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Intermittent blood flow in the murine SCCVII squamous cell carcinoma Trotter, Martin James 1990

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INTERMITTENT BLOOD FLOW IN THE MURINE SCCVII SQUAMOUS CELL CARCINOMA  By MARTIN JAMES TROTTER  B.Sc. The University of British Columbia, 1981 M.D. The University of British Columbia, 1985  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY  in THE FACULTY OF GRADUATE STUDIES (Department of Pathology)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA May, 1990 © Martin James Trotter, 1990  In  presenting this  degree at the  thesis  in  University of  partial  fulfilment  of  of  department  this or  thesis for by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make  It  is  granted  by the  understood  that  publication of this thesis for financial gain shall not be allowed without permission.  Department  of  PATH-oi-ocVy  The University of British Columbia Vancouver, Canada  DE-6 (2/88)  it  agree that permission for extensive  scholarly purposes may be her  for  head of copying  my or  my written  ii  ABSTRACT  Intermittent blood flow in tumour microvasculature is believed to contribute to heterogeneity in tumour oxygen delivery; transient vessel nonperfusion is thought to result in acutely hypoxic cells resistant to conventional radiotherapy.  This thesis describes three main areas of work: (1) the  development of a histologic method capable of detecting intermittent blood flow in experimental tumours at the single vessel level; (2) the quantification and characterization of tumour blood flow fluctuations in the murine SCCVII carcinoma; and (3) the modification of tumour blood flow and the reduction of flow heterogeneity using vasoactive drugs.  A double staining technique involving the sequential intravenous injection of two fluorescent vascular markers was used to detect transient episodes of tumour vessel nonperfusion. The stains employed were Hoechst 33342 and the carbocyanine dye, DiOC (3), both of which have short 7  (< 3 minutes) circulation half-lives and preferentially stain cells adjacent to perfused blood vessels. When injections of the vascular markers are separated by some interval, each stain defines only those tumour vessels which were perfused during the few minutes immediately post-injection; thus, two "pictures" of tumour microvascular flow are obtained and tumour vessels subject to periods of nonperfusion can be easily visualized in frozen sections since they are outlined by one stain but not the other.  Using the double staining technique, in which Hoechst 33342 and then DiOC (3) are administered 7  intravenously 20 minutes apart to unrestrained C3H/He mice, staining mismatch (indicative of transient vessel nonperfusion) is regularly observed in subcutaneous SCCVII carcinoma. Vessels stained with DiOC (3) only (reperfusion of previously nonperfused vessels) or with H33342 only 7  (nonperfusion of previously perfused vessels) are observed in approximately equal numbers. The percentage of tumour vessels subject to intermittent flow is a function of SCCVII tumour size: tumours <100 mg do not exhibit statistically significant amounts of mismatch. At sizes > 100 mg, overall staining mismatch is significantly increased over background levels and maximum  iii mismatch is observed at tumour sizes >400 mg (8.6 ± 2 . 9 % ) .  In most tumours, transient vessel  nonperfusion is more pronounced in central tumour regions. In addition to mismatch observed in individual vessels, large "patches" of unequal staining are also seen. Anaesthesia or restraint do not significantly influence intermittent blood flow.  The above information suggests that transient episodes of tumour vessel nonperfusion occur as a consequence of flow reduction in a feeding vessel; vessels in central regions of large tumours may be susceptible to collapse as a result of elevated tumour interstitial pressure.  In the SCCVII  tumour, a small number of peripheral vessels possess vascular smooth muscle and thus may be capable of vasomotor activity.  The importance of perfusion pressure in the control of tumour microcirculatory flow was examined using vasoactive drugs.  Hydralazine, a vasodilator which lowers blood pressure, causes a  profound reduction in tumour RBC flow to 8.7 + 6.4% of pretreatment values in unanaesthetized mice.  The drug causes collapse of central tumour vessels: following a dose of  10mg/kg  intravenously, 3 6 ± 1 6 % of vessels are completely nonperfused, as detected using the double staining  technique.  Conversely, elevation  of  blood pressure  angiotensin II results in a 2-3x increase in tumour blood flow.  using the vasoconstrictor  In addition, angiotensin II infusion  significantly reduces the number of tumour vessels subject to transient nonperfusion from 8.1 % to 2.0%.  However, intermittent blood flow in the SCCVII carcinoma can also be influenced by  nonvasoactive drugs: nicotinamide, the amide form of vitamin B3, reduces episodes of transient nonperfusion.  In summary, intermittent blood flow has been characterized in a transplanted murine squamous cell carcinoma using a novel fluorescent double staining method which allows the detection of flow fluctuations in solid tumours at the microvascular level. If transient episodes of nonperfusion occur in human tumours and result in impaired oxygen or drug delivery, then such flow fluctuations may be an important factor limiting tumour cure or local control by radiotherapy or chemotherapy.  iv TABLE OF CONTENTS  Page ABSTRACT  ii  TABLE OF C O N T E N T S  iv  LIST OF FIGURES  vii  LIST OF TABLES  ,..x  ACKNOWLEDGEMENTS  xi  1.  INTRODUCTION  1  1.1  Tumour vasculature and blood flow: therapeutic importance  1  1.2  Vascularization of tumours  5  1.2.1  Methods of study  5  1.2.1.1  In vivo methods  6  1.2.1.2  Histologic studies  7  1.2.1.3  Injection techniques  1.3  1.4  10  1.2.2  Tumour angiogenesis  13  1.2.3  Structural abnormalities  17  1.2.3.1  Vascular architecture  17  1.2.3.2  Vascular morphology  19  1.2.3.3  Vessel wall  21  Tumour blood flow  24  1.3.1  Perfusion rate of tumours  24  1.3.1.1  Microvascular pressure  25  1.3.1.2  Viscous resistance  26  1.3.1.3  Geometric resistance  27  1.3.1.4  Tumour interstitial pressure  28  1.3.2  Heterogeneity  30  1.3.3  Intrinsic control of tumour blood flow  32  Tumour hypoxia  35  1.4.1  Evidence for tumour hypoxia  35  1.4.2  Mechanisms of impaired oxygen supply to tumours  37  1.4.2.1  Chronic hypoxia  38  1.4.2.2  Acute hypoxia  39  1.5.  Thesis objectives  41  2.  MATERIALS AND METHODS  43  2.1  Fluorescent perfusion probes  43  2.1.1  43  Hoechst 33342  V  Page  2.2  2.1.2  Carbocyanine dyes  43  2.1.3  Rhodamine6G  45  2.1.4  FITC-dextrans  45  2.1.5  Zinc-cadmium sulphide particles  45  Modulators of tumour blood flow  46  2.2.1  Hydralazine  46  2.2.2  Angiotensin II  46  2.2.3  Nicotinamide and pyrazinamide  46  2.3  Mice and tumours  47  2.4  Pharmacokinetic and physiologic parameters  48  2.4.1  Pharmacokinetics of fluorescent vascular markers  48  2.4.2  Blood pressure and temperature  49  2.4.3  Metabolic parameters  50  2.5  Radiobiology studies  51  2.6  Double staining technique  52  2.7  Quantitative fluorescence measurements  53  2.8  Vascular morphology  54  2.9  Vascular smooth muscle  56  2.10  Oxygen diffusion distance  56  2.11  Theoretical estimates of tumour hypoxia  57  2.12  Laser Doppler flowmetry  58  2.13  Statistical analysis  59  3.  