where more cells are killed than with radiation alone, is therefore often attributed to complementary killing of radioresistant hypoxic cells by TPZ and is suggested as evidence of selective toxicity of TPZ to hypoxic cells in vivo (Zeman et al. 1986; Zeman et al. 1988; Brown and Lemmon 1990; Brown and Lemmon 1991). However, as described above, clonogenic survival studies are unable to provide specific detail regarding the microregional or microenvironmental location of cells that were killed. It is therefore an assumption that the cells killed by TPZ when combined with radiotherapy were killed by selective hypoxic cytotoxic activity, and an indirect effect such as vascular dysfunction cannot be excluded. Indeed vascular damage would not be anticipated in response to TPZ, as the tumour endothelium would typically be regarded as the most well oxygenated population of cells in a tumour due to their proximity to the blood supply, and specific investigations regarding an anti-vascular effect for hypoxic cytotoxins are not typically conducted.    6 Additional support for the hypothesis that TPZ kills hypoxic cells in vivo has been determined in experiments of low oxygen breathing in mice. Greater cell kill is achieved by TPZ when more cells within tumours are made hypoxic through low oxygen breathing (Sun and Brown 1989; Minchinton and Brown 1992a; Minchinton and Brown 1992b; Minchinton et al. 2002). Again, these data suggest that TPZ is more effective in hypoxic tumours, but were not able to specifically confirm selective toxicity to hypoxic tumour cells in lieu of an alternate mechanism such as indirect tumour cell kill via vascular damage.  While the above mentioned studies provide evidence of anti-cancer activity of TPZ in vivo, data from three dimensional tissue model systems such as spheroids (Durand and Olive 1992) and multi-layered cell cultures (Hicks et al. 1998; Kyle and Minchinton 1999) suggest that TPZ does not penetrate well through tissue and may be consumed at intermediate oxygen levels. In animal models, TPZ is delivered to the tumour by blood vessels and must distribute through tumour tissue to reach target hypoxic cells located far from the blood supply (Figure 1.1). The poor tissue penetration profile of TPZ suggests that it will have difficulty reaching hypoxic cells located far from vasculature at concentrations sufficient to be effective. 1.3 TPZ has anti-vascular activity in vivo.  Studies showing limited penetration of TPZ through multi-layered tissue culture models in the Minchinton laboratory at the BC Cancer Agency Research Centre led to the question of how TPZ has anti-cancer activity in vivo if it does not efficiently penetrate through tissue to reach hypoxic cells (Kyle and Minchinton 1999). The activity of TPZ in vivo was assessed using tumour mapping analysis whereby tumours grown subcutaneously in mice were excised for cryosectioning and staining at several time points following treatment. Layered immunohistochemical staining permits identification of cytotoxically damaged tumour cells in the context of the tumour microenvironment, including the presence of hypoxia. A hypothesis of the tumour mapping studies was that TPZ would cause damage to hypoxic cells located far from vasculature. However, results showed a surprising effect whereby TPZ-treated HCT116 tumours had evidence of central vascular damage (Huxham et al. 2006). TPZ mediated vascular dysfunction was characterized as an irreversible loss of perfusion in the centre of tumours at 12-24 hours; by 48-72 hours large areas of necrosis develop. Blood vessels at the tumour periphery appear to be unaffected by TPZ, leaving a   7 viable rim of tumour tissue. TPZ-mediated vascular dysfunction effects are dose-dependent, with approximately 65 % of tumours responding at the near maximum tolerated dose of 60 mg/kg (0.34 mmol/kg) and the remaining 35 % of treated tumours showing no signs of central vascular dysfunction. Vascular dysfunction detected by tumour mapping studies is illustrated in Figure 1.3. Hypoxia is labeled using immunohistochemical (IHC) staining for bound pimonidazole, a bioreductive marker that is administered by intraperitoneal (i.p.) injection 2 hours prior to tumour excision. Pimonidazole is reduced by reductases in areas of low oxygen and binds cellular proteins. The nucleotide analogue BrdUrd is co-administered with pimonidazole 2 hours prior to excision and labels S-phase cells. Vasculature is stained using antibodies for CD31 and perfused blood vessels are labeled with a fluorescent dye (DiOC7(3); carbocyanine) injected intravenously 5 minutes prior to recovering the tumour. An HCT116 tumour with central vascular dysfunction (Figure 1.3B) shows unperfused vessels in the central regions, with tumour margins staining positive for vasculature, perfusion, proliferation and hypoxia. Because BrdUrd and pimonidazole are exogenously administered markers, negative staining in unperfused regions is likely to be an experimental artifact due to vascular dysfunction and consequent poor delivery of the agents. Mapping the entire tumour section using a custom-built robotic microscope enabled analysis of effects on whole tumours, whereas most IHC studies are typically limited to fewer markers and smaller microscope fields. Composite tumour map images of BrdUrd, pimonidazole, carbocyanine and CD31 create an easily identifiable visual representation of central vascular dysfunction in tumours, as shown in Figure 1.3.  Additional tumour models are also sensitive to the anti-vascular effects of TPZ. HCT116 tumours are characterized by relatively low vascular density, with hypoxia located at distances far from vasculature (Figure 1.1). SiHa cervical carcinoma xenografts have a similar pattern of vasculature and hypoxia, and TPZ causes a similar vascular dysfunction effect in these tumours (Huxham et al. 2008). However, murine SCCVII squamous carcinoma tumours have a relatively high density of blood vessels, with some areas of vascularized and perfused tumour tissue that is hypoxic, and these tumours are also sensitive to the anti-vascular effects of TPZ (Huxham et al. 2008).    8  A) Control HCT116 B) Central vascular dysfunction in HCT116  Figure 1.3 Vascular dysfunction visualized using tumour mapping. An untreated control HCT116 colorectal xenograft tumour (A) is compared to a tumour exhibiting vascular dysfunction 24 h following anti-vascular treatment (B). Vascular dysfunction is easily observed by loss of perfusion in central areas indicated by the large number of unperfused CD31 vasculature (red), with a hallmark viable rim of perfused vessels (blue). The peripheral rim also stains positively for exogenously administered markers of hypoxia (green) and S-phase (black) that are only delivered to the perfused tumour tissues. Scale bar 150 µm.  1.4 Tumour hypoxia While the patterns of vasculature and hypoxia are different between HCT116 and SCCVII tumour models, both are similarly sensitive to the anti-vascular effects of TPZ. Poorly oxygenated cells are a common characteristic of solid tumours, indicating that the demand for oxygen in tumours often exceeds supply. Cellular oxygen consumption rates and the availability of blood, which is dependent on the distribution of blood vessels and the blood oxygen carrying capacity, dictate tissue oxygenation levels. Tumours consume oxygen at an intermediate rate that is greater than tissues with low metabolic rates, but lower than tissues with high metabolic rates (Vaupel et al. 1989). Tumour vascular abnormalities (further discussed below in section 1.5) cause poor blood flow and large inter-vascular   9 distances and result in heterogeneous oxygenation of tumour tissue. Normal tissues have considerable variation in tissue oxygenation and hypoxia is defined as oxygen levels that are less than normal. Normal arterial pO2 is typically at 80-100 mmHg, and the median pO2 for tissues ranges from 24 mmHg in the brain to 66 mmHg in the spleen (Vaupel et al. 1989). The median pO2 for tumours is < 20 mmHg and the ratio of normal tissue pO2 to tumour tissue pO2 is almost always >1, with considerable inter-tumour heterogeneity (Vaupel et al. 1989; Brown and Wilson 2004). Causes for tumour hypoxia include severe structural and functional microvessel abnormalities that can cause slow, poor or even static blood flow (perfusion-limited hypoxia; acute hypoxia). Large inter-vascular distances result in tumour cells located far from their nearest blood vessel that are consequently hypoxic (diffusion-limited hypoxia; chronic hypoxia). Cancer patients can be tumour-associated or therapy-associated anemic, which results in reduced oxygen carrying capacity of the blood (anemic hypoxia). Other more minor contributors to tumour hypoxia include reduced oxygen carrying capacity of hemoglobin in smokers due to carbon monoxide binding to hemoglobin (toxic hypoxia). Lower blood oxygen content can also arise from pulmonary diseases or high altitude. Tumour blood vessel supply from the hepatic portal vein system in the liver may also have lower blood oxygen content that can result in tissue hypoxia (hypoxemic hypoxia). For a review of definitions regarding hypoxia see Höckel and Vaupel (2001). 1.5 Hypoxic vasculature in tumours  As described above (section 1.2), TPZ is a prodrug that is reduced to a cytotoxic agent in conditions of hypoxia. It therefore appears counter-intuitive that it can cause damage to vascular endothelial cells, likely the most well oxygenated cell population in solid cancers. Evidence for intravascular hypoxia in tumours comes from cryospectrophotometric studies measuring hemoglobin saturation (HbO2), which directly measure the oxygenation of blood. HbO2 has been measured as low as 0 % and as high as 100 % in tumours (Fenton et al. 1988; Fenton et al. 2001; Måseide and Rofstad 2001). In window chamber models, microelectrode measurements of pO2 found that 25 % of functioning, patent blood vessels had pO2 levels that were indistinguishable from 0 (Dewhirst et al. 1992). Observation of hypoxic perivascular tumour cells despite patency of the blood vessels is indirect evidence of et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al.  11 greater tumour and vascular hypoxia in the central regions of tumours. Data presented in Chapter 2 uses dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) to investigate the impact of vascular function and therefore indirectly, hypoxia, on the sensitivity of tumours to the anti-vascular effects of TPZ. Further studies in Chapter 3 look more directly at the effects of hypoxia, investigating the effects of modulation of blood oxygenation and hypoxia on tumour response to TPZ.  1.6 Abnormal tumour blood vessels In addition to vascular hypoxia, tumour vasculature has many abnormal phenotypic and functional features that could contribute to tumour sensitivity to TPZ. A characteristic of TPZ-mediated vascular dysfunction is the heterogeneity in response. Some tumour blood vessels are sensitive, responding with loss of perfusion, while others are resistant and maintain blood flow despite high doses of TPZ. As described in section 1.3, inter-tumour heterogeneity in response to TPZ is observed in all sensitive tumour models. Approximately 65 % of TPZ-treated tumours respond with central vascular dysfunction and the remaining 35 % are non-responders. This rate of response is similar in each of the HCT116, SiHa and SCCVII tumour models previously examined (Huxham et al. 2006; Huxham et al. 2008). Intra-tumour heterogeneity is reflected by the persistence of a viable rim of undamaged blood vessels even in responding tumours. The inter- and intra-tumour heterogeneity of tumour blood vessel sensitivity to the anti-vascular effects of TPZ is seen despite tumour endothelial cells all deriving from genetically identical host animals, and despite the identical nature of the immortalized tumour models.  Because the cells that make up tumour blood vessels originate from normal tissues, they are hypothesized to be more genetically stable than tumour cells, and should consequently be less likely than tumour cells to develop resistance to chemotherapies (Folkman 2006). Despite the relative genetic normalcy of blood vessel cells, the function and phenotype of tumour blood vessels is affected by the tumour microenvironment, and the resulting tumour vasculature is far from normal. A balance of pro- and anti-angiogenic signalling molecules arising from tumour cells typically initiates growth of blood vessels from neighbouring host tissues in a process known as angiogenesis (Folkman 1971; Ferrara and Kerbel 2005; Folkman 2006). The exact pattern for blood vessel growth varies between   12 tumour types and is highly heterogeneous. Though there are exceptions, like normal tissue vessels, tumour blood vessels are typically comprised of endothelial cells in a tubular structure and the outside of the tubes may be associated with mural support cells such as pericytes (Morikawa et al. 2002) and layers of basement membrane (Baluk et al. 2003). All of these components, in addition to the structure, size, shape, branching pattern, organization and hierarchy can be highly abnormal in tumours (Baluk et al. 2005; Tozer et al. 2005). Severe structural aberrations lead to functional abnormalities such as slow or even static blood flow, as well as high permeability and leakiness due to poor endothelial junctional complexes and large fenestrations (Hashizume et al. 2000; Dewhirst 2003). Tumours also have poor or absent lymphatic systems for drainage of the large volumes of fluid that leak from the sluggish, leaky vessels, and this deficiency can result in a relatively high interstitial fluid pressure (Leu et al. 2000). A central hypothesis presented in this thesis is that cancer-specific features of the tumour vasculature and microenvironment confer sensitivity or resistance to the anti-vascular effects of TPZ in tumours. Tumour vascular function is investigated as a predictor of response to TPZ using DCE-MRI in Chapter 2 of this thesis. Additional functional and phenotype characteristics of blood vessels in tumours that are sensitive or resistant to TPZ- mediated vascular dysfunction are compared and investigated in Chapters 3 and 4.   13 1.7 Vascular disrupting agents (VDAs) As described above, the tumour microenvironment is highly heterogeneous due in large part to the abnormal organization and function of tumour blood vessels. Abnormal vascular features of solid tumours represent tissue-level, targetable features for anti-cancer therapeutics (Baluk et al. 2005). Vascular disrupting agents (VDAs), previously described as vascular damaging agents or vascular targeting agents (VTAs), are a relatively new class of anti-cancer compounds that are distinct to angiogenesis inhibitors. Instead of inhibiting the growth of new vessels, VDAs target and destroy existing tumour vasculature (Thorpe 2004; Tozer et al. 2005). The overall pattern of effect by VDAs is similar to that seen for TPZ. VDA-mediated loss of perfusion is followed by death of dependent tumour cells and the effect is primarily limited to the central regions of tumours with a persisting rim of viable, undamaged tissue at the tumour margins. A flavone acetic acid-derived compound, DMXAA (5,6-Dimethylxanthenone-4-acetic acid, ASA404), is a VDA that interferes with the actin cytoskeleton principally through induction of cytokines (McKeage et al. 2009). Combretastatin phosphate (CA-4-P, ZybrestatTM) is a small molecule VDA that disrupts the cellular cytoskeleton via binding to tubulin (Pettit et al. 1989). A mechanism for VDA- mediated vascular dysfunction proposed by Tozer et al. (2001)describes the effects as similar to those seen in acute inflammatory reactions. The initial action of small molecule VDAs that disrupt the cytoskeleton results in the rounding up of vascular endothelial cells. Inter- endothelial cell gaps result in a substantial, rapid change in vascular permeability. Leaking plasma proteins, edema, leukocyte interactions and infiltration, and an increase in interstitial fluid pressure, all result in blood vessel narrowing and a decrease in blood flow rates.  The similar pattern of vascular damage in tumours by both VDAs and TPZ suggests that there may be some overlap in their mechanisms of action. However, in vitro studies examining the effects of TPZ on endothelial cell cytoskeletal structures showed no effect in hypoxic or normoxic conditions (Huxham et al. 2008). The specific mechanism for TPZ- mediated damage to the tumour vasculature is therefore likely to be distinct to that of the small molecule VDAs. However, the pattern of sensitivity of central tumour blood vessels and insensitivity of the peripheral rim of tumour vessels suggests there may be some overlap in the mechanism for sensitivity of tumour blood vessels to both TPZ and VDAs.    14 A precise mechanism for the specificity of small molecule VDA-mediated damage in tumour vasculature (vs. normal tissue vasculature) remains somewhat unclear, but an overview of hypotheses and supporting data is presented in a review by Tozer et al. (2008). In addition to cell-based mechanisms for selectivity in tumour endothelium, including tubulin modifications and defective inter-cellular junctions, a series of tissue level mechanisms are suggested. These tissue level mechanisms include micro-regional instabilities in blood flow that may sensitize some vessels such that further pharmacologically-induced reductions are catastrophic (Tozer et al. 2001). In addition, high vascular permeability and the resulting high interstitial fluid pressure in tumours may sensitize vessels to collapse following any further disruption in permeability (Beauregard et al. 2001; Tozer et al. 2001; Beauregard et al. 2002) or change in fluid pressure (Boucher et al. 1990). Vascular maturity may also play an important role, as inefficient or inadequate association of mural support cells such as pericytes with tumour blood vessels may reduce vascular stability and the ability of tumour vessels to withstand or recover from vascular damage (Tozer et al. 2008).  The proliferation rate of tumour vascular endothelial cells can be up to 20x greater than in the vasculature elsewhere in the body (Denekamp 1982; Denekamp and Hobson 1982). Proliferating endothelial cells may therefore represent a targetable feature of tumours for anti-cancer drugs, and small molecule VDAs such as CA-4-P are toxic to proliferating endothelial cells in vitro (Kanthou et al. 2004). However, inhibiting proliferation and inducing apoptosis in endothelial cells is likely too slow a process to be responsible for the anti-vascular effects observed within minutes of CA-4-P administration in vivo; disruption of interphase cytoskeletal structures is likely the more important mechanism of action of CA-4- P (Kanthou et al. 2004; Tozer et al. 2005).  Anti-microtubule vinca alkaloid anti-cancer drugs also show vascular targeting effects, though only at near maximum tolerated doses (Hill et al. 1993; Chaplin et al. 1996). Hyperthermia also causes widespread vascular damage in some tumours and the tumour selectivity of these effects have been attributed to the same vascular abnormalities described above (Song 1984). Other strategies for targeting tumour vasculature include targeted antibodies or peptides that use tumour vasculature-specific ligands for delivering toxic payloads to tumours (Thorpe 2004). Photodynamic therapy (PDT) also causes tumour vascular damage, achieving tumour-specificity due to the intravascular nature of the injected   15 prodrug compounds that become activated and cytotoxic only upon exposure to light, which can be limited to tumour regions of interest (Dolmans et al. 2003).  Three main strategies for targeting the tumour vasculature are represented by the examples described above: a) using tumour vascular specific expressions of antigen to which ligands or agents are targeted; b) selective activation of cytotoxic agents when they are located within the tumour region of interest; and c) small molecule VDAs that exploit tumour microenvironmental differences to effect vascular damage. All three strategies work on a similar principal of achieving tumour cell kill through deprivation of oxygen and nutrients via damage to the tumour blood supply. Targeting the tumour vasculature with anti-cancer drugs is a particularly useful strategy due to the accessibility of the target cells to systemically delivered agents. In addition, a relatively small number of affected cells results in the death of a high number of cancer cells that were otherwise dependent on the damaged blood vessels.  As has been described, the hallmark effects of TPZ mediated vascular dysfunction, including loss of central perfusion and persistence of the viable rim, are very similar to those seen with other vascular targeting strategies. While the mechanism of action for TPZ- mediated damage to the tumour vascular endothelium is likely to be distinct to any of the other described anti-vascular strategies, the similarity in pattern of damage suggests some overlap in vessel sensitivity. All or any of the three main targeting strategies described may have relevance to the anti-vascular effects of TPZ. As mentioned in Section 1.6, studies in Chapters 2 and 3 of this thesis investigate aspects of vascular function and phenotype in tumour models that have differential sensitivity to the anti-vascular effects of TPZ. These data may also have relevance to the efficacy and mechanisms for tumour vascular selectivity of the other vascular targeting strategies described here. 1.8 Combining NOS inhibition with hypoxic cytotoxins and vascular disrupting agents (VDAs) While the pattern of TPZ-mediated vascular dysfunction is similar to that of other anti- vascular strategies, the mechanism for damage requires further investigation. A mechanism for TPZ mediated damage in or proximal to endothelial cells may be related to the location of   16 its bioactivation and redox cycling. Many cellular reductases can reduce TPZ, including cytosolic enzymes such as NADPH:cytochrome c reductase (P450R) (Walton et al. 1992; Saunders et al. 2000) and intranuclear enzymes (Evans et al. 1998). TPZ is also reduced by and competitively inhibits nitric oxide synthase (NOS), which has a reductase domain with homology to that of P450R; in conditions of hypoxia TPZ reduction by NOS can result in DNA damage (Garner et al. 1999; Chinje et al. 2003). Neuronal NOS (nNOS, NOSI) and endothelial NOS (eNOS, NOSIII) isoforms of NOS are constitutively expressed and are dependent on calcium/calmodulin for regulation. A third isoform is inducible NOS (iNOS, NOSII). Several studies have identified a correlation between iNOS activity in tumours and poor prognosis, greater metastatic activity or higher tumour grade, including in gastric (Song et al. 2002), head and neck (Gallo et al. 1998) and colorectal carcinoma (Lagares-Garcia et al. 2001; Cianchi et al. 2003). Non-isoform specific NOS expression has also been linked to higher grade tumours in gynecological (Thomsen et al. 1994), breast (Thomsen et al. 1995) and central nervous system tumour studies (Cobbs et al. 1995). NOS has been proposed as a potential therapeutic target for cancer treatment (Fukumura et al. 2006; Fitzpatrick et al. 2008).   