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Fluorescence imaging of tumour hypoxia in xenograft tumour models Sobhanifar, Solmaz 2004

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FLUORESCENCE IMAGING OF TUMOUR HYPOXIA IN XENOGRAFT TUMOUR MODELS  by  SOLMAZ SOBHANlFAR  BSc. (Biochemistry), The University of British Columbia, 2002  A THESIS SUBMITTED IN PARTIAL F U L F I L M E N T OF T H E REQUIREMENTS FOR T H E D E G R E E OF  M A S T E R OF SCIENCE  in  T H E F A C U L T Y OF G R A D U A T E STUDIES  (Department of Experimental Medicine)  T H E UNIVERSITY OF BRITISH C O L U M B I A December 2004  © Solmaz Sobhanifar, 2004  ABSTRACT  Hypoxic cells in solid tumours are known to resist radiotherapy as well as forms of chemotherapy.  Hypoxia is also believed to promote tumour aggressiveness and  metastasis. It is therefore important to develop a practical method to identify hypoxic tumour cells in order to assess which patients could benefit from hypoxic cell-targeted therapies. The goal of this study was to develop an immunohistochemical approach to the detection of hypoxia in xenograft models that may be applied to clinical samples. The hypotheses to be tested were that areas of chronic or transient hypoxia could be distinguished based on specific patterns of hypoxia markers, and that HEF-la could be comparable as a hypoxia marker to pimonidazole. The objectives were: 1) to characterise the kinetics of development under anoxia and loss upon reoxygenation of the exogenous hypoxia marker pimonidazole and the endogenous hypoxia marker HIF-la, 2) to use quantitative fluorescence image analysis to compare patterns of pimonidazole binding and H I F - l a expression in WiDr, SiHa and M006 human tumour xenografts, and to measure marker response under various oxygen- breathing conditions, and 3) to compare hypoxia marker patterns in xenograft tumours with patterns observed in cervical cancer biopsies. H I F - l a and pimonidazole colocalised in regions distant from blood vessels; perinecrotic regions however showed pimonidazole binding but no H I F - l a expression. This lack was not a result of anoxia but likely a result of glucose and serum starvation. With time after pimonidazole administration, the extent of colocalisation with H I F - l a  was reduced from 60% at 90min to 7% at 48hr, consistent with the movement of pimonidazole-labelled cells into necrosis. Patterns of H I F - l a and pimonidazole binding in clinical samples confirmed the dissociation between the markers when pimonidazole was administered 24hr before tumour excision. Overall our xenograft results indicated H I F - l a to be a reliable indicator of tumour hypoxia; however, it over-estimated radiobiological hypoxia (<1% O2) and was not expressed in the most hypoxic regions of tumours.  The kinetics of marker development and loss, and occasional regions  expressing one but not the other marker indicated that H I F - l a in combination with pimonidazole has the potential to measure the presence of transient hypoxia.  -111-  T A B L E OF CONTENTS  Title Page Abstract Table of Contents List of Abbreviations List of Figures List of Tables Acknowledgements  1:  i ii iv vi vii viii ix  INTRODUCTION  1  A: Hypothesis and Objectives B: Background BI: The Tumour Microenvironment B2: Tumour Hypoxia B3: Tumour Pathophysiology B4: Hypoxia and Response to Treatment B5: Hypoxia Directed Therapies B6: Measurement of Hypoxia B7: Signalling Pathways that Influence H l F - l a Expression B8: Using Hypoxia Markers in Combination  2:  MATERIALS & METHODS  32  A: Cell Lines and Culture Conditions B: Xenograft Tumour Models C: Administration of Chemicals D: Gas-Breathing Procedures E: Preparation of Frozen Sections F: Immunohistochemsitry G: Image Acquisition and Analysis H: Western Blot Analysis I: Flow Cytometric Analysis J: Graphical and Statistical Analysis  3:  1 2 2 4 9 15 17 19 25 30  RESULTS  33 33 33 34 35 35 37 39 40 40  42  A: Marker Kinetics A l : H I F - l a Build-up Under Anoxia A2: H I F - l a Half-life Upon Reoxygenation A3: Pimonidazole Binding Under Anoxia A4: Pimonidazole Half-life Upon Reoxygenation B: Marker Colocalisation Studies BI: Marker Colocalisation in Xenograft Tumours B2: Atypical Marker Patterns  -iv-  42 43 43 46 46 49 49 52  C: H I F - l a Nutrient-dependency and Oxygen Requirements CI: In Vitro Oxygen and Nutrient-dependence C2: Reoxygenation of Perinecrotic Cells in Xenografts D: Oxygen Modulation of Xenografts via Gas-breathing E: Hypoxic Cell Half-life in Xenograft Tumours F: Comparison of Patient Biopsies and Xenograft Models  4:  DISCUSSION A: B: C: D:  69  Marker Kinetics Marker Colocalisation Response to Oxygen Modulation HIF-la: Oxygen and Nutrient Effects  E: Hypoxic Cell Half-life and Relation with Patient Biopsies  5:  54 55 56 58 59 67  69 70 72 74 76  CONCLUSION  80  Bibliography  81  -v-  LIST OF ABBREVIATONS  ARCON ARNT bHLH/PAS BrdUrd BOLD  Accelerated radiotherapy with carbogen and nicotinamide aryl hydrocarbon receptor nuclear translocator Basic-helix-loop-helix/Per-ARNT-Sim Bromodeoxyuridine Blood oxygen diffusion imaging  Carbogen  95% 0 + 5% C 0  CA CD31 CDK CoCl DAPI FBS GLUT HIF-1 Hoechst 33342  Carbonic anhydrase Platelet endothelial cell adhesion molecule Cyclin-dependent kinase Cobalt chloride 6'-diamidino-2-phenylindol; nuclear stain Fetal Bovine Serum Glucose transporter hypoxia inducible factor 1 2'-(4-ethoxyphenyl)-5-(4-methyl-l-piperazinyl)-2,5'-bi-lHbenaimidazole trihydrochloride Hypoxia response element intraperitoneal intravenous Mitogen-activated protein kinase Minimum essential medium Microvessel density Non-obese diabetic/severe-combined immunodeficient Formulation of water-soluble glycols and resin Oxygen-dependent degradation domain Phosphate buffered saline Positron emission tomography Phosphatidylinositol 3 kinase l-[(2-hydroxy-3piperidinyl)propyl]-2-nitroimidazole Prolylhydroxylase Oxygen partial pressure PBS containing 1 % B S A and 0.1 % Tween 20 Retinoblastoma protein severe combined immunodeficient Single photon emission computed tomography Tirapazamine Vascular endothelial growth factor Von-Hippel-Lindau  2  HRE i.p. i.v. MAPK MEM MVD NOD/SCID OCT ODD PBS PET PI3K Pimonidazole PH p0 PTN Rb SCID SPECT TPZ VEGF VHL 2  2  2  -vi-  LIST O F F I G U R E S Figure 1: Oxygen-dependent H I F - l a regulation  8  Figure 2: Oxygen-dependency of pimonidazole  23  Figure 3: Signalling pathways in H I F - l a regulation  29  Figure 4: Flow cytometric analysis  39  Figure 5: Stabilisation of H I F - l a under anoxia  43  Figure 6: Loss of H I F - l a after reoxygenation  44  Figure 7: Formation of pimonidazole adducts under anoxia  46  Figure 8A: Loss of pimonidazole adducts after reoxygenation  47  Figure 8B: Cell doubling after reoxygenation  47  Figure 9: Distribution of H I F - l a and pimonidazole in xenograft tumours  49  Figure 10: Colocalisation of H I F - l a and pimonidazole in xenograft tumours  50  Figure 11: Atypical marker patterns in xenograft tumours  52  Figure 12: Effect of oxygen on H I F - l a protein level  54  Figure 13: The effect of glucose and serum on H I F - l a protein level  55  Figure 14: Reoxygenation of distal cells in SiHa xenograft tumours  56  Figure 15: Oxygen modulation of SiHa xenograft tumours  59  Figure 16: Effect of oxygen modulation on percent area staining in xenografts  60  Figure 17: Effect of oxygen modulation of marker colocalisation in xenografts  61  Figure 18: Changes in colocalisation with time after pimonidazole administration  63  Figure 19: Atypical marker patterns with time after pimonidazole administration  64  Figure 20: Hypoxic cell half-life in SiHa xenograft tumours  65  -vii-  LIST O F T A B L E S Table 1: H I F - l a correlation  26  Table 2: List of antibodies  35  ACKNOWLEDGEMENTS First and foremost I would like to thank my wonderful supervisor, Dr. Peggy L . Olive for allowing me the opportunity to work in her laboratory, and for imparting to me her keen interest and endless curiosity, for which I am ever grateful. Her great expertise in the field and generosity with her time was a great benefit, without which this project would not have been possible. I am also very grateful to the members of my laboratory: Susan McPhail for seasoning me on the microscope, Laura Sinnott for her patience in teaching me how to handle and work with mice, Denise McDougle for her invaluable help with flow cytometric analyses, Eric Chu for his help with experiments when more than two hands were necessary, and Bojana Jankovic for helping me interpret my numbers. I also wish to thank Dr. Christina Aquina-Parsons for providing me with clinical biopsies, the Durand, Minchinton, and Karsan laboratories for their valued assistance and for allowing me the use of their equipment, and Will Cottingham for her administrative support. I furthermore thank the members of my supervisory committee, Dr. Ralph Durand, Dr. Vincent Duronio, and Dr. Paul Rennie for their guidance and direction. Lastly, I would like to thank my family for enduring me, and of course for giving me much needed support, and friends for distracting me and thus keeping me functional.  -ix-  1  INTRODUCTION  1A  HYPOTHESIS AND OBJECTIVES The overall goal of this study was to develop an immunohistochemical approach  to the detection of chronic and transient forms of hypoxia in xenograft models that may be applied to clinical samples. The hypotheses to be tested were: 1) areas of chronic or transient hypoxia can be distinguished based on specific patterns of hypoxia marker binding and 2) H I F - l a would show sufficient agreement with pimonidazole as a hypoxic marker in xenograft tumours. The objectives of this thesis were:  1. To characterise  the  kinetics  of development  under anoxia  and loss upon  reoxygenation of the exogenous hypoxia marker pimonidazole and the endogenous marker H I F - l a . 2. To quantify patterns of expression or binding of pimonidazole and H I F - l a in WiDr, SiHa and M006 human tumour xenografts, and to measure marker response under various oxygen conditions.  This included measurement of the degree of marker  colocalisation, and the ability of markers to identify regions of transient as well as chronic hypoxia. 3. To compare hypoxia marker patterns in xenograft tumours with patterns observed in cervical cancer patients.  IB  1B1  BACKGROUND  T H E TUMOUR MICROENVIRONMENT The tumour microenvironment is heterogeneous and often hostile, characterised  by low glucose concentrations, high lactate concentrations, low extracellular pH, and low oxygen tensions (1).  Focal necrosis and regions of hypoxia, as well as energy and  nutrient deficiencies are therefore characteristic of most advanced solid tumours. These features often apply selective pressure for cell populations with survival and growth advantages under severe conditions, leading to tumour progression and metastasis.  l B l a Metabolism The unfulfilled demand for oxygen in tumour cells is a natural consequence of the normal metabolic processes.  Oxygen is necessary in two important steps of glucose  metabolism: "the citric acid cycle" and "oxidative respiration".  Not surprisingly,  tumours often show a greater rate of metabolism than most normal tissue given that tumour cells undergo rapid growth, contain smaller, fewer, and often misshapen mitochondria, and have an almost five-fold increase in O2 consumption (2). It is this high metabolic demand that throws the tumour into a state of hypoxia.  In his influential  studies, Warburg (3) noted that tumours underwent similar metabolic processes as normal tissue. However, he also showed that tumours are able to function both with insufficient glucose levels as well as a compromised vascular supply.  lBlb  Vascular Architecture Although the oxygen partial pressure (p0 ) in normal tissue, depending on tissue2  type, ranges between 10-80mmHg, most tumours tend to have a median p 0 of less than 2  lOmmHg (4, 5). This difference is primarily the result of the abnormal vasculature that develops during tumour angiogenesis.  Tumour blood vessels are shown to have very  irregular vascular architecture whose features include blind ends, arterio-venous shunts, and high angle branching patterns. Furthermore, these vessels tend to be more "leaky" than those in normal tissue, given that they lack smooth muscle and enervation, and may contain an incomplete endothelial lining and basement membrane (6). A further cause of tumour hypoxia may be the phenomenon of "intravascular hypoxia", resulting from the relative lack of arteriolar input into tumours. In most normal tissue, the relative number of arteriolar vessels tends to be quite high. In fact there tends to be a redundancy in arteriolar supply, and the tissue volume supplied by a single arteriole is tightly regulated. However in tumours, the volume of tissue supplied by a single artery is often significantly greater, and the oxygen gradient across tumour cells extends >100-200um away from a single capillary (7).  The anatomical features of these relatively sparse  arterioles result in a steep longitudinal gradient leading to vasculature and tissue hypoxia (8). Measuring the intravascular p 0 in tumour vessels using microelectrodes, Dewhirst 2  et al. (9) were able to demonstrate that the overall average p 0 of tumour vessels was 2  near 20mmHg, and that 25% of tumour blood vessels had p 0 values of less than ImmHg 2  near the centre of the tumour mass, despite the fact that they contained flowing blood. They go further to show using window chamber tumours, that in Fischer-344 rats, the feeding oxygen concentration of arterioles averaged 32mmHg, whereas in arterioles of  healing tissue, this value was 50mmHg (10). Furthermore, it has been estimated that 89% of all tumour microvessels are plasma channels, which do not contain an appreciable red blood cell supply, possibly due to abnormal branching angles and altered rheology of red blood cells (11). Other groups have shown similar results in support of the existence of hypoxia in functional tumour vessels (12, 13).  1B2  TUMOUR HYPOXIA Given the increased metabolic demand of tumour cells in light of an irregular and  often inadequate vascular network, a heterogeneous microenvironment characterised by localised regions poor in oxygen and nutrients would seem the logical outcome.  Cells  have been defined as being modestly hypoxic (-2.5% O2), moderately hypoxic (-0.5% 0 ), and severely hypoxic (-0.1% 0 ) (14). 2  lB2a  2  Chronic vs. Acute Hypoxia Thomlinson and Gray (15) in 1955, were the first to report that some human  tumours grew as nodules within stroma containing blood vessels, and that tumour cells at a distance exceeding 180pm from blood vessels became necrotic. Later another model for tumour hypoxia was described by Tannock et al. (16) that contained a central blood vessel, bordered by a cord of viable tumour cells ranging from 60-120pm, followed at larger distances by necrosis.  