DOUBLE F L U O R E S C E N T STAINING TECHNIQUE  60  3.1  Introduction  60  3.2  Results  62  3.2.1  Perivascular localization of fluorescent stains  62  3.2.2  Pharmacokinetics  66  3.2.3  Toxicity  69  3.2.4  Cardiovascular and metabolic effects of H33342  69  3.2.4.1  Blood pressure/heart rate  69  3.2.4.2  Metabolic parameters  72  3.2.5  Tumour blood flow  72  3.2.6  Radiobiologic effects  80  3.3  Discussion  80  4.  INTERMITTENT BLOOD FLOW  84  4.1  Introduction  84  vi Page 4.2  4.3  Results  86  4.2.1  Tumour size  86  4.2.2  Patches of nonperfusion  90  4.2.3  Implantation site  90  4.2.4  Restraint/anaesthesia  94  4.2.5  Time course  94  4.2.6  Quantitative fluorescence measurements  99  4.2.7  Human tumour xenografts  99  4.2.8  Vascular smooth muscle  103  4.2.9  Vascular morphology  103  4.2.10  Oxygen diffusion distance  103  4.2.11  Intermittent blood flow and acute hypoxia  111  Discussion  113  4.3.1  Mechanisms for tumour blood flow intermittency  113  4.3.2  Duration of tumour vessel nonperfusion  116  4.3.3  Tumour oxygenation: significance of intermittent flow  118  5.  MODULATION OF TUMOUR BLOOD FLOW  121  5.1  Introduction  121  5.2  5.3  5.1.1  Hydralazine  122  5.1.2  Angiotensin II  123  5.1.3  Nicotinamide  124  Results  125  5.2.1  Hydralazine  125  5.2.2  Angiotensin II  130  5.2.3  Nicotinamide  139  Discussion  141  5.3.1  Hydralazine  141  5.3.2  Angiotensin II  147  5.3.3  Nicotinamide  150  6.  SUMMARY AND CONCLUSIONS  153  7.  REFERENCES  156  vii LIST OF FIGURES  Page Figure 1:  Figure 2:  Chemical structures of H33342 and of symmetric carbocyanine dyes  44  Photomicrographs of the Hoechst 33342 and DiOC (3)  63  fluorescent  vascular  markers  ?  Figure 3:  Figure 4:  Photomicrographs of FITC-dextran and zinc-cadmium sulphide particles within tumour vessels  64  Stability of H33342 and DiOC (3) staining of SCCVII tumour cells in 7  vivo  65  Figure 5:  Rate of bleaching of H33342 and DiOC (3)  67  Figure 6:  Blood levels of fluorescent vascular markers following intravenous bolus injection Effect of intravenous DiOC (3) on surviving fraction of SCCVII  68  tumour cells plated in vitro  70  Figure 8:  Effect of H33342 on mouse arterial blood pressure and heart rate  71  Figure 9:  Effect of H33342 on tumour RBC flow  74  Figure 10:  Effect of H33342 on tumour RBC flow: dose response  75  Figure 11:  Effect of H33342 on RBC flow measured in foot skin  77  Figure 12:  Effect of DiOC (3) on tumour RBC flow  78  Figure 13:  Effect of H33342 bolus injection on the response of tumours to 10 Gy x-irradiation  Figure 7:  Figure 14:  Figure 15:  Figure 16:  Figure 17:  Figure 18:  7  7  7  Evidence for "opening" (reperfusion) SCCVII carcinoma microvessels  79  of previously nonperfused 87  Evidence for "closing" (nonperfusion) SCCVII carcinoma microvessels  of previously  Influence of tumour size on overall subcutaneous SCCVII carcinoma  perfusion  Frequency histogram of staining subcutaneous SCCVII tumours  SCCVII  perfused 88  mismatch  mismatch  in 89  "patch"  size  in  Staining mismatch in SCCVII tumours implanted in gastrocnemius muscle  92  93  viii Page Figure 19:  Figure 20:  Figure 21:  Figure 22:  Figure 23:  Figure 24:  Figure 25:  Figure 26:  Figure 27:  Influence of animal restraint or anaesthesia on transient perfusion in subcutaneous SCCVII tumours  95  Effect of the interval between stain injections on vessel "opening" and vessel "closing" in subcutaneous SCCVII carcinoma  98  Quantitative analysis of intermittent fluorescence image processing system  blood  flow  using the 100  Photomicrograph of SCCVII tumour section monoclonal antibody against muscle-specific-actin  stained  with  Frequency histogram of H33342-defined subcutaneous SCCVII tumours <500 mg  vessel diameters for  Frequency histogram of H33342-defined subcutaneous SCCVII tumours >500 mg  vessel diameters for  104  105  106  H33342-defined vascular morphology parameters in subcutaneous SCCVII tumours  107  Digitized image of a "cube" of SCCVII tumour tissue incubated in vitro with the hypoxia probe AF-2  108  Influence of temperature on oxygen diffusion distance in SCCVII tumour cubes  110  Figure 28:  Effect of hydralazine (10 mg/kg i.v.) on SCCVII RBC flow  126  Figure 29:  Effect of hydralazine dose on SCCVII RBC flow  127  Figure 30:  Influence of ketamine/diazepam anaesthesia on hydralazineinduced RBC flow reductions in SCCVII carcinoma  128  Hydralazine-induced reductions in R B C flow in human tumour xenografts grown in athymic nude mice  129  Staining mismatch in a subcutaneous SCCVII carcinoma following administration of hydralazine  131  Evidence for vessel closure in murine SCCVII carcinoma following hydralazine administration  132  Effect of hydralazine dose on functional vasculature in murine SCCVII carcinoma  133  Effect of angiotensin II infusion on mouse arterial blood pressure and heart rate  135  Effect of angiotensin II infusion on RBC flow in mouse skin or in SCCVII carcinoma  136  Figure 31:  Figure 32:  Figure 33:  Figure 34:  Figure 35:  Figure 36:  ix Page Figure 37:  Figure 38:  Relationship of tumour RBC flow to mean arterial blood pressure changes induced by angiotensin II infusion  137  Effect of nicotinamide or pyrazinamide on staining mismatch in SCCVII carcinoma  '.  140  Figure 39:  Effect on nicotinamide on H33342 pharmacokinetics  142  Figure 40:  Effect of nicotinamide on H33342-induced reductions in tumour RBC flow  143  X  LIST O F T A B L E S  Page Table I:  Table II:  Table III:  Table IV:  Table V:  Effect of H33342 on venous blood gases, pH, oxyhemoglobin saturation, and hematocrit  73  Staining mismatch in peripheral subcutaneous SCCVII carcinoma  91  and  central  regions  of  Effect of the interval between stain injections on staining mismatch in subcutaneous SCCVII carcinoma  97  Intermittent blood flow in human tumours grown as xenografts in athymic, nude mice  102  Effect of angiotensin II on intermittent blood flow  .....138  xi ACKNOWLEDGEMENTS  "Among scientists are collectors, classifiers, and compulsive tidiers-up; many are detectives by temperament and many are explorers; some are artists and others artisans.  There are poet-  scientists and philosopher-scientists and even a few mystics." Sir Peter Brian Medawar (1967).  I would like to thank my supervisor, Dr. Peggy Olive, for an office door that was always open and for her whole-hearted support over the past three years.  A great many ideas for work performed in this thesis arose from discussions with Dr. Dai Chaplin. Dai is not only a colleague who freely shares his scientific theories (however improbable) but he has also become a close friend.  I would also like to acknowledge Dr. Ralph Durand, especially for his help with image processing, cell sorting, and computer-related foul-ups.  