Figure 1.4 NO synthesized by nitric oxide synthase (NOS).  Competitive inhibition of NOS by TPZ results in reduced production of nitric oxide (NO), an important messenger molecule synthesized by NOS in a two step oxidation of L- arginine to L-citrulline in the presence of oxygen (Figure 1.4). NO is highly reactive and has a wide array of actions and therefore NOS inhibition has an equally wide array of impacts; for a comprehensive review of the role of NO in tumour biology, see Fukumura et al. (2006). Chronic stimulation by NO from endothelial cells has an important role in recruiting pericytes and is involved in vessel remodeling and maturation (Kashiwagi et al. 2005; Yu et al. 2005). NO can also have a more acute impact on tumour vascular function. Studies   17 pharmacologically inhibiting NOS have resulted in reduced tumour blood flow and decreased tumour vessel diameters (Andrade et al. 1992; Fukumura et al. 1997; Tozer et al. 1997; Ng et al. 2007). Inter- and intra-tumour heterogeneity in vessel response to NOS inhibition has been suggested to be the result of microregional variations in both regional NO production and the inconsistent presence of smooth muscle cells adjacent to tumour vasculature for vasodilatory control (Fukumura et al. 1997).  Reduced tumour blood flow as a consequence of NOS inhibition can cause an increase in tumour hypoxia, and as a result NOS inhibition has been proposed as a complementary treatment for hypoxic cytotoxins in vivo (Wood et al. 1993; Wood et al. 1994). The suggested mechanism for the combination strategy is that an increase in hypoxia due to NOS inhibition will result in greater reduction and bioactivation of the hypoxic cytotoxins, which would ultimately enhance tumour cell kill. Given the observation that TPZ is both a hypoxic cytotoxin and a competitive inhibitor of NOS, Garner et al. (1999) have proposed that TPZ may potentiate itself via this mechanism. The anti-cancer activity of the hypoxic cytotoxin RB6145 was increased when combined with a NOS inhibitor (Wood et al. 1994; Butler et al. 1997). No added systemic toxicity was seen with this combination, suggesting that inhibition of NOS in combination with a bioreductive hypoxic cytotoxin may be therapeutically advantageous. The efficacy of combining RB6145 with NOS inhibition was demonstrated in these studies using growth delay and clonogenic survival assays. As described in section 1.2, using growth delay and clonogenic survival assays it is difficult to conclude with certainty that the mechanism for potentiation of RB6145 by NOS inhibition is an increase in hypoxia and subsequently greater bioactivation of RB6145. The authors noted a time-dependence for cell kill in their clonogenic survival studies. When tumour excision was delayed to 24 hours post treatment with RB6145 or RB6145 in combination with NOS inhibition, a greater cell kill was observed than if tumours were excised at 12 hours (Butler et al. 1997). The timing of tumour excision post treatment is of critical importance if an anti-vascular effect were to have occurred. If tumours are excised and cultured after sustaining loss of perfusion, but prior to onset of tissue death due to the loss of blood vessel supply, then most tumour cells will be rescued by the culture conditions and a high survival rate would be observed. However, if tumours are excised at longer times post treatment, then more cells will have died due to lack   18 of oxygen and nutrient delivery and a dramatically lower number of colonies per gram of tumour tissue would result. The delay in peak cell death measured using clonogenic survival observed in the studies combining RB6145 with NOS inhibition is consistent with an anti- vascular effect. Therefore, a central vascular dysfunction effect, though not assessed in these studies combining the hypoxic cytotoxin RB6145 with a NOS inhibitor, may explain both the time-dependent clonogenic survival results as well as the unexpectedly high rates of cell kill noted by the authors (Butler et al. 1997).  NOS inhibition has also been successfully combined with VDAs in pre-clinical studies. Small molecule VDAs cause losses in perfusion through a mechanism similar to that of an acute inflammatory reaction  (as described in section 1.7), whereas NOS inhibition in tumours primarily results in decreased blood vessel diameters. Therefore, through a mechanism distinct from that seen for VDAs themselves, NOS inhibition enhances the loss of perfusion in tumours treated with a combretastatin (Parkins et al. 2000; Tozer et al. 2009). A protective effect of NO is also suggested by studies overexpressing NOS in tumour models; NOS overexpression attenuates the anti-vascular response of tumours treated with ZD6126, an analogue of colchicine with microtubule destabilizing effects similar to those seen with the combretastatins (Cullis et al. 2006).  There is evidence of TPZ having activity both as a hypoxic cytotoxin and as VDA. Enhancement of both hypoxic cytotoxins and VDAs by inhibition of NOS has therefore led to the work presented in Chapter 4 of this thesis where the anti-cancer activity of TPZ in combination with modulation of NO levels is investigated in multiple tumour models.     19 1.9 Thesis Overview Tirapazamine (TPZ) is a hypoxic cytotoxin that has anti-vascular activity in tumour models grown in mice. The effect of TPZ on tumour vasculature is incomplete, typically leaving a viable rim of undamaged vessels at the tumour periphery. Anti-vascular activity of TPZ has been observed in several tumour types, and in all of these types the effect occurs in 65 % of treated tumours, leaving 35 % unaffected despite identical animal and tumour origins. A mechanism for TPZ damage to select tumour blood vessels remains unclear and represents an important research question.  The purpose of the research presented in this thesis is to explore the mechanism of action of the anti-vascular activity of TPZ, and to investigate features of the tumour microenvironment that confer sensitivity to these effects. Experiments in Chapter 2 explore the utility of dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) to observe the effects of TPZ on vascular function in vivo. DCE-MRI derived biomarkers of vascular function are also examined as potential predictors for tumour response to TPZ-mediated vascular dysfunction. Chapter 3 specifically investigates tumour hypoxia as a relevant microenvironmental feature for tumour vascular sensitivity to TPZ. Tumour mapping analyses are used to examine the in vivo effects of TPZ in combination with decreasing blood vessel oxygenation. IHC staining is also used to compare features of the tumour microenvironment and blood vessels in tumours characterized as either sensitive or resistant to TPZ mediated vascular damage.  The effect of changes in tumour vascular function on tumour sensitivity to TPZ- mediated vascular damage are further examined in tumour mapping studies in Chapter 4. TPZ in combination with NOS inhibition or excess NO supply is investigated in order to determine whether NO has an effect on the anti-vascular activity of TPZ, and whether this activity can be translated to reduced tumour growth.       20 1.9.1 Research hypothesis and objectives The overall hypothesis of this research is that features of the tumour microenvironment confer sensitivity to the anti-vascular activity of tirapazamine.    The main objectives of the work presented in this thesis were to: 1. Use dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) to observe TPZ- mediated vascular dysfunction in vivo.  2. Identify tumour microenvironmental features that confer tumour sensitivity for TPZ.  3. Investigate the impact of modulation of NO in combination with TPZ in tumours.     17 1.10 Chapter summaries  1.10.1 Chapter 2 summary - Detecting vascular-targeting effects of the hypoxic cytotoxin tirapazamine in tumour xenografts using magnetic resonance imaging Purpose: pharmcol mgralimynimcgl cb ita ngimNrnlOoSnh nOimrmiv cb imhnensnumga dfpw( tnra Aaag Smumia9 ic i2cN9muaglmcgnS tmlicScymOnS iaOtgm;oal itni nha mgrnlmra ng9 linimOF fta eohecla cb ita lio9mal ehalagia9 mg ktneiah 1 ml ic 9aiahumga 2taitah rnlOoSnhNinhyaimgy abbaOil cb fpw Ong Aa 9aiaOia9 in vivo olmgy unygaimO halcgngOa munymgy dTz0(F  Methods) Tz munyal cb loAOoingacolSv mueSngia9 .kf77I -agcyhnbil mg umOa 2aha nO;omha9 ni j falSn Aabcha ng9 1R tcohl nbiah mgihneahmicganS mgqaOimcgl cb fpwF xongiminimra 9vgnumO OcgihnliNagtngOa9 Tz0 d,kWNTz0( ngnSvlal 2aha eahbchua9 ic arnSonia Otngyal mg ioucoh rnlOoSnh bogOimcg olmgy i2c Amcunh3ahl) ita rcSoua ihnglbah Ocglingi d4ihngl( ng9 ita mgmimnS nhan og9ah ita OcgOagihnimcgNimua Oohra d0GPk(F ZcraS mueSngia9 bm9oOmnS unh3ahl 2aha ola9 ic cAinmg tmytSv haehc9oOmASa munymgy eSngal bch Scgymio9mgnS Tz munymgy ng9 laSaOimcg cb Ochhalecg9mgy tmlicScymOnS laOimcg F xongiminimra muuogctmlicOtaumOnS uneemgy cb ioucoh rnlOoSniohaB eahbolmcg ng9 gaOhclml agnASa9 OchhaSnimra ngnSvlml Aai2aag itala ng9 Tz munyalF G 6nlOoSnh ,vlbogOimcg 5Ocha d6,5( 2nl 9araScea9 ng9 haechia9 ic agnASa cAqaOimraB ;ongiminimra nllalluagi cb ngimNrnlOoSnh abbaOilF Results) kcgragimcgnS tmlicScymOnS ngnSvlml ltc2a9 Sc2ah bhnOimcgl cb eahbola9 rallaSl ch Sc2ah ehcechimcgl cb rmnASa imlloa mg ita OagihnS haymcgl cb bmra cb amyti fpwNihania9 ioucohlB 2mit ithaa ihania9 ioucohl ltc2mgy gc rnlOoSnh 9vlbogOimcg halecglaF Tz0 9nin habSaOia9 itml haloSi ng9 n lihm3mgy 9aOhanla mg Acit 4ihngl ng9 0GPk rnSoal 2nl laag 2mit ita halecglmra ioucohlF Hcll cb eahbolmcg 2nl bcog9 ic Aa umOhchaymcgnSSv rnhmnASaB 2mit yhaniah Otngyal cAlahra9 mg ita OagihnS nhanl cb ioucohl mg Acit ,kWNTz0 ng9 tmlicScymOnS enhnuaiah ngnSvlalF zaihcleaOimra arnSonimcg cb ehaihaniuagi Tz0 enhnuaiahl haranSa9 itni itcla ioucohl itni 9m9 gci halecg9 ic ita rnlO oSnhNinhyaimgy abbaOil cb imhnensnumga tn9 lmygmbmOngiSv tmytah ehaihaniuagi 4ihngl ng9 0GPk rnSoalF Conclusions) Tz0N9ahmra9 enhnuaiah unel ltc2a9 ycc9 nyhaauagi 2mit tmlicScymOnS ioucoh unelF Tz0 2nl bcog9 ic Aa ng abb aOimra iccS bch gcgNmgrnlmraSv ucgmichmgy imhnensnumgaNua9mnia9 OagihnS rnlOoSnh 9vlbogOimcg ng9 bch eha9mOimgy ioucoh laglmimrmivF   22  CHAPTER: The hypoxic cytotoxin tirapazamine (TPZ) mediates central vascular dysfunction in several pre-clinical tumour models. MRI data from Chapter 2 suggested vascular function as a predictor for tumour sensitivity to the anti-vascular effects of TPZ. The purpose of studies presented in Chapter 3 is to evaluate features of the tumour microenvironment that may confer sensitivity to TPZ, including hypoxia and vascular phenotype.  R4.T8E : Quantitative immunohistochemical (IHC) based tumour mapping was used to evaluate the response to TPZ in a variety of conditions in HCT116 and HT29 colorectal xenograft tumours grown subcutaneously in mice. Markers of vasculature, perfusion, permeability, hypoxia, S-phase, basal lamina and smooth muscle cells were stained and imaged to evaluate vascular dysfunction and characteristics of tumours that are sensitive or resistant to the anti-vascular effects of TPZ. The effect of hypoxia on tumour sensitivity to TPZ was investigated via induction of moderate anemia and exposure of mice to 7-10% oxygen during treatment. Dependence of microvessel sensitivity on oxygenation and TPZ concentration was evaluated using human microvascular endothelial cells (HMECs) grown as tubular structures on Matrigel coated plates.  5REH14ENDHT29 colorectal xenografts were resistant to anti-vascular effects of TPZ, compared to sensitive HCT116 colorectal xenografts. Tumour mapping analyses of the two models revealed quantifiable differences, where HT29 tumours exhibited greater microvessel density and permeability, with thicker layers of CIV and  SMA proximal to vasculature. Decreasing tumour oxygen levels via anemia or low oxygen breathing enhanced TPZ mediated vascular dysfunction, even in otherwise resistant HT29 tumours. HMEC tube structures show evidence of damage at 8h following exposure to clinically relevant TPZ concentrations at 2% oxygen.  ITX21HE9TXENDThese data establish a direct link between the anti-vascular effects of TPZ and hypoxia. Additional tumour specific features of the microenvironment such as vascular architecture and phenotype may also play a role in tumour sensitivity to TPZ mediated vascular dysfunction.   23  CHAPTER: A mechanism for the ability of tirapazamine (TPZ) to mediate central vascular dysfunction in solid tumours remains unclear. Data from Chapters 2 and 3 established hypoxia as a sensitizer to the effect and have implicated vascular function and phenotype as additional, potential predictors for sensitivity. The purpose of studies in Chapter 4 is to evaluate the activity of nitric oxide (NO) on tumour sensitivity to the anti-vascular effects of TPZ.   R4.T8E : HCT116 and HT29 colorectal xenografts and SCCVII murine tumours were grown subcutaneously in mice. TPZ was administered alone or in combination with the non- specific NOS inhibitor L-NNA or with NO donor spermine NONOate. Tumour mapping analyses of vasculature, perfusion, hypoxia, S-phase and NOS expression were used to evaluate TPZ mediated vascular dysfunction. Enhancement of anti-cancer activity of TPZ by NOS inhibition was evaluated by monitoring the growth of HCT116 tumours. 5REH14ENDAdministration of NOS inhibitor L-NNA in combination with TPZ at lower than maximum tolerated doses resulted in reduced HCT116 tumour growth. NOS inhibition in combination with TPZ results in enhanced anti-vascular activity in both HCT116 and SCCVII tumours where the magnitude and frequency of vascular dysfunction were increased. No vascular dysfunction effects were seen in HT29 xenografts treated with the combination of TPZ and L-NNA. Administration of excess NO in combination with TPZ did not protect against TPZ mediated anti-vascular damage, but instead enhanced the vascular dysfunction response.  ITX21HE9TXENDCombining NOS inhibition with TPZ enhances the anti-vascular activity of TPZ and this translates into reduced growth rates in HCT116 tumours. Excess NO also enhances tumour sensitivity to TPZ mediated vascular dysfunction. These data suggest that NO or tumour vascular phenotype may play an important role in tumour sensitivity to TPZ, and that combining TPZ with NOS inhibition warrants further investigation as a therapeutic strategy.    24 1.11 Impact of the Research Data from this thesis provides further evidence of the anti-vascular activity of TPZ in solid tumours and provides direct links between the tumour microenvironment and sensitivity to these effects. Data from Chapters 2 and 3 demonstrate that pre-treatment vascular function or hypoxia can predict the anti-vascular effects of TPZ. These data are evidence of TPZ affecting tumour vasculature through its activity as a hypoxic cytotoxin for therapeutic benefit. Intentionally targeting hypoxic tumour vasculature would represent a novel therapeutic strategy in cancer treatment. This result has relevance to drug development strategies in the field of bioreductive hypoxic cytotoxins, where specific and comprehensive in vivo screening of candidate hypoxic cytotoxins for both anti-vascular and hypoxic cytotoxicity should be undertaken. In addition, pursuit of hypoxic cytotoxic agents that could be limited to and bioactivated within the intra-vascular compartment may represent a useful anti-cancer agent development strategy.  No studies to date have investigated whether TPZ or other hypoxic cytotoxins mediate vascular damage in clinical patients. Experiments in Chapter 2 demonstrate DCE-MRI as a practical technique for observing TPZ mediated vascular dysfunction. DCE-MRI is a method that is translatable to the clinic, and trials examining the activity of TPZ or other hypoxic cytotoxins may consider DCE-MRI for assessment of vascular function in response to treatment. If vascular dysfunction occurs in response to TPZ in the clinic, DCE-MRI would also have useful application in assessing patient response to therapy and may provide applicable biomarkers for predicting response to TPZ. In addition to DCE-MRI biomarkers of vascular function as potential predictors for response, data from Chapter 3 suggest that pre-treatment vascular function, tumour hypoxia or blood oxygenation can have an impact on the frequency and magnitude of the anti- vascular effects of TPZ. These results suggest that in addition to screening candidate patients for tumour hypoxia, it may be useful to assess peripheral blood oxygen levels as potential predictors for sensitivity to TPZ. Other potentially useful predictors of sensitivity to the anti- vascular effects of TPZ suggested in Chapters 3 and 4 include vascular phenotype characteristics such as NOS expression or the maturity of tumour blood vessels.  Experiments in Chapter 4 demonstrate that combining TPZ with a NOS inhibitor enhances the anti-cancer activity of TPZ through greater vascular dysfunction effects. These   25 data provide supporting evidence to the hypothesis that combining bioreductive hypoxic cytotoxins with NOS inhibition is an effective therapeutic strategy, and demonstrate that the mechanism for this effect may involve damage to the tumour vasculature.  Overall, this work emphasizes the importance of specifically examining the effects of anti-cancer drugs on whole tumours in vivo, as the tumour microenvironment is complex and can have a significant impact on drug efficacy and tumour response to treatment.     26 1.12 REFERENCES Anderson, R. F., S. S. Shinde, et al. (2005). 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Int J Radiat Oncol Biol Phys 12(7): 1239-42. Zeman, E. M., V. K. Hirst, et al. (1988). "Enhancement of radiation-induced tumor cell killing by the hypoxic cell toxin SR 4233." Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology 12(3): 209-18.       31   CHAPTER 2 Detecting vascular-targeting effects of the hypoxic cytotoxin tirapazamine in tumour xenografts using magnetic resonance imaging.1                                                  1 A version of this chapter has been published.   Bains* LJ, Baker* JHE, Kyle AH, Minchinton AI, Reinsberg SA. (2009). International Journal of Radiation Oncology Biology Physics 74 (3): 957-965.  *Bains and Baker are co-first authors of this publication.    32 1.Innntroduci oturn 2.1.1 Tirapazamine (TPZ) as a hypoxic cytotoxin with anti-vascular activity The presence of hypoxic cells within tumours reduces the effectiveness of radiotherapy and is a known mechanism for tumour radioresistance (Brown and Wilson 2004). One strategy to combat this resistance is to utilize bioreductive cytotoxins, prodrugs that are reduced under low oxygen conditions to cytotoxic metabolites. Selective toxicity to hypoxic cells complements the preferential toxicity of radiotherapy to oxygenated tumour cells. Tirapazamine (TPZ; 3-amino-1,2,4-benzotriazine-1,4-di-N-oxide; SR 259075; formerly SR 4233) is the most advanced hypoxic cytotoxin under clinical development (for a review of tirapazamine, see Marcu and Olver (2006)). Tirapazamine (TPZ) is reduced by cellular reductases to an oxidizing species that under conditions of low oxygen is further metabolized to oxidizing radicals that cause DNA damage (Anderson et al. 2005). In vivo experiments have shown anti-cancer activity of TPZ in multiple tumour models and in combination with radiotherapy (Zeman et al. 1988; Dorie et al. 1994). However, the anti-cancer activity of TPZ is something of a paradox, as compelling evidence using spheroids and multi-cellular cultures suggests that TPZ is unable to penetrate efficiently through tumour tissue to reach chronically hypoxic cells located far from vasculature (Durand and Olive 1992; Durand and Olive 1997; Hicks et al. 1998; Kyle et al. 1999). This poor penetration of TPZ has been attributed to bioreductive activation at intermediate oxygen tensions more proximal to vasculature (Kyle et al. 1999; Cárdenas- Navia et al. 2007).  We have previously reported an observation of the unexpected ability of TPZ to mediate central vascular dysfunction in murine syngeneic and human xenograft tumours grown in mice (Huxham et al. 2006; Huxham et al. 2008). This anti-vascular activity of TPZ could have a significant impact on its application if found to be clinically relevant.  2.1.2 Dynamic Contrast Enhanced Magnetic Resonance Imaging (DCE-MRI) Monitoring drug effects on tumour blood volume, flow and permeability non- invasively can be accomplished using magnetic resonance imaging (MRI). It has been used extensively in both pre-clinical and clinical situations investigating the effects of vascular   33 disrupting agents (VDAs) such as combretastatin (Galbraith et al. 2003), ZD6126 (Robinson et al. 2003) and DMXAA (McPhail et al. 2006). MRI has also been employed in clinical investigations of vascular disrupting agents (for a review see (O'Connor et al. 2007)). This chapter presents a method for evaluating the effects of TPZ non-invasively using two dynamic contrast-enhanced MRI (DCE-MRI)-derived biomarkers for assessment of anti- vascular agents: initial area under the curve (IAUC) and Ktrans.  