Both such models provide for "chronic" or "diffusion  limited" hypoxia, which occurs as a result of tumour cells having outpaced the growth of their blood supply.  The diffusion distance of oxygen is influenced by several factors  including the rate of blood flow, the presence of intravascular hypoxia, and the rate of  oxygen consumption.  Surprisingly, hypoxia is also occasionally observed in close  proximity to the blood vessels (17, 18). This presents the possibility of a second form of hypoxia termed "acute", "transient" or "perfusion limited" hypoxia, which likely arises from transient and localised changes in vascular perfusion. Chaplin et al. (19, 20) were the first to demonstrate that changes in blood flow and blood oxygen were sufficient to influence radiation response in tumours over 20min intervals. (21),  Furthermore, Hill et al.  using a multi-channel laser Doppler system that can detect micro-regional  fluctuation in perfusion, have shown that transient micro-regional changes in erythrocyte flux are a common feature of both human and xenograft tumours.  Previously it was  assumed that the presence of transient hypoxia required total vascular stasis, and given that this event occurs less than 10% of the time (22, 23) as observed via micro-vessel flow in window chamber tumours, it was also believed that transient hypoxia was a rare event. Evidence suggests, however, that total vascular stasis is not a requirement for transient hypoxia, and that even a two-fold change in fluctuation of red blood-cell flux can reflect a change in vascular p 0 periods of transient hypoxia (24).  2  and could substantially subject tumour regions to  This effect is even more pronounced in the case of  poorly vascularised tumours. It does appear, therefore, that transient hypoxia is perhaps a more common phenomenon than previously assumed. Nevertheless, this categorisation of hypoxia still lends itself to the simplification of a very complex situation. Indeed, the abnormalities in tumour vasculature, as discussed above, lead to temporal shut-down of vessels, changes in oxygen and nutrient gradients in both transversal and longitudinal directions, as well as changes in the direction of blood flow (25).  These changes can  affect the status of both chronic and transient hypoxia, and can easily blur the boundary  between the two.  Although most of the evidence for transient hypoxia thus far comes  from murine models, there is growing evidence suggesting the existence of transient hypoxia in human tumours. These studies include the transient delivery of halogenated pyrimidines in human head and neck tumours (26) and implanted probes that directly measure changes in blood flow and oxygen (27).  lB2b Hypoxic Adaptation and Hypoxia Inducible Factor 1 Mammalian cells have developed adaptive systems that allow them to function under moderate and even severe hypoxia, which involve the expression of genes coding for proteins such as aldolase A and enolase a, lactate dehydrogenase, pyruvate kinase, and glucose transporter one (GLUT-1), all of which are responsible for the anaerobic production of A T P (28). growth  factor  (VEGF),  Hypoxic cells also secrete the angiogenic vascular endothelial which promotes  neovascularisation  (29,  30).  Several  transcription factors have been found to respond to hypoxic stress, such as A P - 1 , N F K B , and Hypoxia-inducible factor-1 (HIF-1), among which HIF-1 appears to be unique with respect to its function as a global regulator of oxygen homeostasis (31).  The Structure of HIF-1 HIF-1  is  a transcription factor for the  activation of genes involved in  erythropoiesis, angiogenesis, and cellular energy metabolism (32, 33).  The protein is a  heterodimer consisting of the HIF-1 a subunit and a constitutively expressed HIF-1 P subunit, known also as the aryl hydrocarbon receptor nuclear translocator (ARNT), both of which belong to a group of transcription factors termed basic-helix-loop-helix/PerA R N T - S i m (bHLH/PAS) proteins (34).  There exist at least two other members of the  HIF-a family, designated as HIF-2a (35) and HIF-3a (36). These HIFs show more cell type-specific expression patterns than HIF-la, although the patterns do partially overlap. HIF-2a has biochemical properties similar to HIF-la, including the ability to bind HIFip and to induce hypoxia-regulated gene expression. HIF-3a has several splice variants, one of which encodes an inhibitory PAS domain which is thought to negatively regulate hypoxia induced gene expression by competing with HIF-la for HIF-lp binding (37). The N-terminal domain of HIF-la contains a basic helix-loop-helix domain and a PAS domain that are essential for dimerisation with HIF-lp, as well as for binding to hypoxia response element (HRE) D N A sequences (38). The C-terminal portion of HIFla  contains  several regulatory domains responsible  for hypoxia-dependent gene  expression, including the T A D - N and T A D - C terminal transactivation domains, which bind general transcriptional coactivators such as CBP/p300, SRC-1 and TIF-2 (39). Nuclear localisation signals are present at both the N and C terminals and translocate HIF-la into the nucleus independent of dimerisation with HIF-ip (40).  A critical  component of the HIF-la protein is its oxygen-dependent degradation domain (ODD) which is involved in protein stability and causes, under normoxic conditions, the rapid degradation of the HIF-la protein through the ubiquitin-proteasomal pathway (41).  Oxygen-dependent Regulation ofHIF-1 a The oxygen dependency of HIF-1 is determined by the expression and activity of its HIF-la subunit, whose levels increase in response to decreases in cellular oxygen concentrations (42).  Under aerobic conditions, HIF-la is bound to the Von-Hippel-  Lindau (VHL) tumour suppressor, causing HIF-la to be ubiquitinated and rapidly degraded (43, 44, 45, 46).  However for this interaction to occur, HIF-la must be  Figure 2. Oxygen-dependent HIF-la regulation (47).  covalently modified to its hydroxylated form by an oxygen-requiring prolyl hydroxylase (PH) (48, 49, 50), and under hypoxic conditions, this modification no longer occurs. Furthermore, under hypoxia, HIF-1 a dissociates from heat shock protein Hsp90 (51), a molecular chaperone, and is translocated to the nucleus (52).  Nevertheless, to be fully  active, it requires suitable redox conditions (53), as well as interaction with coactivators, namely C R E B binding protein/p300 (CBP/p300), and steroid receptor coactivator-1 (SCR-1) (19). The subsequent increase in HIF-1 a then allows the protein to bind HREs, which are sequences found in the promoter regions of genes involved in the increased delivery of oxygen or metabolic adaptation to oxygen deprivation (54).  1B3  TUMOUR PATHOPHYSIOLOGY The progression of a malignant tumour is largely promoted by an increase in  neoplastic cell load, microenvironment-induced phenotypic changes in neoplastic and stromal cells, as well as genotypic changes and clonal selection (55).  Much evidence  suggests that hypoxia, in addition to adapting cells to a hostile tumour microenvironment, also promotes  genetic instability through point mutations,  deletions,  and gene  amplification. These events may arise from errors in D N A repair and/or replication due to impairment of the activity of enzymes such as topoisomerases, helicases, and ligases (56, 57), as well as through reperfusion injury and oxidative damage to the D N A (58). The resulting genetic instability consequently may lead to an increase in the number of genomic variants selected to survive and proliferate under low oxygen conditions. This in turn may result in the clonal selection of p53-deficient tumour cells, due to the complex interaction of the p53 oncogene with HIF-1, whose relationship is discussed  below. To this effect, Kim et al. (59) have shown that in cervical cancer, hypoxia greatly accelerates the selection pressure for tumours with a compromised apoptotic potential. In addition, hypoxia can contribute to tumour progression by causing upregulation of V E G F , a most potent promoter of neoangiogenesis (60).  Furthermore, hypoxia is  involved in a number of other major tumourigenic pathways mainly through the actions of HIF-1, some of which are described below.  lB3a  H I F - l a and Pathophysiology Under physiological hypoxia, HIF-la  acts as a sensor and leads to the  upregulation of hypoxia-inducible genes to adjust the cell to oxygen deprivation conditions and to attenuate the insult. Nevertheless in solid tumours, these benefits are largely lost, given that HIF-1 simply cannot respond to fully resolve hypoxic stress (61). Rapid tumour growth constantly produces new regions of hypoxia such that a uniform normoxic environment is never established. modifications often lead to increased pathogenesis.  Indeed, as described above,  such  HIF-1 contributes to the pathogenic  modulation of various processes leading to increased angiogenesis, local invasion, distant metastasis, and alteration of apoptotic programs. Metabolism HIF-1 has been shown to upregulate various genes involved in metabolic processes, inducing a shift from oxidative to anaerobic glycolytic pathways. This switch is crucial for cell survival, providing an alternative energy pathway during oxygen shortages. Expression studies performed in HIF-la deficient embryonic stem cells have shown decreased expression of 13 different genes encoding both glucose transporters and  -10-  glycolytic enzymes, and HIF-1 has also been shown to upregulate fructose-2,6-bisphosphate, a regulator of glycolytic flux (62). Anaerobic glycolytic end-products such as lactate and pyruvate have been shown to promote the stability of HIF-la and HIF-1dependent gene expression, possibly forming a feed-forward loop and propagating the effects of HIF-la (63).  Tumours are furthermore characterised by having an acidic  extracellular microenvironment, much of this owing to the lactate produced during anaerobic glycolysis.  Nevertheless, pH levels inside the cell are tightly regulated by  transmembrane proteins such as carbonic anhydrases CA9 and CA12, which catalyse the reversible hydration of carbon dioxide to bicarbonate and protons (64).  This process is  thought to maintain intracellular homeostasis, while further aggravating extracellular acidosis as bicarbonate is exchanged for intracellular chloride.  C A 9 and CA12 have  interestingly been identified as a new class of HIF-1 regulated proteins (65). In this way, HIF-1 appears not only to relieve hypoxic stress by allowing a switch to the anaerobic glycolytic pathway, but also to allow maintenance of the crucial physiological pH in cells, despite the acidity resulting from this switch. Angiogenesis  and  Metastasis  Under normal physiological conditions, endothelial cells are tightly regulated as a result of the delicate balance between various pro- and anti-angiogenic factors. However, in solid tumours, metabolic stress, acidosis, mechanical strain, hypoglycemia, and hypoxia (the major physiological stimulus), disrupt this balance and turn on an "angiogenic switch", which leads to neoangiogenesis, allowing tumours to establish their own vascular networks (66).  One of the key mediators of angiogenesis is V E G F , a  protein responsible for the proliferation, survival, and migration of endothelial cells, and  -11-  an essential component for blood vessel formation (67).  HIF-1 has been shown to be a  key factor in the regulation of V E G F , its receptor, VEGFR, and many other angiogenic factors (68).  Immunohistochemical analysis of human tumour biopsies has revealed the  overexpression of HIF-la in many common cancers and its association with tumour V E G F expression and vascularisation (69, 70).  Furthermore, V E G F overexpression in  several tumours has been correlated with high vascularity, lymph node metastasis, and liver metastasis (71, 72). The process of angiogenesis influences metastasis, and certain members of the V E G F family have been implicated in lymphangiogenesis (73, 74, 75). Lymphangiogenesis is the production of lymphatics, small thin channels similar to blood vessels which do not carry blood, but carry tissue fluid from the body to ultimately drain back into the blood-stream. Metastasis arises as a result of tumour cells gaining access to lymphatics, in theory by one of two ways: the first is that tumours metastasise solely by invasion of pre-existing tissue lymphatics at their margin, while the second theory proposes that metastasis can also occur via formation of new lymphatics within the body of the tumour itself (76). Nevertheless, V E G F plays a part not only in the formation of lymphatics, but also induces plasma protein leakage (77), stimulates basement-membrane degradation (78, 79), and induces endothelial cell migration and proliferation (80, 81). Apoptosis As stated previously, hypoxia has been shown to select for malignant cell clones having increased resistance to hypoxia-mediated apoptosis. The wild-type p53 is a multifunctional transcription factor that mediates responses to various cellular stresses, such as D N A damage and hypoxia, and is an integral component of the surveillance system that arrests cell cycle progression under unsuitable conditions (82). Somatic mutations in the  -12-  p53 tumour suppressor are the most common alterations in human tumours, occurring in approximately 50 percent of all cancers, and such defects are strongly associated with metastasis and tumour progression (83).  There is increasing evidence that mutations in  p53 play an important role in regulating both the survival and the angiogenic response of tumour cells to hypoxia. Various studies suggest that p53 negatively regulates V E G F induction (84, 85, 86). In agreement, it has been observed that when overexpressed, the wild-type p53 blocks transcription from numerous promoters lacking p53-binding sites, including those induced by HIF-1 (87).  The body of evidence now available suggests a  delicate balance in the interaction of HIF-la and p53.  p53 increases in response to  hypoxia (88) and maintains a role as a tumour suppressor, where it is thought to either bind and lead to the proteosomal degradation of HIF-la, independent of the V H L pathway (89, 90), or to out-compete HIF-1 for the p300 coactivator (91), thereby inhibiting the induction of genes required for angiogenesis. However, under prolonged and severe hypoxia, HIF-la appears to bind and stabilise the p53 protein, facilitating apoptosis under unsuitable oxygen levels (92).  A recent report by Kaluzova et al. (93)  further suggests that hypoxia alone does not lead to significant overexpression of p53, but rather D N A damage in combination with hypoxia is necessary for p53 to lead to H I F - l a degradation. In light of these results, it would appear that p53 inactivation in cancers not only inhibits hypoxia-induced apoptosis, but also, through failure to inhibit HIF-la, provides a potent stimulus for the activation of the angiogenic switch in tumours.  