Technicians and fellow graduate students at the B.C. Cancer Research Centre helped me prepare and perform many experiments and I would like to particularly acknowledge Douglas Aoki, Sandy Lynde, Bill Grulkey, Carrie Peters, Carissa Toth, Charlene Vikse, Nancy LePard, and Denise MacDougal. I would like to thank Fred Jensen for expert care of the animals.  Colleagues beyond the confines of the Research Centre also assisted me in my research work and I wish to acknowledge their help:  Dr. Brian Acker initiated the projects with laser Doppler  flowmetry; Bev Thomas performed the immunohistochemistry; and Betty Pearson spent many hours teaching me microsurgical techniques.  My wife, Theresa, has carried most of the domestic load especially in the past year and this thesis would simply not have been completed without her encouragement and her unique understanding of the many personal sacrifices which must always be made when one's spouse insists on "going to school" forever.  1  1.  1.1  INTRODUCTION  TUMOUR VASCULATURE AND BLOOD FLOW: THERAPEUTIC IMPORTANCE  Advancements in cancer research, specifically in the field of tumour biology, are realized at several levels of inquiry: molecular, cellular, organ/tissue, and whole animal. The rapid evolution of the broad discipline of molecular biology has shifted the experimental emphasis from the pathophysiological realm to that of important cellular and subcellular disease mechanisms. Nevertheless, structure/function abnormalities of neoplasia at the tissue level remain only partly understood and continued research is essential. Indeed, therapeutic exploitation of aberrant solid tumour structure, physiology, and metabolism may provide important adjuncts to conventional and novel treatment modalities.  Solid malignant tumours are composed of both cancerous cells and normal host components. Tumour growth, resulting from uncontrolled neoplastic cell division, is absolutely dependent on a parallel proliferation of the nonmalignant cells which comprise the tumour vasculature.  Thus,  neovascularization, uncommon in normal adult tissues, is a universal characteristic of all solid tumours larger than 1-2 mm diameter (Folkman, 1986; 1990). In nonneoplastic tissue, the vascular supply of an organ is balanced with the need for blood flow imposed by the metabolic activity of its cells. In general terms, Weibel (1984) called this principle "symmorphosis", defining it as "a state of structural design commensurate to functional needs resulting from regulated morphogenesis". Neoplasms, by definition, exemplify abnormal morphogenesis and there is a disproportionate relationship between tumour tissue and its vascular supply. Tumours are said to "outgrow" their blood supply; neovascularization lags behind the increase in the number of neoplastic cells (Tannock, 1970) and consequently the vasculature is unable to meet the increasing nutrient  2  demands of the expanding tumour mass. In solid tumour tissue, the principle of symmorphosis is not operative.  Since the geometry of a capillary network is dictated by the shape and arrangement of the cells it supplies (Baez, 1977; Weibel, 1984), it is not surprising that tumour vasculature, like the cancerous tissue it serves, is morphologically and functionally abnormal. Tumour blood supply is characterized by spatial and temporal heterogeneity in both structure and function. A decline in average blood flow with tumour growth can lead to randomly distributed regions of altered microenvironment (Vaupel et al., 1981; Kallinowski et al., 1989).  The development of hypoxia,  acidosis, and nutrient depletion can appreciably alter the tumour response to nonsurgical therapy (Moulder and Rockwell, 1987; Wike-Hooley et al., 1984; Teicher et al., 1981).  For example,  radioresistant hypoxic cells exist in some solid human tumours and can influence the response to radiation therapy (Cater and Silver, 1960; Urtasun et al., 1976, 1986b; Henk and Smith, 1977; Denekamp et al., 1977; Gatenby et al., 1988). Thus, tumour vascular insufficiency, with resultant nutrient failure, has important clinical relevance.  Deficiencies in tumour blood supply also limit the clinical efficacy of some chemotherapeutic agents as a result of inadequate drug uptake and nonoptimal distribution in tumour tissue (Jain, 1989). The physiological factors affecting drug delivery have not generally been addressed in development of new anticancer agents.  Methods to manipulate tumour perfusion, especially to  increase flow and reduce heterogeneity or to suppress flow after an agent has been delivered, may be expected to improve tumour localization of, for example, drugs, monoclonal antibodies, biological response modifiers, and lymphocytes. Modulation of tumour blood flow for therapeutic benefit is therefore an area of active experimental and clinical investigation (Denekamp et al., 1983; Denekamp, 1984; Hirst, 1986a; Brown, 1987; Siemann, 1987; Smyth et al., 1987; Jirtle, 1988; Stratford et al., 1988; Chaplin, 1988; 1989; Guichard, 1989).  3 Inadequate vascularization of neoplastic tissue, while clearly detrimental to treatment success, can, conversely, be viewed as a potential target for therapy.  In contrast to the endothelium of  normal blood vessels, vascular endothelium of tumour vessels is actively proliferating (Hobson and Denekamp, 1984) as a result of angiogenic stimuli (Folkman, 1986). Such cells may provide a target, perhaps antibody mediated, for tumour selective vascular attack (Denekamp, 1984). Evidence is also accumulating that vascular damage is an important component of several forms of cancer therapy (Song, 1984; Murray et al., 1987; Wieman et al., 1988; Nelson et al., 1988; Watanabe et al., 1988; Bibby et al., 1989; Zwi et al., 1989). Measures to enhance this vascular injury are therefore a meaningful consideration in the development of new treatment strategies.  Tumour physiology, rarely considered as an element in the design of cancer therapy, has recently received renewed attention as a parameter modifiable for therapeutic benefit and as a target for treatment. Tumour vasculature is of particular interest since it differs from that of normal tissue both in morphology and function.  Tumour blood flow is the principal modulator of tumour  metabolism and microenvironment and plays a crucial role in optimal delivery of anticancer agents and in tumour response to radiotherapy or hyperthermia. An understanding of tumour vascular function, especially at the microcirculatory level, can provide a therapeutic advantage and is an important component in our knowledge of tumour biology.  The study of tumour blood flow and its relevance to cancer therapy can be addressed using many conceptual and experimental approaches. In this thesis, major emphasis will be placed on the relationship between vascular structure and function.  Structural abnormalities of tumour  vasculature influence, directly and indirectly, the factors which determine tumour blood flow and thus the tumour microenvironment.  This thesis introduction will review tumour vascularization,  tumour blood flow, and tumour hypoxia, the latter a therapeutically relevant consequence of abnormal tumour physiology.  