2.1.3 Current study outline The experiments presented in this chapter follow the schematic presented inF igure F 1.4. HCT116 tumour-bearing mice received baseline MR scans, were subsequently treated with TPZ, and received a follow-up scan at 24 hours. Cryosections of tumour slices corresponding to MR imaging regions of interest were obtained using fiducial markers. Tumours were evaluated for TPZ-mediated vascular dysfunction using staining data for vasculature, perfusion and necrosis. These histological tumour mapping analyses were used to confirm and validate observations seen in DCE-MRI data and parameter maps. DCE-MRI has the potential to be highly beneficial for determining whether TPZ-mediated loss of perfusion occurs in patients, and whether this is a contributing mechanism for the clinical anti-cancer activity of TPZ.     34  Subcutaneous tumours grown to ~150 mm3 BrdUrd admin i.p. 7T MRI 24 h 60 mg/kg TPZ 2 h DiOC7(3) admin i.v.; tissue harvest MRI  # 1 MRI  # 2 A) image for DiOC7(3)  (perfusion) stain for CD31  (vasculature) & BrdUrd (s-phase) stain for hematoxylin (necrosis) tumour mapping analysis B) Tumour cryosections stained & imaged robotic microscope with slide loader  Figure 2.1 Experiment overview.  A) HCT116 tumours grown subcutaneously in the dorsal region of mice were imaged using a 7T MR scanner. A second MR scan was obtained 24 h following 60 mg/kg treatment with TPZ. S-phase marker BrdUrd was administered 2 h prior to an intravenous injection of perfusion marker DiOC7(3) administered 5min prior to euthanasia. Tumour tissues were excised with fiducial markers attached. (B) A robotic microscope with automated slide loader obtains tiled images of whole tumour cryosections at a resolution of 1.5 µm/pixel. Fluorescent images of DiOC7(3) were obtained prior to staining and imaging for CD31 (vasculature), BrdUrd (S-phase cells) and hematoxylin. Overlayed images were cropped and analyzed; corresponding MR images and histological images were compared.   2: 2.2   METHODS 2.2.1 Mice, fiducial markers and tumours Theypoxe ictn ranz m ( PP Znn)ds Zncalcva Pu f- .M as ZnRn IRnz cv -CR cvdfcfCfc-vrg rvcirg 3rtcgcf,Q nHbnRcinvfd cv flcd tlrbfnR ZnRn rbbR-wnz I, fln 1vcwnRdcf, -3 6Rcfcdl o-gCiIcr rvz cfd 9vcirg orRn o-iicffnn k9bbnvzctnd 6s os eS7 0czCtcrg irR)nRd kFigure 2.2S ZnRn t-vdfRCtfnz -3 b-g,nfl,gnvn fCIcva kcvvnR zcrinfnR %7:m iiS rvz 3cggnz Zcfl brRr33cv ZrH rvz drgcvn tRnrfcva rv DE(wcdcIgn cvfnR3rtn flrf Zrd rgd- znfntfrIgn cv tR,-dntfc-vd7 DrR)nR fCInd ZnRn cibgrvfnz dCItCfrvn-Cdg, kd7t7S cv fln drtRrg Rnac-v -3 ictns tRrvc-trCzrg -Rcnvfrfc-vs fZ- zr,d bRc-R f- d7t7 cibgrvfrfc-v -3 m H P%q VoAPPq tnggd7 Vcdf-g-actrg dntfc-vd rvz DEx dgctnd ZnRn fr)nv dCtl flrf fln 3czCtcrg irR)nR gr, bnRbnvzctCgrR f- fln ciracva bgrvn -3 nrtl dgctnQ fln 3cHnz vrfCRn -3 fln irR)nR nvdCRnz dirgg rvaCgrR zc33nRnvtnd InfZnnv dntfc-vcva bgrvnd 3-R lcdf-g-actrg rvz dnRcrg DE dgctnd7 Aln inrv Zncalf 8 dfrvzrRz nRR-R kd7n7S -3 nHtcdnz fCi-CRd Zrd :P. 8 qm ia7      Figure 2.2 Fiducial markers. A) 2 cm sections of tubing were filled with paraffin (green) and saline (purple), and implanted subcutaneously in the sacral region of mice. 2 days later, 8 x 106 HCT116 cells were implanted in the same region. Tumours typically grew around tubes and could be identified in histological sections, as shown in a magnified CD31 image (B) (scale bar 150 µm) and MR images, white arrow (C) (scale bar 500 µm). The locations of fiducial markers are also indicated in Figure 2.5 and Figure 2.6 by blue stars.    36 2.2.2 Treatments Mice were anaesthetized with isoflurane, received an MRI scan and were administered either TPZ or vehicle one hour after removal from anaesthesia. TPZ (synthesized by Dr. L.A. Huxham (Huxham et al. 2006)) was administered by intraperitoneal (i.p.) injection at 60 mg/kg (0.34 mmol/kg) using a 1.25 mg/ml solution in saline. A second MRI scan was obtained 24 hrs after treatment; mice awakened promptly following anaesthesia, however some TPZ-treated mice required longer periods to recover to their pre- anaesthesia fitness. No correlation was found between length of recovery time and response to TPZ treatment. Mice that recovered within 35 min post-anesthesia received 500 mg/kg i.p. 5-bromo-2-deoxyuridine (BrdUrd, Sigma Chemical, Oakville, ON) as a back-up perfusion marker (Janssen et al. 2005), with tumour excision one hour post-BrdUrd administration. All mice received a 35 µl intravenous dose of 0.6 mg/ml carbocyanine, DiOC7(3), (Molecular Probes, Eugene, OR) in 75 % DMSO 5 minutes before euthanasia. Excised tumours were embedded and frozen with fiducial markers at -20ºC.  2.2.3 DCE-MRI MR images were acquired using a 7T horizontal-bore Bruker BioSpec 65/35 with a transmit/receive solenoid coil. A fast low-angle shot sequence (FLASH) (6 slices, 1mm thickness, 0.5 mm gap, 312.5 µm in-plane resolution) with a repetition time TR = 226 ms was performed, followed by a FLASH with a TR of 113 ms; both used a flip angle of 75º. Contrast agent (35 mM Gd-DTPA, 10 µl/g) was injected via tail vein catheter at 18 µl/s using a power injector. Ten pre-contrast and 124 post-contrast image stacks were acquired at time resolution of 14.5 s.  2.2.4 Immunhistochemical staining and image aquisition Serial step 10 µm cryosections were cut at 0.5 mm intervals, imaged for carbocyanine fluorescence and fixed in acetone-methanol for 10 min. Vasculature and proliferation were stained using antibodies for CD31 and incorporated BrdUrd respectively (Kyle et al. 2003) and slides were counterstained with hematoxylin. Whole tumour sections were imaged as previously reported (Kyle et al. 2007) using a system which allowed for tiling of adjacent   37 microscope fields of view such that images of entire tumour cryosections were captured at a resolution of 1.5 µm per pixel.  ChChap ter 2prsrumy-yp Similar image analysis methods have been previously reported with additional detail provided below (Huxham et al. 2006; Huxham et al. 2008). Using both fiducial and anatomical landmarks, six cryosections per tumour were identified that matched the MRI slices. Using NIH-Image and user-supplied algorithms, digital images were superimposed. Tumours were manually cropped to tumour tissue boundaries with staining artefacts removed; necrosis was subsequently cropped from hematoxylin images. The viable fraction (VF) is derived from the proportions of necrotic and whole tissue. Values reported in this thesis reflect the proportion of necrosis as (1-VF). Positive fluorescence for CD31 and carbocyanine images was obtained by applying a threshold (>5 SDs above background) with neighbouring positive pixels grouped as `objects'. The perfused vessel fraction (PF) was calculated as the proportion of CD31 positive objects that were also at minimum 20 % overlapped with positive carbocyanine pixels on the overlaid image.  Each histological section was manually aligned with the corresponding MR slice using custom IDL software (Interactive Data Language, 2007). For MRI/histological image correlative analyses, carbocyanine images with a pixel size of 1.5 µm x 1.5 µm were thresholded and the number of carbocyanine positive objects in an area corresponding to an MRI pixel (0.3125 mm x 0.3125 mm) was calculated. A 0.3 mm x 0.3 mm resolution `carbocyanine map' depicting pixels at intensities representative of the number of perfused vessels in the histological sections was produced. Data for individual tumours are displayed as means for six analyzed slices per tumour ± s.e. ChChDp cing-s pvryilurgpfmyolsix-nspg2yznsy2p A combined Vascular Dysfunction Score (VDS) was used to determine an objective, quantifiable assessment of vascular dysfunction response to TPZ; see Figure 2.3 for a rationale for the VDS.      38  The VDS reported here incorporates both the viable fraction (VF) and perfused fraction (PF) within a tumour section: VDS = 1-(VF x PFc) The PFc was calculated as the proportion of perfused vessels for a tumour (PF; see above) divided by the maximum proportion of perfused vessels as observed in controls. For individual tumours a VDS will be 0 if all the tumour tissue is viable (VF = 1) and vessels are all perfused (PF = 1). It will be higher for cases of increased necrosis or reduced perfusion. This objective score permits quantitative evaluation of tumours that may have loss of perfusion measurable using either CD31 staining combined with a perfusion marker, or necrosis in tumours that have no remaining CD31. Tumours with a VDS of  > 2 SDs higher than the mean control score (VDSmin) are considered strong-responders to TPZ and remaining tumours are described as non-responders.     39 A) Control HCT116 B) Vascular dysfunction in HCT116: lots of unperfused CD31 C) Vascular dysfunction in HCT116: unperfused tissue becoming necrotic  Figure 2.3 Rationale for combined Vascular Dysfunction Score (VDS) Images of a representative HCT116 control tumour (A) show typical corded architecture with perfusion marker DiOC7(3) labeling of CD31 stained vasculature (blue) surrounded by BrdUrd labeled S-phase cells (black) and regions of hypoxia labeled with pimonidazole (green). Vascular dysfunction may manifest as in tumour (B) where unperfused CD31 stained vascular objects (red) remain in central areas of otherwise viable tissue identified using hematoxylin counterstaining (grey). An alternate manifestation of vascular dysfunction may appear as in tumour (C) where CD31 staining is disappearing and tissue is becoming necrotic. Quantification of the proportion of perfused vessels (PF) would indicate vascular dysfunction for tumour (B). However, fewer CD31 stained objects may remain in a tumour (C), and describing both the loss of perfusion and the proportion of viable tissue (VF) more robustly represents vascular dysfunction.    vi  2.2.7 DCE-MRI analysis Fgure4 .1N ueOrSnhb Snu gubt re onaoganrb ocns-b ls fT guls- ul-sna lsrbsulrP l4n-bu Ohe4 ZhbHlsCborles 6fm ; qqd4u9 nst 3nubalsb 6fm ; TT24u9 uonsu re ohbnrb oesobsrhnrles rl4b oghpbuE mbanxlplrP eO rcb oesrhnur n-bsr Snu tbrbh4lsbt ls n Zcnsre4 urgtP guls- tlagrbt oesrhnur n-bsr un4ZabuE .slrlna Dhbnu gstbh rcb Fesobsrhnrles fl4b Fghpbu 6.DwFu9 Sbhb onaoganrbt 3P lsrb-hnrls- rcb oesobsrhnrles rl4b oghpbu Ohe4 rcb 3b-lssls- eO bscnsob4bsr re rcb Oeghrc Ohn4b 6di u Zeur oesrhnur nhhlpna9E D rSeH oe4Znhr4bsr 6Figure 1.49 7brP 4etba Snu gubt re Olst rcb rhnsuObh oesurnsr 67rhnsu9 tbuohl3ls- 4epb4bsr eO oesrhnur n-bsr Ohe4 3aeet Zanu4n 65Z9 lsre rcb bxrhnpnuoganh bxrhnobaaganh rluugb uZnob 65b9 6feOru nst 7bh4etb TIIT9E fcb hbanrlesuclZ 3brSbbs rcb oesobsrhnrlesu eO oesrhnur n-bsr ls rcb rluugb nst rcb 3aeet Zanu4n nhb tbuohl3bt 3P rcb bygnrles L  Scbhb Fr6r9 nst FZ6r9 nhb rcb rl4bHtbZbstbsr oesobsrhnrles eO oesrhnur n-bsr ls rcb rluugb nst pnuoganh Zanu4nL hbuZborlpbaPE  2.2.8 Radial analysis Fgure4 hntlna lsrbsulrP nsnaPulu .1N ueOrSnhb Snu ohbnrbt re ygnsrlOP bnoc Znhn4brbh nu n Ogsorles eO tlurnsob Ohe4 rcb rg4egh bt-bE Arnhrls- Ohe4 rcb egrultb bt-bL 2TqE8 µ4 hl4u Sbhb ohbnrbt nst Znhn4brbh 4bnsu Zeeabt Oeh naa ulx ualobu ls n rg4eghE Du n 4bnughb eO rg4egh hntlna cbrbhe-bsblrPL n cbrbhe-bsblrP lstbx 6V.9 Snu onaoganrbt nu rcb Zbhobsr tlOObhbsob 3brSbbs rSe Sbl-crbt 4bnsu0 rcb egrbh TM2 nst rcb lssbh qM2 eO rcb rg4eghE   41 2.2.9 Statistics All statistical analyses were performed using the GraphPad Prism software (Version 4.0c for Macintosh, 2005). Non-parametric tests were used in all instances: typically a Kruskal-Wallis analysis of variance rank test, followed by Dunn's post tests between groups. P values < 0.05(*) and < 0.01(**) are reported. A Spearman's rank correlation was used for IAUC mean values and a Pearson's correlation coefficient with 95 % confidence intervals is reported for regression analyses for IAUC radial profiles vs. carbocyanine fluorescence.  V Gd-DTPA contrast agent Red blood cells Endothelium Tumour cells VV e ip  Figure 2.3 Ratonrlteflnc tamecifg ab dRVsDyS (aflrn)l ngefl. The two compartment Kety model of contrast agent delivery comprises the vascular plasma volume (Vp), and the extravascular extracellular tissue volume (Ve). Gd-DTPA does not penetrate the cellular membrane and is therefore excluded from the intracellular compartment (Vi). A single intravenous bolus administration of contrast agent (Gd-DTPA, green stars) is delivered to the tumour via the vasculature (blue). The amount of contrast agent in either of the compartments depends on the surface area and permeability of the endothelium, the concentration difference between the two compartments, as well as blood flow, heart rate and blood pressure. 2.3 RESULTS Figure 2.5 Figure 2.6 2.3.1 Histological analysis identifies tumours with an increase in necrosis or a decrease in perfusion in response to TPZ Figure 2.7A Figure 2.7B Figure 2.7C et al.    43   Figure 2.5 DCE-MRI and histological parameter data maps and images of HCT116 tumours.  Parameter data maps reflect the calculated IAUC values for individual voxels for baseline day 0 (row 1) and day 1 (row 2), and the fraction of perfused vessels measured using histology displayed at MR resolution (row 3). Histological tumour maps are shown as composite images of carbocyanine fluorescence (red) overlaid on the hematoxylin counter-stained tissue background (grey), with BrdUrd staining in black (row 4). Boxed areas indicate magnified regions (row 5); N = necrotic; blue stars = fiducial markers. Row 4 scale bar is 1250 µm, row 5 is 125 µm.   44   Figure 2.6 DCE-MRI and histological parameter data maps and images of HCT116 tumours  Parameter data maps reflect the calculated IAUC values for individual voxels for baseline day 0 (row 1) and day 1 (row 2), and the fraction of perfused vessels measured using histology displayed at MR resolution (row 3). Histological tumour maps are shown as composite images of carbocyanine fluorescence (red) overlaid on the hematoxylin counter-stained tissue background (grey), with BrdUrd staining in black (row 4). Boxed areas indicate magnified regions (row 5); N = necrotic; blue stars = fiducial markers. Row 4 scale bar is 1250 µm, row 5 is 125 µm.     45  Figure 1.4 NirOsOyOnite hneziOdez bcOtgeo it xOobuaOr (utbdiSt neOourez uoitg ciodSaSgibOa OtOa)oio.  Amounts of necrosis (1-VF) (A) and perfusion (PF) (B) in sections of individual control and tirapazamine treated tumours. Vascular Dysfunction Scores (VDS) are reported (C), incorporating both necrosis and perfusion values. Tumours 7-9 are classed as non-responders; tumours 10-14 are strong-responders.  2.3.2 BrdUrd data not included BrdUrd is a nucleotide analogue that is useful as a marker of cells in S-phase. BrdUrd is delivered to tumour tissue via tumour blood vessels, which means that it can function as a perfusion marker (Janssen et al. 2005). However, in cases of vascular dysfunction, it is difficult to differentiate between poor delivery of the marker and a decrease in proliferation in areas of low BrdUrd incorporation. In our previous studies, low BrdUrd staining reflected a decrease in proliferation in areas around perfused blood vessels following TPZ administration, with large areas of unperfused tissue also showing a complete lack of BrdUrd incorporation, likely due to a combination of decreased proliferation and poor delivery of the marker (Huxham et al. 2006; Huxham et al. 2008). In data presented in the current chapter, BrdUrd was administered at a lower dose to some mice to assist in determination of large areas of perfusion loss using the tumour mapping images, however carbocyanine was successfully injected intravenously in every animal, providing a more reliable marker for perfusion assessment. Our MR imaging protocol is unable to evaluate proliferation for useful comparisons between tumour mapping and imaging, making proliferation data of relatively low value for this study and it is consequently not included.      46 2.3.3 DCE-MRI analysis shows decreases in IAUC and Ktrans for TPZ-treated tumours with central vascular dysfunction Control tumours show no significant changes in their pre- and 24h post-treatment IAUC values, while TPZ-treated tumours show a combined average reduction. Whole tumour mean IAUCs for all tumours are shown in Figure 2.8A, with retrospective classification of TPZ-treated tumours into non- and strong- responders based on histological data (VDS). Strong-responding tumours have significantly lower post-treatment IAUC values relative to controls  whereas non-responders are similar to controls (Figure 2.8C). Non-responders have greater aim eimtelm.e IAUC values relative to strong- responders. Pre-treatment IAUC and VDS in response to TPZ are correlated using the Spearman's rank test (R = -0.667, one-sided p = 0.042) (Figure 2.11B). Spearman's rank correlation is also significant between pre- and post- treatment IAUC values in all treated tumours (R = 0.929, one-sided p = 0.001) (Figure 2.11A). These data indicate that higher pre- treatment IAUC predicts for higher post-treatment IAUC and for a low VDS score. Note that, prior to retrospective separation into non-and strong- responders, the mean baseline IAUC value of the treated group is not significantly different from the control group. Mean Ktrans values exhibit similar trends; strong-responders show marked reduction of average Ktrans (73 % reduction) while non-responders show a non-significant (p = 0.1) average loss from pre-treatment values (59 % reduction). As shown in Figure 2.8B, elevated pre-treatment Ktrans values are seen in those tumours that did not respond to TPZ treatment relative to the strong responders, and only the strong-responders have a significantly lower post-treatment Ktrans value relative to controls. Therefore, both DCE-MRI derived biomarkers IAUC and Ktrans assessed prior to TPZ treatment predicted which HCT116 colorectal xenografts responded with vascular dysfunction.    47   Figure 2.8 Tirapazamine-mediated changes in vascular function measured using DCE-MRI parameters IAUC and Ktrans.  DCE-MRI derived biomarkers IAUC (A) and Ktrans (B) are shown where bars represent individual tumours, horizontal lines indicate group means. TPZ-treated groups were retrospectively classed as non- and strong- responders based on histological data (*p < 0.05) (**p < 0.01).     48   Figure 2.9 Tumour mapping microregional heterogeneity in response to TPZ.  Radial analyses of images show small amounts of perfusion (A) and large amounts of necrosis (B) in the central regions of TPZ-treated strong responding tumours.  2.3.4 Microregional heterogeneity of response to TPZ MRI and histological maps (Figures 2.4, 2.5) illustrate that some TPZ-treated tumours experienced a loss of perfusion in central regions but retained a peripheral rim of perfused tissue. Figure 2.9A shows the average density of carbocyanine-positive vessels as a function of distance from the tumour margin. Control (left) and non-responders (middle) show comparable amounts of carbocyanine throughout their tissues, while strong-responders (right) show only a few remaining perfused vessels at the rim. Figure 2.9B shows the radial distribution of necrotic tissue; as is typical for HCT116 xenografts, necrosis is observed in treatment-naive tumours (left), particularly in the central regions. This necrosis is elevated slightly for non-responders (middle) and is increased dramatically in strong-responders (right), reaching nearly 100 % as close as 1 mm to the tumour margin in some tumours.   iT rapazmn memz(PaP Z)n sbod aP Pu)ce ae Figure 2.10 cutnt vut nmgamz gaPvnawhva)e )Z sbod ae mzz vhp)hnP wtZ)nt vntmvptev xby tluawavP m Pza,uv ,nmgatev cavu ua,utn -mzhtP mv vut .tna.utn( ntzmva-t v) gaPvmeCtP Zhnvutn Zn)p vut pmn,aef bZvtn vntmvptev cavu kDEM vhp)hnP CzmPPaZatg mP Pvn)e,RntP.)egtnP xIM na,uvy Pu)c ptme sbodP m..n)mCuae, jtn) v)cmngP vut Ctevnt )Z vhp)hnPM cuazt e)eRntP.)egtnP Pu)c sbodP cuaCu mnt ntghCtg Zn)p .ntRvntmvptev zt-tzP whv g) e)v ntmCu jtn) xIM paggztyf NOs .mnmptvtn pm.P ctnt C)p.mntg cavu uaPv)z),( vhp)hn pm.P v) gtvtnpaet cutvutn gmvm meg apm,tP ctnt PhCCtPPZhzz( )wvmaetg Zn)p Papazmn mntmP )Z vhp)hnPf se mggava)e v) -aPhmz C)eZanpmva)e )Z C)Rnt,aPvnmva)e xFigures 2.5, 2.6yM m Ahmevavmva-t C)p.mnaP)e cmP g)et w( C)nntzmvae, vut utvtn),tetav( aegaCtP xGsy Z)n .)PvR vntmvptev sbod .mnmptvtn pm.P meg Cmnw)C(meaet uaPv)z),aCmz pm.P xFigure 2.11Cy cutnt m .)Pava-t C)nntzmva)e cmP )wPtn-tg xDtmnP)e C)nntzmva)e O 7 1f0TM cavu T% S C)eZagteCt aevtn-mzP 1fi% R 1fTVyf      Figure 2.10 MRI microregional heterogeneity in response to tirapazamine.  Losses in perfusion occur predominantly in the central regions of strong responding tumours, as shown with radial analyses of IAUC parameter maps before (A) and after (B) treatment with TPZ.   34 Strong responders Non responders Controls C) Heterogeneity Index Correlation: Post-IAUC values vs carbocyanine Po st - tr ea tm en t I AU C  he te ro ge n ei ty  in de x -25 0 25 50 75 100 125 25 50 75 100 Carbocyanine heterogeneity index A) Pre-treatment IAUC vs Post-treatment IAUC 0 10 20 30 40 50 60 70 0 10 20 30 40 Po st - tr ea tm en t I AU C Pre-treatment IAUC Non responders Strong responders B) Pre-treatment IAUC vs Vascular Dysfunction Score 0 10 20 30 40 50 60 70 0.0 0.2 0.4 0.6 0.8 1.0 Vascular Dy sfun ction S co re (VDS ) Pre-treatment IAUC Non responders Strong responders  Figure 2.11 Correlation of MRI and histology features. Evidence for pre-treatment MRI parameter IAUC as a useful predictor for tumour response to TPZ comes from correlations of pre- and post-treatment IAUC values (A) and of pre-treatment IAUC values with the Vascular Dysfunction Score (B). Successful co-registration of MRI and histological parameter maps is suggested by positive correlation of post-treatment IAUC heterogeneity index with the carbocyanine heterogeneity index (C).    51 2.4 DISCUSSION Although the effectiveness of tirapazamine (TPZ) has been attributed to its cytotoxicity to hypoxic tumour cells, its poor tissue penetration profile (Durand and Olive 1992; Durand and Olive 1997; Hicks et al. 1998; Kyle et al. 1999) presents a paradox regarding its mechanism for anti-cancer activity in vivo. Using immunohistochemistry IHC) based tumour mapping, we have previously shown that TPZ has an unexpected vascular- targeting effect in multiple tumour models (Huxham et al. 2006; Huxham et al. 2008), leading to the question of whether this effect also occurs in patients. Studies in this chapter investigate the use of DCE-MRI as a clinically relevant tool for observing TPZ-mediated vascular dysfunction. Studies followed three major steps: a baseline DCE-MRI scan followed by TPZ treatment, a follow up scan at 24 h post-treatment, and finally, tumour excision and histological analysis.  