Proliferation Cells under prolonged hypoxia are generally believed to be slow or nonproliferating, as revealed by studies in human tumour xenografts using pimonidazole  -13-  (marker of hypoxia), and BrdUrd (marker of proliferation) labelling, demonstrating that cells were very seldom marked by both (94).  Furthermore, previous studies have  indicated that HIF-1 activation prevents G l / S transition through at least two different mechanisms (95); HIF-1 may lead to the upregulation of cyclin-dependent kinase inhibitors  (CKIs),  p21  Cipl  ,  and p27  K,pl  ,  whose  sustained  expression  suppresses  cyclin/CDK2 activity, leading to the dephosphorylation of retinoblastoma protein (Rb), resulting in cell cycle arrest at the G l / S transition point. HIF-1 may also down-regulate cyclin E levels known to bind CDK2, again leading to arrest in G l / S . In fact, cyclin E/CDK2 kinase activity in HIF-1 a-deficient cells was shown to be increased, resulting in only partial retardation, but still substantial cell growth under hypoxic conditions. Furthermore, hypoxic cells in vitro were shown capable of proliferation when the gene for HIF-la was inactivated, again demonstrating its important role in this pathway (95). Nevertheless, experiments using exogenous markers of proliferation and hypoxia have shown the co-existence of a small but distinct number of proliferating cells in hypoxic tumour compartments (96, 97, 98). uncoupled from acidosis, clonogenicity (99).  One also observes from in vitro studies that, when  hypoxia actually enhances tumour cell  viability and  Moreover, an increased overlap between proliferative and hypoxic  marker binding was observed after radiation in canine tumours, which is interesting in itself and of relevance to radiotherapy (100).  It is possible that these groups of  proliferating hypoxic cells may represent transient hypoxia, in that they are hypoxic enough for marker binding, but are able to proliferate in short windows of oxygenation, such as would be provided by reoxygenation after clearance of normoxic cells by  -14-  radiotherapy.  Further investigation into this area would be valuable, in that it may  provide information on methods to identify therapeutically relevant hypoxic cells.  1B4 lB4a  HYPOXIA AND RESPONSE TO TREATMENT Radioresistance Hypoxic cells have long been notorious for their resistance to ionising radiation,  and the survival of this fraction of cells is thought to lead to the reappearance of tumours after radiotherapy. Schwartz first observed the oxygen effect in 1912, and noted that the erythema reaction on the forearm was reduced when the radium applicator was pressed hard to the skin. In 1953, Gray et al. established the radioresistance of hypoxic tumour cells. It was later shown that these cells require three times the radiation dose of nonhypoxic cells to achieve the same biological effect (101); oxygen is highly electron affinic and is able to "fix" or make permanent radiation-induced radicals produced in D N A that might otherwise undergo charge recombination (102). Furthermore, hypoxiainduced radioresistance has been clearly shown in clinical studies on soft tissue sarcomas (103, 104), uterine cervical carcinomas (105, 106, 107), and head and neck carcinomas (108, 109, 110), where the presence of hypoxic regions in tumours has had adverse effects on locoregional control and/or disease-free survival after radiotherapy.  lB4b Chemo-resistance It has been shown that hypoxia (and accompanying nutrient deprivation) reduces the proliferation rate or arrests progress through the cell-division cycle (111), and this occurs as a function of distance from the blood vessel (112).  Given that most cancer  drugs such as alkylating agents act by causing damage to D N A , thereby inducing  -15-  apoptosis during the synthesis phase of the cell cycle (113), such drugs lack efficacy on non-cycling hypoxic cells. Furthermore, drug delivery is also problematic since the drug is often metabolised by the intermediate layers of cells before reaching the hypoxic areas, causing a reduction in dose (114, 115).  Drug uptake may also be a concern given that  tumour cells are known to exhibit a lower extracellular pH than normal cells, but maintain a constant intracellular pH (116).  This creates a p H gradient, leading to  decreased uptake of weakly basic drugs such as vinblastine (117). Hypoxia in addition often leads to the production of stress proteins responsible for resistance to drugs such as doxorubicin (118), etoposide (119), and methotrexate (120).  lB4c  Chronic vs. Transient Hypoxia The effects of hypoxia on therapy described above are applicable primarily to  chronic hypoxia, whereas the presence of transient hypoxia may yield still greater difficulties in obtaining an adequate treatment response.  Firstly, the time course of  transient hypoxia could have serious implications, such that if at the time of therapy, blood vessels are closed, cells may be protected from both radiation and chemotherapyinduced damage (121).  Tumour cells, moreover, have been shown to undergo  intermittent growth arrests (122) possibly due to transient changes in oxygen, which could render them still less susceptible to certain chemotherapeutic agents. It has also been reported that a great number of hypoxic cells in xenograft systems retain their viability and growth potential as a result of the intermittent availability of oxygen and nutrients (123, 124). It is thus very possible that following therapy, the surviving fraction  -16-  of these transiently hypoxic cells, upon reperfusion, may proliferate and result in tumour repopulation.  1B5  HYPOXIA-DIRECTED THERAPIES  lB5a Physiology-based Therapies To date, breathing of hyperoxic gases has been the simplest method of decreasing diffusion-limited hypoxia. The breathing of carbogen gas, consisting of 95% 0  2  and 5%  CO2, has been shown effective in raising the arterial partial pressure of oxygen (pCh) in both tumour models and patient biopsies (125, 126, 127, 128, 129), although a few studies have found no significant effect or worsening of oxygenation status (130, 131). The rationale behind the small fraction of CO2 is to induce an increase in respiratory drive, as well as improved blood perfusion, due to COvinduced vasodilation (132). However, when the tumour vasculature lacks responsive smooth muscle and is parallel to vasculature of the surrounding host tissue, carbogen can have a reverse effect; blood perfusion may be increased in the surrounding normal tissue but reduced in the tumour (the "steal phenomenon") (132). Furthermore, some patients lack tolerance for the 5% CO2, due to feelings of dyspnoea. Thus, the CO2 fraction in most patient studies has been reduced to a more tolerable 2% (133), which has been shown as effective as the 5% in increasing tumour oxygenation.  A novel approach in radiotherapy is A R C O N  (accelerated radiotherapy with carbogen and nicotinamide).  This approach combines  accelerated fractionated radiotherapy to counteract cellular repopulation, carbogen to reduce diffusion-limited hypoxia, and nicotinamide, a vasoactive agent believed to reduce perfusion-limited hypoxia. Nicotinamide, the amide derivative of vitamin B 3 , has been  -17-  studied extensively for its radiosensitising properties, and is likely to act by preventing intermittent vascular shut-down, as observed in a number of experimental and human tumours (134, 135).  In clinical trials, the use of A R C O N has been shown most effective  for improved local control and survival for head and neck and bladder cancers (136, 137, 138), and phase 1 and 2 trials have shown the feasibility and tolerability of A R C O N in various tumour types; the dose used, nevertheless, may be limited by the gastro-intestinal toxicity of nicotinamide. In addition, bioreductive drugs that require hypoxic conditions and the production of the appropriate set of reductase enzymes for activation have been used as hypoxia-selective  cytotoxins  (139).  These drugs include indolequinones  mitomycin C and E09, SR4233 (tirapazamine; TPZ), RSU1069, AQ4N, and CB1954, from which mitomycin C and TPZ, in combination with radiotherapy, have produced significant improvements in locoregional control and overall survival (140, 141).  lB5b  Molecular-based Therapies Given the wide array of oncogenes that are hypoxia-regulated, hypoxia-targeted  molecular-based therapies have received much attention.  The major role of HIF-1 in  resistance to therapy and cancer progression has led to the development of various HIF-1 targeted therapies.  One of these is the development of peptides that hinder interaction  between HIF-1 and the co-activator p300/CREB, thereby disrupting the transcriptional activity of HEF-1 (142).  Other techniques include the use of anti-sense technology,  leading to the down regulation of H I F - l a via mRNA silencing (143), as well as highthroughput screening of small molecular inhibitors of the HIF-1 transcriptional pathway (144).  -18-  HRE-directed gene therapy involves the construction of therapeutic agents under control of hypoxia-inducible promoters (HREs), which restrict the therapeutic effect to hypoxic regions.  A n example is the enzyme cytosine deaminase, which although not  toxic on its own, can convert the prodrug 5-fluorocytosine (5FC) into a cytotoxic agent, a process called gene-directed enzyme/prodrug therapy (GDEPT) (145, 146).  This method  could also be used to deliver pro-apoptotic, anti-proliferative, or anti-angiogenic genes to hypoxic tumour areas. Hypoxia-targeted gene delivery has been achieved using vehicles such as retro-and adenoviruses, liposomes, and naked D N A injections, along with novel vectors such as bacteria and macrophages known to accumulate in hypoxic regions (147).  1B6  MEASUREMENT OF HYPOXIA Clinically relevant techniques used to assess the presence of hypoxia in individual  human tumours can be classified as either direct or indirect, whereby direct techniques measure oxygen levels in tissue or blood, and indirect techniques provide a measure of the extent of "hypoxia" as opposed to measuring p 0  2  levels. Each of these methods  presents a set of advantages and disadvantages that are addressed below.  lB6a Direct measurement of Tumour Oxygenation Direct assays for measuring tumour oxygen levels include those applied to circulating blood such as oxyhemoglobin saturation measurements (148) or blood oxygen diffusion imaging (BOLD) (149), or those applied to tissues using needle electrodes. This latter approach will be the focus of this discussion due to its widespread use. The first needle electrodes used widely in the clinic were small polarographic needle  -19-  electrodes developed by the Eppendorf Company (KIMOC 6650, Sigma-pCVHistograph, Eppendorf, Hamburg, Germany). This instrument functions by being mechanically and progressively moved through tissue in a ratcheting motion, while p 0 measurements are 2  collected via an electrolytic reaction that occurs at the cathode placed in the tip of the needle.  Although this method has been widely and successfully used in many clinical  studies, it does present significant limitations.  These include the invasiveness of the  technique, the difficulty in accessing the tumour, the failure to distinguish necrosis from hypoxia, the inability to provide information regarding hypoxic patterns or hypoxia at the single cell level, as well as the cost and dependence on a technically-skilled user (150, 151).  lB6b  Indirect Measurement of Tumour Oxygenation Indirect measurements of tumour oxygen levels involve the use of chemical or  biological reporters, which provide a signal in the absence of oxygen.  Non-invasive  molecular imaging techniques include single photon emission computed tomography (SPECT) imaging and positron emission tomography (PET) imaging, which are based upon the emission and subsequent detection of photons emitted from the isotope decay of administered drugs.  PET imaging is believed to be a more powerful technique than  SPECT for several reasons, including ease of preparation of radiopharmaceuticals, higher resolution, and accuracy in quantitation of measurement; nevertheless, the advantage of PET comes at the cost of greater radioactivity (152). The resolution of PET imaging is inversely limited by the energy of the emitted positron, with the most widely studied imaging agent being F-Miso, a relatively low energy positron emitter. 18  -20-  Modern PET  instruments can provide high quality images with moderate spatial resolution and provide accurate quantitative results, although the ability to detect small percentages of hypoxic cells is questionable. Moreover, the cost and availability of such facilities currently limit their use. Invasive molecular imaging techniques include reporters that can be categorised as exogenous or endogenous markers of tumour hypoxia.  Exogenous markers are  bioreductive drugs that were developed in the 1970s initially as hypoxic-cell sensitisers, and later proposed as chemicals that could be used to identify hypoxic cells.  These  agents form intracellular covalent bonds with thiol-containing molecules as a function of decreasing oxygen (153), and can then be detected using liquid scintillation methods (154), autoradiography (155), and more recently, specific antibody detection techniques (156).  Although these markers can be used in the clinic and have the advantage of  marking hypoxia with high resolution in the context of the tumour microenvironment, a disadvantage of using exogenous markers is that they must be administered before biopsy which may be costly and does not allow for the study of archival material. A possible alternative that may overcome these problems is the use of endogenous markers such as hypoxia-regulated genes or gene products. As mentioned previously, these include H I F - l a and effectors such as GLUT-1, GLUT-3, and CA9. The advantage of these endogenous markers is their use in retrospective studies on archival material. Furthermore, CA9, GLUT-1, and GLUT-3 are non-diffusible and membranous (unlike targets such as VEGF), providing the added advantage that they need not be fixed for staining, and can be used for flow cytometry and sorting of viable cells.  -21-  In this study,  the focus is placed on the exogenous marker pimonidazole and the endogenous marker HIF-la.  Pimonidazole as a Hypoxia Marker The use of pimonidazole as a hypoxia marker, developed by Dr. James A . Raleigh, has been validated in preclinical studies (157, investigation (159, 160, 161).  158) and is under current clinical  Pimonidazole is a lipophilic 2-nitroimidazole compound  that binds to thiol-containing hypoxic cells and forms adducts at oxygen partial pressures of less than lOmmHg (162, 163).  This process requires the presence of one electron  nitroreductases such as NADPH.xytochrome p450 reductase, xanthine oxidase, and aldehyde dehydrogenase, which reduce and activate the 2-nitroimidazoles in two separate one-electron reactions (figure 2).  