4  The section on tumour vascularization will be subdivided into several parts: (1) methods of study: in this thesis a novel methodology was employed to investigate microregional perfusion in solid tumours and, therefore, methods of study are reviewed in some detail: (2) tumour angiogenesis, a prerequisite for expansive tumour growth; and (3) structural abnormalities of tumour vasculature vascular architecture, vascular morphology, and abnormalities of the blood vessel wall. Following this, blood flow in tumours will be reviewed:  (1) perfusion rate and the factors which determine  tumour blood flow - microvascular pressure, viscous resistance, geometric resistance, and tumour interstitial pressure; (2) flow heterogeneity, both spatial and temporal (the thesis work attempted to characterize, for the first time, intermittent blood-flow in solid experimental tumours); and (3) loss of intrinsic microcirculatory control mechanisms. In the final introductory section, the problem of tumour hypoxia in conventional cancer therapy, especially radiotherapy, and possible mechanisms responsible for the development of hypoxia, including fluctuations in tumour blood flow, will be discussed.  5  1.2 VASCULARIZATION OF TUMOURS  1.2.1 METHODS OF STUDY  Studies of tumour vasculature have been motivated by three general concerns (Warren, 1979): (1) measurement of tumour vascular patterns and vascular density as a diagnostic or prognostic indicator, (2) importance of vascular morphology in drug delivery to tumours, and (3) role of the vascular system in tumour biology, specifically tumour growth, metastasis, and the development of necrosis and tumour hypoxia.  The macroscopic architecture of tumour vasculature and vascular morphology (defined as the "spatial and temporal distributions of vascular volume, length, diameter, and vascular surface area" (Jain, 1988)), have been studied both qualitatively and quantitatively using a variety of techniques, including: (1) in vivo observation of vasculature using tumours grown as thin twodimensional sheets in transparent observation chambers, direct photographic observation (eg. culpophotography), or angiographic methods; (2) histologic examination of tumour sections using conventional stains or stains specific for vascular elements; these methods allow examination of the total vascular bed; (3) injection techniques using intravascularly administered agents which are retained in or demarcate blood vessels and can subsequently be detected in tumour sections; such techniques measure perfused or functional tumour vasculature.  Each of these methodologies will be described in some detail in the following sections but particular emphasis will be placed on the use of injection techniques, since, in the study of solid tumours growing in three dimensions, such methods provide both structural and functional information about tumour vasculature at the microvascular level. The application of injection techniques to study dynamic changes in tumour perfusion is described in Sections 3 and 4.  6  1,2.1.1 In vivo methods  Since the pioneering work of Algire (Algire, 1943; Algire and Chalkley, 1945), many investigators have analyzed vessel growth and vascular morphology using in vivo microscopy of tumours grown as thin two-dimensional sheets in transparent observation chambers (see reviews by Reinhold, 1979 and Jain, 1988). Transparent chamber techniques allow vascular patterns to be followed over time and they permit functional studies of perfusion dynamics under direct in vivo observation. There are several major disadvantages to the approach however, the most serious of which is the problem of elevated tissue pressure due to proliferation of tumour cells in a confined space. This pressure increase is in part responsible for the vascular stasis and necrosis observed in such "sandwich" preparations and indeed, blood flow in many nonperfused vessels can be restored by releasing pressure in the chamber (Eddy and Casarett, 1973; Reinhold, 1979). For these reasons and the problems of tearing, fibrosis, or infection in chambers left in situ for more than a few weeks, only the early stages of tumour growth can be studied. A final restriction is the requirement that tumours must be transplanted into subcutaneous tissues, and other sites are not easily studied.  Angiography (radiography following intravascular administration of radiopaque contrast media) is, apart from some specialized photographic techniques such as culpophotography (Koller, 1963), the only noninvasive method for the study of the vascular architecture in unrestricted threedimensional tumours and consequently has been used extensively in the diagnosis of human malignancies and in the macroscopic study of vascular patterns in both human and experimental tumours  (Ekelund,  1979).  Vasoactive agents have been employed in combination with  angiography (pharmacoangiography) in an effort to improve visualization of tumour vasculature and to differentiate malignant tumours from benign lesions or granulation tissue (Ekelund, 1979). Conventional angiographic methods lack the resolution required for the discrimination of tumour microvasculature although radiopaque agents can be used in invasive injection techniques requiring subsequent removal and sectioning of the tumour tissue (Section 1.2.1.3).  7  1.2.1.2 Histologic studies  Blood vessels can be identified in tissue sections by a variety of methods which can be broadly classified into three groups:  conventional histologic stains, specific stains for structural  components of blood vessels, and injection techniques. Histologic methods are invasive; since tumour tissue must be removed from the host, the major disadvantage of such methods is that vascular morphology cannot be followed at the same tumour location over time.  Conventional  histologic stains  Large blood vessels are easily identified in routine hematoxylin and eosin stained sections of tumour tissue. However, small thin-walled vessels such as capillaries often collapse during tissue preparation and cannot be visualized. Vessel staining can be enhanced using periodic acid Schiff (PAS) or trichrome stains, but these techniques are not specific for blood vessels. Tannock and Steel (1969) used a combination of PAS, Luxol fast blue, and hematoxylin to stain fixed sections of a murine mammary carcinoma and noted that the PAS-positive vascular endothelium gave good contrast with blue-stained erythrocytes and hematoxylin-stained nuclei. The Masson-trichrome technique has been used to estimate vascular density in cervix carcinoma (Siracka et al., 1988).  In addition to light microscopy, tumour vessels have also been examined using transmission or scanning electron microscopy (Warren, 1979; Grunt et al., 1985; 1986a, 1986b; Kaido and Uehara, 1987; Ahlstrbm et al., 1988).  Such ultrastructural studies formed the basis for a  classification of tumour vessels by Warren (1979), later modified by Jain (1987).  