2.4.1 DCE-MRI parameters IAUC and Ktrans DCE-MRI is a noninvasive method of quantitatively measuring vascular function by monitoring tumour uptake of a systemically administered contrast agent. Interpretation of DCE-MRI-derived data is complex and sensitive to several physiological parameters, including blood flow, vascular surface area, vascular permeability, tumour extravascular space and patient physiological values such as heart rate and blood pressure (Figure 1.4) (O'Connor et al. 2007). Initial Area Under the contrast agent Concentration time-curve (IAUC) is a model-free value that reflects uptake of the contrast agent in tumours. The volume transfer coefficient (Ktrans) reflects the transfer of contrast agent from the blood vessels and the extravascular space, and is interpreted as an indication of vascular permeability or tumour blood flow depending on whether contrast agent delivery is limited by blood flow or permeability (Tofts et al. 1999). IAUC and Ktrans are distinct biomarkers that are reasonably reproducible and are used in assessing tumour vascular function in both pre-clinical and clinical studies (Roberts et al. 2006). Unfortunately, standardized methods for the use of DCE-MRI are not yet agreed upon. Methods for measurement, analysis and interpretation of DCE-MRI data that would be useful in multiple centres and for use in   52 clinical trials to provide meaningful, biologically validated endpoints continue to be developed (Leach et al. 2005; Roberts et al. 2006; O'Connor et al. 2007). Several pre-clinical studies have used MRI in the investigation of vascular disrupting agents (VDAs), including examinations of ZD6126 (Breidahl et al. 2006), DMXAA (McPhail et al. 2006) and CA-4-P (Beauregard et al. 2001). MRI has also been employed in clinical investigations of VDAs (see (O'Connor et al. 2007) for a review of MRI evaluation of VDAs in the clinic). Post-treatment examination of TPZ-mediated vascular dysfunction was done at 24h, in contrast to other VDAs where follow up MR imaging may be done much earlier, at 30 min to 3h following administration of the compounds. The anti-vascular effects of TPZ do not manifest in HCT116 tumours for several hours post-treatment, with peak loss of perfusion occuring at 24h, making this timepoint a rational choice for the present studies (Huxham et al. 2006). While most experimental designs involve pre- and post-treatment evaluations of T1 weighted images, the application of contrast agents and the use of calculated biomarkers varies considerably between studies. Data presented here in Chapter 2 reports TPZ-treatment induced changes in vascular function using biomarkers IAUC and Ktrans derived from the two-compartment model (Figure 2.8) (Tofts et al. 1999). These data were obtained using a pre- and post- imaging strategy that was facilitated by an implanted fiducial marker. Positive correlation of post-treatment IAUC values with the biological treatment response indicator, VDS, validates DCE-MRI biomarkers as useful indicators of TPZ mediated vascular dysfunction. These data therefore suggest IAUC and Ktrans as useful biomarkers of TPZ effect that can be used in the clinic. Additionally, new data analysis techniques including microregional analysis of parameter maps and the creation of the vascular dysfunction score (VDS) were developed and reported in these studies.  2.4.2 Analysis of DCE-MRI and histological images The use of simple implanted fiducial markers (Figure 2.2) greatly reduced variations in MR image slice angulation and position which were observed in preliminary studies using a detachable external marker. This suggests that internal markers are a more reliable solution for longitudinal studies of tissue that moves as easily as a subcutaneous tumour. With the assistance of these fiducial markers, tumour cryosections were obtained to closely approximate the MRI slices, and histological tumour maps for vascular perfusion and tumour   9d istancec rsas uiulhosgx yfs mec.ul tnaasluNenic vsNrssi Nfs Nfass bsuc.asbsiNc np Osap.ceni uas 2.eNs cNae8ei0 -Figures 2.5, 2.61 OuaNet.lualh ei le0fN np Nfs sHOsaebsiNul geppet.lNesc eifsasiN ei c.tf u tnbOuaecnix  ,i ull np Nfs N.bn.a bngslc asOnaNsg ei Oasmen.c cN.gesc4 Nfs NhOetul auNs np muct.lua ghcp.itNeni ascOnics Nn y6( cssi ec uOOanHebuNslh V9 )4 reNf asbueiei0 N.bn.ac fumei0 leNNls na in nvmen.c tsiNauleosg lncc np Osap.ceni sppstNc bsuc.auvls reNf Nfs cNuNet N.bn.a buOc -T.Hfub et al. <wwV> T.Hfub et al. <ww;1x y.bn.ac tluccsg uc BcNani0WascOnigsacB Nn y6( NasuNbsiN sHfeveN u ce0iepetuiN lncc np Osap.csg msccslc na 0asuNsa lsmslc np istancec ei Nfs tsiNaul as0enic np Necc.sx BCniWascOnigsacB uas N.bn.ac NfuN buh fums c.cNueisg u gstasucs ei bsuc.auvls muct.lua p.itNeni .cei0 LqkWFö, na fecNnln0etul uiulhcec4 v.N Nfncs tfui0sc rsas seNfsa eic.ppetesiN ei gs0ass na Nnn fsNsan0sisn.clh gecNaev.Nsg Nfan.0f Nfs N.bn.a Necc.sc Nn asc.lN ei u c.cNueisg4 tsiNauleosg lncc np Osap.ceni Nn u lua0s Necc.s uasux ,p gubu0s ntt.ac ei u cei0ls msccsl4 v.N isuavh msccslc tniNei.s Nn p.itNeni uig uas uvls Nn c.OOlh Nfs N.bn.a tsllc reNf ugs2.uNs i.N aesiNc uig nHh0si4 Nfsi Nfs muct.lua gubu0s buh inN asc.lN ei gsuNf np N.bn.a tsllcx Afsi buih ise0fvn.aei0 ms ccslc uas gubu0sg4 ei u tsiNauleosg as0eni4 Nfsi Nfs N.bn.a tsllc buh bnas sppstNemslh vs gsOaemsg np Nfsea nHh0si uig i.NaesiNc uig rell inN c.amemsx P tnbveisg muct.lua ghcp.itNeni ctnas -ZLI1 Oanmegsc ui nvEstNems4 2.uiNeNuNems bsuc.as np Nfs muct.lua geca.ONei0 sppstNc np y6( uig egsiNepesg N.bn.ac %4 ; uig M uc iniWascOnigsac uig Nfs asbueiei0 N.bn.ac åw W åR uc ascOnigsac4 reNf 2.uleNuNems sHubeiuNeni np Nfs N.bn.a buOc tnipeabei0 Nfscs nvcsamuNenic -Figure 2.7C1x  Fö, uig fecNnln0etul OuaubsNsa buOc 2.uleNuNemslh gsOetN muct.lua tfui0sc ntt.aaei0 OaspsasiNeullh ei Nfs tsiNas as0enic np N.bn.ac uig 2.uiNeNuNems augeul uiulhcsc tnipeabsg Nfscs nvcsamuNenic pna ,P5q4 DNauic4 istancec uig fecNnln0etullh uccsccsg Osap.ceni uiulhcsc -Figure 2.9, Figure 2.101x yfs fsNsan0siseNh eigsH tasuNsg vh tnbOuaei0 OsaeOfsaul uig tsiNaul as0enic np N.bn.ac OsabeNNsg u tnbOuaecni vsNrssi Nfs Nrn ebu0ei0 bnguleNesc> OuaubsNsac panb fecNnln0etul N.bn.a buOOei0 -tuavnthuieis fsNsan0siseNh1 uig Fö, -OncNWNasuNbsiN ,P5q fsNsan0siseNh1 rsas pn.ig Nn fums u OnceNems tnaasluNeni -Figure 2.11C1x  2.4.3 Pre-treatment perfusion as indicator of non-response P ce0iepetuiN ugmuiNu0s np .cei0 Fö, ec Nfs uveleNh Nn Osapnab lni0eN.geiul bsuc.asbsiNcx Afsi guNu panb Nfncs N.bn.ac egsiNepesg Nn vs iniWascOnigsac -N.bn.ac %4 ;   54 and 9) based on their histologically-derived VDS were examined retrospectively, it can be seen that prior to TPZ treatment these tumours exhibited greater IAUC and Ktrans values relative to the rest of the as yet treatment-naive tumours. A strong correlation is found between pre-treatment IAUC and tumour response (Figure 2.11). Post-treatment MRI data shows that the values for non-responding tumours did decrease, but not to the same degree as seen in the strong-responders. An overall positive correlation was found between pre- and post-treatment measurements for MRI parameter IAUC (Figure 2.11). These data suggest that tumours more likely to be sensitive to the vascular-targeting effects of TPZ may be identified prior to treatment as those which have low levels of vascular function, which may indirectly suggest greater tumour hypoxia. Although the sample size is small in this study, the observation is important considering the hypoxic cytotoxic mechanism of TPZ, and suggests that the vascular targeting mechanism of TPZ may be related to hypoxia.  Support for a hypoxia-associated predictor for the clinical success of TPZ treatment comes from findings by Rischin et al. (2006) who found that patients with hypoxic primary tumours, as identified on 18F-fluoromisonidazole positron emission tomography (PET), more often benefit from tirapazamine/cisplatin treatment than do patients with non-hypoxic primaries; 2 of 3 non-hypoxic patients failed locally whereas only 1 of 19 hypoxic patients exhibited loco-regional failure when treated with TPZ. No data were presented to indicate whether or not the anti-cancer activity of TPZ was a consequence of vascular targeting effects. Gd-based contrast agents are in clinical use and would therefore be practical for determining whether a vascular targeting mechanism for TPZ could be responsible for its clinical efficacy. The MRI protocol used in these experiments is directly transferable to the clinic. The biomarkers for treatment response identified here, IAUC and Ktrans, are often used in clinical investigations of other vascular disrupting agents, and should be further examined for their diagnostic utility in assessing treatment response to TPZ (O'Connor et al. 2007). In particular, trials studying the efficacy of TPZ should be accompanied by DCE-MRI studies with sufficient time and spatial resolution to create IAUC maps and examine heterogeneities in tumour perfusion.    88 2.5 CONCLUSIONS Whiletp ea rnsohtf v ecciphfsht hnsh hiudif yspxicsf biaxhedaw lthtfueatl HC T16r sal S hfsap utspiftutahpw ep s xsalelsht eusVeaV HedusfItf bdf hiudif ftpodapt hd hftshutah mehn ,-PZ ,nt ipt db adytc euocsahtl belix esc usfItfp sal sascCpep db uexfdftVedasc nthtfdVtatehC pndmtl hnsh .gT lshs xdfftp odal mtcc mehn nephdcdVexsc bealeaVpZ ,iudifp ptapeheyt hd hnt yspxicsf(hsfVtheaV tbbtxhp db ,-P piphseatl utspifsHct cdpptp ea otfbipeda sal dapth db atxfdpep ea hnt xtahfsc ftVedap db heppitw sal hntpt tbbtxhp mtft scpd ptta ea ada( eayspeytw cdaVehileasc 9r)(.gT sascCptpZ ,ndpt btm hiudifp hnsh lel adh ftpodal mtft bdial hd nsyt t5neHehtl peVaebexsahcC neVntf ofthfts utah otfbipeda yscitp sp utspiftl HC T16r sal S hfsapZ     56 1.2R EFNFEFCSF R Anderson, R. F., S. 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"Tirapazamine causes vascular dysfunction in HCT-116 tumour xenografts." Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology 68(2): 138-45. Huxham, L. A., A. H. Kyle, et al. (2008). "Exploring vascular dysfunction caused by tirapazamine." Microvasc Res 65(2): 247-55. Janssen, H. L., A. S. Ljungkvist, et al. (2005). "Thymidine analogues to assess microperfusion in human tumors." Int J Radiat Oncol Biol Phys 21(4): 1169-75. Kyle, A. H., C. T. Chan, et al. (1999). "Characterization of three-dimensional tissue cultures using electrical impedance spectroscopy." Biophys J 62(5): 2640-8. Kyle, A. H., L. A. Huxham, et al. (2003). "Tumor distribution of bromodeoxyuridine-labeled cells is strongly dose dependent." Cancer Res 23(18): 5657-11. Kyle, A. H., L. A. Huxham, et al. (2007). "Limited tissue penetration of taxanes: a mechanism for resistance in solid tumors." Clin Cancer Res 03(9): 2804-10. Leach, M. O., K. M. Brindle, et al. (2005). "The assessment of antiangiogenic and antivascular therapies in early-stage clinical trials using magnetic resonance imaging: issues and recommendations." Br J Cancer 41(9): 1599-610. Marcu, L. and I. Olver (2006). "Tirapazamine: from bench to clinical trials." Current clinical pharmacology 0(1): 71-9. McPhail, L. D., D. J. McIntyre, et al. (2006). "Rat tumor response to the vascular-disrupting agent 5,6-dimethylxanthenone-4-acetic acid as measured by dynamic contrast-enhanced magnetic resonance imaging, plasma 5-hydroxyindoleacetic acid levels, and tumor necrosis." Neoplasia 8(3): 199-206.   57 O'Connor, J. P., A. Jackson, et al. (2007). "DCE-MRI biomarkers in the clinical evaluation of antiangiogenic and vascular disrupting agents." Br J Cancer 96(2): 189-95. Rischin, D., R. J. Hicks, et al. (2006). "Prognostic significance of [18F]-misonidazole positron emission tomography-detected tumor hypoxia in patients with advanced head and neck cancer randomly assigned to chemoradiation with or without tirapazamine: a substudy of Trans-Tasman Radiation Oncology Group Study 98.02." J Clin Oncol 24(13): 2098-104. Roberts, C., B. Issa, et al. (2006). "Comparative study into the robustness of compartmental modeling and model-free analysis in DCE-MRI studies." Journal of magnetic resonance imaging : JMRI 23(4): 554-63. Robinson, S. P., D. J. McIntyre, et al. (2003). "Tumour dose response to the antivascular agent ZD6126 assessed by magnetic resonance imaging." Br J Cancer 88(10): 1592-7. Tofts, P. S., G. Brix, et al. (1999). "Estimating kinetic parameters from dynamic contrast-enhanced T(1)-weighted MRI of a diffusable tracer: standardized quantities and symbols." Journal of magnetic resonance imaging : JMRI 10(3): 223-32. Tofts, P. S. and A. G. Kermode (1991). "Measurement of the blood-brain barrier permeability and leakage space using dynamic MR imaging. 1. Fundamental concepts." Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine 17(2): 357-67. Zeman, E. M., V. K. Hirst, et al. (1988). "Enhancement of radiation-induced tumor cell killing by the hypoxic cell toxin SR 4233." Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology 12(3): 209-18.       58      CHAPTER 4 Decreased blood oxygen tension sensitizes tumours to the anti-vascular effects of tirapazamine. 2                                                    2 A version of this chapter has been submitted for publication and is under review.  Baker JHE, Kyle AH, Flanagan EJ, Methot SP, Bartels KL and Minchinton AI. Decreased blood oxygen tension sensitizes tumours to the anti-vascular effects of tirapazamine.  3.1 INTRODUCTION et al. in vitro et al. in vitro in vivo et al. et al. et al. et al.  in vitro in vivo et al.et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al.3.2 METHODS  3.2.1 Mice and tumours 3.2.2 Treatments et al. et al. µ et al. µ et al.3.2.3 Immunohistochemistry (IHC) et al. µ et al.  3.2.4 Image acquisition and analysis et al. inltvrlo    27 De nkaknkmp ,J. ,d,BH mobD.s d( 1978 dD).",E tdEk,ka. (ds ,J. E."dmnBse bBsh.s De ,J. ,d,BH mobD.s d( 1978 dD).",El iJ. t.s(oE.n (sB",kdm -crf kE "BH"oHB,.n BE ,J. x d( 1978 dD).",E ,JB, Bs. noBH tdEk,ka. (ds 9ku1C-7fl iJ. Ba.sBp. km,.mEk,e ds tsdtds,kdm d( tk5.HE BDda. ,Js.EJdHn (ds da.sHBe.n kbBp.E :BE n.,.sbkm.n (ds kmnkaknoBH bBsh.sE Bmn kE s.tds,.n km 8l0 µb km"s.b.m,E (sdb m.Bs.E, aBE"oHB,os.l 3.2.5 Vascular Dysfunction Score (VDS) iJ. w9M E"ds. 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Dd,J ,J. wr Bmn cr Bs. yl iobdosE (sdb ,s.B,b.m, psdotE E"dskmp JkpJ.s ,JBm ,J.ks "dm,sdH w9Mbkm Bmn ,JB, EJd:.n (d"oE.n Bs.BE d( aBE"oHBs neE(om",kdm km ,obdos bBtE :.s. "dmEkn.s.n tdEk,ka. (ds aBE"oHBs neE(om",kdml 3.2.6 Endothelial Tube Assay WP :.HH tHB,.E -rkEJ.sf :.s. "dB,.n km Wyy µH d( 0yx FB,skp.H -39 cJBsbkmp.mf km F1936878 b.nkB Bmn BHHd:.n ,d E., B, 7C N1 (ds 8 z W Jl SobBm bk"sdaBE"oHBs .mnd,J.HkBH ".HHE -SF41L tsdakn.n De 9sl RHe IBsEBm -Rn.E et al. 8ggWL K.dmp et al. WyyWff :.s. Bo,J.m,k"B,.n DBE.n dm in vitro "oH,os. tB,,.smE Bmn D.JBakdosQ km"Honkmp ,J.ks BDkHk,e ,d (dsb ,oDoHBs E,so",os.E dm FB,skp.H "dB,.n tHB,.El 1.HHE :.s. E..n.n -8 5 8y0 ".HHE Y :.HHf km 8l0 bH F1936878 b.nkB -MkpbBf EottH.b.m,.n :k,J 8y x r3MQ 8y µpYbH 4Gr -MkpbBf Bmn 8y mpYbH pHo,Bbkm. -MkpbBfl 1.HHE (dsb.n ,oDoHBs E,so",os.E De WP J tdE,6E..nkmp Bmn :.s.   64 subsequently treated at indicated concentrations of TPZ; minimum 5 wells per treatment. Plates were brought to indicated gas concentrations in 10.3 min using a custom built aluminum chamber sealed and equilibrated via 8 x 1 min cycles of evacuation and pressurization and final pressure was maintained for 1 h at 37 ºC. Wells were then rinsed and replaced with supplemented MCDB-131 and were incubated at 37 ºC in 5 % O2 / 5 % CO2 / 90 % N2. For fluorescent imaging Calcein AM (Invitrogen), 10 µM in media, was added 1 h prior to endpoint and was rinsed with phosphate buffered saline (PBS, HyClone) prior to fixation in 10 % formalin (Sigma) overnight.  3.2.7 Statistics All statistical analyses were performed using GraphPad Prism software (version 4.0e for Macintosh). Nonparametric Mann-Whitney U tests were used for comparisons between groups; p values *<0.05, **<0.01 and **<0.001 are reported. Where appropriate charts display values for individual tumours as means for analysis of whole tumour sections; combined means are reported for 4-8 tumours per group ± standard error (s.e.).   CHCA PTER 4EA 3.3.1 HT29 colorectal xenograft tumours are resistant to vascular dysfunction effects of TPZ HT29 tumours treated with TPZ show no difference in their proportion of perfused vasculature (PF), however a small but significant increase in the proportion of non-viable tissue (1-VF) is seen (Figure 3.1A, B; Table 3.1). Only 1 of 10 HT29 tumours scored a VDS value greater than the VDSmin (0.505) (Figure 3.1C). HT29 tumour maps confirm no evidence of central vascular damage in response to TPZ, with exception to the single tumour scoring >VDSmin which has a central area of necrosis; representative images are shown (Figure 3.2). TPZ treatment in HCT116 tumours resulted in 3 of 5 tumours exhibiting vascular dysfunction and scoring greater than the VDSmin (0.782), although no significant differences were found in PF and 1-VF values between groups (Figure 3.1C, Table 3.1).   47    A) Perfused vasculature (PF) B) Non-viable fraction (1-VF) C) Vascular Dysfunction Score (VDS) %CD31 obje cts + ve for  ca rb o  in  vi ab le  tis su e % Ne cr o tic  & No n - pe rfu se d tis su e Vascular Dy sfun ctio n Sc o re (VDS)  0 25 50 75 100 0 25 50 75 100      0 0.25 0.50 0.75 1.00 HCT116 HCT116 HCT116 Controls 60TPZ %CD31 obje cts + ve for  ca rb o  in  vi ab le  tis su e 0 25 50 75 100 0 25 50 75 100 HT29 HT29 HT29 Controls 60TPZ Controls 60TPZ % Ne cr o tic  & No n - pe rfu se d tis su e Controls 60TPZ Controls 60TPZ Controls 60TPZ     0 0.25 0.50 0.75 1.00 Vascular Dy sfun ctio n Sc o re (VDS ) * Pe rfu se d fra ct io n  (PF ), %  %CD31 obje cts + ve for  ca rb o  in  vi ab le  tis su e Pe rfu se d fra ct io n  (PF ), %  Ne cr o tic  fra ct io n  (1- VF ), %  % Ne cr o tic  & No n - pe rfu se d tis su e Ne cr o tic  fra ct io n  (1- VF ), %   Figure 3.1 HT29 and HCT116 colorectal xenograft sensitivity to anti-vascular effects of TPZ. Quantitative histological data displays the perfused fraction (PF) (A), non-viable fraction (1-VF) (B) and a combined VDS (C) for HCT116 and HT29 colorectal xenografts treated with 60 mg/kg TPZ. Individual tumours represented by bars and group means by horizontal lines; *p<0.05.   66 A) HT29, Untreated control B) HT29, 60 mg/kg TPZ, 24h   Figure 3.2 HT29 tumour maps. Representative tumour maps of (A) control and (B) 60 mg/kg TPZ-treated HT29 colorectal xenografts at 24h. Staining shows unperfused vasculature (CD31, red), perfused blood vessels (CD31 overlapped with DiOC7(3), blue), perfusion dye (DiOC7(3), cyan) and hypoxia (pimonidazole, green) overlayed on hematoxylin background staining (grey). Scale bar 150 µm.   67  Table 3.1 TPZ-mediated vascular dysfunction in HT29 and HCT116 colorectal xenografts. Decras d bslsox s y bslsoxs yxoslsoxs yxo gnt s iszrgcreus msyxo gnt s h-vfs Control 65.2 ± 10.8 97.8 ± 3.4 0.364 ± 0.09 0.549 n/a 60TPZ 65.3 ± 13.4 88.4 ± 17.9 0.406 ± 0.20 - 1/10 hp-..2s Control 70.6 ± 9.5 60.1 ± 12.4 0.572 ± 0.10 0.782 n/a 60TPZ 49.5 ± 23.0 42.8 ± 31.6 0.762 ± 0.26 - 3/5 PF = perfused fraction; SD = standard deviation; VF = viable fraction; VDS = vascular dysfunction score; VDSmin = (mean control VDS) + (2 x SD); 60TPZ = tirapazamine at 60 mg/kg.  2.2. T reatmns3etmsDnsCE-TMeRRntnsanIT4nCunnsThiocclTEsMTho gTam-mtnaCE-TdnsmqtERCIT Vascular density is lower in HCT116 relative to HT29 tumours, as shown by a greater mean distance of tissue to nearest CD31 object (Figures  2 .5s ,e67). The average size of CD31+ve objects is similar in both models (Figures  2 .5s Ci99,e). The proportion of vessels positive for the perfusion marker DiOC7(3) is significantly lower in HT29 tumours relative to HCT116 (Figures  2 .5s rig17). Proliferating cells and hypoxia were also mapped in untreated HCT116 and HT29 colorectal xenografts. The overall proportion of S-phase cells is reflected by the % pixels positive for incorporated BrdUrd, where no difference is seen between models (Figures 2 05s,e67). However, HT29 xenografts show a trend of fewer proliferating cells proximal to vasculature compared to HCT116 (Figures  2 05s Ci99,e). The amount of bound pimonidazole is somewhat lower in HT29 tumours, particularly at distances further from vasculature (>75 µm) (Figures 2 05srig17). Mann-Whitney U tests were performed on BrdUrd and pimonidazole staining data at distances from vasculature as indicated on graphs by addition of s.e. bars, with no significant differences found (Figures 2 0 ).    34 B) Proliferation & Hypoxia A) Vascular density & Perfusion Br dU rd+ ve pi xel s, % ± s e Distance from vasculature, µm Pi m o n id az o le  st ai n in g  in te n sit y Distance from vasculature, µm C) Tumour Maps HT29HCT116 0 5 10 15 20 25 0 25 50 75 100 125 150 0 25 50 75 100 125 1500 10 20 30 0 5 10 15 20 HCT116 HT29 HCT116 HT29 0 25 50 75 Si ze of CD31 obje cts , pi xel s, ± s e HCT116 HT29 M ean distan ce to nea rest  CD31 stainin g, µm ± s e 0 10 20 30 40 50 60 70 80 HCT116 HT29 ** HCT116 HT29 0 20 40 60 80 100 ** HCT116 HT29P er fu se d  fra ct io n  (P F),  %  ±  se Br dU rd+ ve pi xel s, % ± s e  Figure 3.3 Tumour microenvironment of HCT116 and HT29 colorectal xenografts. CD31 staining data in HCT116 and HT29 tumours reflects microvessel density as the average distance of tissue to its nearest CD31 positively stained object (A, left) and average vessel size (A, middle). The fraction of perfused vessels (PF) is calculated as the proportion of DiOC7(3)+ve CD31 objects (A, right). Quantitative analysis of incorporated BrdUrd staining as a marker for S-phase cells is displayed as whole tumour means (B, left) or as a function of distance from nearest vascualture (B, middle). Tumour hypoxia is reflected by pimonidazole labeling as a function of distance from vasculature (B, right). Magnified tumour map images reflect representative staining patterns of HCT116 (C, left) and HT29 (C, right). Staining shows unperfused vasculature (CD31, red), perfused blood vessels (CD31 overlapped with DiOC7(3), blue), perfusion dye (DiOC7(3), cyan), hypoxia (pimonidazole, green) and S-phase (BrdUrd, black) overlayed on hematoxylin background staining (grey).Scale bar 150 µm; (**p<0.01).   