The first reaction is reversible in the presence of  molecular oxygen, and the second can only take place in the presence of low oxygen concentrations (half-maximal inhibition at 4pM as measured by microelectrodes) (164). The first step in the reaction appears to be responsible for the hypoxic specificity in bioreductive activation and binding. The hydroxylamine that forms from the radical anion is the species believed to bind thiol-containing peptides to from stable adducts. However it must be considered that contrary to 1-electron reductases,  2-electron  reductases such as D T diaphorase can bypass this first oxygen-dependent reductive step and cause reduction of pimonidazole irrespective of its oxygen status.  To this effect,  Janssen et al. (165) have demonstrated that pimonidazole staining of frozen sections under normoxia gave rise to some pimonidazole labelling, mainly in keratinising areas, suggesting that the concentration of non-specific oxygen-independent nitroreductases  -22-  Figure 2. Oxygen-dependency of pimonidazole. Pimonidazole is reduced by nitroreductases in a two-step oxygen regulated process, and is activated for adduct formation (166).  -23-  may influence pimonidazole labelling, as detected in histological sections. Furthermore, it is important to be aware that the intracellular/extracellular concentration ratio and local concentration of pimonidazole adducts are pH-dependent, and enhanced at acidic pH (167). Despite these limitations, pimonidazole has proven to be an effective hypoxia marker both in animal and human studies, with no associated toxic effects (168, 169, 170). Pimonidazole is administered to patients intravenously, is well distributed throughout the entire body, including the brain, and is excreted in part via the urinary tract with a plasma half-life of approximately 6 hours (171). A biopsy of the tissue is then taken typically 24 hours later, and pimonidazole adducts can be detected via immunohistochemistry, enzyme-linked immunosorbent assay, or flow cytometry.  HIF-1 a as a Hypoxia Marker Given the importance of HIF-1 in hypoxia and tumour progression, recent efforts have been made to use HIF-la as a hypoxia marker that could be used in predicting response to therapy. Various studies, using archival formalin-fixed tumour biopsies, have correlated HIF-la expression to outcome as well as parameters such as micro vessel density (MVD) and Eppendorf electrode measurements. Table 1 shows that in 13 recent outcome studies performed on a range of tumour types, 8 correlated HIF-la expression to poor survival, and/or local failure, 4 revealed no correlation, while 1 studies reported a positive correlation between overexpression and survival. Taken together, HIF-la does appear to be useful as a prognostic marker. The correlation of HIF-la expression with other measures of hypoxia, such as Eppendorf electrode measurements, pimonidazole  -24-  binding, and CA9 expression, has nevertheless yielded mixed results in patient studies. A source of these discrepancies may be sample handling. For example, a scattered rather than focal expression pattern may be due to the improper treatment of archival material; given the rapid stabilisation of HIF-la under hypoxia, tumour biopsies or resections which become hypoxic upon removal must be prepared promptly to avoid high background levels of HIF-la. Differences in kinetics between markers may also result in areas of mismatch due to transient changes in tumour oxygenation. HIF-1 in addition plays an important role in tumourigenesis; the loss of HIF-la in embryonic stem cells leads to dramatic retardation in the growth of solid tumours (172), and furthermore, HIF-la is upregulated in the majority of human cancers (173). Given HIF-la's important role in tumour progression, it is not surprising that its stabilisation promotes the survival of both hypoxic and non-hypoxic tumour cells under severe microenvironment conditions.  Figure 3 illustrates some of the major tumourigenic  pathways involved in HIF-la expression, of which only some are hypoxia related.  1B7  SIGNALLING PATHWAYS THAT INFLUENCE HIF-la EXPRESSION  lB7a The PI3K Pathway The disregulation of signal transduction from tyrosine kinase and its effectors, which occurs via autocrine-stimulation or inactivation of the tumour suppressor PTEN, is a common phenomenon in many human cancers. Numerous studies have reported nonhypoxic stimuli such as growth factors including insulin (174), IGF (175), EGF (176), HGF/scatter factor (177), PDGF and TGFp (178), as well as the inflammatory mediators NO and TNFP (179) to induce HIF-1 activity through the PI3K/Akt pathway, and many  -25-  Table 1. HIF-la Correlation. Overview of patient tumour studies on HIF-la status and its prognostic value as well as its correlation to other parameters Tumour site (patient cohort) Breast (n=206) Cervix (n=91) Cervix (n=68)  Tumour stage  Treatme nt modality T1-2;N+ • CT/HT/ Surg pTl-b CT/RT/ Surg FIGOI-IV CT/RT/ Surg  HIF-la expression pattern  Analytic Parameters  Outcome and Correlations  Reference  Nuclear  Point system: scored # (+) cells and intensity Point system: scored # (+) cells and intensity Quantitative: positive nuclear fraction  Independent prognostic factor Strong independent prognostic factor No corr w/ survival; no corr w/ oxgen tesion and hypoxic fraction via eppendorf No corr w/ prognosis; corr w/ Eppendorf Corr w/ poor prognosis, MVD, VEGF Worse response to PDT; no response to salvage RT; no corr w/ survival. Corr w/ poor prognosis, MVD, VEGF  Schindl M et al. 2002 (180) Birneretal 2000 (181) Mayer et al. 2004(182)  Corr w/ local failure and poor overall survival  Koukourakis et al. 2002 (187)  Over-expression corr w/ increased survival Trend for poor survival.; corr w/ CA9, VEGF  Giatromanolaki et al. 2001 (188) Hui et al. 2002 (189)  Corr w/ shorter overall survival, MVD Corr w/ worse disease free and local failure survival No corr w/ survival unless combined w/ p53 expression data; corr w/ MVD  Birner et al 2001 (190) Aebersold et al. 2001 (191) Birner et al. 2002 (192)  Nuclear Nuclear  Cervix (n=42) Endometrium (n=81) Esophagus (n=37)  FIGO IBIII FIGO I  RT  Nuclear  % (+) tumour area labelled  Surg/RT  Tis; TI  PDT/RT  Nuclear/ scattered cytoplasmic Nuclear/ cytoplasmic  Semi-quant: absent/weak/strong staining Qualitative: strong exp (+); weak/no exp (-)  Gastrointestine (n=53)  Benign; borderline; malignant T3-4; N2b-3  Surg  Nuclear  distinct nuclear labelling: <10% (-); >10% (+)  CT/RT  6% nuclear; 36% cytoplasmic  Tl-2;NO-l  Surg  Nl;N2-3; Tib  CT/RT  Nuclear/ cytoplasmic Nuclear  Low grade T3-4; N1+  CT/RT/ Surg CT/RT  FIGO I-IV; LMPIA  Surg/CT  % labelling above/below mean scored (-)/(+) respectively Point system: scored # (+) cells and intensity. Semi-quant: scored based on staining pattern, not intensity Semi-quant: weak/strong staining Point system: scored # (+) cells and intensity Point system: scored # (+) cells and intensity  H&N (n=75) NSCLC (n=108) Nasopharynx (n=90) Oligodendroglioma (n=51) Oropharynx (n=98) Ovary (n=102)  Nuclear/ Cytoplasmic Nuclear: focal and scattered Nuclear  Haugland et al. 2002(183) Sivridis et al. 2002 (184) Koukourakis et al. 2001 (185) Takahashi et al. 2003 (186)  of these studies have identified mTOR/FRAP to work down-stream of Akt. Furthermore oncogenes such as V-SRC, H-RAS, erB2, and receptor tyrosine kinases, as well as loss of the P T E N tumour suppressor, have all led to the increase in activity of both the PI3K/Akt pathway and HIF-1. The mechanism by which PI3K induces HIF-la, is independent of the V H L pathway that is functional under hypoxia (193), and appears to act by increasing the translation of HIF-la mRNA (194, 195).  Studies however have shown that the  involvement of the PI3K/Akt pathway in the growth factor and oncogenic induction of HIF-la is neither sufficient for the activation of HIF-la nor essential for its induction under hypoxia, and is of a much lower magnitude than that caused by hypoxia (196). The involvement of a positive feedback loop has also been suggested, where V E G F has been shown to both activate and be activated by the PI3K/Akt pathway (197, 198). Therefore, the PBK/Akt could possibly have an indirect role in the activation or maintenance of HIF-la, which to varying degrees may be dependent on the initial activation of V E G F by a hypoxic stimulus.  lB7b The MAPK Pathway Three well characterised M A P K signalling pathways in mammalian cells include the extracellular signal-related kinase (ERK) 1/2 (also known as p42/p44 MAPKs), the jun amino-terminal kinase (JNK/SAPK), and the p38 M A P K .  The ERK-1/2 signalling  pathway is activated primarily by mitogens, whereas the JNK/SAPK and the p38 M A P K pathways are regulated by pro-inflammatory cytokines, as well as a diverse array of cellular stresses, such as radiation, hydrogen peroxide, and heat and osmotic shock. Interestingly, the activation of all three pathways has been implicated in the regulation of  -27-  HIF-la in response to various signals.  It has been shown thus far that the hypoxic  activation of the MAPKs is dependent on both cell type as well as the severity of the hypoxic stress (199).  Although the E R K pathway is not primarily stress-responsive  (200), it has been shown to be involved in the hypoxia-induced transactivation of HIF-la (201, 202, 203, 204, 205), likely by indirect phosphorylation (206). Although MAPKs appear to play a role in the hypoxic activation of HIF-la, there are still many HIF-la inducing pathways that are hypoxia-independent.  Hepatocyte growth factor (HGF), a  cytokine that modulates mitogenesis (207), stimulation and promotion of matrix invasion (208), and angiogenesis (209) has been shown to enhance the D N A binding activity of HIF-la  via post-translational  stabilisation  through the  stress kinase  pathways,  JNK/SAPK and p38 M A P K (210). Furthermore, it has been shown that transformation by viral oncogenes such as the G protein-coupled receptor (GPCR) from the Kaposi's sarcoma-associated herpes virus, can lead to the activation of HIF-la as well as V E G F overexpression through the induction of both the p38 and E R K pathways (211). Oncogenic Ras mutations, which are very common in human cancers, can also lead to V E G F expression via either the PI3K pathway, as mentioned earlier, or the M A P K pathway, depending on cell type (212). The regulation of HIF-la appears to occur on multiple levels.  It involves a  number of mediators which in turn, may respond to various and at times, multiple physiological and pathological stimuli, often in a cell-type and context-specific manner. In light of these observations, HIF-la may not be strictly predictive of the pattern of hypoxia in certain tumour types. Nevertheless, the majority of studies indicate that the upregulation of HIF-la in response to such pathways is much less significant compared  -28-  EGF,HGF, PD GF, TGFp, V-SRC, H-RAS,erB2  Hypoxia?? —± P I 3 K  1  ^  A k t  NFkB  HIF-la—  p53 — WDM2  HIF-la transcription  Hypoxia transcrv tional actfraton HPG Oncogenes  >  JNK/SAPK  Pro teo sont Degradation  p38 MAPK HRE  Figure 3. Signalling Pathways in HIF-la regulation. The schematic illustrates the regulation of HIF-la by both hypoxia and non-hypoxia-related stimuli, on levels of trascription and post-trascription.  -29-  to that of hypoxia, such that HIF-la should in theory be applicable for the identification of regions of hypoxia in most tumour types.  The involvement of such pathways,  however, may lead to an increase in background levels of HIF-la, in such a way thatproper thresholding must be carefully applied to minimise the measurement of hypoxia-independent HIF-la expression.  1B8  USING H Y P O X I A M A R K E R S IN C O M B I N A T I O N Given the prognostic value of HIF-la, it is useful to investigate how well this  marker relates to other hypoxia markers. By comparing pimonidazole binding and HIFl a expression in tumours under various oxygen conditions, we hope to determine whether the use of H I F - l a presents an adequate measure of tumour hypoxia.  The  combination of hypoxia markers may also provide the opportunity to distinguish between chronic and transient forms of hypoxia based on differences in marker kinetics, and may allow one to follow the turnover of hypoxic cells in solid tumours. The current study aims to use the endogenous marker H I F - l a and the exogenous marker pimonidazole, believed to have different kinetics, for these purposes. Given that studies investigating the dynamics of the two markers in patient samples would be problematic due to both the lack of availability and heterogeneity of the material, studies are performed first using human tumour xenografts grown in immunodeficient mice. The rationale behind the use of this system follows the recognition that human cancers which are transplanted into mice very often retain many characteristics of the original tumour, including histology, chromosomal abnormalities, surface antigen expression, and vascular structure (113). Nevertheless, a limitation of this model remains that mice and humans have considerably  -30-  different physiology, and such differences must be taken into consideration both with rates of oxygen consumption and marker development. Using  a xenograft  tumour model, an immunohistochemical method  was  developed to combine and to study H I F - l a and pimonidazole marker patterns in relation to each other.  To do this, the development and loss kinetics of marker binding and  expression were first determined, and the oxygen and nutrient dependency of the endogenous marker, H I F - l a were studied.  These patterns were studied in a range of  xenograft tumour types, and as a function of changing oxygen in SiHa xenografts via oxygen breathing experiments.  The ability of the combination of the two markers to  identify regions of transient hypoxia were examined, and the life-time of hypoxic cells was determined by measuring the progression of pimonidazole-labelled cells from areas of viability to those of necrosis as a function of time after chemical administration. The feasibility of this fluorescence immunohistochemical approach was then tested in cervical cancer biopsies, and the patterns obtained compared to those in the xenografts.  -31-  2.  MATERIALS AND METHODS  2A  C E L L LINES A N D C U L T U R E CONDITIONS  1) SiHa: human cervical squamous cell carcinoma cell-line; adherent; contains HPV-16 (1-2 copies per cell); pRb and p53 compromised; doubling time 20-22hr; obtained from the American Culture Collection (ATCC) 2) WiDr: human colon adenocarcinoma cell-line; adherent; expresses mutant p53 (273G-273A); expresses EGF; doubling time ~24hr; obtained from A T C C 3) M 0 0 6 human astrocytoma cell-line; low mitotic index; prominent endothelial proliferation and extensive necrosis; doubling time ~24hr; obtained from Dr Alan Franko (214), Cross Cancer Institute, Edmonton A B The above cell-lines were chosen for their ability to grow in monolayer culture and as xenografts in immunodeficient mice, and to represent a range of tumour types.  