8  Specific stains for blood vessels (a) Enzyme histochemistry Histochemical staining techniques for alkaline phosphatase activity are a standard method for demonstrating endothelial cells in normal tissues. Alkaline phosphatase is found in the endothelial cells of all types of blood vessels (Gomori, 1939) but some authors claim that endothelial cell alkaline phosphatase activity is confined to the arterial side of the terminal vascular system (Mlynek et al., 1985).  Several studies have demonstrated that alkaline phosphatase activity is  reduced in tumour blood vessels compared to those of normal tissues (Manheimer and Seligman, 1948; Murray et al., 1989) but the method has been successfully utilized to demonstrate and quantify vasculature in human colorectal carcinoma (Mlynek et al., 1985) and in carcinoma of the cervix (Awwad et al., 1986).  A disadvantage of this technique is the occasional reactivity of  nonvascular stromal elements (Monis and Rutenburg, 1960); this positive reaction of connective tissue can make exact quantitation of vasculature impossible (Porschen et al., 1989). In addition, histochemical markers in general do not effectively distinguish blood vessel capillaries from lymphatic vessels (Barsky et al., 1983).  Enzyme histochemical methods have also been used to detect 5'-nucleotidase, an enzyme which is present on the surface of endothelial cells, and like alkaline phosphatase is active against phosphate monoesters such as AMP (Murray et al., 1989). Nucleoside diphosphatase activity can also be demonstrated on endothelial cells if ADP is used as a substrate for the enzyme. Murray et al. (1989) concluded that in murine tumours, ADPase activity is a good marker for vascular endothelium but that 5'-nucleotidase and alkaline phosphatase are poor markers since their activity was low or absent in the tumours studied.  (b) Lectin binding methods A lectin is defined as a sugar-binding protein or glycoprotein of non-immune origin which agglutinates cells and/or precipitates complex carbohydrates (glycoconjugates).  The most  9  promising application of lectins is in the analysis of glycoconjugates at the cell surface (Allison, 1986). Vascular endothelium can be identified by lectin-immunoperoxidase or lectin-fluorochrome staining using Ulex europaeus agglutinin I, a lectin with strong affinity for a-L-fucose (Holthofer et al., 1982). This lectin is not a specific marker of vascular endothelial cells since it has been shown to bind to lymphatic vessels (Fujime et al., 1984), squamous epithelia (Holthofer et al., 1982), and colorectal carcinomas (Yonezawa et al., 1982; Matsushita, et al., 1985). Its usefulness has been established as a specific and sensitive indicator of the endothelial origin of many soft tissue tumours (Allison, 1986). Ulex europaeus agglutinin I does not appear to stain murine endothelial cells (Murray et al., 1989).  (c) Immunological methods Antibodies which recognize antigenic determinants of structural components of blood vessels are extremely useful in the identification of tumour vasculature in histological sections. Antibodies raised against endothelial cell or basement membrane antigens have been used.  Human  factor Vlll-related antigen is a well established marker of vascular endothelium at least in human tissue (Mukai et al., 1980; Stephenson et al., 1986).  Several endothelial cell-specific antigens  have also been employed to identify vascular endothelium in human (Schlingemann et al., 1985; Porschen et al., 1989) and murine tissues (Murray et al., 1989). Antibodies against components of vascular basement membrane can also be used to detect blood vessels in histological sections. Antibodies to Type IV collagen and to laminin are said to distinguish lymphatic vessels from blood vessels (Barsky et al., 1983). Anti-laminin antibodies have also been used successfully in murine tumours (Murray et al., 1987; Murray et al., 1989). Use of anti-laminin antibodies may be restricted to vessel identification in sarcomas since some carcinoma cells produce extravascular laminin (Murray et al., 1989).  10  1.2.1.3 Injection techniques  Most conventional histological staining methods give relatively poor contrast between blood vessels and other tissue elements. Intravascular injection of coloured substances retained in the vascular space has been used in an attempt to overcome this problem. Injection of dyes, labelled erythrocytes, or particulate matter allows visualization of perfused blood vessels and therefore is useful for quantitative morphological studies of functional tumour vasculature at the microscopic level in three-dimensional tumours.  The major disadvantage of such techniques is their  invasiveness; that is, they require removal of tumour tissue, and this limits their applicability in the study of vascular morphology in most human tumours.  The literature describes many different  particulate solutions and coloured substances which have been used to visualize perfused vasculature.  Particulate matter Injection methods were first used by Virchow (1863) and Thiersch (1865) to demonstrate the capillary network in the stroma of tumours.  Historically, the most commonly used injectable  substance for the study of tumour vasculature has been India ink.  India ink is a particulate  suspension of colloidal carbon ("carbon black") in which the carbon particles are approximately 20-50 nm in size. In many studies, the suspension is perfused into the animal under pressure.  Many qualitative studies have employed injection of particulate matter. Goldmann (1907) used intracardiac injections of India ink to ascertain whether vessels in spontaneous and transplanted mouse tumours were patent to flowing blood and he compared the vascularity of carcinomas and sarcomas.  Lewis (1927) injected 3% solutions of India ink to study the vascular pattern in five  different spontaneous rat tumours and the method has been used to determine the origin of the blood supply (arterial versus portal) to experimental hepatic neoplasms (Krishna Murthy, 1959; Fisher, et al., 1961). Waters and Green (1959) studied the vascular system of two transplantable mouse granulosa tumours by standard histological sections and India ink-injected (intra-aortic or  11  intravenous), cleared, whole mounts. An India ink-gelatin mixture has also been used to fill the tumour vascular system (e.g. Tiboldi, et al., 1968).  Several investigators have used colloidal carbon for morphometric measurements of tumour microvasculature (Vogel, 1965; Hilmas and Gillette, 1974).  In these studies, colloidal carbon  suspension was injected under pressure into a central vein of anaesthetized mice.  Quantitative  morphometric analysis (Chalkley, 1943) was used to examine vascular volume, vessel diameter, and vascular surface area and length in formalin-fixed, cleared sections of solid tumours.  Colloidal carbon is also a precise marker for identifying leaky blood vessels at the ultrastructural level.  Colloidal carbon particles are able to penetrate hyperpermeable endothelium but are  retained by the underlying basement membrane if it is intact.  