69 3.3.3 Differences in vascular function between HCT116 and HT29 colorectal xenografts Quantitative analysis of the presence of 500 kDa FITC-dextran as a function of distance from nearest blood vessels in HCT116 and HT29 tumours show a difference between the two models. In HCT116 the intensity of FITC at distances far from vasculature is similar when examined at 2 and 20 min post i.v. administration (Figure 2.8A). However, in the HT29 model the marker was able to diffuse further from vasculature with time (Figure 2.8C), suggesting greater extravasation of the high MW marker in HT29 tumours. 3.3.4 Differences in vascular phenotype between HCT116 and HT29 colorectal xenografts The proportion of CD31+ve objects positive for the smooth muscle cell marker is significantly lower in the HCT116 model than in HT29 tumours, and thicker layers of  SMA are seen proximal to vasculature in HT29 tumours (Figure 2.1C). The proportion of collagen type IV basal lamina (CIV)+ve CD31 objects was similar in both HCT116 and HT29 colorectal xenograft models (Figure 2.BA). However, thicker layers of CIV are found proximal to vasculature in HT29 tumours.    34 A) HCT116 B) HT29 2 min 20 min 0 10 20 30 40 50 0 10 20 30 Distance to vasculature,  µm FI TC Fluo res cen ce  In te n sit y Distance to vasculature,  µm 0 10 20 30 40 50 0 10 20 30 FI TC Fluo res cen ce  In te n sit y 2 min 20 min 20 min 2 min 20 min 2 min  Figure 3.4 Vascular function in HCT116 and HT29 colorectal xenografts. Fluorescence intensity of 500 kDa FITC-labeled dextran in HCT116 (B, left) and HT29 (B, Right) xenografts is displayed as a function of distance from nearest vasculature. Tumours were harvested at 2 or 20 min after intravenous injection of FITC-dextran; images show FITC fluorescence (black) and CD31 stained vasculature (red). Scale bar 150 µm.      39 0 10 20 30 40 0 20 40 60 80 100     0     5   10   15   20   25   30   35 Distance from vasculature, µm α SMA+ ve CD31 obje cts , % α SMA+ ve pi xel s, % HCT116 HT29 A) Maturity, αSMA HCT116 HT29 B) Tumour maps ** HCT116 HT29  Figure 3.5 CD31 and  SMA staining in HCT116 and HT29 colorectal xenografts. Staining of smooth muscle is shown as the proportion of CD31 objects dual labeled for  SMA (A, left) and the amount of  SMA as a function of distance from CD31+ve objects (A, right). Representative tumour maps (B) show  SMA staining (black) on hematoxylin (grey) background for HCT116 (left) and HT29 (right) tumours. Scale bars 150 µm; (**p<0.01).    39 A) Collagen Type IV CIV+ ve CD31 obje cts , % HCT116 HT29 0 25 50 75 100 0 50 100 150   0 10 20 30 40 50 60 70 80 CIV+ ve pi xel s, % Distance from vasculature, µm HCT116 HT29 HCT116 HT29 B) Tumour maps  Figure 3.6 CD31 and Collagen Type IV (CIV) staining in HCT116 and HT29 colorectal xenografts.  Staining for basement membrane is shown as the proportion of CD31 objects dual labeled for CIV (A, left) and the amount of CIV staining as a function of distance from CD31 (A, right). Representative tumour maps (B) show CIV staining (black) on hematoxylin background (grey) for HCT116 (left) and HT29 (right) tumours. Scale bars 150 µm.    73 3.3.5 10% O2 breathing or induction of anemia enhances the anti-vascular effects of TPZ in HCT116 colorectal xenografts Subcutaneous HCT116 colorectal xenografts were made more hypoxic by induction of moderate anemia, where blood was removed 30 min prior to treatment with 60 mg/kg TPZ. Anemic mice had 34.0 % fewer red blood cells (RBCs) per unit volume relative to unbled animals. Bleeding-induced anemia resulted in a slightly reduced but not significant proportion of perfused vessels in the anemic group relative to unbled controls (Table 3.2). Induction of anemia did not increase hypoxia in these tumours, as indicated by the amount of bound pimonidazole (Figure 3.7A). The anemic TPZ-treatment group resulted in a higher frequency of tumours, 6 of 7 (86 %), scoring greater than the VDSmin (0.622) relative to tumours from unbled TPZ-treated animals where only 4 of 7 (57 %) tumours were > VDSmin (Figure 3.8A). No significant difference was found between mean VDS for the anemic vs unbled control TPZ-treated groups. TPZ-treated HCT116 tumours obtained from mice breathing 10 % O2 / 5% CO2 / balance N2 for 30 min prior and for 6 h post TPZ-administration had a greater magnitude and frequency of vascular dysfunction relative to tumours from mice breathing room air as seen in mean VDS scores (Figure 3.8A, Table 3.2). Low (10 %) O2 did result in increased tumour hypoxia at all distances from vasculature (Figure 3.7B).    vi 1.8Combini g N.OSmhtm hwyC.Sphxmhtm8Chhwmhcsaox.Sphxmhxm1dvu owp.Sowml.rOyC.em wsrtyxOSphxmpxm(V1DDAm.xwm(1n)mOhCheoOS.Cmcoxhae.tSrim Group PF% ± SD VF% ± SD VDS ± SD VDS min # tumours > VDS min HCT116 Air Control 67.4 ± 6.6 68.8 ± 10.2 0.534 ± 0.10 0.731 n/a Air 60TPZ 53.3 ± 9.3 55.0 ± 16.3 0.701 ± 0.13 - 4/7 Low (10%) O2 Control 66.0 ±10.9 79.3 ± 8.1 0.475 ± 0.11 - 0/5 Low (10%) O2 60TPZ 33.0 ± 12.3 38.9 ± 5.5 0.872 ± 0.54 - 7/7 Anemic Control 60.4 ± 5.5 60.9 ± 11.0 0.628 ± 0.09 - 1/5 Anemic 60TPZ 42.8 ± 8.9 53.2 ± 11.5 0.771 ± 0.08 - 6/7 HT29 Air Control 46.1 ± 5.1 91.5 ± 8.4 0.581 ± 0.03 0.632 n/a Air 60TPZ, 12h 60.3 ± 6.7 99.1 ± 1.1 0.402 ± 0.07 - 0/5 Low (7%) O2 Control 55.4 ±10.3 95.2 ± 4.1 0.471 ± 0.11 - 0/5 Low (7%) O2 60TPZ, 8h 28.2 ± 17.8 60.6 ± 19.0 0.823 ± 0.14 - 4/5 Low (7%) O2 60TPZ, 12h 49.3 ± 17.8 85.9 ± 19.8 0.564 ± 0.21 - 2/5 Ta b le3.1PeZ .3-mdtscu ry b Pd-cZ-3Z Zeft-dtscu na b ft-oHe .3-mdtscu nyr b f-Pm1H-3 Z2P.1cmdtsc Pms3eu nyr9tc  b C9e-c mscd3sH nyr6 x Cg 7 ry6u Ip4TN b dt3-l-O-9tce -d Ip 9ShVS0   0 50 100 150 0 10 20 30 40 50 A) Anemic HCT116 xenografts C) 7% O2 HT29 xenografts Pi m o n id az o le  st ai n in g, av g in te n sit y Pi m o n id az o le  st ai n in g, av g in te n sit y 0 50 100 150 0 10 20 30 40 50 Air 10 % O2 Air 7 % O2 Distance from vasculature, µm Distance from vasculature, µm ** ** *** ** ** * 0 50 100 150 0 10 20 30 40 50 Pi m o n id az o le  st ai n in g, av g in te n sit y Distance from vasculature, µm Air Anemic B) 10% O2 HCT116 xenografts  Figure 2.6 DCEu-MRiCI Ca RunCur dhsCtiM iI olcppm MIE ocfH TC-CreTRM- teICgrMaR1. Tumour hypoxia detected using bound pimonidazole staining is shown as a function of distance from nearest vasculature for HCT116 xenografts in anemic mice (A) and in mice breathing 10% O2 (B) relative to air breathing controls. Similar data is shown for HT29 tumour bearing mice breathing 7% O2 (C); (*p<0.05) (**p<0.01) (***p<0.001).    47 A) TPZ in low oxygen HCT116 xenografts B) TPZ in low oxygen HT29 xenografts Vascular Dy sfun ction S co re , VD S 12h air 60TPZ C) Tumour maps Air HT29, 60 mg/kg TPZ 7 % O2 HT29, 60 mg/kg TPZ     0 0.25 0.50 0.75 1.00 12h 7% O2  controls 8h 7% O2   60TPZ 12h 7% O2 60TPZ 0.00 0.25 0.50 0.75 1.00 Vascular Dy sfun ction S co re , VD S anemic controls anemic 60TPZ air controls air 60TPZ  10% O2  controls 10% O2   60TPZ ** * **  Figure 3.8 Hypoxia enhances anti-vascular effects of TPZ in HCT116 and HT29 xenografts.  Central vascular dysfunction scores (VDS) are shown for 60 mg/kg TPZ (60TPZ) treated anemic or 10% O2 HCT116 (A) and 7% O2 HT29 (B) colorectal xenografts relative to their control groups. Representative tumour maps of air breathing control (C, left) and 7% O2 breathing hypoxic (C, right) HT29 tumours treated with 60 TPZ are shown; unperfused vasculature (CD31, red), perfused vasculature (DiOC7(3) labeled CD31, blue), S-phase cells (BrdUrd, black) and hypoxia (pimonidazole, green).  Scale bar 150 µm; (*p<0.05) (**p<0.01).    76 3.3.6 7% O2 breathing enhances the anti-vascular effects of TPZ in HT29 colorectal xenografts HT29 xenograft-bearing mice were exposed to 7% O2 / 5% CO2 / balance N2 for 30 min prior and 6 h after receiving 60 mg/kg TPZ. Tumour hypoxia was increased in HT29 tumours in similar proportions to 10% oxygen breathing in HCT116 colorectal xenografts (Figure 3.7C). As seen in earlier analysis (Figure 3.1, Table 3.1), HT29 tumours treated with 60 mg/kg TPZ from mice in room air did not show any signs of vascular dysfunction, with 0 of 5 scoring greater than the VDSmin (0.699)  (Figure 3.8B, Table 3.2). However, tumours harvested from mice in reduced oxygen conditions do show central vascular dysfunction with 4 of 5 tumours (80 %) > VDSmin at 8 h and 2 of 5 (40 %) > VDSmin at 12 h. This effect of hypoxia sensitizing HT29 tumours to the anti-vascular effects of TPZ is demonstrated in representative tumour maps (Figure 3.8C).   3.3.7 Tirapazamine mediates damage to endothelial tube structures in vitro in a time, concentration and oxygen-dependent manner HMECs grown as tubular structures on Matrigel-coated 24 well plates were subsequently exposed to TPZ under 2% or 0.2% O2 / 5% CO2 / balance N2 for 1 h, were rinsed, and then 8 or 24 h later were imaged using fluorescent Calcein AM cell viability indicator. Tubular structure networks formed prior to treatment with TPZ are comprised of multiple cells in linear structures. Exposure of the tubes to 10 µM TPZ at 2% O2 for 1 h had minimal effect on the network or cellular makeup of the structures, whereas in 25 µM the cell density is much reduced, resulting in deteriorated networks (representative images, Figure 3.9A). At lower 0.2% pO2, tubes exposed to 25 µM TPZ were even further deteriorated at 24 h post-treatment, however wells imaged at 8 h post-treatment show a less severe difference in cell density and network structures relative to controls (Figure 3.9B). These results demonstrate that a 1 h exposure to TPZ at clinically relevant concentrations, 25 µM, in 2 % O2 can cause vascular damage that does not manifest for several hours post-treatment.   44  Control, 24h 10µM TPZ, 24h 25µM TPZ, 24h A) 2% Oxygen B) 0.2% Oxygen Control, 24h 25µM TPZ, 8h 25µM TPZ, 24h  Figure 3.9 TPZ mediates damage to endothelial tube structures in a concentration, time and oxygen dependent manner.   xi 3.2 METHOTTEDS vAe rsonftsh fctnapfhbulfd s::sbth Bc t*s et al.g  LlBBo pshhslh nc turBudhJ fco osh,nts t*s blBcBkscnbntH B: nr,lfctso turBudhJ hBrs EK y B: turBudh fds  n iltcfn utlc M Iftf ,dshsctso *sds nc R*f,tsd E :ncoh :sftudsh B: t*s turBud pfhbulftuds t*ft fds onhtncbt nc turBud rBoslh t*ft fds hschntnps fco dshnhtfct tB t*s fctnapfhbulfd s::sbth B: vAeJ fco h*Bgh t*ft dsoubnck LlBBo BSHksc lspslh t*dBuk* lBg BSHksc Ldsft*nck Bd ncoubtnBc B: fcsrnf bfc hschntn.s spsc dshnhtfct turBudh tB vAearsonftso pfhbulfd oHh:ucbtnBcM (v20 bBlBdsbtfl SscBkdf:th fds dshnhtfct tB t*s pfhbulfd tfdkstnck s::sbth B: vAeJ g*nls (Rv99) bBlBdsbtfl SscBkdf:th gsds fkfnc h*Bgc tB Ls hschntnps OFigure 3.1P O(uS*fr tao.gp 277)4 3fnch fco 3f5sd tao.gp 2770PM -t*sd lfLBdftBdnsh *fps h*Bgc (v20 hschntnpntH tB vAe LBt* h oyhaln fco h oyhynJ *Bgspsd cBcs B: t*s fhhfHh uhso ondsbtlH fhhshhso :Bd fctna pfhbulfd s::sbth O6nnr tao .gp 900x4 (fH tao .gp 277xPM (Rv99) fco (v20 bsll lncsh fds LBt* bBlBdsbtfl bfdbncBrf rBoslh fco LBt* gsds kdBgc fh SscBkdf:th nc kscstnbfllH nosctnbfl rnbsM v*s scoBt*slnfl bsllh g*nb* bBr,dnhs t*s pfhbulftuds nc LBt* t*shs turBudh h*Bulo t*sds:Bds Ls t*s hfrsJ hukkshtnck t*ft nt nh t*s turBud rnbdBscpndBcrsct fco nth nr,fbt Bc turBud pfhbulftuds t*ft bBc:sd hschntnpntH tB vAeM  3.4.1 Tumour vasculature and TPZ sensitivity vurBud pfhbulftuds nh fLcBdrfl gnt* dsh,sbt tB nth Bdkfcn.ftnBcJ htdubtuds fco :ucbtnBcJ gnt* bBchnosdfLls *stsdBkscsntH BLhsdpso nc LBt* nctsda fco nctdfaturBud bBr,fdnhBch OIsg*ndht 277E4 3flu5 tao .gp 277K4 3dudLsdk tao .gp 277iPM vurBud rf,,nck fcflHhsh ,dshsctso *sds nc R*f,tsd E bBr, fdnck t*s (v20 fco (Rv99) bBlBdsbtfl SscBkdf:t rBoslh h*Bg t*ft (v20 turBudh *fps kdsftsd pfhbulfd oschntH fco f lBgsd ,dB,BdtnBc B: ,sd:uhso pshhslh OFigure 3.3PM v*s ,dshscbs B: t*nb5sd lfHsdh B: Lfhsrsct rsrLdfcsJ RmwJ fco hrBBt* ruhblsJ  6%; O Figure 3.5PJ huddBuconck LlBBo pshhslh nc (v20 turBudh rfH Ls dsh,BchnLls :Bd t*s ,dshscbs B: :sgsd ,dBln:sdftnck turBud bsllh ,dBSnrfl tB pfhbulftuds bBr,fdso tB (Rv99) turBudh O Figure 3.3PM Fdsftsd frBucth B: hrBBt* ruhbls bsllh Bd ,sdnbHtsh ,dBSnrfl tB pfhbulftuds *fps ,dspnBuhlH Lssc h*Bgc tB bBddslfts gnt* dshnhtfcbs tB LBt* fctnafcknBkscnb fco pfhbulfd onhdu,tnck fkscth O3sc1frnc tao .gp 900i4 3sdksdh tao .gp 277E4 vB.sd tao.gp 277iPM (BgspsdJ nctdfaturBud *stsdBkscsntH B: turBud rf,,nck pfhbulfd fcflHhsh nh lBg fco t*sds:Bds gBulo ln5slH cBt sS,lfnc pfdnftnBc nc dsh,Bchs gnt*nc t*s   79 HCT116, SiHa and SCCVII tumour models, where ~ 35 % of tumours show no centralized loss of vascular function (Huxham et al. 2006; Huxham et al. 2008; Bains and Baker et al. 2009).  Poor vascular function is another possible predictor for tumour sensitivity to TPZ, as demonstrated in studies presented in Chapter 2 using DCE-MRI derived biomarkers initial area under the curve (IAUC) or Ktrans (Bains and Baker et al. 2009). Vascular permeability has been suggested to be a predictor for VDA sensitivity, based on studies using intravital microscopy and MRI (Beauregard et al. 2001; Tozer et al. 2008). A rationale for the correlation between pre-treatment permeability and sensitivity to VDAs is that tumour permeability permits greater delivery of the VDAs to tumours through leakage, and that loss of perfusion in response to the drugs results in even greater trapping of the drugs within tumours (Tozer et al. 2008). Additionally, high permeability of tumour blood vessels is at least partially responsible for greater interstitial fluid pressure (IFP) observed in solid tumours (Boucher et al. 1990; Leu et al. 2000), and greater IFP may make tumour blood vessels more vulnerable to collapse in response to VDAs (Tozer et al. 2001; Tozer et al. 2008).  Studies evaluating tumour permeability, including the DCE-MRI derived biomarkers IAUC and Ktrans, are typically unable to determine if observations are specifically the result of differences in vascular perfusion, permeability, blood flow, vascular volume, or rates of extravasation or extravascular diffusion (O'Connor et al. 2007). Quantitative analysis mapping the microregional location of high molecular weight FITC-dextran suggests that the vasculature of HT29 tumours is more permeable than that of HCT116 tumours (Figure 3.4), which is consistent with previous observations using DCE-MRI where increased vascular function predicted for decreased sensitivity to TPZ. However, as is the case with most assays measuring vascular permeability, these results may also be the consequence of any of the other vascular physiological parameters previously mentioned.  3.4.2 Tumour hypoxia and TPZ sensitivity Decreasing tumour blood oxygenation either via induction of anemia or low oxygen breathing resulted in enhancement of the anti-vascular effects of TPZ (Figures 3.7, 3.8). A greater magnitude and frequency of TPZ-mediated vascular dysfunction is seen in the anemic   80 and lower oxygen tumours, and previously resistant HT29 tumours were sensitized to TPZ- mediated vascular dysfunction. This data establishes a direct link between hypoxia and sensitivity to the anti-vascular effects of TPZ, and suggests that the bioreductive hypoxic cytotoxic mechanism of TPZ may be relevant in its ability to mediate vascular dysfunction. Evidence for a hypoxic cytotoxic mechanism of TPZ in vivo has previously been suggested to come from studies finding decreased clonogenic survival in tumours treated with TPZ in combination with radiotherapy (Zeman et al. 1988). Radiotherapy kills oxygenated cells and additional cell kill by a complementary agent is thought to have effectively targeted the hypoxic cell fraction, or to have radiosensitized cells. Low oxygen breathing has also been shown to sensitize tumours to the anti-cancer activity of TPZ (Minchinton and Brown 1992). However, these studies were not able to specifically confirm in vivo that TPZ kills hypoxic tumour cells and they did not assess for an anti-vascular effect. The effects of moderate anemia and low O2 breathing on the tumour microenvironment were evaluated using pimonidazole, which optimally labels cells at extremely low (< 10 mmHg) pO2 (Gross et al. 1995; Arteel et al. 1998). Anemia did not change tumour hypoxia as indicated by pimonidazole binding (Figure 3.7), despite effecting a drop in perfusion that was sufficient to score > VDSmin for 1 of 5 tumours (Table 3.2). None of the non-TPZ treated anemic tumours with VDS > VDSmin showed central vascular dysfunction upon visual inspection of tumour maps. While direct measurements of tumour blood oxygenation were not obtained in these studies, the sensitizing effects of anemia and low O2 breathing on TPZ-mediated vascular dysfunction effects suggest blood oxygenation is relevant to the activity of TPZ in vivo. The anti-vascular effects of TPZ in vitro were shown to be oxygen dependent, with more severe effects observed at 0.2 % than 2 % oxygen, but deterioration of tubular structures is clearly seen even in 2 % oxygen conditions with 25 µM TPZ (Figure 3.9). Damage to endothelial tube structures was not as evident at 8 hours post-treatment as that seen at 24 hours. The time frame for effects is consistent with that seen in mice, where vascular dysfunction is delayed several hours from TPZ administration and exposure (Huxham et al. 2006; Huxham et al. 2008; Bains and Baker et al. 2009). The effects in vitro were also concentration-dependent, with greater damage at 25 µM vs 10 µM (4.5 µg/ml vs 1.8 µg/ml). The 4.5 µg/ml concentration is a clinically relevant exposure, as a clinical study   2T he tramen ofcloin itmi m amshara ilpfomifg glnf l: uy, ad.a L zhfpgn m afme cfmb cpmnam vlevfeiomihle wMams- l: kxT µd.ap Ohit me fphahemihle tmp:(ph:f l: 19 ahe w8omtma et al. 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L,,P-x Wfotmcn m ofiolncfvihGf mempznhn l: cof( iofmiafei tfaldplIhe pfGfpn :l o cmihfein itmi ghg lo ghg eli ofncleg il )WB vlrpg colGhgf nlaf fGhgfevf mn il Otfitfo Ipllg lszdfemihle hn ofpfGmei il itf vphehvmp mvihGhiz l: )WBx  29A3 785716408543 Zf tmGf ofcloifg meg vlacmofg iralro algfpn itmi Ofof nfenhihGf meg ofnhnimei il itf meih(Gmnvrpmo f::fvin l: )WB meg :lreg iralro ahvolfeGholeafeimp gh::fofevfn itmi amz vle:fo nfenhihGhiz il )WBx Vfnhnimei iralron tmGf dofmifo Gmnvrpmo gfenhiz itmi hn Ilit alof amirof meg alof cfoafmIpf itme itmi l: nfenhihGf iralronx Flgrpmihle l: iralro tzclshm meg Ipllg lszdfe nhdeh:hvmeipz fetmevfg itf meih(Gmnvrpmo f::fvin l: )WBE fnimIphnthed m ghofvi pheb IfiOffe itf tzclshv vzililshvhiz  l: )WB meg hin meih(Gmnvrpmo f::fvin in vivox )ldfitfoE itfnf gmim colGhgf :roitfo fGhgfevf l: meih(Gmnvrpmo mvihGhiz l: )WB in vivo meg hgfeih:z nlaf :fmirofn l: iralron itmi vlrpg If :roitfo heGfnihdmifg il gfifoahef h: )WB( afghmifg Gmnvrpmo gzn:revihle lvvron he cmihfeinx    82 3.6 REFERENCES Ades, E. 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(1986). "SR-4233: a new bioreductive agent with high selective toxicity for hypoxic mammalian cells." Int J Radiat Oncol Biol Phys 12(7): 1239-42. Zeman, E. M., V. K. Hirst, et al. (1988). "Enhancement of radiation-induced tumour cell killing by the hypoxic cell toxin SR 4233." Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology 12(3): 209-18.           85      CHAPTER 4 Inhibition of nitric oxide synthase enhances tirapazamine-mediated vascular dysfunction in pre-clinical tumours.3                                                  3 A version of this chapter will be submitted for publication. Baker JHE, Kyle AH, Balbirnie A, Gabriel E, Cran J and Minchinton AI.  Inhibition of nitric oxide synthase enhances tirapazamine-mediated vascular dysfunction in pre-clinical tumours.   86 CHAP TER 3.58RT3EP 3.5.5 10%O2ObOr0ea t1hin Og O od2uc0f fdmumuc0e s0mo Oem0-vOgflTO% Ofm0v0md 0e v0vu The presence of hypoxic cells in tumours was initially hypothesized more than 50 years ago, and tumour hypoxia has since been found to be a characteristic of many solid tumours (Thomlinson and Gray 1955). Hypoxic cells are resistant to radiation and chemotherapy, however they are also a target for anti-cancer therapies (Brown and Wilson 2004). Tirapazamine (TPZ; SR4233; 3-amino-1,2,4-benzotriazine 1,4-dioxide) is one of the most advanced hypoxic cytotoxins in clinical trial; for a review see Marcu and Olver (2006). Cellular reductases reduce and bioactivate TPZ and in the absence of oxygen TPZ is further metabolized to oxidizing radicals capable of causing DNA damage (Anderson et al. 2005). TPZ has greater toxicity to hypoxic cells than to oxygenated cells in vitro and enhances cell kill by radiotherapy and cisplatin in vivo (Zeman et al. 1986; Brown and Lemmon 1990; Dorie and Brown 1993).  TPZ also has the ability to effect dose-dependent, catastrophic vascular dysfunction in the central regions of tumours (Huxham et al. 2006; Huxham et al. 2008; Bains, Baker et al. 2009). TPZ-mediated vascular dysfunction is characterized by a loss of perfusion that is irreversible, occurs in only 65 % of treated tumours, and leaves a hallmark viable rim of undamaged vessels in affected tumours. In Chapter 2, DCE-MRI studies suggested that tumours with greater pre-treatment vascular function levels are less responsive to TPZ- mediated vascular damage (Bains, Baker et al. 2009). Follow up studies in Chapter 3 characterized vascular phenotype differences between sensitive and resistant colorectal xenografts. Decreasing blood oxygenation in vivo through low oxygen breathing or induction of moderate anemia increased the magnitude and frequency of anti-vascular effects of TPZ, establishing a direct link between hypoxia and TPZ mediated vascular dysfunction. 3.5.P 1hi %aZlfaZ Hd C0m%0f 6c0Za xdemoOga tC6xn  Sensitization of tumours to the anti-vascular effects of TPZ via decreasing blood oxygenation in vivo suggests that the anti-vascular effect of TPZ is related to its hypoxic cytotoxic activity, however a detailed mechanism for the effect remains unclear. The reductase enzyme responsible for bioreduction and activation of TPZ and its location in the   54 and9n) dbs)9eotb)9odeoa s9nhi re  bdl9)agoa bo ace 9rye)tei goab-tgysnhg) gsabtbav 9V DSwx Dce)e g)e yete)gh )einsagyey acga cgte reeo bdlhbsgaei bo )einsbom DSwp boshnibom sva9y9hbs eo,vdey ynsc gy TPZSuIsva9sc)9de s )einsagye ASUCKf. A(gha9o et al. MRRkz 2gnoie)y et al. kKKK. goi boa)gonsheg) eo,vdey A0tgoy et al. 