These  cell-lines were also chosen due to having been well-characterised in our laboratory. The SiHa cervical carcinoma cell-line in particular was chosen for comparison with patient cervical cancer biopsies. A l l three cell-lines were maintained in exponential monolayer growth in Eagle's minimum essential medium (MEM) (Sigma-Aldrich, Oakville, ON, Canada containing 10% (v/v) fetal bovine serum (FBS) (Gibco B R L , Grand Island, N Y , USA).  The cells were grown as monolayers in 100mm plastic tissue culture plates  (Falcon 3003) at 37°C in a humidified atmosphere of 5% CO2, 95% air. Cells were subcultivated twice weekly to maintain exponential growth, and were recovered from culture plates by removing the culture medium, rinsing the plates in 0.1% trypsin in citrate phosphate saline buffer (Gibco), leaving a thin layer of trypsin remaining after the  -32-  second rinse, and incubating cells for ~6min at 37°C. Cell counts were performed using a Coulter Counter cell analyser (Beckman/Coulter, Fullerton, CA).  2B  XENOGRAFT  TUMOUR  MODELS  Xenograft tumours were grown from SiHa, WiDr, and M 0 0 6 cell lines. Tumour cell suspensions (0.1ml at 10 cells/ml) were subcutaneously injected in the back of 7-8 6  week old NOD/SCID (non-obese diabetic/severe combined immunodeficient) mice. The tumour lines were maintained by serial implantation of the tumours in the flanks of NOD/SCID mice.  NOD/SCID mice were kept in a pathogen-free unit in an air-  conditioned facility, approved by the Canadian Council of Animal Care. Approval of the local animal care committee was obtained for all protocols. To implant tumours, mice were shaved over the back. NOD/SCID mice were then anaesthetised with isoflurane, the areas swabbed with alcohol, and injected once subcutaneously with a minced tumour suspension or with single cell suspensions using a 20g needle. The area was swabbed again with alcohol and mice were allowed to recover in a warm environment before returning to the animal holding room. Tumours were used when they reached a size a 0.25 - 0.6g in approximately 3-4 weeks.  2C  ADMINISTRA  TION  OF  CHEMICALS  Pimonidazole  Pimonidazole-hydrochloride (hypoxyprobe-1; Chemicon Int. Temecula, C A , USA),  a bioreductive marker for hypoxia, was administered to mice intraperitoneally  (i.p.) at lOOmg/kg (215). Mice were sacrificed by cervical dislocation 90min to 48hr  -33-  following pimonidazole administration and the tumours excised.  Pimonidazole was  administered to patients intravenously (i.v.) at 500mg/m and biopsies taken 24hr after 2  administration.  Informed consent was obtained from all patients and approval for the  study was given by the British Columbia Cancer Agency and the University of British Columbia clinical investigations committee.  The plasma half-life of pimonidazole is  ~30min in mice (216), and ~6hr in humans (171).  Hoechst 33342 Hoechst 33342 (Sigma/Aldrich, St. Louis M O , USA), a fluorescent dye used as a marker for vascular perfusion, was administered to mice i.v. at a volume of 0.1ml and a concentration of 4mg/ml in PBS. Given its relatively short plasma half-life of 110s, Hoechst 33342 binds in the first few minutes after administration, and only produces extensive fluorescence in cells adjacent to perfused vasculature (217).  Mice were  sacrificed by cervical dislocation lOmin following Hoechst 33342 administration. Given that a perfusion marker in humans has not yet been approved, the endothelial cell marker CD31 was used to identify vasculature in patient biopsies.  2D  GAS-BREATHING PROCEDURES Gas-breathing experiments were performed using a PROOX model 110 hypoxic  chamber (BioSpherix, Redfield New York, USA), which equilibrates ambient air with nitrogen or oxygen gasses to create the desired gas concentration. Mice were placed in the chamber 5min prior to pimonidazole administration, and were returned to the chamber for 1.5-2hr at the desired oxygen concentration.  Mice were then removed  briefly, administered Hoechst 33342, and placed in the chamber for lOmin, followed by  -34-  sacrifice.  For carbogen (95% O2, 5% C 0 ) , mice were placed inside a plexi-glass 2  chamber and gassed with carbogen (PRAXAIR, ON, Canada).  2E  PREPARA TION OF FROZEN SECTIONS Mice were sacrificed by cervical dislocation and the tumour excised within 30s.  The tumour sample was embedded and frozen at -20°C in O.C.T.(Tissue-Tek).  10pm  sections were obtained using a Cryostar HM560 (Microm International GmbH, Walldor, Germany).  Frozen sections were melted for 30s on glass slides in preparation for  immunohistochemistry and fluorescence microscopy.  2F  IMMUNOHISTOCHEMISTRY Sections were fixed in cold 2% paraformaldehyde (Sigma-Aldrich) in PBS for 15  minutes at 4°C. The sections were then rinsed sequentially in PBS, M e O H (-20°C), PBS, and immersed in P T N (PBS, 1% bovine serum albumin, 0.1% Tween) for 5min at room temperature. 30-40pl of antibody solution (table 2) were then applied to each slide and covered with 1cm paraffin strips (to prevent evaporation) for 30min in humidified 2  containers at ambient temperature. The primary antibody was rinsed off in PBS and the slide immersed in PTN for 5min before addition of the fluorescent secondary antibody. In double-staining procedures, the slides were fixed in 2% paraformaldehyde before addition of the second primary antibody to prevent cross-reaction. Following all staining procedures, sections were rinsed for 5min in PBS, drained, covered with lOul Fluorogard mounting medium (BioRAD, Mississauga, O N , Canada) and sealed with a cover-slip. Appropriate secondary antibody controls were performed for each experiment.  -35-  Table 2. List of antibodies.  Primary (1°) and secondary (2°) antibodies are indicated.  Typically, the H I F - l a primary antibody and Alexa 594 secondary antibody were administered  before  the  anti-pimonidazole  antibody  and Alexa 488  secondary.  Antibodies were diluted in PTN. Antibody H I F - l a (1°)  Origin Mouse anti-human  Pimonidazole-HCl  Mouse anti-human  (1°) CD31 primary (1°)  Mouse anti-human  Alexa 488 (2°) Alexa 594 (2°)  Goat anti-mouse IgG F(ab') Goat anti-mouse IgG F(ab')  -36-  Source BD Transduction Laboratories, Canada Natural Pharmaceuticals International Inc., N C  Dilution 1:100  Dakocytomation, Mississauga, ON Molecular Probes, U S A  1:50  Molecular Probes, U S A  1:200  1:200  1:200  2G  IMAGE ACQUISITION AND ANALYSIS Many studies use a semi-quantitative method of analysis in evaluating the  presence and extent of maker binding, as displayed in table 1.  However, much more  information may be gained from a quantitative analysis, and the ease in evaluation due to image quality and definition in fluorescence imaging, as well as the possibility of a double staining protocol allowed us to evaluate marker patterns quantitatively.  Slides  were viewed with 488nm excitation (green light emission), 594nm excitation (red light emission), and U V excitation (blue light emission) using a lOx neofluor objective with a Zeiss Axioplan 2 epifluorescence microscope with an attached Retiga Exi mono cooled 12 bit C C D camera (Q Imaging, Canada). The images were digitised using Northern Eclipse 5.0 software (Empix, Toronto, ON, Canada). When possible, three sections were obtained from each tumour (proximal, mid, and distal), and five to ten lOx magnification images were obtained from each section. lOx magnification grayscale images (1360x1024 pixels, lpixel/ja, 8bit) of sections showing HIF-la, pimonidazole, and Hoechst 33342 were pseudocoloured, superimposed and analysed for degree of colocalisation or mismatch between the colours using software written for the Northern Eclipse 5.0 software by Hart Lambur. The mismatch software measured the number of pixels designated red (R; HIF-la), green (G; pimonidazole), and yellow (RG; both), and the number of R G pixels were divided by the total number of R or G pixels to provide % colocalisation with respect to HIF-la and pimonidazole respectively.  To do this, each grayscale image was displayed individually and  thresholded. This required manual application of thresholds to determine positive regions  -37-  of labelling. The thresholds chosen were typically 2x and 3x the level of background labelling for H I F - l a and pimonidazole respectively.  A higher threshold was used for  pimonidazole, which showed a lower level of background labelling.  The number of  pixels from each individual image was automatically counted and entered into a spreadsheet. The images were then superimposed, and matching pixels from both images were counted and entered into the spreadsheet, from which the % colocalisation was calculated, as described above. Values for H I F - l a and pimonidazole development (start-point and end point with respect to distance from the blood vessel, as marked by Hoechst 33342) were obtained using ImageJ software (NIH Image: http://rsb.info.nih.gov/ij). Three to four regions from each image were selected by drawing a line from the centre of a blood vessel through a tumour cord. The regions were selected such that they encompassed one or a small group of blood vessels on one side, and were adjacent to a region of obvious necrosis on the other.  Selection was necessary in order to model a simplified system, where the cord  would start from an area of highest oxygen concentration (area stained with Hoechst 33342) and extend through to that of the lowest oxygen concentration (region of necrosis). The ImageJ RGB profiler function then produced a colour histogram of each selected region, from which the start and end points of H I F - l a and pimonidazole staining with respect to the blood vessel were obtained and calibrated in microns.  Given the  variation in the size of the tumour cords, the results were normalised. Values for the H I F - l a start points were first divided by the corresponding pimonidazole start points, and values for the H I F - l a end point were accordingly divided by the pimonidazole end points to obtain start and end point ratios.  -38-  These ratios were then multiplied by the  average pimonidazole start and end points over all samples, to obtain relative H I F - l a start and end values, respectively.  2H  WESTERN BLOT  ANALYSIS  Approximately 6xl0 cells were lysed in RIPA lysis buffer (50mM Tris-HCL pH 6  7.4, 150mM NaCI, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, ImM EDTA) to which ImM PMSF, lug/ml aprotinin, lp,g/ml leupeptin, ImM Na3VC>4, and ImM NaF were added before use (all reagents were obtained from Sigma Chemical Co., Oakville ON, Canada). (BioRad).  Protein concentrations were standardised using the D C Protein Assay  Proteins were separated on a 10% polyacrylamide precast gel system  (BioRAD) and transferred to a nitrocellulose membrane (BioRAD). The membrane was immersed in 5% non-fat dry milk in TBS-T (25mM Tris-HCL, pH 7.04, NaCI, KCI, 0.05% Tween 20) for one hour at room temperature with shaking. Membranes were next incubated in anti-mouse monoclonal H I F - l a antibody (1:1000 dilution) overnight. After several washes in TBS-T, membranes were incubated in horseradish peroxidase antimouse antibody (1:5000 dilution; Sigma) for lhr and developed using the E C L detection kit  (Amersham, Oakville, O N , Canada).  Membranes were  exposed under a  chemiluminescence imager (Multilmage Light Cabinet, Alpha Innotech Corp., CA) for 4lOmin.  Images were analysed for band density using FluorChem software (v.3.04A,  Alpha Innotech). Relative band density was obtained by first adjusting (by division) to the band density of cells in air-equilibrated medium, and then to the highest band density obtained in each sample. Experiments were repeated twice.  -39-  21  FLOW CYTOMETRIC ANALYSIS Approximately 10 to 10 cells were fixed in 70% EtOH in PBS. Cells were 5  6  centrifuged, washed in PBS, and rehydrated in PST (PBS with 4% FBS and 0.1% triton X-100) for 15min.  Cells were centrifuged and incubated in pimonidazole antibody  (1:200) for 2hr on a shaker. Cells were then centrifuged, washed in PST, and incubated in Alexa 488 antibody (1:200) for 45min with shaking. Cells were again centrifuged, washed twice in PBS, and incubated in DAPI (1:50 dilution of lOOug/ml) for nuclear staining.  Cells were then analysed using a dual laser Epics Elite-ESP flow cytometer  (Coulter Corp., Hialeah, FL). Gates were set to discriminate against debris on the basis of cell size and time of flight. List mode files were collected and post processed to determine parameters of interest using the WINLIST software package (Verity Software House Inc., Topsham, ME). Figure 4 depicts typical read-outs obtained from flow cytometric analyses. The means of the peak intensities, shown in Figures 4C and D were used as a measure of the amount of pimonidazole adducts. Experiments were repeated twice.  2J  GRAPHICAL AND STA TISTICAL ANAL YSIS For statistical analysis of data, the mean and SD values were determined. The  one-way A N O V A followed by the Tukey-Kramer test was used to compare multiple means (JMP Start Statistics 2  nd  Ed., SAS Institute Inc.). p values of less than 0.05 were  considered to represent significant changes.  Graphical analysis of kinetics data was  performed by fitting each curve to an appropriate exponential function using Gnu Plot software (Thomas Williams, Collin Kelly; 2004).  -40-  DNA Content -->  L O G FITC-PINIO -->  Figure 4. Flow cytometric analysis. SiHa cells were first gated on forward light scatter and time of flight to eliminate debris. Panels A and C are control cells, panels B and D are SiHa cells after 4hr incubation with pimonidazole under anoxic conditions.  -41-  3  RESULTS  Given the importance of tumour hypoxia in both tumour progression and response to therapy, hypoxia should be measured routinely in tumour biopsies.  A method  applicable to formalin-fixed paraffin-embedded sections could be easily incorporated into pretreatment analysis of patient biopsies. In this study 1) we examined the ability of an endogenous hypoxia marker, HIF-la, to mark the same regions as an established chemical marker for hypoxia, pimonidazole. 2) The rate of development of the markers under hypoxic conditions and their rate of loss upon reoxygenation were examined, and 3) the role of oxygen and nutrients (glucose/serum) in marker binding were determined. 4) The localisation of the two markers within tumour cords and their response to oxygen modulation were also studied. 5) Lastly, two patient biopsies were examined to confirm the feasibility of using the staining protocol on human frozen-sections.  3A  MARKER KINETICS All marker kinetics experiments were performed using cultured SiHa cells. Cells  were removed from culture plates using trypsin and transferred as cell suspensions to medium-containing (MEM/10%FBS unless otherwise specified) pre-gassed glass spinner culture flasks (150 rpm) equilibrated with 0% O2 in nitrogen and with 5% carbon dioxide to maintain the pH of the bicarbonate buffer.  