Sites of vascular leakage are  therefore identified in electron micrographs as deposits of colloidal carbon in the vascular basement membrane. Such a technique has been employed by Dvorak et al. (1988) to identify and characterize tumour blood vessels that are leaky to circulating macromolecules.  Tannock and Steel (1969) have outlined the drawbacks of particulate matter injection. Essentially, the major disadvantage is that the vascular system is not demonstrated under physiological conditions. Depending on the applied injection pressure and perfusion time, all of the following may occur: opening of normally collapsed vessels, vascular dilation, incomplete filling of smaller vessels, and vessel rupture.  Finally, the section thickness required for adequate visualization of  the intravascular material is usually in excess of 100/ym.  Coloured or fluorescent dyes Intratumour localization of systemically administered coloured or fluorescent compounds has been used extensively to study the blood supply of experimental solid tumours (reviewed by Goldacre and Sylven (1962) and by Warren (1979)). The distribution of most of these dyes is not restricted to  12  the vascular space; the majority diffuse rapidly into cells or into the interstitial space, and therefore they do not act as specific markers of blood vessels. Tumour localization by fluorescein has been used in human patients (Moore, 1947; Bierman et al., 1951) and elegant studies by Goldacre and Sylven (1962) employed a variety of blood-borne coloured substances as indicators of tumour vascular function. These stains included lissamine green, Evans blue, trypan blue, eosin, neutral red, methylene blue, and fluorescein, none of which were confined to the vascular compartment. Very high doses of the light-absorbing dyes were required in order to achieve sufficient contrast with unstained tissue. Fluorescent dyes have the advantage of a high contrast and a high light yield at very low concentrations  (Reinhold,  1979).  Although most  intravenously-injected  fluorescent dyes also diffuse rapidly from the vascular space, they can be visualized within tumour blood vessel if tumours are removed and sectioned immediately after stain administration (Reinhold,  1965).  Fluorescent  compounds have  also  been  used for  rapid  sequence  microangiograms of tumours grown in observation chambers (Reinhold, 1979).  More recently, fluorescent stains have been employed which do not rapidly diffuse throughout the interstitial space but rather bind avidly to endothelial cells and perivascular tumour cells and thus outline perfused tumour blood vessels (Reinhold and Visser, 1983; Chaplin et al., 1985).  The  utilization of this class of stains will be discussed in detail in Section 3.  Other methods Casts of tumour vasculature can be made by intravascular injection of a polymer (eg. silicon rubber, neoprene latex, vinyl acetate) or other substance (eg. gallium) which fills the vascular tree (Jain, 1988). Tumour tissue can then be digested with, for example, a strong base or acid, and a cast of the vascular system is revealed (Gullino and Grantham, 1962; Shivas and Gillespie, 1969). Preparation of vascular casts is employed as a preliminary step to ultrastructural studies of normal (Hodde and Nowell, 1980) and tumour vessels using scanning electron microscopy (Grunt et al., 1985; 1986a, 1986b; Kaido and Uehara, 1987; Ahlstrom et al., 1988). A detailed study of vascular  13  branching patterns in three-dimensional tumours has been carried out using vascular casts (Jain etal., 1989).  Microangiography has also been used by many investigators to study tumour vascular architecture (reviewed by Warren, 1979). A number of radiopaque media have been employed, usually injected under pressure, under anaesthesia or after the tumour-bearing animal has been sacrificed. Microangiography adequately demonstrates fine vascular structure in malignant tissue but is invasive and requires radiographic equipment. The technique does, however, provide a measure of perfused vasculature, albeit under nonphysiological conditions.  Radioactive microspheres (10-25 um  diameter), when injected into the vascular system, are  trapped in capillary beds and have been used to quantitate tumour blood flow (Endrich et al., 1981; Jirtle and Hinshaw, 1981). Fluorescent microspheres or particles have also been used, and can be visualized within tumour vessels in histologic sections (Chaplin et al., 1987; Jirtle, 1988). However, microspheres are trapped in only a small fraction of perfused vessels and, while they can provide an estimate of local blood flow, they give no information about tumour vascular morphology.  1.2.2 TUMOUR ANGIOGENESIS  A review of tumour vascular structure must begin with a discussion of the origin of tumour blood vessels.  In the early phase of solid tumour growth, tumours are usually thin with a limited cell  population (eg. carcinoma in situ). Nutrients are delivered and waste products removed by diffusion, a relatively ineffective transport system, and thus tumour size is restricted. Uncontrolled growth and expansion can only be realized with the support of a vascular system capable of meeting the metabolic needs of the malignant cells.  The concept that solid tumours are  angiogenesis-dependent, that is, tumour growth beyond a diameter of several millimeters is dependent on vascularization of the neoplastic mass, is based on several observations (Folkman,  14  1986; 1990).  For more than 100 years, pathologists have observed that growth of most solid  tumours is associated with the proliferation of blood vessels in the surrounding host tissues. Examination of vascularization in isografts of normal and neoplastic tissue revealed that grafts of tumour tissue stimulated the proliferation of host vessels more than did grafts of normal tissue (Ide et al., 1939; Algire and Chalkley, 1945; Merwin and Algire, 1956).  Algire and Chalkley (1945)  suggested that the capacity to elicit formation of new capillaries, tumour angiogenesis, might be required for continuous tumour growth. Further evidence for the interdependence of angiogenesis and tumour growth was obtained from experiments by Folkman et al. (1966). Tumours implanted into isolated perfused thyroid glands did not become vascularized and stopped growing at a diameter of 1-2 mm. The tumour cells remained viable however, and produced rapidly growing nodules when re-transplanted into the original host.  Swelling of vascular endothelium was  observed in the perfused thyroid glands and it was postulated that the subsequent lack of endothelial cell proliferation prevented the new capillary formation necessary for growth of the tumour implant.  A summary of the evidence supporting the hypothesis that tumour growth is  angiogenesis-dependent is given by Folkman (1990).  