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MRR4z Tm et al. kKK4.x 'ocbrbab9o 9V TE2 cgy reeo 9rye)tei a9 bos)egye and9n) cvl9Fbg goi cgy ace)eV9)e reeo ynmmeyaei gy g ynbagrhe s9dlhedeoag)v a)egadeoa V9) cvl9Fbs sva9a9Fboy A(99i et al. MRRUz 7nahe) et al. MRR4.x Dce l)9l9yei descgobyd V9) acby s9drboei a)egadeoa by acga TE2 bocbrbab9o sgnyey go bos)egye bo and9n) cvl9Fbg acga )eynhay bo m)egae) rb9gsabtgab9o 9V cvl9Fbs sva9a9Fbs l)9i)nmy a9 aceb) sva9a9Fbs V9)d V9) m)egae) and9n) sehh 6bhhx 1bteo ace 9rye)tgab9o acga DSw by g s9dleababte bocbrba9) 9V TE2p 1g)oe) et al. AMRRR. cgte l)9l9yei acga DSw dgv l9aeoabgae bayehV tbg acby descgobydx Dce ln)l9ye 9V yanibey bo acby /cglae) U bya9 boteyabmgae ace l9aeoabgh eVVesay 9V TE d9inhgab9o 9o ace )eyl9oye 9V and9n)y a9 DSwx   11  2.4 DISCUON 4.2.1 Reagents 6itsbsosaicv nsd drcexvdiovy lr gt. M.u. TPZxsa ,TPZxsa et al. zmmhN scy syaicidevtvy se hm At Dm aH:(H ,m.4D At m.z4 aaA5:(HN lr icetsbvtieAcvs5 ,i.b.N ic0vReiAc. M) WWu ,9iHasN nsd syaicidevtvy se 21m aH:(H lr i.b. ic0vReiAc ,TPZxsa et al. zmm1N. W; yAcAt dbvtaicv W;W;sev ,ScEietAHvcN nsd btvbstvy ic 2m a8 Ws;T scy yi5Pevy ic pO9 eA m.z a8 Ws;T iaavyisev5r btiAt eA icetsEvcAPd ,i.E.N ic0vReiAc sRRAtyicH eA ascPwsRePtvt icdetPReiAcd. ScetsEvcAPd ic0vReiAcd Aw 2m aH:(H W;W;sev nvtv yAcv diaP5escvAPd5r niex scy vEvtr 4m aic wA55AnicH 6pf syaicidetseiAcG wAt s eAes5 Aw D W;W;sev ic0vReiAcd ,pvts5es et al. 2CCjN. 6xv 9)bxsdv ast(vt 3)OtAaA)z)yvAZrPtiyicv ,OtyItyG 9iHas /xvaiRs5N nsd syaicidevtvy se 2mmm aH:(H s5AcH niex hm aH:(H Aw exv xrbAZis ast(vt biaAciysoA5v ,btAEiyvy lr gt. k Ls5viHxN sd sc i.b. ic0vReiAc z x btiAt eA eiddPv xstEvde. 6xv w5PAtvdRvce yrv gi;/ j ,4N ,8A5vRP5st ptAlvdNG m.h aH:a5 yiddA5Evy ic j3 - ,E:EN yiavexr5 dP5wAZiyv : z3 - devti5v Tz;G nsd syaicidevtvy icetsEvcAPd5r sd s ast(vt Aw Evddv5 bvtwPdiAc 3 aic btiAt eA eiddPv xstEvde.  4.2.2 Mice and tumours Fvas5v W;g:9/Sg scy /4T:TvW aiRv nvtv lt vy scy asicesicvy ic APt icdeiePeiAcs5 9pF scias5 wsRi5ier ic sRRAtyscRv niex exv /scsyisc /APcRi5 Ac ucias5 /stv HPiyv5icvd. qZbvtiavced ic exid Rxsbevt nvtv sbbtAEvy lr exv IciEvtdier Aw Otieidx /A5Palis scy ied ucias5 /stv /Aaaieevv ,ubbvcyiRvd OG /G gN. 8iRv nvtv s55Anvy wtvv sRRvdd eA descysty 5slAtseAtr tAyvce wAAy scy nsevt. /v55d nvtv asicesicvy sd aAcA5srvtd PdicH aiciaPa vddvceis5 avyisG 8q8:qO99 ,Tr/5AcvN dPbb5vavcevy niex 2m - lAEicv HtAnex dvtPa ,Tr/5AcvN scy bsddsHvy vEvtr 4 eA D ysrd. T/622h ,1Z2mh Rv55dG ic W;g:9/Sg aiRvNG T6zC ,3Z2mh Rv55dG ic W;g:9/SgN scy 9//KSS ,3Z2m3 Rv55dG ic /4T:TvWN nvtv iab5scevy dPlRPescvAPd5r iceA exv dsRts5 tvHiAc Aw aiRv. FAt ePaAPt asbbicH vZbvtiavced aiRv nvtv tscyAa5r sddiHcvy eA vZbvtiavces5 HtAPbd nxvc sEvtsHv ePaAPt EA5Pavd tvsRxvy 2mm eA 23m aa 4 sd avsdPtvy PdicH Rs5ibvtd Aw extvv AtexAHAcs5 yisavevtd ,sG l scy RN PdicH exv wAtaP5s EA5Pav Y  :h,slRN. FAt HtAnex sddvddavce ePaAPtd nvtv tscyAa5r sddiHcvy wAt etvseavce 2D 4.2.3 Immunohistochemistry et al. µ et al. 4.2.4 Image acquisition and overlay et al. µ inltvrlo 4.2.5 Image analysis   76 HT29Tx HennTo g9Trafxeon fra nHferert fxHe-fbHn xo29ioam cobx9nen wfn bx9ppoa fwfs gfnoa 9r do2fH9%sOer nHferoa nobHe9rn/ r9r5pox-Tnoa HennTo wfn bx9ppoa fwfs gfnoa 9r 9ioxOfsoa e2fton 9- ClN30 lehC vkNP fra do2fH9%sOerm Zdo iefgOo -xfbHe9r k.uP 9- HT29Txn wfn bfObTOfHoa gfnoa 9r Hdo xfHe9 9- Hdo H9HfO rT2gox 9- pe%oOn er bx9ppoa e2fton gs Hdo H9HfO rT2gox 9- pe%oOn er wd9Oo HT29Tx fxofnm  yoxborH p9neHeio nHferert wfn 9gHferoa Tnert Hdo px9p9xHe9r 9- pe%oOn fH erHorneHeon 2ooHert 9x o%booaert f Hdxond9Oa ifOTo fg9io gfb1tx9Tram u9x aenHxegTHe9r frfOsnen 9- pe29reaf(9Oo xoOfHeio H9 ifnbTOfHTxo0 ofbd pe%oO er f pe29reaf(9Oo e2fto wfn n9xHoa gfnoa 9r eHn aenHfrbo xoOfHeio H9 ClN35p9neHeio ifnbTOfHTxo fra Hdo fioxfto HennTo erHorneHs er 3m) µ2 erbxo2orHn -x92 ifnbTOfHTxo wfn aoHox2eroam u9x aTfO p9neHeio nHferert frfOsnen 9- ClN3 er b92gerfHe9r weHd faaeHe9rfO 2fx1oxn0 Hdxond9Oan woxo noH H9 eaorHe-s nHferert fg9io gfb1tx9Tra fra f 2ere2T2 A6 , 9ioxOfp wfn xoVTexoa H9 bOfnne-s ClN3 9gDobHn fn aTfO OfgoOoam Zdo px9p9xHe9r 9- pox-Tnoa kyuP fra ochS 4io ionnoOn wfn 9gHferoa gs aeieaert Hdo H9HfO rT2gox 9- ClN3 9gDobHn p9neHeio -9x lehCvkNP 9x ochS xonpobHeioOs gs Hdo H9HfO rT2gox 9- ClN3 9gDobHnm 4.2.6 Vascular Dysfunction Score (VDS) Zdo .lS nb9xo dfn pxoie9TnOs goor xop9xHoa er CdfpHox A k8fern fra 8f1ox et al. A667P fra wfn Tnoa ftfer doxo weHd f 29ae-ebfHe9r fn xop9xHoa>  .lS z 35 k.u % yuP wdoxo .u kiefgOo -xfbHe9rP fra yu kpox-Tnoa -xfbHe9rP fxo bfObTOfHoa fn aonbxegoa fg9iom lobxofnoa pox-Tne9r kO9w yuP 2fs go ernT--ebeorH H9 xop9xH ifnbTOfx asn-TrbHe9r er bfnon wdoxo HennTo dfn fOxofas px9booaoa H9 robx9nen fra erfaoVTfHo rT2goxn 9- ClN3 nHferoa ionnoOn xo2fer H9 xo-OobH O9nn 9- -TrbHe9rfO ifnbTOfHTxo kFigure 2.3Pm Zdoxo-9xo eH en robonnfxs H9 erbOTao g9Hd Hdo .u fra yu er Hdo .lS aTo H9 Hdo pfHd9pdsne9O9ts 9- ifnbTOfx asn-TrbHe9rm MrnHofa 9- bfObTOfHert eraeieaTfO HT29Tx .lS Tnert 2f%e2fO b9rHx9O ifOTon kfn er CdfpHox AP0 .lS er CdfpHoxn N fra E en bfObTOfHoa eraoporaorHOs -9x ofbd HT29Tx Tnert 9rOs yu fra .um Zdono eraoporaorH ifOTon fxo Hdor b92pfxoa H9 Hdo .lS2er bfObTOfHoa fn Hdo b9rHx9O 2ofr pOTn A % Slm q ifOTo 9- 3 eraebfHon b92pOoHo ifnbTOfx asn-TrbHe9r0 wdoxo g9Hd Hdo .u fra yu fxo 6m ZT29Txn -x92 HxofH2orH tx9Tpn nb9xert detdox Hdfr Hdoex b9rHx9O   91 VDSmin and that showed focused areas of vascular dysfunction in tumour maps were considered positive for vascular dysfunction. 4.2.7 Statistics All statistical analyses were performed using GraphPad Prism software (version 4.0e for Macintosh). Nonparametric Mann-Whitney U tests were used for comparisons between groups; p values *<0.05, **<0.01 and **<0.001 are reported. Where appropriate charts display values for individual tumours as means for analysis of whole tumour sections; combined means are reported for 4-8 tumours per group ± standard error (s.e.).     17 2. M ETHODSHM 4.3.1 L-NNA enhances TPZ-mediated vascular dysfunction in HCT116 xenografts pharmcol glamcmiyngymhc hb chctiNoOmbmO Sve mcsmrmyhn utSSd fmys w( aA92A ;Fk noiTzyol mc g imAcmbmOgcyz0 mcOnogiol agAcmyTlo gcl Anogyon bno)TocO0 hb gcymt.giOTzgn noiNhcio mc Ip;--j yTahTn RochAngbyi oRgamcol gy 7w s nozgym.o yh w( aA92A ;Fk gzhco qFigure 4.1, Table 4.1x, W3e .gzToi fono Anogyoiy mc yTahTni ynogyol fmys -4( aA92A utSSd mc Oharmcgymhc fmys omyson j( aA92A hn w( aA92A ;Fk, -4( aA92A utSSd G j( aA92A ;Fk sgl w hb P q4( Zx hb yTahTni iOhnmcA BW3eamc6 gcl utSSd G w( aA92A ;Fk gzih sgl w hb P q4( Zx noiNhcio ngyo qFigure 4.1, 4.26 Table 4.1x, utSSd glamcmiyonol gzhco gy -4( aA92A  nolTOol yso NonbTiol bngOymhc qF5x nozgym.o yh Tcynogyol Ohcynhzi6 Am.mcA ihao mclm.mlTgz yTahTni mc ysmi AnhTN g W3e iOhno Anogyon ysgc yso W3eamc qOhcynhz aogc G 7e3ix qTable 4.1x, Ihfo.on6 none hb yso utSSd ynogyol yTahTni ishfol g OocyngzmHol zhii hb NonbTimhc mc yTahTn agNi gcl fono ysonobhno not classed as responders qnoNnoiocygym.o yTahTn agN ishfc mc Figure 4.2iiix, ;Fk ynogyaocy gzhco gy j( aA92A OgTiol w hb P yTahTni q4( Zx yh iOhno B W3eamc gcl loahciyngyo bhOTiol gnogi hb gcymt.giOTzgn obboOyi mc yTahTn agNi, d zhfon lhio hb w( aA92A ;Fk lml chy noiTzy mc gc0 mclm.mlTgz yTahTni iOhnmcA Anogyon ysgc yso W3eamc,    93  B) Vascular Dysfunction Score (VDS) in HCT116 tumours Vascular Dy sfun ction S co re , VD S Carbo+ ve CD31 obje cts (PF) , % A) Perfused vasculature in HCT116 tumours controls 60TPZ 60TPZ + L-NNA 40TPZ 40TPZ + L-NNA L-NNA 0 25 50 75 100      0 0.25 0.50 0.75 1.00 controls 60TPZ 60TPZ + L-NNA 40TPZ 40TPZ + L-NNA L-NNA *** *** *  Figure 2.8 Tap izmini-idz ezmtzceh tz-isvthcultr effec-h df oDC iz EMo88R -uIdurhA Uutz-i-t-ive Kt-t. The perfused fraction (PF) (A) and VDS (B) are reported for individual tumours (bars); group means are represented by horizontal lines. (*p<0.05) (**p<0.01).    Table 4.1. Impact of modulation of NO on TPZ-mediated vascular dysfunction in  HCT116 colorectal xenografts. Group PF% ± SD VF% ± SD VDS ± SD VDS min # tumours > VDS min Control 64.7 ± 8.4 74.0 ± 12.7 0.519 ± 0.11 0.734 n/a 60TPZ 39.7 ±18.0 46.5 ± 26.8 0.779 ± 0.21 - 4/5 60TPZ +  180L-NNA 30.1 ± 24.3 32.0 ± 22.9 0.878 ± 0.15 - 4/5 40TPZ 63.4 ± 7.4 86.6 ± 16.9 0.446 ± 0.15 - 0/5 40TPZ + 180L-NNA 27.5 ± 18.3 35.5 19.4 0.873 ± 0.11 - 4/5 180L-NNA 48.4 ± 7.9 63.9 ± 14.6 0.688 ± 0.11 - 2/5 NONOate 57.8 ± 9.4 80.2 ± 10.7 0.513 ± 0.10 - 0/4 60TPZ + NONOate 28.4 ± 18.9 33.8 ± 23.0 0.872 ± 0.15 - 5/6 PF = perfused fraction; SD = standard deviation; VF = viable fraction; VDS = vascular dysfunction score; VDSmin = (mean control VDS) + (2 x SD); 60TPZ = tirapazamine at 60 mg/kg; 40TPZ = tirapazamine at 40 mg/kg; 180L-NNA = l-nitro-l-arginine at 180 mg/kg; NONOate = spermine NONOate at 10 mg/kg every 30 min for total 4 injections.   47  HCT116 Tumour maps i) Control iv) 60 mg/kg TPZ + 180 mg/kg L-NNA vi) 40 mg/kg TPZ + 180 mg/kg L-NNAv) 40 mg/kg TPZ iii) 180 mg/kg L-NNA ii) 60 mg/kg TPZ  Figure 2.8 Tap izmini-idz ezmtzceh tz-isvthcultr effec-h df oDC iz EMoRRI -uAdurhU -uAdur AtKh. Tumours maps show staining for unperfused vasculature (CD31, red), perfused vasculature (DiOC7(3)+ve CD31, blue), S-phase cells (BrdUrd, black) and hypoxia (pimonidazole, green). Representative tumours are shown for each group, with exception to ii), iv) and vi) where displayed tumour maps are representative of central vascular dysfunction effects observed within indicated treatment groups. Scale bars 150 µm.   12  3. .Di fernccisvinaularncituniolcrbwlHiCTc1bart6dain11nrtcid1i29xi6aig42pphi enadyHl1tci tumorr yp esandioi hg l u .T xc,kc dvnv reosxdbo ypypfwo idi baw rdcbd(dmfbwMg m0fbco w0o eos()roi (sfmwdab B3Az db 5;S..j w)xa)sr soMfwdno wa mabwsaMr BTable 4.1zv HMw0a)c0 baw rwfwdrwdmfMMg rdcbd(dmfbwD ypypfwo db maxhdbfwdab Ldw0 jT xc,kc S3- idi dbmsofro w0o xofb PZC rmaso fbi w0o (soE)obmg a( w)xa)sr RPZCxdb soMfwdno wa S3- wsofwxobw fMabo BFigure 4.3B, Table 4.1zv S)xa)s xfer r0aL w0o o((omw fr f rwsdkdbc Marr a( eos()rdab db w0o mobwsfM socdabr a( f((omwoi w)xa)sr BFigu e 4.3Czv tumorr yp w0oso(aso idi baw esawomw fcfdbrw w0o fbwdInfrm)Mfs o((omwr a( S3- fbi dbrwofi feeofsoi wa ob0fbmo w0o o((omwr rdxdMfsMg wa ypC db0dhdwdab hg 4IyyHv    47 C) HCT116 tumour maps A) Perfused vasculature in HCT116 tumours 0 25 50 75 100     0 0.25 0.50 0.75 1.00 Vascular Dy sfun ction S co re , VD S i) 4 x 10 mg/kg NONOATE ii) 60 mg/kg TPZ + (4 x 10 mg/kg NONOATE) 60TPZ + NONOate NONOate Carbo+ ve CD31 obje cts (PF) , % 60TPZ + NONOate NONOate B) Vascular Dysfunction Score (VDS) in HCT116 tumours * *  Figure 3.6 CD1eaa nd eolTo1ea ToyipITa1uVTr e((e1ya )( stH io 29sccx yuf)ura. The perfused fraction (PF) (A) and VDS (B) are reported for individual tumours (bars); group means are represented by horizontal lines. Tumours maps (C) show staining for unperfused vasculature (CD31, red), perfused vasculature (DiOC7(3)+ve CD31, blue), S-phase cells (BrdUrd, black), hypoxia (pimonidazole, green) and tissue background (hematoxylin, grey). Scale bars 150 µm; (*p<0.05).    iT 4.3.3 Decrease in proliferating cells in response to NOS inhibition in combination with TPZ rap zpmne(Pp Zz)Z)ze()s )b odZanup cpmmu (s tvrggw ehx)hzu ynu xpnuhzpl hu(s, eap p-),ps)hu xnz.pz Czlfzlk nlx(s(uepzpl e) pnca ns(xnm D a Zz()z e) ehx)hz anzPpue ne n l)up )b gEEE x,M.,R rhx)hz czI)upce()su cz)ZZpl e) zpx)Pp s)sdZpzbhupl nsl spcz)e(c nzpnu ypzp nsnmIupl b)z Czlfzl uen(s(s, nsl ua)y eane wE x,M., rjN (u nOmp e) zplhcp Zz)m(bpzne()s (s eapup nzpnuk Ohe yaps c)xO(spl y(ea eap AGo (sa(O(e)z 7dAA1 eap pbbpce (u (sczpnupl e) u(,s(b(cnscp  0Figure 4.4%R o(x(mnzmIk SE x,M., rjN l)pu s)e u(,s(b(cnsemI zplhcp Zz)m(bpzne()s yapzpnu (s c)xO(sne()s y(ea eap gVE x,M., 7dAA1 eapzp (u n u(,s(b(cnse zplhce()s zpmne(Pp e) c)sez)mu )z 7dAA1 nm)spR rap AG l)s)z uZpzx(sp AGAGnep nmu) anu s) pbbpce )s Zz)m(bpzne()s yaps nlx(s(uepzpl nu n u(s,mp n,psek yapzpnu (s c)xO(sne()s y(ea rjN (e zplhcpu Zz)m(bpzne()s e) znepu u(,s(b(cnsemI m)ypz eans ea)up upps y(ea rjN nm)sp )z rjN (s c)xO(sne()s y(ea 7dAA1R      Br dU  + ve  pi xe ls,  %  ±  s. e.  0 3 6 9 12 15 18 controls 60TPZ 60TPZ + L-NNA 40TPZ 40TPZ + L-NNA L-NNA NONOate 60TPZ + NONOate ** *** ** **  Figure 4.4 Decrease in S-phase cells when TPZ combined with NO modulation. Staining for S-phase marker BrdUrd is reported as a group mean ± s.e. Analysis was performed  only in tumour regions that contained perfused vasculature; *p < 0.05.    98 4.3.4 L-NNA enhances growth inhibition effects of TPZ in HCT116 xenografts L-NNA in combination with TPZ results in inhibited tumour growth relative to that seen for either treatment alone (Figure 4.5). HCT116 tumours were treated with 40 mg/kg TPZ, 180 mg/kg L-NNA or a combination of both drugs on day 0 when tumours reached an average size of 59.0 ± 7 mm3. Repeat volume measurements show separation of the combined treatment from all groups, with significant differences relative to untreated controls on days 5, 7 and 9. Mean weight loss was greater in the combination treatment (9.9 ± 2.3%) relative to 40 mg/kg TPZ alone (6.7 ± 1.7%) but this difference was not significant. Data represented as means for minimum n = 7 tumours per group, ± standard error.    Tumour  V olum e, mm 3 Control L-NNA 40mg/kg TPZ 40mg/kg TPZ + L-NNA 0 1 2 3 4 5 6 7 8 9 0 100 200 300 Days from treatment * * *  Figure 4.5 Inhibition of NOS by L-NNA enhances growth inhibition effects of TPZ in HCT116 tumours. HCT116 tumours were treated on day 0 with 180 mg/kg L-NNA, 40 mg/kg TPZ or a combination of both drugs. Statistically significant differences in tumour volumes are indicated for the (40 mg/kg TPZ + 180 mg/kg L-NNA) group (black, dashed), relative to untreated controls (grey, solid); (*p < 0.05). Volumes shown ± s.e.   33 4.3.5 L-NNA enhances the vascular dysfunction effects of TPZ in SCCVII murine but not in HT29 xenografts 2Theprhsp ncpo fyxxi lewtht e uTmd cs vmpo poh dhTawthu aTelpcms k(BW esu 0cev4h aTelpcms k)BW ma .OO)gg pwrmwTt esu dTmuwlht tmrh amlwthu eThet ma 0etlw4eT ubtawslpcms thhs cs pwrmwT redtz , ma S fyxxi pThephu pwrmwTt oeu )P. Z )P. rcs ; 2oh espcy0etlw4eT haahlp ct 4htt th0hTh poes cs 2(-ypThephu .OO)gg pwrmwTt1 nohTh NR r597 2(- Thtw4phu cs , ma M pwrmwTt kNR 6W tlmTcs7 Z)P.rcs  esu pwrmwT redt uhdlpcs7 4eT7hT amlwthu eThet ma 0etlw4eT ubtawslpcms kFigure 4.61 Table 4.2W; Omrvcscs7 DAR r7597 fyxxi ncpo NR r7597 2(- uTerepcle44b cslThetht poh 0etlw4eT ubtawslpcms Thtdmsth cs .OO)gg pwrmwTt1 ncpo M ma M kDRR 6W ma pwrmwTt Z)P.rcs  esu poh rhes )P. tlmTh amT poh 7Tmwd htthspce44b ep poh re8crwr ma D; Hsoeslhrhsp ma 2(-yrhucephu 0etlw4eT Thtdmsth vb fyxxi cs .OO)gg pwrmwTt e4tm mllwTt ep 4mnhT umtht1 nohTh SR r7597 2(- e4msh Thtw4pt cs R ma S pwrmwTt Z)P. rcs 1 omnh0hT nohs lmrvcshu ncpo DAR r7597 fyxxi poct Thtdmsth cslThetht pm , ma S pwrmwTt kKM 6W;  gs C2á3 lm4mThlpe4 8hsm7Teapt sm hsoeslhrhsp ma 2(- rhucephu 0etlw4eT ubtawslpcms vb fyxxi ct thhs cs pwrmwT redt k Figure 4.7, Table 4.2W; Cmnh0hT1 Iwespcpepc0h ese4btct Thtw4pt cs D ma M 2(-ypThephu pwrmwTt1 á ma N fyxxi pThephu pwrmwTt1 esu S ma M lmrvcsepcms pThephu pwrmwTt tlmTcs7 e )P. Z)P.rcs ; xm amlwthu eThet ma wsdhTawthu mT shlTmpcl pcttwht nhTh mvthT0hu cs pohth pwrmwTt1 esu pohb eTh pohThamTh l4etthu et smsyThtdmsuhTt kFigure 4.7W; 2Theprhsp net eddeThsp4b ev4h pm haahlp 4mtt ma 0etlw4eT awslpcms cs 4eT7h swrvhTt ma 0htth4t ncpocs poh pwrmwTt1 vwp pohth haahlpt nhTh smp twvtpespce4 hsmw7o1 mT nhTh smp twaaclchsp4b dTm8cre4 pm helompohT pm lewth lhspTe4 0etlw4eT ubtawslpcms esu twvthIwhsp pwrmwT pcttwh uhepo;    100 Table 4.2. Impact of NOS inhibition on TPZ-mediated vascular dysfunction in SCCVII  and HT29 tumours. Group PF% ± SD VF% ± SD VDS ± SD VDS min # tumours > VDS min SCCVII Control 27.1 ± 7.5 99.3 ± 1.5 0.731 ± 0.07 0.878 n/a 60TPZ 20.0 ± 12.1 73.3 ± 39.2 0.817 ± 0.13 - 2/5 60TPZ + 180L-NNA 0.62 ± 0.61 1.2 ± 1.2 0.999 ± 0.00 - 5/5 40TPZ 27.3 ± 7.1 95.7 ± 3.3 0.740 ± 0.06 - 0/4 40TPZ + 180L-NNA 16.6 ± 18.9 26.1 ± 28.9 0.916 ± 0.12 - 3/4 180L-NNA 17.1 ± 9.1 77.7 ± 35.1 0.868 ± 0.11 - 3/4 HT29 Control 57.5 ± 4.0 100 ± 0 0.425 ± 0.40 0.505 n/a 60TPZ 62.6 ± 11.3 92.4 ± 8.1 0.415 ± 0.15 - 1/5 60TPZ + 180L-NNA 45.5 ± 11.5 98.8 ± 2.8 0.551 ± 0.11 - 4/5 180L-NNA 54.8 ± 10.5 98.3 ± 3.2 0.459 ± 0.11 - 2/6 PF = perfused fraction; SD = standard deviation; VF = viable fraction; VDS = vascular dysfunction score; VDSmin = mean control VDS + 2 x SD; 60TPZ = tirapazamine at 60 mg/kg; 40TPZ = tirapazamine at 40 mg/kg; 180L-NNA = l-nitro-l-arginine at 180 mg/kg.    343 B)  SCCVII tumour maps controls 60TPZ 60TPZ +L-NNA 40TPZ 40TPZ +L-NNA A) Vascular Dysfunction Score (VDS) in SCCVII tumours L-NNA     0 0.25 0.50 0.75 1.00 Vascular Dy sfun ction S co re , VD S iv) 60 mg/kg TPZ +      180 mg/kg L-NNA v) 40 mg/kg TPZ +      180 mg/kg L-NNA ii) 180 mg/kg L-NNAi) Control iii) 60 mg/kg TPZ ** *  Figure 4.6 NOS inhibition enhances the anti-vascular effect of TPZ in SCCVII tumours. Vascular dysfunction scores (VDS) are reported for TPZ-treated SCCVII tumours (A); individual tumours represented by bars, group means by horizontal lines. Tumours maps (B) show staining for unperfused vasculature (CD31, red), perfused vasculature (DiOC7(3)+ve CD31, blue), tissue background (hematoxylin, grey) and hypoxia (pimonidazole, green). Representative tumours are shown for each group, with exception to ii), iii), iv) and v) where displayed tumour maps are representative of vascular dysfunction effects observed within indicated treatment groups. Scale bars 150 µm; (*p<0.05) (**p<0.01).   102 B) HT29 tumour maps controls 60TPZ 60TPZ +L-NNA A) Vascular Dysfunction Score (VDS) in HT29 tumours L-NNA Vascular Dy sfun ction S co re , VD S iv) 60 mg/kg TPZ + 180 mg/kg L-NNA ii) 180 mg/kg L-NNAi) Control     0 0.25 0.50 0.75 1.00 iii) 60 mg/kg TPZ  Figure 4.7 NOS inhibition does not enhance anti-vascular effects of TPZ in HT29 tumours. VDS scores are reported for TPZ-treated HT29 tumours (A); individual tumours represented by bars, group means by horizontal lines. Tumours maps (B) show staining for unperfused vasculature (CD31, red), perfused vasculature (DiOC7(3)+ve CD31, blue),  hypoxia (pimonidazole, green) and tissue background (hematoxylin, grey). Representative tumours are shown for each group. Scale bars 150 µm. 4.3.6 L-NNA and spermine NONOate effects on hypoxia in colorectal xenografts Figure 4.8 0 50 100 150 0 4 8 12 16 0 50 100 150 0 30 60 90 A) HCT116 colorectal xenografts Pi m o n id az o le  st ai n in g in te n sit y Distance from vasculature, µm Control 180 mg/kg L-NNA Pi m o n id az o le  st ai n in g in te n sit y Distance from vasculature, µm B) HT29 colorectal xenografts Control 180 mg/kg L-NNA4 x 10 mg/kg NONOate Figure 4.8 Modulation of NO effects on hypoxia in HCT116 and HT29 tumours. Pimonidazole labeling of hypoxic cells is displayed as a function of distance from nearest CD31 stained blood vessel for HCT116 (A) and HT29 (B) colorectal xenografts. NOS inhibition using 180 mg/kg L-NNA (black, dashed) increases hypoxia in both tumour models, relative to controls (black, solid) (A, B). Excess NO using 4 doses of spermine NONOate also results in an increase in pimonidazole labeling in HCT116 tumours (A).    45B 4.3.7 Differential NOS expression in colorectal xenografts that are sensitive (HCT116) and resistant (HT29) to TPZ mediated vascular dysfunction rdUisaUsn ucl44e adn ulot ghfhisgUaf msdhkiaSU- kihpd -.vg.Uadsh.-fb ,d w(J20c)J H,gs psis ,HH.dhy,-UhgysH,gaffb -Ua, dsn Shi cJI4 adn Uys w(0 sdTbHs .-,dk vhUy a dhdP-Zsg,S,g ZadPw(0 adU,vhnb x.w(06 adn ad adU,vhnb -Zsg,S,gaffb favsf,dk sdnhUysf,af w(0 xsw(06; 8mZis--,hd hS aff ,-hShiH- hS w(0j isSfsgUsn vb -Ua,d,dk ,dUsd-,Ub naUa Shi .w(0j ,- -,H,fai ,d ucl44e  adn ulot x Table 4 .1Fig4 u r36; 0Zsg,S,g smZis--,hd hS sw(0 -Ua,d,dk ,- smZis--sn a- Uys ZihZhiU,hd hS cJI4 OMs Ms--sf- UyaU ais aU H,d,H.H o5 R n.af favsfsn Shi sw(0 -Ua,d,dk; ulot U.Hh .i- yaMs -,kd,S,gadUfb Sspsi sw(0OMs cJI4 Ms--sf- isfaU,Ms Uh ucl44e U.Hh.i- xTable 4 .1Fig4 Ba,,u 6; uhpsMsij Uysis ,- dh -,kd,S,gadU n,SSsisdgs ,d Uys hMsiaff sw(0OMs cJI4 Ms--sf nsd-,Ub vsUpssd U.Hh.i-j a- isSfsgUsn vb Uys aMsiaks n,-Uadgs hS U,--.s Uh Uys dsais-U n.af sw(0 adn cJI4 OMs hvqsgU ,d ucl44e adn ulot x Table 4.1Fig4eabC36;    105 A) Quantitative assessment of NOS expression in HCT116 and HT29 colorectal xenografts Av g in te n sit y N OS  st ai n in g eNOS + ve CD31 obje cts , % Di st an ce  to n ea re st  eN OS  + ve  CD31  obje ct,  µm ii) HT29 0 5 10 15 20 25 HCT116 HT29 0 5 10 15 HCT116 HT29 0 50 100 150 200 250 HCT116 HT29 ** i) HCT116 B) Tumour maps of vasculature and NOS expression in HCT116 and HT29 colorectal xenografts  Figure 4.9 NOS expression variation between HCT116 and HT29 tumours. No significant differences in overall NOS expression are seen between the HCT116 and HT29 colorectal xenograft models (A, left). Specific assessment of eNOS+ve CD31 staining shows that a greater proportion of HCT116 vessels are positive for eNOS (A, middle). However, the overall density of eNOS +ve CD31 vasculature is similar in both HCT116 and HT29 (A, right). Representative images of HCT116 and HT29 colorectal xenografts illustrate CD31 staining (alone, red; overlapped with eNOS, black), non-specific NOS (grey) and eNOS (overlapped with CD31, black). 