Cells were maintained with continuous  gassing at 0% O2 as required at 37°C. High cell density (2xl0 cells/ml) compensated for 5  any small oxygen leakage into the flasks to maintain oxygen concentrations as close as possible to 0%.  -42-  3Al  HIF-la BUILD-UP UNDER ANOXIA Cell suspensions were incubated under anoxia as described above for up to 16hr,  after which the cells were rapidly removed into ice-cold medium containing cobalt chloride (C0CI2; final concentration of lOOuM) to inhibit H I F - l a degradation. C0CI2 binds the oxygen-dependent degradation (ODD) domain of H I F - l a and inhibits V H L binding, thereby inhibiting degradation (218). As shown using western analysis, H I F - l a was stabilised quickly under anoxia, and by 2hr, the half-maximal signal was reached (figure 5). This is consistent with the presence of a steady state level of H I F - l a that is ready to be stabilised upon exposure to hypoxic stress.  3A2  HIF-1 a HALF-LIFE UPON REOXYGENATION Cell suspensions were incubated under anoxic conditions as described for 4hr.  Subsequently, cells were removed and reoxygenated by immersion in air-equilibrated medium.  Further degradation of H I F - l a was inhibited by adding C0CI2.  lysed and analysed by western blot analysis (figure 6). reoxygenation was found to be < lmin.  -43-  Cells were  The half-life of H I F - l a upon  1.2  1.0  0.8  T 3 C ID  JS 0.6  y = 0.97- 0.84 e  53  (-0.24x)  > « 0.4  0.2  0.0 0  2  4  1  r~  8  10  Time (hr)  Figure 5.  Stabilisation of H I F - l a under anoxia.  ~l  12  1  1  1  14  16  18  SiHa cells were incubated under  anoxia, and cell lysates were subjected to western blot analysis. Relative band densities were obtained as described (refer to 2H), and an exponential function was fitted to data points.  -44-  1.2 - i  0  2  4  6  8  10  12  Time (min)  Figure 6. Loss of H I F - l a after reoxygenation. SiHa cells were incubated for 4hr under anoxia and were reoxygenated.  Cell lysates were subjected to western blot analysis.  Relative band densities were obtained as described (refer to 2H), and an exponential function was fitted to data points.  -45-  3A3  PIMONIDAZOLE BINDING UNDER ANOXIA Cell suspensions were equilibrated under anoxia as described, and incubated with  2, 10, and 50u.g/ml pimonidazole hydrochloride. Cells were then sampled from each flask at various times, fixed in 70% ethanol, immunostained with anti-pimonidazole antibodies, and analysed for fluorescence by flow cytometry (figure 7).  At high  concentrations of pimonidazole, the initial rate of pimonidazole adduct formation was greater than at the lower concentrations, possibly related to the K m of the system. At 10 and 50u.g/ml of pimonidazole, the curves start to level at ~2hr, likely due to saturation, although cell toxicity effects cannot be excluded.  The half-maximum signal was  obtained at 20min and 40min for 50u.g/ml and lOjag/ml, respectively.  As mice were  administered lOOmg/kg pimonidazole, the higher doses are probably more typical of the in vivo exposure.  3A4  PIMONIDAZOLE HALF-LIFE UPON REOXYGENATION Cells were incubated under anoxia as described in 50jag/ml of pimonidazole for  4 hours, after which they were removed, placed in culture dishes and allowed to recover under ambient oxygen.  Cells were trypsinised and removed from the plates at the  indicated times, fixed in ethanol, and analysed by flow cytometry. The doubling time of the same cell population was also assessed by counting the cells after typsinisation. The half-life of the pimonidazole adducts was 45hr (figure 8A) and the doubling time of the cell line was 21hr (figure 8B). Although one would expect adduct half-life to be equal or less than cell doubling time, the observed effect may be due to anti-body binding affinity. Therefore, these results still suggest that loss of pimonidazole adducts may largely be a  -46-  Figure 7. Formation of pimonidazole adducts under hypoxia. Different concentrations of pimonidazole were added to SiHa cells that were incubated in spinner flasks under anoxia for various times.  Mean fluorescence was detected using flow cytometry.  Relative values were obtained by dividing by the mean fluorescence of cells incubated in air-equilibrated medium for each time point. Appropriate functions were fitted to data points.  -47-  Figure 8A.  Loss of pimonidazole adducts after reoxygenation.  SiHa cells were  incubated in spinner flasks under 50 pg/ml pimonidazole and anoxia for 4hr, and then cultured under ambient oxygen.  Mean fluorescence was detected by flow cytometry.  Relative values were obtained by division by the mean fluorescence of cells incubated in air-equilibrated medium for each time point. reoxygenation.  Figure 8B.  Cell doubling after  Cells from the same experiment were allowed to recover in ambient  oxygen in vitro to examine the relation between loss of pimonidazole adducts and cell division. Exponential functions were fitted to data points.  -48-  result of cell division. Furthermore, the doubling time of 21hr, which is the same for cells under normal conditions, suggests that pimonidazole at 50pl/ml is likely not very toxic.  3B  MARKER COLOCALISATION STUDIES Three different human tumour cell lines, WiDr colon carcinoma cells, SiHa  cervical carcinoma cells, and M 0 0 6 glioma cells, were grown as xenografts for use in the following experiments.  3B1  M A R K E R COLOCALISATION IN XENOGRAFT TUMOURS Frozen sections from xenograft tumours were incubated with antibodies to HIF-  l a and pimonidazole as described, with Hoechst 33342 used as a marker of perfusion. H I F - l a showed a nuclear labelling pattern as expected, and pimonidazole showed a cytoplasmic labelling pattern, confirming no cross-reaction between the two monoclonal antibodies (figure 9). HIF-la  Regarding the pattern of labelling, in all three xenograft types,  appeared closer to the Hoechst 33342-labelled perivascular regions than  pimonidazole.  Unexpectedly, the region binding pimonidazole antibody extended  beyond that labelled for HIF-la.  Occasionally hypoxic regions of the tumour showed  very low levels of, or lacked altogether only one of the two markers. The colocalisation between H I F - l a and pimonidazole was determined for each xenograft type. The mean co localised fraction with respect to H I F - l a in SiHa, WiDr, and M 0 0 6 xenografts was 60 ± 11%, 59 ± 17%, and 50 ± 14%, respectively, and the mean colocalised fraction with respect to pimonidazole was 45 ± 13%, 23 ± 9%, and 27 ± 12%, respectively (figure 10).  -49-  Figure 9. Distribution of HIF-la and pimonidazole in xenograft  tumours.  Tumour bearing mice were  administered pimonidazole (lOOmg/kg from a 20mg/ml stock, given i.p.). After 1.5hr, mice were administered Hoechst 33342 (0.1 ml of a 4mg/ml solution, given i.v.), and sacrificed after lOmin. Frozen sections were stained with appropriate antibodies, and fluorescent immimohistochernistry was used to visualise areas of HIF-la  (red), pimonidazole (green), and Hoechst  33342 (blue) at lOx magnification.  A Depicts clear  nuclear HIF-la labelling, Cytoplasmic pimonidazole labelling, and nuclear Hoechst 33342 labelling in a SiHa xenograft tumour. B, C and D represent SiHa, WiDr, and M 0 0 6 xenografts respectively.  -50-  Figure 10.  Colocalisation of H I F - l a and pimonidazole in xenografts.  Data were  obtained by studying antibody staining patterns in SiHa (n=5), WiDr (n=5) and M 0 0 6 (n=5) xenografts using lOx magnification fluorescent images. The fraction colocalised was determined by dividing the number of colocalised pixels of red and green (HIF-la and pimonidazole respectively: RG) by the total number of red (R) or green (G) pixels (refer to 2G). RG/R represents fraction of total H I F - l a colocalised with pimonidazole, and RG/R represents the fraction of total pimonidazole colocalised with HIF-la.  -51-  No significant difference was observed in the mean colocalised fraction with respect to H I F - l a in the tumour types, but the mean colocalised fraction with respect to pimonidazole in SiHa xenografts was significantly higher than both WiDr and M 0 0 6 xenografts (p = 0.05; Tukey-Kramer).  It should be noted that given the different  localisation of the two markers (nuclear vs. cytoplasmic), the colocalised fraction may have been underestimated.  The thickness of the frozen sections (lOju) however, may  have compensated for this difference; most cells were imaged in three dimensions, so that the cytoplasmic staining anterior and posterior to that of the nucleus would provide sufficient staining signal to coincide with the latter.  Atypical Marker Patterns Marker colocalisation studies above revealed occasional hypoxic regions in all three xenograft types where labelling of only one of the two markers was very low or altogether missing.  Figure 11A shows an area where H I F - l a labelling occurs in the  absence of pimonidazole labelling. The reverse was also observed, where pimonidazole labelling occurred in the absence of H I F - l a labelling (figure 11B).  Both of these  patterns could be indicative of transient changes in perfusion (transient hypoxia), whereas areas labelled with both markers, as are more commonly observed, would represent primarily chronic hypoxia. Often, H I F - l a and pimonidazole labelling occurred inside areas labelled with Hoechst 33342, possibly indicating a very low oxygen gradient despite plasma flow through the vessel (figure 11C).  These patterns however were  relatively rare (<5% blood vessels), as observed by others (219), so that they did not greatly contribute to decreases in marker colocalisation.  -52-  Figure 11.  Atypical marker patterns in xenograft  tumours. Fluorescent immunohistochemistry was used to visualise  areas of H I F - l a  (red), pimonidazole  (green), and Hoechst 33342 (blue). A H I F - l a staining with little pimonidazole labelling and lack of Hoechst 33342 staining (SiHa).  B Pimonidazole and Hoechst  33342 labelling with little H I F - l a labelling (M006). C Pimonidazole and H I F - l a labelling very close to the Hoechst 33342 labelled area (SiHa).  -53-  3C  HIF-1 a: OXYGEN-DEPENDENCY AND NUTRIENT  REQUIREMENTS  3C1  IN VITRO OXYGEN AND NUTRIENT DEPENDENCE The lack of H I F - l a labelling in perinecrotic tumour regions was investigated by  studying the oxygen and nutrient-dependency of H I F - l a expression. SiHa cultured cells were incubated in spinner flasks for 4hr at various oxygen concentrations.  The cells  were then removed and H I F - l a degradation was inhibited by adding C o C l and placing 2  cells on ice. Figure 12 shows that as oxygen concentration was decreased, the relative amount of H I F - l a increased accordingly, and that the highest level of H I F - l a was seen under anoxic conditions. Cells from the same population were incubated for 4hr at 0% O2 in serum and glucose-free medium, glucose-free medium with 10% serum, 1% serumcontaining medium, and 10% serum containing medium (figure 13).  H I F - l a was not  observed under the first two conditions, but increased as the percent serum was increased.  3C2  REOXYGENATION OF PERINECROTIC CELLS IN XENOGRAFTS To test whether the lack of H I F - l a in regions distal to the blood vessels was due  to in vivo changes in oxygen, tumour oxygen levels were modified by increased oxygenbreathing; this would increase oxygen diffusion distances and oxygen  levels in  perinecrotic areas, possibly allowing stabilisation of H I F - l a in these regions.  Mice with  SiHa xenografts were administered pimonidazole and after 2hr (by which time the pimonidazole should be metabolised and largely eliminated from the blood) were given 60% O2 to breathe for another 2hr (experimental group), or were left in air for a further 2hr (control group).  Mice were then administered Hoechst 33342.  -54-  The tumours  Oxygen  0% 0.5%  2% Air  HIF-1a (120KD) Actin  Figure 12.  Effect of oxygen on H I F - l a protein level.  SiHa cells were incubate in  spinner flasks under various oxygen concentrations for 4hr, and the cell lysates subjected to western blot analysis. Band densities were adjusted as described (refer to 2H).  -55-  Glucose Serum  +  1%  HIF-1a (120KD)  +  10%  Actin  Figure 13.  Effect of glucose and serum on H I F - l a protein level.  incubated in spinner flasks at % 0  2  SiHa cells were  for 4hr in glucose-free M E M , or in M E M containing  0%, 1% or 10% FBS. Cell lysates were subjected to western blot analysis and band densities were adjusted as described (refer to 2H).  -56-  Pimonidazole i.p  I ;  Hoechst i.v  ; I •  2hr air  Tumour excision  ;I  2hr 60 % (exp) 2hr air (control)  Figure 14. Reoxygenation of distal cells in SiHa xenograft tumours. Tumour bearing mice were administered pimonidazole (lOOmg/kg at 20mg/ml i.p.).  After 2hr, mice  were given 60% oxygen (B; n=4) or air (A: control; n=3) to breathe, administered Hoechst 33342 (4mg/ml  lOOpl i.v.), and sacrificed after lOmin.  Fluorescent  immunohistochemistry was used to visualise areas of HIF-la (red) and pimonidazole (green). lOx magnification images were analysed by measuring start and end points of marker-labelled regions with respect to the blood vessel, using histograms of the marker profiles (refer to 2G).  -57-  were excised and frozen sections were stained for pimonidazole, HfJF-la, and Hoechst 33342. The average distance from the blood vessels to the cord edge (~300|i) is larger than those normally encountered in most solid tumours (~100-200u.).  These large  distances are a result of cord selection. Larger cords rather than small ones were selected for the reason that when mice were give 60% oxygen to breath, oxygen diffusion in smaller cords resulted in perinecrotic oxygen concentrations which were likely too high for H I F - l a stabilisation, as indicated by the overall absence of H I F - l a in the entire tumour cord, including regions that had previously bound pimonidazole.  Since these  perinecrotic regions were of interest in this study, larger cords were necessary so that the level of oxygen in these regions would increase, but would still be low enough to allow H I F - l a stabilisation.  In the 60% oxygen-breathing mice, the area stained for H I F - l a  was reduced past the region that stained for pimonidazole under ambient air breathing conditions, but still did not extend into the perinecrotic regions (figure 14). This result indicates that increasing oxygen level alone is not sufficient for the stabilisation of HIFl a in perinecrotic xenograft regions.  3D  OXYGEN MODULATION OF XENOGRAFTS VIA GAS-BREATHING Mice carrying SiHa xenografts were administered pimonidazole and exposed to  either air (21% 0 ), 10% 0 , or carbogen (95% 0 , 5% C0 ) for 1.5hr. They were then 2  2  2  administered Hoechst 33342 and sacrificed after lOmin.  