Proliferation of new vessels around a tumour is thought to be induced by diffusable angiogenic factors, released continuously by the tumour cells, that can stimulate capillary growth over distances of 2-5 mm. The first direct evidence for such factors was provided by experiments in which melanoma or choriocarcinoma cells were separated by a Millipore filter from the normal stromal tissue of the hamster cheek pouch (Greenblatt and Shubik, 1968; Ehrmann and Knoth, 1968). The tumour implants induced a vasoproliferative response despite having no direct cell-cell contact with the host tissue. An angiogenic factor isolated from tumours (Folkman et al., 1971) and from transformed cells growing in tissue culture (Klagsbrun et al., 1976) was termed "tumour angiogenesis factor" (TAF) by Folkman who proposed that the factor was probably present in most human and animal tumours. Several peptides with angiogenic capability have been fully purified, their amino acid sequences determined, and their genes cloned. Many less well characterized  15  factors, some of which are lipids, have also been isolated (reviewed by Folkman and Klagsbrun, 1987; Pawaletz and Knierim, 1989). Angiogenic factors appear to fall into two categories based on their putative targets: those that act directly on vascular endothelial cells to stimulate migration or proliferation and those that act via an indirect pathway.  Folkman and Klagsburn (1987) have  proposed several possible indirect pathways. Indirect angiogenic factors may act by mobilizing host cells, such as macrophages, and activating them to secrete growth factors and/or chemotactic factors for vascular endothelial cells.  Angiogenic factors could also cause the  release of endothelial growth factors from extracellular matrix.  Whatever the mechanism, the final target for angiogenic factors is the endothelial cell lining normal host vessels, and thus this cell is of central importance in tumour neovascularization (Pawaletz and Knierim, 1989). Migration and proliferation of vascular endothelial cells forms the framework for new vessel development. The sequential events in capillary growth in response to an angiogenic stimulus have been studied by serial electron microscopy in tumours implanted into the rabbit cornea (Ausprunk and Folkman, 1977). New vessels originate only from capillaries or from small venules which lack smooth muscle. Vessels with a muscular coat such as arterioles do not appear to participate in the angiogenic response. Local degradation of vascular basement membrane and migration of endothelial cells through the defect leads to the establishment of a capillary sprout. Thus, the first steps in angiogenesis occur without proliferation of endothelial cells. Endothelial cell proliferation then takes place in the midsection of the sprout allowing further elongation. In the presence of neoplastic cells, surrounding normal vascular endothelium has an increase in the thymidine labelling index from 0.7% to 8% (Ausprunk and Folkman, 1977; Cavallo et al., 1972). The solid cord of endothelial cells develops a lumen by forming intracellular vacuoles and intercellular gaps (Pawaletz and Knierim, 1989). The tip of one sprout joins with another to form a capillary loop through which blood begins to flow. New sprouts originate from the capillary loop.  These new, "immature" vessels do not possess a continuous basement membrane and  16  where a basement membrane is found, it is thin and often perforated (Warren, 1966).  Further  structural abnormalities of tumour blood vessels will be discussed in the following section.  Tumour cells surround newly formed capillary loops (Tannock, 1968; 1972) or, conversely, vessels encircle tumour cells (Thomlinson and Gray, 1955), both geometries resulting in a cylinder of tumour cells with a radius of 60-150//m (equivalent to the estimated diffusion distance of oxygen). The thymidine labelling index for tumour cells decreases with increasing distance from the capillary (Tannock, 1968; 1970). These observations provide further evidence that tumour growth is dictated by and dependent upon the vascular system. However, it is possible that the contiguity of tumour cells to a capillary may not depend entirely on diffusion of oxygen and nutrients. Nicosia et al. (1983) found that when capillary sprouts from a plasma clot approached a tumour focus on the periphery of the clot, tumour cells grew rapidly around the capillary as a cylindrical cuff. There is no blood flow in this system. One interpretation is that tumour growth can be directly facilitated by capillary endothelial cells or perhaps by extracellular matrix components which these cells produce.  It is generally believed that vessels in host tissue adjacent to the tumour are induced to sprout and these sprouts gradually penetrate into the tumour mass. However, it has also been demonstrated that tumours can infiltrate surrounding connective tissue and expand around newly formed vessels, illustrating that tumours can acquire their vascularity, not by vessel ingrowth, but by vessel incorporation (Thompson et al., 1987). The origin of tumour vessels from host tissue capillaries and venules (Kligerman and Henel, 1961) implies a lack of a direct arterial supply to the neoplastic tissue.  Many observations suggest, however, that after formation of the microcirculatory  component, an arterial supply is acquired, perhaps when sprouts or loops anastomose with arteriolar vessels, although mechanisms by which this might occur are not clear. Kligerman and Henel (1961) claimed that the arterial supply originated from existing somatic vessels, an observation confirmed by Gullino and Grantham (1962) who stated that "the main branches of the  17  vascular tree of the host organ become the main branches of the vascular tree of the transplanted tumor; they do not increase in number, only in length and caliber". Peters et al. (1980) noted that mammary adenocarcinoma grown in a transparent chamber was supplied by 1 -4 main arterioles of 35-70 jjm diameter and that the number of arterioles only increased when tumour incorporated arterioles of the previously normal surrounding area.  Incorporation of such pre-existing normal  host vessels has important consequences for tumour vascular function, as will be discussed later (Gullino, 1975; Jain, 1988).  1.2.3 STRUCTURAL ABNORMALITIES  Tumour blood vessels, the rapid development of which is induced by tumour-derived angiogenic influences, are structurally immature and abnormal. Structural abnormalities can be considered at three levels: (1) the overall pattern of vascularization i.e. vascular architecture, morphology,  i.e.  vascular volume  and vessel diameter,  length  and  surface  (2) vascular area,  and  (3) ultrastructural changes in the vessel wall.  1.2.3.1 Vascular architecture  In very general terms, the purpose of the microcirculation is to deliver blood to tissue parenchyma in balance with the metabolic needs of the cells.  Each normal tissue in the body has its own  characteristic vascular system but all circulatory beds share certain common structural features which are modified by the particular arrangement and function of the parenchymal tissue. Malignant tissue, as a consequence of abnormal morphogenesis, has a structurally abnormal blood supply. Lewis (1927) noted that each tumour type had a characteristic vascular pattern and that "the blood vessels do not determine the growth of the tumour; but the tumour determines the growth and pattern of blood vessels". The structurally diverse nature of neoplastic growth leads to a wide variation in the organization of tumour vasculature and a common pattern cannot be recognized. In his exhaustive review, Warren (1979) concludes, "...each tumor type, and in some  18  cases each tumor, tends to be a law unto itself.  