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T reatmns3D3TCtETEt-sTtCTMRITD4Tushni4D3uTCtEToclTusgDimsgTdi3hq-iETge3Cq4hmDt4T I mlrhrmt. nh,z ri pt.ruthriv hyrn yozbhy,nrn rn u,h,l-ririv rV hy, ptnmf.tl htlv,hriv ,VV,mhn bV Bk2 tl, thh,ifth,u 0o hy, zl,n,im, bV ,am,nn 5s( Byrn wtn ibh hy, mtn, ri nhfur,n mb-0ririv 43 -v;Pv Bk2 wrhy l,z,th ubn,n bV nz,l-ri, 5s5sth,( Zinh,tu bV zlbh,mhriv hf-bfl ptnmf.thfl, Vlb- ut-tv, g ,abv,ibfn 5s tzz,tl,u hb sensitize hf-bfln hb hy, tihrc ptnmf.tl ,VV,mhn bV Bk2(  5s wtn zlbpru,u ri ,am,nn Vbl fz hb d y ri hy, z.tn-t tiu nybf.u ytp, w,.. ,am,,u,u T a Bk2 z.tn-t yt.Vc.rp,ng wyrmy rn hybfvyh hb 0, t0bfh dM -rifh,n ri lbu,ihn X6rimyrihbi et al. d33dS( Byrn zlb.biv,u ,azbnfl, mbf.u ytp, ,am,,u,u hyth i,,u,u hb zlbh,mh ptnmf.thfl, Vlb- baruthrp, nhl,nn rirhrth,u 0o Bk2 tiu -to tmhft..o ytp, u,nht0r.rK,u hy, ptnmf.thfl, nfmy hyth rh wtn -tu, pf.i,lt0., bl .,nn t0., hb l,mbp,l Vlb- ut-tv, riV.rmh,u 0o Bk2( Zi t uurhrbig 5s ubibln ytp, zl,prbfn.o 0,,i Vbfiu hb l,nf.h ri ti 9baov,i nh,t. ,VV,mhC wy,l, 5s u,ml,tn,n nonh,- rm 0.bbu zl,nnfl, tiu mbin,xf,ih.o hf-bfl 0.bbu V.bw rn u,ml,tn,u ri t ubn,cu,z,iu, ih -tii,lg zlbufmriv t i,h ulbz ri hf-bfl baov,ithrbi u,nzrh, hy, ptnbur.thblo , VV,mhn bV 5s ri ibl-t. hrnnf,n X)yti et al. 188MS( kr-birutKb., .t0,.riv bV yozbarm m,..n ri LD B114 hf-bfln hl,th,u wrhy nz,l-ri, 5s5sth, uru nybw n.rvyh.o riml,tn,u nhtririv th urnhtim,n Vtl Vlb- ptnmf.thfl,g hybfvy hy, urVV,l,im, wtn ibh nrvirVrmtih tiu wtn .,nn hyti hyth n,,i wrhy hy, 5s) riyr0rhblg Hc55I X Figure 3.1S( By,l,Vbl, utht Vlb- hy, nhfur,n zl,n,ih,u y,l, ri Dytzh,l T mtiibh mbiVrl- t nz,mrVrm -,mytirn- Vbl Bk2 -,urth,u ptnmf.tl uonV fimhrbig 0fh ub nfvv,nh hyth 5s) tiu 5s -to z.to lb.,n ri rhn ,VV,mhn bi hf-bfl ptnmf.thfl,( Zh l,-trin fim.,tl rV hyrn rn uf, hb .bmt.rK,u tmhrpthrbi bV Bk2 0o 5s) ri ,iubhy,.rt. m, ..n tiu mbin,xf,ih.o vl,th,l t-bfihn bV s de c tiu l,ufm,u Bk2 XBk2eS wrhyri hy, ,iubhy,.rf-( Lbw,p,lg LDB114 hf-bfln wrhy vl,th,l n,inrhrprho hb Bk2 w,l, nybwi hb ytp, vl,th ,l ,azl,nnrbi bV ,5s) ri hy,rl ptnmf.tl ,iubhy,.rf- l,.thrp, hb l,nrnhtih LBd8 hf-bfln X Figure 3.5S( I.h,lithrp,.og l,ufm,u .,p,.n bV   2:T hey pohxicxtpoh nr pa p zyam(h iP nrZ xoex) hxio )d slv upd fzi(io- hey yPPy.ha iP MrZ fzitm.yt )d slvR rzI uitm(phxio iP nr (yCy(a )d xoex)xhxio iP nrZ )d slv upd zyam(h xo tyahp)x(xgyt pot ayoaxhxgyt Cpa.m(phmzyI 3ex.e .im(t p(ai i..mz hezim-e yc.yaa amff(d iP nrR ,pah(dI edficxp f(pda po xufizhpoh zi(y xo slv p.hxCxhd pot zytm.yt )(iit icd-yophxio epa )yyo aei3o hi yoepo.y hey pohxQCpa.m(pz p.hxCxhd iP slvI pa aei3o xo Hepfhyz bR woex)xhxio iP nrZ upd zytm.y )(iit P(i3 zphyaI .pmaxo- ty.z ypayt )(iit icd-yophxio iz xo.zypayt hmuimz edficxp hi zyam(h xo yoepo.yuyoh iP slv uytxphyt Cpa.m(pz tdaPmo.hxioR 1mzheyz 3iz6 xa zy9mxzyt hi tyhyzuxoy 3ex.e iP heyay yPPy.haI iz 3eph .iu)xophxio iP heyay yPPy.haI xa zy(yCpoh hi hey uy.epoxau Piz slv uytxphyt Cpa.m(pz tdaPmo.hxioR Hiu)xophxioa iP slv 3xhe nrZ xoex)xhxio iz yc.yaa nr p(ai zyam(hyt xo p ty.zypayt Pzp.hxio iP ZQfepay hmuimz .y((a kFigure 4.4SR sey zytm.yt hmuimz -zi3he i)ayzCyt 3xhe slv pot nrZ xoex)xhxio xo .iu)xoyt hzyphuyoh upd heyzyPizy )y p .ioay9myo.y iP )ihe zytm.yt fzi(xPyzphxio pot yoepo.yt pohxQCpa.m(pz yPPy.haR 7yafxhy )ihe 0Hs22% pot ZHHDww aei3xo- ahzx6xo- yoepo.yuyoh iP hey pohxQCpa.m(pz yPPy.ha iP slv 3eyo .iu)xoyt 3xhe nrZ xoex)xhiz ,QnnE k Figure 4.1I Figure 4.6SI hey 0sqT hmuimz zyupxoyt zyaxahpoh kFigure 4.7SR sey zy(phxCy xoayoaxhxCxhd iP 0sqT .i(izy.hp( cyoi-zpPha hi slvQuytxphyt Cpa.m(pz tdaPmo.hxio epa )yyo tyuioahzphyt xo Hepfhyz bI 3eyzy heyxz zyaxahpo.y 3pa iCyz.iuy )d ayoaxhxgphxio Cxp VA rq )zyphexo-R 0dficxpI )mh oih nrZ xoex)xhxio p(ioyI .po yoepo.y hey pohxQCpa.m(pz yPPy.ha iP slv xo 0sqT hmuimzaR wP hey zi(y iP nrZ xoex)xhxio xo yoepo.xo- slvQuytxphyt Cpa.m(pz tdaPmo.hxio xa xo.zypaxo- hmuimz edficxpI heyo fyzepfa nrZ xoex)xhxio xa oih p)(y hi fzitm.y hey apuy yPPy.ha xo 0sqT hmuimza pa xo iheyz uity(aR E(hyzophxCy(dI fyzepfa hey fzyayo.y iz p)ayo.y iP nr xa (yaa xufizhpoh hepo hey p)x(xhd iP Cpa.m(phmzy hi zyafiot hi nr P(m.hmphxioaR sexa i)ayzCphxio xa amffizhyt )d hey Pxotxo-a xo Hepfhyz b iP zy(phxCy(d hex.6 .iphxo-a iP HwD pot  Z8E xo 0sqT .i(izy.hp( cyoi-zpPhaI 3ex.e upd up6y he yu (yaa ayoaxhxCy iz uizy p)(y hi zy.iCyz Pziu Cpa.m(pz tpup-y zy(phxCy hi hey ayoaxhxCy 0Hs22% .i(izy.hp( cyoi-zpPha heph ept (yaa HwD pot Z8ER sey Cpzxphxio xo ux.zizy-xiop( nrZ ycfzyaaxio xo heyay .iohzpahxo- hmuimz uity(a k Figure 4.9S p(ai iPPyza p fihyohxp( ycf(pophxio pa hi 3ed 0sqT hmuimza upd )y (yaa ayoaxhxCy hi nr (yCy(aI pa 0sqT hmuimza epCy p (i3yz Pzy9myo.d iP Cpa.m(phmzy ahpxoxo- fiaxhxCy Piz ynrZR    110 4.4.3 Bioreductive hypoxic cytotoxic targeting of tumour vasculature enhanced by NOS inhibition Combining NOS inhibition with hypoxic cytotoxins for enhanced anti-cancer activity has been previously suggested and has been shown as a therapeutically beneficial strategy (Wood et al. 1994; Butler et al. 1997). However, most studies examining the effects of bioreductive hypoxic cytotoxins have not examined whole tumour sections histologically and may therefore miss opportunities to observe anti-vascular activity. Butler et al. (1997) observed that the hypoxic cytotoxin RB6145, when combined with NOS inhibitor L-NNA, produced an effect that far exceeded that expected for a bioreductive agent selectively killing hypoxic tumour cells. The clonogenic cell survival was decreased if tissues were harvested at > 12 h post administration (Butler et al. 1997). Earlier studies using clonogenic survival studies in SCCVII/Ha tumours showed that L-NNA enhanced the anti-cancer activity of RB6145 and caused extensive necrosis in treated tumours when examined at 24 h post- treatment (Wood et al. 1994). The timing of tissue harvest in clonogenic survival studies is crucial when detecting an anti-vascular effect (see section 1.2). If tissues are harvested too early following anti-vascular treatment the tumour cells that would otherwise be doomed to die due to lost blood vessels would be effectively rescued by the excision and plating of cells in clonogenic survival assays. The time-dependent survival effects observed when combining hypoxic cytotoxin RB6145 with NOS inhibition may have occurred due to an anti-vascular effect, which would also account for the greater than expected cell kill in those studies (Wood et al. 1994; Butler et al. 1997). RB6145 was also combined with the NOS inhibitor L- NNA in subcutaneous SaF tumours to produce a growth delay that, although only a 1.5 day effect, was significantly greater than that seen for RB6145 alone (Horsman et al. 1996). No assessment for anti-vascular effects were undertaken in studies examining NOS inhibition treatment combined with RB6145, but we propose that a large, irreversible anti-vascular effect may have occurred that would explain enhancement of the bioreductive hypoxic cytotoxin RB6145 by the NOS inhibitor L-NNA.    111 2.4 DISDCUONISO TPZ is reduced by NOS to result in reduced available NO and increased oxidative stress. Tumour vasculature is already under greater oxidative stress than most tissues due to sluggish and intermittent blood flow. In addition, tumour vasculature may be selectively unable to withstand or recover from vascular damage. Therefore, it seems plausible that targeting tumour vasculature with bioreductive cytotoxins that can competitively inhibit NOS may represent a viable therapeutic approach with an advantageous mechanism for selectively targeting tumour tissue for damage.  The studies presented here in Chapter 4 demonstrate a therapeutic benefit for combining TPZ with NOS inhibition and emphasize the importance of ascertaining whether the vascular dysfunction effects of TPZ occur in clinically treated patients. In addition, assessment of an anti-vascular effect is strongly suggested for other hypoxic cytotoxins alone or in combination with NOS inhibition.      229 LIST  OFO OGUORT Peti,G.eR SB nBR gB gB gh(etiR et al. 1988)"B ISst(osl a,.ai,d(iG r.ui,e(er dhi hca.f(swGiliod(ui ocd.d.f(o(dc .b sed(dmA., -wsA(e.w2R9R4wEiex.d,(sx(ei 2R4wt(.f(tiGBI p,r :(.A.l 0hiA E122"y 92KWwW4B Pet,stiR gB TBR zB SB vs,dR et al. 12779"B Izeh(E(d.,G .b e(d,(o .f(ti GcedhsGi Giliod(uilc ,itmoi bl.M (e dmA.,wsGG.o(sdit ei.usGomlsdm,iBI :,(d(Gh V.m,esl .b Ths,Aso.l.rc CHA14"y 2879w)B :s(eGR NB VBR VB vB :sDi,R et al. 19887"B IHidiod(er usGomls,wds,rid(er ibbiodG .b dhi hca.f(o ocd.d.f(e d(,sasxsA(ei (e dmA., fie.r,sbdG mG(er Asreid(o ,iG.eseoi (Asr(erBI zed V Sst(sd peo.l :(.l ThcG AL1-"y 7)WwK)B :ioDAseR VB gBR 5B 3B :ioDAseR et al. 12778"B IPaas,ied hct,.fcl ,st(osl a,.tmod(.e Ec ai,.fce(d,(diy (Aal(osd(.eG b., iet.dhil(sl (eJm,c b,.A e(d,(o .f(ti set Gmai,.f(tiBI T,.o Csdl Post go( 6gP  PA14"y 2K98w4B :,.MeR VB FB set FB VB NiAA.e 12778"B IT.died(sd(.e Ec dhi hca.f(o ocd.d.f(e gS 49-- .b oill D(ll(er a,.tmoit Ec b,sod(.esdit (,,st(sd(.e .b A.mGi dmA.,GBI 0seoi, SiG  1H194"y WW4)w7B :,.MeR VB FB set 3B SB 3(lG.e 19884"B Ijfal.(d(er dmA.m, hca.f(s (e oseoi, d,isdAiedBI Csdm,i Siu(iMG 0seoi,  L1K"y 4-Ww4WB :mdli,R gB PBR TB VB 3..tR et al. 1277W"B IjehseoiAied .b E(.,itmod(ui t,mr d.f(o(dc (e Am,(ei dmA.m,G Ec (eh(E(d(.e .b dhi sod(u(dc .b e(d,(o .f(ti GcedhsGiBI :, V 0seoi, AS14"y 4-Zw44B 0h(eJiR jB 0BR SB NB 0.MieR et al. 1988-"B IC.ewemolis, l.osl(xit hmAse Cpgzz iehseoiG dhi E(.sod(usd(.e set d.f(o(dc .b d(,sasxsA(ei 1gS49--" (e u(d,.BI F.l Ths,Aso.l  SE1K"y 294Zw ))B 0mll(GR jB SBR 5B NB OslEi,R et al. 1988K"B I5mA.m, .ui,ifa,iGG(.e .b (etmo(Eli e(d,(o .f(ti GcedhsGi 1(Cpg" (eo,isGiG ser(.rieiG(G set Asc A.tmlsdi dhi sed(wdmA.m, ibbiodG .b dhi usGomls, t(G,mad(er sried kHK29KBI F(o,.usGo SiG AC19"y WKwZ4B Hsu(GR TB HBR ?B FB 5.xi,R et al. 19889"B IjehseoiAied .b usGomls, ds,rid(er Ec (eh(E(d.,G .b e(d,(o .f(ti GcedhsGiBI zed V Sst(sd peo.l :(.l ThcG  1L1)"y 2)-9wKB H.,(iR FB VB set VB FB :,.Me 1277-"B I5mA.,wGaio(b(oR Gohitmliwtiaietied (edi,sod(.e EidMiie d(,sasxsA(ei 1gS 49--" set o(Galsd(eBI 0seoi, SiG  1E127"y 4K--wKB juseGR VB 3BR OB Lmt.hR et al. 1277Z"B I5(,sasxsA(ei (G AidsE.l(xit d. 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(211)"o awvScIclcxv x- vclycu xFcpe tnvlShte cvp.uet h tedeulcOe yep.ulcxv cv l.ixy Idxxp -dxH lShl ct yeOeytcIde HclS RzhyMcvcveoa mhvuey 3et 57(7"f 1b4z77o GxDeyr qo Por No Yo Wycter et al. (0551"o a:clycu xFcpe tnvlShte cvScIclcxv evShvuet lSe l.ixy Ohtu.dhyzphihMcvM e--eult x- uxiIyelhtlhlcv hzb 6zxzgSxtgShle hl udcvcuhddn yedeOhvl pxtetoa mdcv mhvuey 3et 15(22"f 6)42z15o jhdlxvr Po wor mo 3o jxd-r et al. (2110"o aGSe yxde x- unlxuSyxie Wb75 hvp unlxuSyxie Wb75 yep.ulhte cv lSe yep.ulcOe IcxhulcOhlcxv x- lSe vxOed IevDxlychDcve pcz:zxFcpe SngxFcu unlxlxFcv 6zhicvxz2r0rbzIevDxlychDcvez2rbzpcxFcpe (T3 b066r jw: 715)7" In ix.te dcOeyoa CcxuSei WShyihuxd 44(0"f 072z1o jxxpr Wo 9or 9o Po Thvtxir et al. (211b"o awvp.ulcxv x- SngxFch cv eFgeycievlhd i.ycve l.ixyt In lSe vclycu xFcpe tnvlShte cvScIclxyr :qzvclyxzRzhyMcvcveoa mhvuey 3et 54(0b"f kb74zk6o Zeihvr Yo Por 9o Po CyxHvr et al. (214k"o aT3zb066f h veH Icxyep.ulcOe hMevl HclS ScMS tedeulcOe lxFcucln -xy SngxFcu ihiihdchv ueddtoa wvl 9 3hpchl Jvuxd Ccxd WSnt 12()"f 2061zb0o   339           CHAPTER 5 Conclusions     551 5.1 Research hypothesis  Alt houhget gf clie utetvunl sve cg iratecipvct clt antiai- ztnlvriez fgu vrcimnvrntu vnciaic( gf clt TigutPonciat l(hgZin n(cgcgZir ciuvhv)vzirt bAdyx, Ady lve putvctu cgZinic( cg l(hgZin ntDDe clvr gZ(ptrvctP ntDDe antiavs-O Toc nvr Pvzvpt clt ntrcuvD TDggP ateetDe gf cozgoue pugsr ir zint antiai-, Altut ie ngreiPtuvTDt irctum vrP ircuvmcozgou ltctugptrtic( ir cozgou utehgret cg AdyO sicl 921 ; gf cutvctP cozgoue elgsirp rg vrcimavenoDvu tfftnce sltr cutvctP sicl rtvu zvZizoz cgDtuvctP Pgete, 7atr ir utehgrPirp cozgoueO v aivTDt uiz gf orPvzvptP ateetDe vrP cieeot htueiece fgDDgsirp cutvcztrc sicl Ady, Alt zvir l(hgclteie gf clt sguH ir clie clteie ie clvc ftvcoute gf clt cozgou zinugtraiugrztrc ngrftu cozgou etreiciaic( cg clt vrcimavenoDvu tfftnce gf ciuvhv)vzirt, Alt zinugtraiugrztrcvD ftvcoute gf cozgoue clvc stut etreiciat gu uteiecvrc cg clt vrcimavenoDvu tfftnce gf Ady stut ngzhvutP, kcuvctpite fgu etreici)irp cozgoue cg clt vrcimavenoDvu tfftnce gf Ady stut vDeg veeteetP ir clie tffguc cg nlvuvnctui)t clt ztnlvriez fgu AdymztPivctP avenoDvu P(eforncigr ir egDiP cozgoue,  5.2 Assessing the anti-vascular effects of TPZ  Alt vrcimavenoDvu tfftnce gf Ady stut huizvuiD( veeteetP ir clie clteie sguH oeirp 8K-mTvetP cozgou zvhhirp vrvD(eie, .v(tutP 8K- ecvirirp gf zoDcihDt zvuHtue nutvcte ghhgucoric( fgu zoDcihDt ftvcoute cg Tt veeteetP ir utDvcigr cg grt vrgcltu, 8zvpte gf slgDt cozgou etncigre stut ptrtuvctP T( ciDirp zinugen gh( sltut vPUvntrc fitDPe vut vocgzvcinvDD( ecicnltP cgptcltu, IatuDv(tPO ngzhgeict izvpte gf zoDcihDt zvuHtueO PtenuiTtP ve vcu-cst ulr O nutvct aieovD uthutetrcvcigre gf clt tfftnce gf Puope ir cozgoueO vrP Covrcicvciat Pvcv zv( vDeg Tt gTcvirtP, Alie vhhugvnl ie pvirirp hghoDvuic( fgu iratecipvcigre gf zvr( vrcim nvrntu Puope ant iai-O vrP ugTgcin zinugenghte sicl vocgzvctP ftvcoute vut ngrciroirp cg izhugat clt vnnteeiTiDic( vrP vhhDinvcigr gf cozgou zvhhirp fgu zvr( DvTguvcguite bwvvrPtue yvtle. 0660q Strcgr yvtle. 066Eq Suvrng yvtle. 066Mq AutPvr yvtle. 066Rx, 8r vPPicigr cg tZvzirirp clt zinugutpigrvD tfftnce gf AdyO  cozgou zvhhirp lve Tttr oetP eonnteefoDD( ir gou DvTguvcgu( cg veetee clt ant iai- tfftnce gf etatuvD cltuvhtocine irnDoPirp cuveco)ozvT bjvHtu yvtle. 066:vxO DihgegzvD iuirgctnvr bjvHtu yvtle. 066:TxO vrcimztcvTgDicte bKoZlvz yvt le. 066ExO clt cvZvrte bw(Dt yvtle. 0664x vrP kmhlvet zvuHtu juP'uP bw(Dt yvtle. 0662x,   vvi Fgguggrue1 .H 1yu pe1oxcpglsnpt uHHul1g .H hm3 2ugltoMu2 oe 1yog 1yugog sgoed 1sr.st rpffoed pepnTgug Ppg 2.eu MT g1poeoed ltT.gul1o.eg .H 1sr.stg dt.Pe gsMls1peu.sgnT oe roluZ mto.t 1. 1oggsu yptcug1 rolu Putu p2roeog1utu2 ua.due.sg rptb utg4 Pyoly npMunu2 yTf.aol Vfor.eo2pz.nu8 pe2 Nxfypgu VOt2(t28 lunngZ F Hns.tuglue1 2Tu VlptM.lTpeoeu4 So)6 EVw88 Ppg p2roeog1utu2 oe1tpcue.sgnT 1. npMun Hsel1o.eoed4 futHsgu2 cpglsnp1stuZ N1poeoed pe2 Hns.tuglue1 orpdoed .H 1yugu rptbutg4 oe l.rMoep1o.e Po1y npMunoed .H 1sr.st cpglsnp1stu V6Swv8 pe2 1oggsu Vyurp1.aTnoe84 pnn .e 1yu gpru gul1o.eg dueutp1u2 1yu 1sr.st rpfg Pyutu lue1tpn cpglsnpt 2TgHsel1o.e l.sn2 upgonT Mu o2ue1oHou2 Vguu Figure 1.28Z kspe1o1p1ocu pepnTgog Ppg plyoucu2 goronptnT 1. ftuco.sg tuf.t1g .H hm3 ru2op1u2 cpglsnpt 2TgHsel1o.e V5saypr et al. 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RDDi9 5saypr et al. RDDC8Z 7e p22o1o.e4 oe2oco2spn 1sr.stg ptu l.rfptu2 1. l.e1t.n rupeg 1. 2u1utroeu p HtuUsuelT H.t 1yu esrMut .H 1sr.stg 1yp1 gsg1poeu2 lp1pg1t.fyol lue1tpn cpglsnpt 2TgHsel1o.eZ O.1y 1yu HtuUsuelT pe2 rpdeo1s2u .H tugf.egu ptu sgu2 oe 1yog 1yugog 1. oecug1odp1u Pyu1yut r.2oHolp1o.e .H 1tup1rue1 l.e2o1o.eg4 2.gu .t p2roeog1utoed oe l.rMoep1o.e Po1y l.rfnurue1ptT 2tsdg lpe oeltupgu .t 2ultupgu 1yu lue1tpn cpglsnpt 2TgHsel1o.e uHHul1g .H hm3Z F 1.1pn .H R0 56hvvi 1sr.st aue.dtpH1g Putu 1tup1u2 Po1y iD rd,bd hm3 pe2 ptu tuf.t1u2 oe 1yog 1yugog4 .H Pyoly vi yp2 ISN gl.tug dtup1ut 1ype 1yuot uafutorue1pn ISNroe 4   117 an average response rate of 64%. This rate of response is similar to those previously reported for TPZ  (Huxham et al. 2006; Huxham et al. 2008).   While the VDS was successful for evaluating the anti-vascular effects of TPZ in tumours, tumour mapping data are by nature static, with samples only analyzable at one time point in an experiment. Additionally, the exogenous markers and large samples required make application of this technique limited in the clinical setting. Magnetic resonance imaging (MRI) technology is used in clinical oncology applications for diagnosis and monitoring response to treatment, with dynamic contrast-enhanced (DCE-MRI) being increasingly used for the assessment of vascular function (Jackson et al. 2007; O'Connor et al. 2007). Drug effects on tumour blood volume, flow and permeability can be accomplished non-invasively using MRI. Several pre-clinical studies have used MRI in the investigation of other vascular disrupting agents, including examinations of ZD6126 (Robinson et al. 2003), DMXAA (McPhail et al. 2006) and CA-4-P (Beauregard et al. 2001). MRI has also been employed in clinical investigations of vascular disrupting agents; for a review see O'Connor et al. (2007).  Chapter 2 presents a method for evaluating the effects of TPZ non-invasively using two DCE-MRI derived biomarkers for assessment of vascular function: initial area under the curve (IAUC) and Ktrans. IAUC and Ktrans were determined for tumours before treatment with TPZ and these values were retrospectively correlated with histological determinations of tumour sensitivity to TPZ-mediated vascular dysfunction (Figure 2.11). These data show that pre-treatment measures of vascular function IAUC and Ktrans may be useful predictors of sensitivity to TPZ. Both IAUC and Ktrans are related measures of vascular function. IAUC is a model-free value that reflects uptake of Gd-DTPA, the contrast agent used in these studies. The volume transfer coefficient (Ktrans) reflects transfer of the contrast agent from the blood vessels to the extravascular space. Ktrans may be interpreted as an indication of vascular permeability or tumour blood flow depending on whether contrast agent delivery is limited by blood flow or permeability in the individual sample being analyzed (Figure 2.4) (Tofts et al. 1999). Therefore, discrimination between blood flow and permeability are difficult using these analyses due to the small molecule nature of the Gd-DTPA tracer that may not be completely intravascular due to the high permeability of tumour blood vessels (Leach et al. 2005). Further studies using macromolecular contrast agents may be able to determine more   118 specifically which aspects of vascular function can predict for tumour sensitivity to the anti- vascular effects of TPZ. Measures of vascular function may indirectly reflect levels of oxygenation which may be of more direct physiological relevance for predicting sensitivity to TPZ-mediated vascualr dysfunction. Positron emission tomography (PET) scanning using radio-labeled hypoxia indicators is an alternative method for non-invasively monitoring hypoxia levels in tumours, and has been successfully used in studies attempting to predict which tumours may have greater sensitivity to TPZ (Rischin et al. 2006). It is unclear if this type of study would be sufficiently sensitive to detect differences in the levels of intermediately oxygenated tumour blood vessels, or if overall tumour hypoxia levels would be a useful predictor for sensitivity to TPZ-mediated vascular dysfunction. Due to the vascular damage effects of TPZ, PET imaging of radio-labeled tracers may have limited application in monitoring increases in tumour hypoxia following treatment, as loss of perfusion in the tumour would lead to reduced delivery of the tracer molecules. In addition to their potential as predictors for sensitivity to TPZ, the DCE-MRI derived biomarkers IAUC and Ktrans were demonstrated as useful measures of tumour response to TPZ (Figure 1.3(B1.3) . Determining if the anti-vascular effects of TPZ observed in pre-clinical models occurs in clinically treated patients is of high importance. In Chapter 2, the use of DCE-MRI to non-invasively detect the effects of TPZ in tumours was validated. A clinical trial based on the MRI protocol presented in Chapter 2 has been undertaken by the gynecological oncology group (GOG) at the BC Cancer Agency in Vancouver, where patients with cervical cancer are being recruited for a randomized Phase III trial examining radiotherapy and cisplatin alone or in combination with TPZ. Patients recruited to the DCE- MRI trial are being imaged prior to their therapy that may include TPZ, with follow up DCE- MRI scans obtained at two timepoints following treatment. Results from this trial may indicate whether TPZ is able to mediate central vascular dysfunction in clinical cervical cancers. Further studies examining TPZ or other hypoxic cytotoxins in clinical trials could also investigate the potential of DCE-MRI as a tool for either assessment of response, or for predicting response to therapies that may have anti-vascular activity.   119 In Chapter 2, MRI-derived biomarkers IAUC and Ktrans were useful in predicting which HCT116 tumours would be more sensitive to the anti-vascular effects of TPZ. Tumour blood vessel phenotype as a predictor for sensitivity to TPZ was further explored and is described in Chapter 3. HCT116 colorectal xenografts are sensitive to the anti-vascular effects of TPZ and appear to be less permeable to extravasation of high MW FITC-dextrans than HT29 colorectal xenografts, which are resistant to TPZ. This result is consistent with DCE-MRI data from Chapter 2, where greater vascular function predicts for less sensitivity to TPZ. However, the distance of the FITC-dextran from vasculature could be the result of many features, including vascular permeability, extravasation rates, blood flow rates and tumour microenvironment-specific features that regulate extravascular diffusion of agents in solid tissue. The same confounding variables exist in most permeability assessments, including DCE-MRI and intravital microscopy. Interpretation of these results must therefore be carefully considered in context of further data.  Additional data described in Chapter 4 suggests that poor tumour vascular function can predict for tumour sensitivity to TPZ-mediated vascular dysfunction. Decreasing levels of NO via inhibition of NOS caused a drop in perfusion in HCT116 and SCCVII tumours and sensitized them to greater damage by TPZ. However, inhibition of NOS did not effect a significant change in the perfused fraction of HT29 tumour blood vessels, and these tumours remained resistant to the anti-vascular effects of TPZ. The vasculature in HT29 tumours also have thicker layers of  SMA and collagen type IV relative to HCT116 tumours, which may suggest a greater capacity to regulate blood flow or decreased sensitivity to modulation of signaling molecules such as NO.  While a detailed mechanism remains unclear, it appears that poor vascular function can predict for tumour sensitivity to the anti-vascular effects of TPZ. Greater vascular permeability has been suggested to be a predictor for sensitivity to other vascular disrupting agents (VDAs) based on studies using intravital microscopy and MRI (Beauregard et al. 2001; Tozer et al. 2008). A rationale for the correlation between greater pre-treatment permeability and greater sensitivity to VDAs is that tumor vascular permeability permits greater delivery of the VDAs to tumors through leakage, and loss of perfusion in response to the VDAs results in even greater trapping of the drugs within tumors (Tozer et al. 2008).   