2  The tumour was excised and  frozen sections were stained for pimonidazole, HIF-la, and Hoechst 33342 (figure 15). To quantify markers with respect to oxygen changes, 60% 0 used, given that 95% 0  2  2  instead of carbogen was  decreased staining to such an extent that accurate quantification  -58-  became problematic. Images were manually thresholded and analysed for the fraction of marker mismatch as already described. The percent area labelled by H I F - l a showed a significant decrease (p = 0.05; Tukey-Kramer) with increase in oxygen. The percent area labelled by pimonidazole however showed a significant decrease (p = 0.05; Tukey Kramer) from air-breathing to 60% 02-breathing mice, but shows no significant difference from air breathing to 10% 02-breathing mice (figure 16). Changing oxygenbreathing conditions also affected colocalisation ratios (figure 17).  The colocalised  fraction with respect to H I F - l a significantly differed (p = 0.05; Tukey Kramer) between the air breathing and the 10 and 60% 02-breathing conditions and the colocalised fraction with respect to pimonidazole differed significantly (p = 0.05; Tukey-Kramer) between the 10% 02-breathing and the air and 60% 02-breathing conditions. pattern in these changes suggests these differences  Lack of a consistent  to possibly be the result of  inconsistent increase in the percent pimonidazole staining upon decrease in oxygen level.  3E  HYPOXIC CELL HALF-LIFE IN XENOGRAFT TUMOURS Mice with SiHa xenografts were administered pimonidazole and at times up to  48hr later, were administered Hoechst 33342 lOmin before sacrifice.  Frozen sections  were stained with antibodies against H I F - l a and pimonidazole and analysed for colocalised fraction. Fluorescent images showed that over time, mismatch between the two markers increased and pimonidazole-labelled cells appeared to move further into necrotic regions (figure 18). Furthermore, with time after pimonidazole administration, there was an increase in areas lacking one marker all together (figures 19A, B and C).  -59-  Figure  15.  Oxygen  modulation  of  SiHa  xenograft tumours. Tumour bearing mice were administered  pimonidazole  (lOOmg/kg  at  20mg/ml i.p.) and were given A: air (21% O2,) B: 10% 0 , or C : carbogen (95% 0 , 5% C0 ,) to 2  2  2  breathe.  After 1.5hr, mice were administered  Hoechst  33342  sacrificed  (4mg/ml  after  lOmin.  lOOul  i.v.),  and  Fluorescent  immunohistochemistry was used to visualise areas of H I F - l a (red), pimonidazole (green), and Hoechst 33342 (blue).  -60-  60  Air Figure 16.  10%  60%  Effect of oxygen modulation on percent area staining in xenografts.  Tumour bearing mice were exposed to 10% O2 (n=3), air (21% 0 ; n= 5) and 2  60% 0  2  (n=3) for 1.5hr. lOx magnification fluorescent images were analysed to  obtain the number of red pixels (HIF-la) and green pixels (pimonidazole) over the total number of pixels to obtain % area staining.  -61-  10%  60%  Figure 17. Effect of oxygen modulation on marker colocalisation in xenografts. Tumour bearing mice were exposed to 10% O2 (n=3), air (21% O2; n= 5) and 60% O2 (n=3) for 1.5hr. Data were obtained by studying mismatch patterns using lOx magnification fluorescent images. The fraction colocalised was determined by dividing the number of colocalised pixels of red and green (HIF-la and pimonidazole respectively: RG) by the total number of red (R) or green (G) pixels (refer to 2G).  RG/R represents the fraction of H I F - l a colocalised with  pimonidazole, and RG/R represents the fraction of pimonidazole colocalised with HIF-la.  -62-  Figure 19D shows an area where pimonidazole appears to have accumulated in perinecrotic areas, and 19E a region where very little pimonidazole is observed, very likely due to loss of necrotic tissue upon slide processing. distinguished from viable cells based on morphology.  Necrotic cells were  In viable hypoxic regions,  pimonidazole shows cytoplasmic and nuclear staining and appears to cover nearly the whole cell (figure 19A). In necrotic areas however, pimonidazole shows a "honey-comb" morphology, where the nucleus appears unlabelled and the staining looks moe membranous (figures 19B, C, and D).  The rate at which pimonidazole stained cells  moved into regions of necrosis was taken as a measure of hypoxic cell life-time, given that cells capable of metabolising pimonidazole must have been viable at the time of pimonidazole administration. Hypoxic cell half-life was obtained by measuring changes in the fraction of H I F - l a colocalised with pimonidazole, to measure the rate at which pimonidazole moved away from the H I F - l a labelled region (considered viable) to regions of necrosis.  H I F - l a levels remained constant throughout the images at the  various times, and did not contribute to changes in co localised fraction. Images were manually thresholded and analysed for the fraction of marker mismatch as already described (2G). The hypoxic half-life was found to be 16hr (figure 20).  -63-  Figure 18. Changes in colocalisation with time after pimonidazole administration. Tumour bearing mice were administered pimonidazole 1.5hr (A), 12hr (B), 24hr (C), and 48hr (D) before biopsy, and the mismatch between pimonidazole and HIF-la was studied as a function of time.  -64-  Figure 19.  Atypical marker patterns with time after pimonidazole administration.  Fluorescent  immunohistochemistry  was  used to visualise  pimonidazole (green), and Hoechst 33342 (blue).  areas of HIF (red),  A, B, and C reveal very typically  observed areas of mismatch in xenografts excised 12, 24, and 48hr after pimonidazole administration, respectively.  D and E show the accumulation of pimonidazole-labelled  cells in obvious necrotic areas, and the loss of pimonidazole labelling due to slide processing respectively, in xenografts excised 24hr after pimonidazole administration.  -65-  Figure 20. Hypoxic cell half-life in xenograft tumours.  Tumour bearing mice were  administered pimonidazole at 1.5hr and tumours were excised up to 48hr later. The colocalised fraction with respect to H I F - l a was determined as described (2G).  Each  point shows the mean and standard deviation for up to 8 sections per tumour for 3-5 tumours. A n exponential function was fitted to data points.  -66-  3F  COMPARISON OF PA TIENT BIOPSIES AND XENOGRAFT MODELS Having established the staining protocols and kinetics for the human tumour  xenografts, a feasibility study was performed using frozen sections obtained from biopsy samples of patients with cancer of the cervix being treated at the B.C. Cancer Agency. Patients were part of a study headed by Dr. Christina Aquino-Parsons to examine expression of pimonidazole and correlation of this marker with outcome. Patients were administered pimonidazole 24hr before biopsy, and the biopsy was placed in ice-cold buffer, returned to the Research Centre on ice and immediately frozen. Two consecutive sections were stained for H I F - l a and pimonidazole, and for pimonidazole and CD31 respectively. Images from the two sections were superimposed using the pimonidazole staining pattern, and a triple stained image was then constructed. Figure 21 shows that the patient biopsy displayed similar patterns of H I F - l a and pimonidazole staining as the xenograft tumours. The amount of mismatch between the two markers, however, appears greater than that seen for the cervical xenografts, possibly due to the 24hr separation between pimonidazole administration and biopsy.  -67-  Figure 21.  The distribution of HIF-la and pimonidazole in cervical tumour biopsies. A.  Patients were administered pimonidazole (500mg/m i.v.) 24hrs before biopsy. Fluorescent 2  immunohistochemistry was used to visualise areas of HIF-la (red) and pimonidazole (green), and the vascular marker CD31 (blue). B is an image of a cervical tumour biopsy cord, and C and D are SiHa cords from xenografts excised 1.5hr and 24hr after pimonidazole administration, respectively.  -68-  4.  DISCUSSION  4A  MARKER KINETICS . The nature of hypoxia, whether chronic or transient, poses different challenges in  both tumour progression as well as in therapy, and may require different strategies for treatment.  Therefore markers with kinetics that, when applied together, allow one to  identify these two forms of hypoxia, would be advantageous.  Kinetics studies of H I F - l a  in SiHa cells revealed that the protein is quickly stabilised (half-max ~2hr) and rapidly degraded (t!/ <lmin). In accordance, other studies reveal H I F - l a half-life to be on the 2  order of minutes (220, 221), whereas there appear to be differences in kinetics of development.  Similar to the results from this study, Vordermark et al, using U87 M G  human glioblastomas observed H I F - l a levels to peak at 6hr and to maintain these levels up to 18hr (222), and in Fadu and HT1080 cells, to increase sharply at lhr and continue to increase up to 24hr (223).  In contrast, Jewell et al. (221), studying HeLaS3, cells  observed a half-maximum level of H I F - l a as early as 13.3min and a maximal level at lhr. A possible explanation for these variations may be cell-type differences (224), or differences in assay technique; H I F - l a is a very labile molecule, such that small differences in protocol can affect its stability. As observed from SiHa cells, the quick response to changes in oxygen make this marker an ideal candidate for the study of transient changes in perfusion. Although pimonidazole adducts form very quickly in the absence of oxygen (half-max = 20-40min), they are lost very slowly (iVi ~2dy). Therefore once pimonidazole adducts are formed, they will not respond to subsequent changes in oxygen. These characteristics allow pimonidazole to be predictive of hypoxic past, and when combined with the HIF-la, to allow the determination of areas where  -69-  only one of the markers maybe mainly present (transient hypoxia), and regions where both markers are present (mostly chronic hypoxia).  4B  MARKER COLOCALISATION Fluorescent imaging revealed H I F - l a labelling to be strictly nuclear and focal,  and pimonidazole labelling to be focal and cytoplasmic.  Furthermore, mismatch was  found to occur close to the blood vessel, where H I F - l a was stabilised in the absence of pimonidazole binding. The principle explanation for this is that pimonidazole requires lower oxygen levels (-1%)  for binding than does H I F - l a (-2%), and an additional  possibility may be that H I F - l a expression is higher in more metabolically active cells, located closer to the blood vessels. Mismatch was also observed at the perinecrotic edge, where pimonidazole adducts were found in the absence of HIF-la. Possible explanations for this observation are discussed in section 4D. Areas showing atypical marker patterns, defined as those deviating from the normal pattern as shown in figure 9, were a further source of mismatch.  In all three xenograft types, there were occasional cords where  labelling of one or the other marker was low or absent, suggesting that transient changes in perfusion may have occurred in the short time frame of about l-2hr.  However,  tumour cords are three dimensional, and so the contribution of out-of-plane blood vessels cannot be excluded. The two most common patterns of this type seen were: a) H I F - l a labelling in the presence of little or no pimonidazole and an absence of Hoechst 33342, and b) pimonidazole labelling close to the centre of the tumour cord in the presence of Hoechst 33342 but little or no H I F - l a labelling. The first pattern may be suggestive of a tumour cord having been well perfused at the time of pimonidazole administration and  -70-  later becoming much less perfused, possibly due to vessel closure.  This would allow  H I F - l a to become stabilised very near the centre of the cord in the absence of both pimonidazole and Hoechst 33342.  The latter pattern may be indicative of the recent  reopening of the blood vessel, where the tumour cord may have been sufficiently hypoxic at time of pimonidazole administration, but may have become reperfused sometime before Hoechst 33342 administration. This would result in most of the H I F - l a being degraded, whereas pimonidazole, which does not respond to subsequent oxygen changes, would remain stable.  The most commonly observed atypical pattern in the xenografts  occurred where both H I F - l a and pimonidazole maintained their normal pattern relative to each other, but labelled very near, and in the case of HIF-la, within a brightly-labelled Hoechst 33342 area. This is suggestive of the existence of hypoxia in functional blood vessels, possibly due to a steep longitudinal oxygen gradient inside the vessel, or the presence of plasma channels, having a low supply of red blood cells. Despite these occasional patterns, studies revealed that the two markers generally showed a reasonable degree of colocalisation, as observed in three different xenograft types.  This result agrees with the findings of Vukovic et al. (225) who showed that  overlap between H I F - l a and EF5, another exogenous hypoxic marker, was statistically significant in SiHa xenografts, although associations in the present study were stronger. Vordermark et al. (222), however, found low colocalisation between H I F - l a and pimonidazole in U87 M G glioblastoma xenografts using a double staining protocol for flow cytometry.  Discrepancies may result from differences in methods used (flow  cytometry vs. fluorescence imaging), as well as thresholding techniques, which are to some extent subjective.  -71-  The use of endogenous markers such as H I F - l a would allow the study of archival material and would be more convenient and less expensive, given no administration of costly drug would be necessary.  It would, therefore, be useful to assess the hypoxic  marker pattern in the xenograft model in relation to the pattern seen in the patient biopsies, as will be discussed in section 4E.  4C  RESPONSE TO OXYGEN MODULA TION To see how our xenograft model system responded to changes in oxygen, mice  bearing xenograft tumours were subjected to carbogen, ambient oxygen, and 10% 02breathing. The response of the system was very significant in the carbogen -breathing mice to the extent that the range of labelling of both markers decreased dramatically and in many regions, disappeared completely. For this reason, 60% O2 rather than carbogen was used in order to allow quantification of marker labelling, given that the latter images contained so little of the markers that analysis of representative sections became problematic. In the 60% 02-breathing mice, the system showed a significant response, and the labelling of both markers decreased approximately to the same extent (figure 16). In the 10% 02-breathing mice, however, the range of labelling of H I F - l a significantly increased, while that of pimonidazole remained largely unchanged. Similar observations were made by Vordermark et al. (222) who showed in flow cytometry studies performed on U87 glioblastoma xenografts that the percentage of pimonidazole-positive cells did not increase following 10% oxygen-breathing, despite the increase in the percentage of H I F - l a positive cells. Olive et al. (226) showed in SCC7 xenograft tumours using a flow cytometric assay, that -15% of cells were labelled under air-breathing, -35% labelled  -72-  under 10% 02-breathing, and -90% in clamped tumours.  The flow cytometry data  suggests 10% 02-breathing to have a much lesser effect on hypoxic fraction than clamping.  Interestingly, Raleigh et al. (227), using fluorescence imaging in C3H  mammary xenograft tumours, showed that clamping greatly increased the extent of pimonidazole staining from -10% in air-breathing to -40%, but that immunostaining did not completely cover perivascular regions.  This again suggests, in light of the flow  cytometry data, that since clamping only produced 40% immunostaining, decreasing oxygen breathing from 21% (air) to 10% would not have had a drastic effect in the percentage of pimonidazole labelling (on a tumour that is already quite hypoxic) using a fluorescence imaging method. One possible explanation for these results may be that reducing oxygen-breathing by half produces a greater population of intermediately hypoxic than highly hypoxic cells; given that vascular oxygen levels in tumours average from 2-3%, a drop from 20% to 10% in oxygen breathing may easily push vascular oxygen levels below 2%, allowing H I F - l a to be stabilised, but may not be as effective in dropping oxygen levels below 1% in order to allow pimonidazole adduct formation. Another explanation may involve the concentration of pimonidazole administered to mice. Durand et al. (215) observed that the percentage of pimonidazole-positive tumour cells increased as a function of dose in the range of 0-200mg/kg. In the current study and that of Vordermark et al., pimonidazole was given i.p. at lOOmg/kg, and in the study by Raleigh et al., the dose was administered i.v. at 60mg/kg. The possibility thus may exist that the doses used in these studies may have been insufficient to label the increased number of hypoxic cells. Mismatch studies revealed that the amount of overlap between the markers was not maintained among the different oxygen-breathing conditions,  -73-  although there were greater similarities observed between the air-breathing and 60% (Inbreathing mice. This likely reflects differences in the oxygen thresholds of the markers, and further suggests that the relationship between change in the number of intermediately and highly hypoxic cells as a function of oxygen-breathing levels may not be spatially and/or numerically proportional.  4D  HIF-la: OXYGEN AND NUTRIENT EFFECTS The observation that pimonidazole adducts form in perinecrotic areas in the  absence of H I F - l a led us to question whether certain conditions in these regions are suitable for H I F - l a stabilisation. Jiang et al. (228) have previously suggested that the stabilisation of H I F - l a peaks at 0.5% 0 and diminishes under anoxic conditions. It was 2  therefore necessary to investigate whether anoxia in perinecrotic regions may be the reason for the absence of HIF-la. However, both our in vitro and in vivo studies suggest that this is unlikely, since H I F - l a showed greatest stabilisation at 0% O2, and when the oxygenation of perinecrotic regions of xenograft tumours was increased by having mice breath 60% O2, H I F - l a still failed to appear in these regions. The lack of agreement in the in vitro results may be due to differences in experimental design.  Whereas the  experiments in this study were performed in spinner culture flasks, Jiang et al. used vessels wherein cells, immersed in a thin film of medium, were equilibrated with gasses. It is possible that these cells may have exhausted their nutrient supplies given the 4hr incubation time and the small amount of medium available to them, so that not only hypoxia, but also lack of nutrients may have contributed to the observations.  We next  investigated nutrient requirements for H I F - l a stabilisation. In vitro experiments under  -74-  anoxia revealed that the decrease in stabilisation may be due to lack of serum factors and glucose in these regions.  With respect to serum levels, this observation appears  reasonable given that certain growth factors are responsible for the transcriptional and translational induction of the steady state level of H I F - l a via the PI3K and M A P K pathways respectively. Furthermore, although glucose deprivation alone did not seem to affect H I F - l a stabilisation, its combination with serum factors may be important in HIFl a induction; glucose levels in anoxic regions would be most critical, given that the lack of oxygen forces cells to rely on anaerobic glycolysis alone, which is a much less efficient method of A T P production than oxidative reduction. Therefore, in the absence of both serum and glucose, the production of H I F - l a may be very costly and the upregulation of other genes by HIF-1 may not be possible with limited cellular resources. Pimonidazole, nevertheless, which is easily metabolised by cells with little cost in energy, led to adduct formation in nutrient-deprived perinecrotic cells, although there is evidence that cells incubated under very low glucose levels (0.015mM) bind 2.5 times less misonidazole, a chemically related drug (229). This raises the question of whether hypoxic perinecrotic cells marked by pimonidazole are of radio-biological significance; given that H I F - l a is a major survival protein under hypoxia and is believed to be a prognostic marker for survival, cells that have ceased to produce H I F - l a may be very sick and of little threat to repopulation following therapy. It is possible, however, that upon clearance of normoxic cells, these perinecrotic cells, upon reperfusion, may recover sufficiently to once more produce HIF-la.  There are difficulties in investigating this  theory in vivo. Flow cytometric analysis of disaggregated SiHa xenografts, where pimonidazole-labelled cells were sorted based on intensity of Hoechst 3334 staining,  -75-  revealed that cells with the lowest Hoechst 3334 staining  (i.e., pimonidazole-labelled  cells furthest from the blood vessel) were able to divide and form colonies when plated (230). This cell population, however, may not represent the perinecrotic pimonidazole labelled cells in the xenograft, since these could have been lost upon enzyme digestion. Perhaps one approach is to use a double staining protocol where cells stained for pimonidazole  and not  HIF-la  are  sorted,  but the  problem arises that upon  homogenisation of the xenograft, the resultant reoxygenation would lead to H I F - l a degradation, and even minimal loss in the presence of C0CI2 may make the data difficult to interpret.  Thus whether this population of cells is relevant to therapy remains  unknown and will impact on the interpretation of hypoxia marker binding in clinical samples.  4E  HYPOXIC CELL HALF-LIFE AND RELA TION WITH PA TIENT BIOPSIES To see how well the xenograft model system represented patient biopsies, cervical  tumour biopsies from two patients were stained for HIF-la, pimonidazole, and CD31. The pattern seen in the patient biopsies was very similar to that observed in the xenografts, where H I F - l a and pimonidazole showed focal nucleic and cytoplasmic labelling respectively, both occurring at a distance from blood vessels.  Although the  spatial patterns of H I F - l a and pimonidazole were maintained with respect to the blood vessels, there was little overlap between the two markers; H I F - l a was expressed closer to the blood vessels as expected, but pimonidazole appeared to be present further out along the edges of the tumour cord. One possibility for this lack of overlap could be the 24hr time-period between administration of pimonidazole and biopsy; dividing tumour  -76-  cells from the centre of the cord may have pushed the once viable pimonidazole-labelled cells further into regions of perinecrosis and necrosis.  To investigate this, mice with  SiHa xenografts were administered pimonidazole 1.5, 12, 24, and 48hrs prior to tumour excision.  Images from these xenografts  revealed that longer intervals between  pimonidazole administration and tumour excision caused a decrease in the degree of overlap between the two markers, and that pimonidazole-labelled cells appeared to be progressively driven into necrotic areas. Accumulated changes in perfusion resulted in even greater mismatch between the two markers. It was also observed that on some slides, necrotic areas were lost during the staining process, consequently resulting in the loss of a considerable percentage of pimonidazole-labelled cells. Indeed the colocalised fraction with respect to H I F - l a decreased from -60% at 1.5hr to -30% at 24hr, and -7% at 48hr. One concern in these studies was that the plasma half-life in humans is considerably longer than that in mice (6hr vs. 30min) so that pimonidazole is available and metabolised over a longer period of time. Nevertheless a large degree of mismatch was observed even at the 12hr time point, so that the longer plasma half-life of pimonidazole does not compensate for the long wait period between pimonidazole administration and biopsy. Furthermore, colocalisation between the 12hr and 24hr time points was very similar, which may owe to the fact that mice at the 12hr time point were administered pimonidazole overnight, and murine rates of metabolism and blood flow increase nocturnally (231, 232).  Overall, the hypoxic half-life in SiHa xenografts was  measured to be ~16hr. This result agrees with those of Durand et al. who, using flow cytometric measurement of pimonidazole labelling in SiHa xenografts, found a tumour cell half-life of ~4dy (233), and an average hypoxic cell half-life of ~17hr (unpublished  -77-  data).  Ljungkvist et al., using a fluorescent imaging techniques with two different  chemical hypoxic markers, found hypoxic turnover rates to vary between tumour types, and to be related to both the organisation of hypoxic cells as well as their potential doubling times (in press). In an experiment conducted on patients with squamous cell carcinoma of the head and neck, Janssen et al. (234) found no significant colocalisation between H I F - l a and pimonidazole.  In their study, nuclear H I F - l a staining was observed in 0-18% of the  tumour tissue, and colocalisation with respect to H I F - l a varied from 0.02-25%. Furthermore, from the 6 evaluable tumours, H I F - l a labelling was greatest distal from the blood vessel in only 1. Nevertheless, Aebersold et al. (191) in a study of 98 patients with squamous cell cancer of the oropharynx found focal expression of H I F - l a distal from the blood vessel in 65% of the biopsies, where as diffuse staining independent of vessel proximity was observed in 35%.  Other studies have also found increased focal  expression of H I F - l a distal from the blood vessels (235, 236). The very low fraction of biopsies having focal staining in Janssen's study therefore may be the result of a small sample size.  The diffuse H I F - l a staining observed in both studies may be due to  improper handling of biopsies, since the material becomes hypoxic upon removal, and H I F - l a stabilises within minutes of hypoxic exposure.  A further reason may be the  genetic heterogeneity of the biopsies, where in some tumours, pathogenic pathways involved in H I F - l a regulation may be active.  The low colocalisation of H I F - l a and  pimonidazole in the above study may also be due to the patients having been administered pimonidazole the day before biopsy, which may, as observed in the current study, result in even less overlap between the two markers.  -78-  Evidence from xenograft studies, as well as results obtained from a small number of cervical cancer biopsies in our study suggest that H I F - l a may be a good endogenous marker for the detection and quantification of hypoxia in cervical tumours, provided care is taken to promptly fix or freeze tumours after biopsy. Results also indicate that waiting too long after administration of pimonidazole to take a biopsy may provide an inadequate representation of the hypoxia in the tumour, and may lead to underestimation if pimonidazole-labelled  necrotic  areas  are  lost  during  slide  processing.  The  recommendation is therefore made that if possible, biopsies be taken no more than 12hr following pimonidazole administration. This is the time course typically used by the Nordsmark et al. (237), so it will be of interest to compare our results in an ongoing pimonidazole study with theirs. A larger study examining the relationship of H I F - l a and pimonidazole in cervical cancer tumours would be furthermore necessary to properly assess the suitability of the use of H I F - l a as a hypoxia marker in these patients.  -79-  CONCLUSIONS  Given the importance of hypoxia in tumour progression and the obstacles it poses to therapy, effective methods to measure hypoxia in order to identify patients who would benefit most from hypoxia-directed therapies should be established.  The present study  supports the use of H I F - l a as reliable endogenous hypoxic marker in cervical tumours. Xenograft models reveal substantial overlap in antibody staining of H I F - l a and pimonidazole, and also suggest that H I F - l a may be a more immediate representation of the hypoxia within a tumour due to its rapid stabilisation under anoxia and rapid loss upon reoxygenation.  In considering whether H I F - l a and pimonidazole label the same  cells within a tumour, it is apparent from these results that mismatch between these markers is related not only to differences in oxygen dependency of binding/expression, notably in perivascular regions, but also to differences in nutrient requirements for binding/expression, notably in perinecrotic regions.  A further consideration is that  mismatch can occur if there is a long delay between pimonidazole administration and biopsy.  Regions labelled with pimonidazole can progress quite quickly into areas of  necrosis. The type of hypoxia within a tumour is also of great importance since transient and chronic hypoxia may present different challenges upon the effectiveness of tumour therapy, as well as choosing an appropriate hypoxia-directed therapy. The use of both markers together with a perfusion marker suggested the presence of regions of transient hypoxia, a method that once validated can possibly be applied to patient biopsies, given that an appropriate time course for marker stabilisation or binding is permitted.  -80-  BIBLIOGRAPHY 1. Vaupel P. Tumour microenvironmental physiology and its implications for radiation oncology. Semin Radiat Oncol 2004;14:198-206. 2. Hoberman H D . Is there a role for mitochondrial genes in carcinogenesis? Cancer Res 1975;35:3332-3335. 3. Warburg O. The metabolism of tumours. London, Costable & Co. 1930. 4. Hockel M , Schlenger K , Arad D, Miltze M , Shaffer U , Vaupel P. 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