xiI woouludkettha numn ysprsevutulh dD l.rdp vtddo AsMMst  uM el tseMl yepluetth psMydkMuvts Ddp mpselsp uklspMluluet Dt.uo ypsMM.ps cfgb, dvMspAso uk Mdtuo l.rdpM cTd.Knsp anti-v xLLIH zs. ant i-v iIII,a eko mpselsp fgb reh rejs l.rdp vtd do AsMMstM rdps A.tkspevts ld KdtteyMs uk psMydkMs ld -:wM cBdEsp anti-v iIIxH BdEsp anti-v iIIF,S  fk D.l.ps Ml.ousM ul Wd.to vs Aet.evts ld ph eko .Ms etlspkels :GJCqRf lddtM M.Kn eM numnsp rdtsK.tep Wsumnl KdklpeMl emsklM ld oslspruks rdps MysKuDuKetth Wnslnsp ul uM vtddo DtdW dp AeMK.tep ysprsevutulh lnel Kek ypsouKl Ddp BbN MskMuluAulhS :GJCqRf uM dD yepluK.tepth numn Aet.s o.s ld ulM ydlskluet ld vs eyytuso eM e ypsouKldp dD psMydkMs uk KtukuKet yelusklMS woouludket sDDdplM Kd.to tddj el rdps l.rd.p rdostM Wuln ouDDspskluet MskMuluAulh ld BbNa sYerukukm Wnslnsp lnsps eps nuMldtdmuKet Dsel.psM M.Kn eM lns ypsMskKs dD  !qw lnel KdkMuMlsklth Kdppstels Wuln MskMuluAulh ld BbNS B.rd.p vtddo AsMMst repjspM dD ynskdlhys eko D.kKludka Wnuts kdl ypsAud.Mth sAet.elso eM pstsAekl Ddp BbN dp dlnsp nhydYuK KhldldYukMa reh ypdAuos .MsD.t ukouKeldpM dD l.rd.p MskMuluAulh ld lnsMs op.mMS foskluDuKeludk eko KnepeKlspuEeludk dD ypsouKldpM dD psMydkMs ld ekluCKekKsp lnspeyh eps dD mpsel Aet.sS &nsk eKK.pels ypsouKldpM dD psMydkMs ld lpselrskl eps eA utevtsa yelusklM reh vs rdps KepsD.tth MstsKlso Ddp lpselrsklM lnel Kd.to vs vsksDuKuet eko Kek eAduo Dp.ultsMM ldYuK dp sYyskMuAs lnspeyusM lnel lnsh Wd.to kdl vsksDul DpdrS  BbN neM mpselsp ldYuKulh ld nhydYuK KsttM lnek dYhmskelso KsttM sc usnlr eko ulM sctusur ldYuKulh neM vssk eMM.rso ld vs dD e Murutep rsKnekuMr cMss MsKludk xSi,S JAuoskKs Ddp e nhydYuK KhldldYuK rsKnekuMr dD BbN sctusur neM ypsAud.Mth vssk M.mmsMlso ld Kdrs Dpdr Ml.ousM Dukoukm osKpseMso KtdkdmskuK M.pAuAet uk l.rdpM lpselso Wuln BbN uk Kdrvukeludk Wuln peoudlnspeyhS Reoudlnspeyh juttM dYhmskelso KsttM eko eoouludket Kstt jutt vh e Kdrytsrskleph emskl uM lnd.mnl ld neAs sDDsKluAsth lepmslso lns nhydYuK Kstt DpeKludka dp ld neAs peoudMskMuluEso KsttM cNsrek anti-v xLFF,S zdW dYhmsk vpselnukm neM etMd vssk MndWk ld MskMuluEs l.rdpM ld lns ekluCKekKsp eKluAulh dD BbN cqukKnukldk eko TpdWk xLLi,S OdWsAspa lnsMs Ml.ousM Wsps kdl evts ld MysKuDuKetth KdkDupr sctusur lnel BbN juttM nhydYuK l.rdp KsttM eko lnsh ouo kdl eMMsMM Ddp ek ekluCAeMK.tep sDDsKlS :ele Dpdr lnuM lnsMuM M.mmsMlM lnel yddp dYhmskeludk dD lns l.rd.p AeMK.tel.ps MskMuluEsM l.rd.pM ld lns icns uiye.-il  sDDsKlM dD BbNS wM MndWk uk Gneylsp Pa skodlnstuet   121 tube structures in vitro showed damage in response to clinically relevant TPZ concentrations at 2% oxygen, showing that the endothelium is sensitive to TPZ damage at intermediate oxygen levels (Figure 3.9). Induction of moderate anemia or low oxygen breathing increased the magnitude and frequency of vascular dysfunction in sensitive HCT116 tumours and low oxygen breathing sensitized otherwise resistant HT29 tumours to catastrophic central vascular damage in response to TPZ (Figure 3.8, Table 3.2). Further evidence from Chapter 4 showed that both excess NO and decreased NO resulted in somewhat greater HCT116 tumour hypoxia, and both strategies resulted in enhanced anti-vascular activity of TPZ (Figures 4.1 - 4.3, Table 4.1). In addition, the suggestion that poor tumour vascular function (further described above in section 1.3) predicts for tumour sensitivity to TPZ-mediated vascular dysfunction may be indirect support for lower tumour oxygenation as a predictor or sensitizer to the effect.  Intravascular hypoxia was not directly measured in studies in this thesis, but could provide valuable data and may be useful in further investigations regarding TPZ mediated vascular dysfunction. Pimonidazole labeling was used in this thesis as an indicator of tumour hypoxia, but pimonidazole has been suggested to be most effective at labeling cells at < 10 mmHg (1 % O2) (Gross et al. 1995; Arteel et al. 1998) and therefore may not be able to reflect tumour or blood oxygenation at intermediate oxygen levels (> 1 % O2). It would be useful to know whether microregional blood oxygenation varies in a pattern that correlates with TPZ sensitivity in HCT116 tumours, particularly with respect to intra-tumour heterogeneity. This would help confirm whether tumour blood oxygenation is primarily responsible for tumour sensitivity to the anti-vascular effects of TPZ. Other reports of intra- and inter-tumour heterogeneity with respect to blood vessel oxygenation suggest a consistent pattern of lower oxygenation in the central regions of tumours relative to the more oxygenated peripheral margins. This pattern reflects the pattern of TPZ-mediated vascular dysfunction, providing additional evidence that the anti-vascular effects of TPZ in vivo are related to its status as a hypoxic cytotoxin. Microregional variation can be amplified in the presence of low or sluggish blood flow, as M. Dewhirst et al. (1999) have shown that fluctuating blood flow can induce a disproportionate increase in regional hypoxia in areas of low vascular density (Kimura et al. 1996; Dewhirst et al. 1999; Lanzen et al. 2006). The microregional pattern of tumour hypoxia suggests a possible mechanism for TPZ selectivity   133 Asd ecr irbedao drv,sbt sA ehushdtm nhdecrd ecrsdre,iao tehg,rt ly przc,dte et al. f3..-( attrttrg ecr Bserbe,ao ,uBaie sA ,berdurg,aer sd Aohiehae,bv skyvrb sb l,sdrghie,2r avrbet0 abg ecr ahecsdt thvvrte ecae ec,t crerdsvrbr sht ehushd u,idsrb2,dsburbeao Araehdr uay lr Bade,aooy drtBsbt,lor Asd Bssd cyBsk,i iyesesk,b Brbredae,sb ecdshvc tso,g ehushd e,tthr abg Asd ecr abe,52atihoad rAAriet sA )VD in vivo fTwdgrbat5Ia2,a et al. 3..-(m   xhBBsde Asd a cyBsk,a5attsi,aerg Bdrg,iesd Asd ecr io,b,iao thiirtt sA )VD edraeurbe isurt Adsu 4,tic,b et al. f3..;(0 zcrdr Bae,rbet z,ec vdraerd 1Pn5Aohsdsu,tsb,gaZsor l,bg,bv grerierg ht,bv VM) zrdr usdr o,Xroy es lrbrA,e Adsu )VD ecab Bae,rbet z,ec bsb5cyBsk,i Bd,uady ehushdtm 6aby sA ecr io,b,iao ed,aot BrdAsdurg z,ec )VD ca2r lrrb hbalor es Bdr5 tidrrb Bae,rbe ehushdt Asd cyBsk,am Sgrbe,Ay,bv trbt,e,2r ehushdt Asd )VD edraeurbe latrg sb cyBsk,i teaeht eyB,iaooy atthurt ecae vdraerd ehushd cyBsk,a z,oo drthoe ,b ,bidratrg )VD5 urg,aerg cyBsk,i iyesesk,i,ey es  hypoxic tumour cellsm Vdr2,sht tehg,rt tcsz,bv ,uBds2rg abe,5iabird rAAriet ghr es osz skyvrb ldraec,bv g,g bse attrtt ecr uricab,tu Asd ecr rbcabirg )VD aie,2,ey abg atthurg ecr hB es  1.K vdraerd iosbsvrb,i iroo X,oo zat ghr es ,bidratrg iyesesk,i,ey sA )VD es ehushd iroot f6,bic,besb abg 9dszb 1CC38 6,bic,besb abg 9dszb 1CC3(m SA ehushd lossg skyvrbae,sb cat ab ,uBaie sb ehushd trbt,e,2,ey es )VD0 at thvvrterg ly Bdr5io,b,iao tehg,rt ,b TcaBerd L0 ,e uay lr lrbrA,i,ao es ossX ae ecr lossg skyvrbae,sb sA Bae,rbet ,b ed,aot z,ec )VDm Vae,rbet z,ec abru,a sd osz lossg BH 3 or2rot Asd secrd dratsbt uay ca2r vdraerd trbt,e,2,ey es )VD0 abg grerie,sb sA Bae,rbe lossg skyvrbae,sb uay a,g ,b trorie,bv Bae,rbet o,Xroy es lr uste  drtBsbt,2r es edraeurbem xidrrb,bv Asd abru,a sd lossg skyvrb iaddy,bv iaBai,ey zshog BrdcaBt lr a usdr rAArie,2r abg usdr Bdaie,iao tidrrb Asd )VD Bae,rbet0 ,b o,rh sA ecr sAerb usdr isuBo,iaerg aBBdsaic sA ,uav,bv Asd ehushd cyBsk,am nhehdr tehg,rt abg io,b,iao ed,aot z,ec )VD sd secrd cyBsk,i iyesesk,bt uay ,berbe,sbaooy attrtt lossg skyvrbae,sb at a Bstt ,lor Bdrg,iesd Asd drtBsbtrm Sb agg,e,sb0 dredstBrie,2r abaoytrt sA isbiohgrg io,b,iao ed,ao gaea uay lr Bdaie,iaom )d,aot attrtt,bv Bae,rbe drtBsbtr es )VD eyB,iaooy r2aohaer lossg cruaesid,e ,b sdgr d es ro,u,baer Bae,rbet zcs adr grrurg ess abru,i es lr agu,eerg es ecr ed,aotm Se uay ecrdrAsdr lr Bstt,lor es gs dredstBrie,2r isddroae,2r r2aohae,sbt sA ecrtr tehg,rt es ,b2rte,vaer zcrecrd Bdr5edraeurbe Bae,rbe cruaesid,e isddroaert z,ec ehushd drtBsbtrt es )VD edraeurbem   123 In addition to tumour blood vessels and tumour blood oxygenation, the impact of nitric oxide (NO) was examined as a potential feature of the microenvironment that may impact tumour sensitivity to TPZ. Data presented in Chapter 4 suggests that decreased NO availability may sensitize tumour blood vessels to the anti-vascular effects of TPZ. Evidence from the literature demonstrates that TPZ competitively inhibits NOS to reduce its production of NO. TPZ is also bioactivated by NOS. When TPZ is administered in combination with a non-specific NOS inhibitor such as L-NNA, the vascular dysfunction effects of TPZ are enhanced in HCT116 and SCCVII  tumour models (Figures 4.1, 4.6). This enhancement is particularly evident at lower 40 mg/kg doses of TPZ that do not typically cause vascular dysfunction, but that can result in catastrophic anti-vascular effects when combined with L-NNA. The combination results in reduced tumour growth in HCT116 xenografts (Figure 4.5). However, NOS inhibition in combination with TPZ was not able to cause central vascular dysfunction in the resistant HT29 model. Staining for the endothelial isoform of NOS, eNOS, in HT29 colorectal xenografts showed that a lower proportion of HT29 tumour vasculature was positive for eNOS staining relative to HCT116 tumours (Figure 4.9). This data suggests that perhaps TPZ is bioactivated by NOS at greater frequency in the endothelium of HCT116 tumours, or that inhibiting the production of NO may have a more dramatic effect in HCT116 tumours to result in their greater sensitivity relative to HT29 tumours. Interestingly, inhibition of NOS by L-NNA alone does produce some moderate vascular dysfunction, particularly in SCCVII tumours (Figure 4.6), but appears to be less potent than the effects seen with TPZ. Low doses of L-NNA have resulted in growth delay effects in SCCVII tumours in other laboratories (Horsman et al. 1996). Perhaps NOS inhibition can cause some vascular dysfunction and TPZ is more potent than non-specific NOS inhibitors such as L-NNA, but when combined the TPZ and NOS inhibition can produce stronger anti-cancer effects. It is unclear if it is bioactivation of TPZ by NOS or reduced levels of NO in the tumour vasculature, or both effects in combination, that is responsible for the anti-vascular activity of TPZ in tumours. Provision of excess NO in combination with TPZ did not protect tumour vasculature from the anti-vascular effects of TPZ (Figure 4.3). This lack of protection may not reflect the desired control conditions where excess NO provides the opposite of   124 NOS inhibition. Instead, excess NO may have an alternate effect where systemic blood pressure drops so much that blood flow is actually removed from the tumour in contrast to extra blood flow elsewhere (Shan et al. 1997). Therefore, in the tumour, excess NO actually results in increased hypoxia, which can enhance the effects of TPZ. This may suggest that hypoxia is a more potent sensitizer than inhibition of NOS, which is also supported by the finding that in resistant HT29 tumours low oxygen breathing produced a sensitizing effect whereas inhibition of NOS in combination with TPZ was unable to have this effect.  Future studies investigating other hypoxic cytotoxins may find benefits in combinations with NOS inhibitors and should be investigated for anti-vascular effects as well as anti-cancer effects. It would also be interesting to look at isoform-specific inhibitors of NOS and their individual abilities to effect vascular dysfunction in tumours alone or in combination with TPZ. Perhaps analogues of TPZ or other hypoxic cytotoxins could be assessed for their ability to be reduced by NOS, their ability to inhibit NOS production of NO and their ability to effect central vascular dysfunction. These data would help select drugs that may have greater efficacy.   125 1.In troduci roin While a clear mechanism for activity of TPZ in the tumour vasculature remains elusive, the work described in this thesis has provided evidence that TPZ mediates anti- vascular effects in specific microregions of tumours. The anti-vascular effects of TPZ are detectable using DCE-MRI, and some tumour models are inherently resistant to its effects. Modulation of blood oxygenation levels had the most potent impact on conferring sensitivity to otherwise resistant tumours, and tumour vascular function and pre-treatment hypoxia may be useful predictors for response to TPZ. Combining TPZ with a NOS inhibitor enhances tumour vascular dysfunction, which translates to reduced tumour growth in HCT116 tumour xenografts. The direct impacts of microenvironmental features such as hypoxia and vascular phenotype on tumour sensitivity to the anti-vascular effects of TPZ are difficult to prove with certainty. Experimental data confirms the relevance and impact that tumour hypoxia may have with respect to the anti-vascular effects of TPZ. Vulnerable, destabilized or hypoxic vasculature that is prevalent in solid tumours may be responsible for conferring the selective activity of TPZ and for the inter- and intra-tumour heterogeneity of response to TPZ. Further work with TPZ should focus on determining whether anti-vascular effects occur in clinical tumours. Assessment of effective pre-treatment predictors of sensitivity would be of high value. These suggestions also apply to other bioreductive hypoxic cytotoxins that have not previously been investigated for anti-vascular activity.   Ultimately the target cell for TPZ in mediating vascular dysfunction remains to be identified. TPZ may inhibit NOS production by tumour cells, or may have direct cytotoxic effects to intermediately oxygenated, or intermittently hypoxic tumour cells located proximal to the tumour vasculature. Damage to these cells may result in vascular damage sufficient to cause the losses in perfusion seen in these studies. Alternatively the target cell for damage by TPZ may be the endothelial cells or vascular support cells lining intermediately oxygenated, or intermittently perfused tumour blood vessels. Cellular damage and stress may be a consequence of mitochondrial dysfunction, the presence of reactive oxygen species and oxidative stress due to redox cycling of TPZ and production of the byproduct superoxide, or direct damage sustained in response to reduced and bioactivated TPZ. Further work   126 investigating the mechanism for anti-vascular activity may focus around identifying the target cell and the chemical species responsible for damage.  Based on the studies described in this thesis, it is recommended that TPZ treatment in the clinic should endeavour to assess vascular function prior and post-treatment using MRI or another indicator. 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"Rat tumor response to the vascular-disrupting agent 5,6-dimethylxanthenone-4-acetic acid as measured by dynamic contrast-enhanced magnetic resonance imaging, plasma 5-hydroxyindoleacetic acid levels, and tumor necrosis." Neoplasia 8(3): 199-206. Minchinton, A. I. and J. M. Brown (1992). "Enhancement of the cytotoxicity of SR 4233 to normal and malignant tissues by hypoxic breathing." Br J Cancer 66(6): 1053-8. Minchinton, A. I. and J. M. Brown (1992). "Improving the effectiveness of the bioreductive antitumor agent SR 4233 by induced hypoxia." Adv Exp Med Biol 317: 177-81. O'Connor, J. P., A. Jackson, et al. (2007). "DCE-MRI biomarkers in the clinical evaluation of antiangiogenic and vascular disrupting agents." Br J Cancer 96(2): 189-95. Rischin, D., R. J. Hicks, et al. (2006). "Prognostic significance of [18F]-misonidazole positron emission tomography-detected tumor hypoxia in patients with advanced head and neck cancer randomly assigned to chemoradiation with or without tirapazamine: a substudy of Trans-Tasman Radiation Oncology Group Study 98.02." J Clin Oncol 24(13): 2098-104. Robinson, S. P., D. J. McIntyre, et al. (2003). "Tumour dose response to the antivascular agent ZD6126 assessed by magnetic resonance imaging." Br J Cancer 88(10): 1592-7. Shan, S. Q., G. L. Rosner, et al. (1997). "Effects of diethylamine/nitric oxide on blood perfusion and oxygenation in the R3230Ac mammary carcinoma." Br J Cancer 76(4): 429-37. Tofts, P. S., G. Brix, et al. (1999). "Estimating kinetic parameters from dynamic contrast-enhanced T(1)-weighted MRI of a diffusable tracer: standardized quantities and symbols." Journal of magnetic resonance imaging : JMRI 10(3): 223-32. Tozer, G. M., C. Kanthou, et al. (2008). "Tumour vascular disrupting agents: combating treatment resistance." The British journal of radiology 81 Spec No 1: S12-20. Tozer, G. M., V. E. Prise, et al. (2001). "Mechanisms associated with tumor vascular shut-down induced by combretastatin A-4 phosphate: intravital microscopy and measurement of vascular permeability." Cancer Res 61(17): 6413-22. Tredan, O., A. Garbens, et al. (2009). "The Hypoxia-Activated ProDrug AQ4N Penetrates Deeply in Tumor Tissues and Complements the Limited Distribution of Mitoxantrone." Cancer Res 69(3): 940-947. Zeman, E. M., V. K. Hirst, et al. (1988). "Enhancement of radiation-induced tumor cell killing by the hypoxic cell toxin SR 4233." Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology 12(3): 209-18.    129  APPENDIX  A. List of Publications - Manuscripts   *Denotes publications that are included as part of the thesis.  *Baker JHE, Kyle AH, Balbirnie A, Cran J, Gabriel E and Minchinton AI. Inhibition of nitric oxide synthase enhances tirapazamine-mediated vascular dysfunction in pre-clinical solid tumours. In preparation for submission for publication.  *Baker JHE, Kyle AH, Bartels KL, Flanagan EJ, Methot SP and Minchinton AI. Decreased blood oxygen tension sensitizes tumors to the anti-vascular effects of tirapazamine. Submitted for publication October 27, 2009.  Wong LY, Franssen Y, Qadir M, Baker JHE, Kapanen A, Masin D, Minchinton AI, Gorski S, Gelmon K, Dr. Bally M and Dragowska V. Gefinitib and RAD001 in combination enhance cytostasis and impede tumor growth in trastuzumab sensitive and resistant HER2 overexpressing breast cancers. Submitted for publication October 2, 2009.  *Bains LJ*, Baker JHE*, Kyle AK, Minchinton AI and Reinsberg ST.  (2009).  Detecting vascular-targeting effects of hypoxic cytotoxin tirapazamine in tumour xenografts using MRI.  Radiation Oncology Biology Physics, 74 957-965. *Co-first authors.  Baker JHE, Lam J, Kyle AH, Sy JT, Oliver T, Co S, Dragowska W, Ramsay E, Anantha M, Ruth T, Adam M, Yung A, Kozlowski P, Minchinton AI, Ng S, Bally M, Yapp DTT.  (2008). Irinophore CTM, a novel nano-formulation of irinotecan, alters tumor vascular function and enhances the distribution of 5-FU and doxorubicin.  Clinical Cancer Research, 14 7260-7271.  Baker JHE, Lindquist KE, Huxham LAH, Kyle AK, Sy JT and Minchinton AI.  (2008).  Direct visualization of heterogeneous distribution of trastuzumab in human epidermal growth factor receptor type 2 overexpressing xenografts.  Clinical Cancer Research, 14 2171-2179.  Huxham LAH, Kyle AK, Baker JHE, McNicol KL and Minchinton AI.  (2008).  Exploring vascular dysfunction of tirapazamine. Microvascular Research, 75 247-255.  Baker JHE, Huxham LAH, Kyle AK, Lam K and Minchinton AI.  (2006).  Vascular - specific quantification in an in vivo Matrigel chamber angiogenesis assay.  Microvascular Research,  71 69-75.  Huxham LAH, Kyle AK, Baker JHE, McNicol KL and Minchinton AI.  (2006).  Tirapazamine causes vascular dysfunction. Radiotherapy and Oncology, 78 138-145.   ixS    MAalp hsm otllpucle nsm m cBprlp dUm 5-bb nom 5A2ey-TT UZm zACyl Um dp-Tl +4m P-bTAF f BF2 +AgFAI-C cV ODSSERV fFXpBI-XBb -tBe-Fe A, X6tA6p IBTC6bBX6pl FlXNApyT 6T-Fe t6bX-u(1AXAF ,b6AplTClFCl  t-CpATCA(HV  Advanced Drug Delivery Reviewsm E) ixEu iEDV  5631Bt zom GHbl o5m m wHy-bC16y zG BF2 s-FC1-FXAF ofV ODSSvRV  s-CpAple-AFBb l,,lCXT A, eltC-XBr-Fl -F 5LMuiiq 3lFAepB,XTV  Cancer Researchm qv qEx)u qEviV  GHbl o5m 5631Bt zom m c6pTXAF 54 BF2 s-FC1-FXAF ofV ODSSxRV  M6tA6p 2-TXp-r6X-AF A, rpAtA2lA3H6p-2-Fl bBrlbl2 ClbbT -T TXpAFebH 2ATl 2l(lF2lFXV Cancer Researchm qx E)S)uE)iiV    131 List of Publications - Selected Meeting Abstracts (presented by Baker JHE)  *Denotes presented abstracts that are relevant to the thesis.  *Targeting the tumour microenvironment: anti-vascular effects of the hypoxic cytotoxin tirapazamine. Baker JHE, Kyle AH, Bartels KL Flannagan EJ, Methot SP, Balbirnie A, Cran J, Gabriel E, Bains LJ, Reinsberg SA and Minchinton AI. Presented as a poster, EORTC-AACR-NCI Annual Meeting, Boston MA, 2009.  *Investigating the vascular targeting effects of the bioreductive radiosensitizer tirapazamine. Baker JHE, Kyle AH, Flannagan E, Methot S, Balbirnie A and Minchinton AI. Presented as a poster, Radiation Research Annual Meeting, Savannah GA, 2009.  *Mechanisms associated with vascular targeting in cancer mediated by tirapazamine Baker JHE, Kyle AH, Flannagan E, Methot S, Balbirnie A and Minchinton AI. Presented as a poster, Keystone Dissecting the Vasculature Meeting, Vancouver, 2009.  *Lower tumour perfusion and permeability in tumours with stronger sensitivity to vascular targeting effects of tirapazamine  Baker JHE, Bains L, Reinsberg ST, Kyle AH, Methot S, Flannagan E and Minchinton AI. Presented as a talk, Tumour Microenvironment Meeting, Miami, 2008.  *Differential sensitivity to vascular targeting effects of tirapazamine in HCT116 and HT29 xenografts  Baker JHE, Bains L, Huxham LAH, Kyle AH, Lindquist KE, Reinsberg S, Minchinton AI. Presented as a talk, Vascular Targeted Therapies in Oncology (European Society of Hematology), France, 2007.  Liposomal CPT-11 changes the vasculature and oxygenation status of HT29 tumours Baker JHE, Sy J, Co S, Yung A, Dragowska W, Ruth T, Adam M, Ng S, Minchinton A, Kozlowski P, Ramsay E, Bally M, Yapp D.   Poster, Tumour Microenvironment:  Progression, Therapy and Prevention, Florence,  2007.  Distribution of Herceptin in tumour xenografts overexpressing HER2/neu. Baker JHE, Lindquist KE, Huxham LAH, Kyle AH, Sy J, Minchinton AI.   Poster, AACR Annual Meeting, Washington DC, 2006.  Characterization of in vivo anti-angiogenic activity using the Matrigel Chamber Angiogenesis Assay and Tumour Blood Vessel Mapping. Baker JHE, Huxham LA, Kyle AH, Lam K, Minchinton AI.   Poster, Pathobiology of Cancer Workshop hosted by AACR, Aspen, 2006.  A quantitative in vivo Matrigel chamber angiogenesis assay:  Anti-angiogenic activity of thalidomide and LL/M27 primary tumours. Baker JHE, Huxham LA, Kyle AH, Lam K, Minchinton AI.   Presented as a talk, Tumour Microenvironment Meeting, Oxford, 2005.    132 B. Biohazard Approval Certificate: B06-0187     133 1.5Hypoxi51xc 51 cvpapsxv u5Hltrenmn5 5 5 5 5  132 D. Animal Care Certificate: A07-0404  

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