It has to be recognized, therefore, that the  vascular morphology of tumors, like other characteristics, has to be studied for each tumor type and that generalizations may be difficult to make." Differences in the vascular patterns of various tumours are governed by a combination of at least three factors (Reinhold and van den Berg-Blok, 1983):  (1) the growth pattern and growth rate of the tumour cells,  (2) the effectiveness of the  angiogenic stimulus released by the tumour, and (3) the influence of tumour interstitial pressure on compression of blood vessels.  Since a malignant tumour is an expansile, growing mass, its vascular supply is constantly changing, adapting to requirements of the tumour or being compressed and destroyed by the tumour cells (Falk, 1978). Tumour vascular architecture, then, is difficult to depict accurately in static, permanent terms but rather must be envisioned as a dynamic entity involving development, destruction, and rearrangement of blood vessels.  In a typical vascular tree, blood flows successively through arteries, arterioles, terminal arterioles, capillaries, postcapillary venules, venules, and veins. In malignant tissue, this hierarchy is often lost and the vessel network is most commonly described as "chaotic" (Reinhold and van den BergBlok, 1983).  Tumour blood supply can be broadly described under two idealized categories:  peripheral and central (Rubin and Casarett, 1966).  In tumours with peripheral vascularization,  dilated, tortuous host vessels and newly formed vessels are located at the tumour periphery; penetrating vessels may or may not be present. As the tumour grows it incorporates within itself the peripheral vasculature it has induced. In larger tumours with this vascular pattern, central necrosis occurs. In tumours with central vascularization, host vessels supply the tumour core and branches anastomose with a rich peripheral vascular net. Observations by Falk (1978) agreed with this concept and he described a "lax" type of tumour with central supply and drainage and a "tense" type with peripheral supply and drainage.  Most tumours actually consist of many  "modules", each having one of these two types of idealized vascular patterns (Jain, 1988).  19  Apart from loss of hierarchy, several other abnormalities of tumour vascular architecture are commonly recognized.  Arterioles supplying tumour tissue are recognized as thin, straight  channels that give off branches at right angles (Reinhold and van den Berg-Blok, 1983) but the venous system dominates the macroscopic appearance of the tumour vascular network. In some instances, if newly formed venular sprouts anastomose with other venules, the region served may be both supplied and drained by veins (Warren, 1979; Vaupel et al., 1989). Capillary and venous vessels in and around tumours are dilated, tortuous, saccular and elongated or even "stretched". Abnormal branching patterns may also occur: Jain et al. (1989) have described many vessel trifurcations which they claim are uncommon in normal tissue. Finally, tumour blood flow often passes directly from the arterial to venous side of the tumour circulation via arteriovenous shunts, anatomically distinct channels with a high perfusion rate that do not contribute significantly to nutritive flow (Reinhold, 1971; Jain, 1988). In a rat mammary adenocarcinoma, Peters et al. (1980) observed frequent direct connections between 30-40 /um arterioles and venules of the same size via aneurysm-like bulges.  The general characteristics of tumour vasculature reviewed above are descriptive and qualitative in nature.  Quantitative measurements of tumour vascular morphology using transparent  chambers and injection techniques, and detailed histologic and ultrastructural study of the tumour vessel wall reveal further structural abnormalities, many of which directly influence function and control of tumour circulation.  1.2.3.2 Vascular morphology The spatial and temporal distributions of vascular volume and vessel diameter, length, and surface area are defined as the vascular morphology of tumours (Jain, 1988). Quantitative studies of these parameters have been undertaken using tumours grown as two-dimensional sheets in observation  20  chambers or using histological methods on three-dimensional tumours, most often employing injection techniques.  Transparent chamber techniques allow the observation of early stages in vessel growth.  Most  studies show an increase in tumour vascular space in the first few days after tumour implantation (Algire and Chalkley, 1945; Yamaura and Sato, 1974; Peters et al., 1980). Tumour capillaries are, on average, of much larger diameter than normal tissue capillaries and this enlargement is most pronounced in rapidly growing tumours (Algire and Chalkley, 1945). Capillary diameter and length increase in all stages of tumour growth (Vogel, 1965; Hilmas and Gillette, 1974; Asaishi et al., 1981).  In malignant neurilemmomas grown in the hamster cheek pouch (Eddy and Casarett,  1973), and in rat mammary adenocarcinomas (Peters et al., 1980), vessels of up to 200 fjm in diameter have been observed.  Most quantitative studies of vascular morphology in three-dimensional tumours miss the early stages of vessel growth. All such studies show that vascular surface area and length decrease during the later stages of growth (reviewed by Gullino, 1975; Vaupel, 1977; Jain, 1988). Estimation of vascular volume and changes in this parameter as a function of tumour growth appear to depend on the tumour type as well as the experimental method employed. Two techniques have been utilized to measure the vascular space of tumours: (1) determination of the percentage of tumour weight occupied by blood present in the vascular system (labelled with a marker confined to the intravascular space), or (2) morphometric analysis of tumour serial sections. The vascular space in tumours varies from 1 to 27% (Jain, 1988), for example: 0.9 to 2.2% in human melanoma xenografts grown in nude mice (Solesvik et al., 1982); 15 to 18% in murine mammary carcinoma (Vogel, 1965); and 27.5% in a spontaneous canine lymphosarcoma (Straw et al., 1974). Gullino and Grantham (1964) found that vascular space increased linearly with size when tumours (Walker carcinoma 256) were small but that there was no such correlation for larger tumours.  Other  investigators found no change in vascular volume with tumour growth (transplanted mammary  21  adenocarcinoma) (Vogel, 1965; Hilmas and Gillette, 1974) but both Song and Levitt (1971) using Walker carcinoma 256 and Vaupel (1977) using DS-carcinosarcoma noted a decrease in vascular space as tumours enlarged.  Several quantitative studies have measured an increased tumour intercapillary distance as tumours grow (Tannock and Steel, 1969: Vaupel, 1977).  In DS-carcinosarcoma the mean  intercapillary distance increased threefold during tumour growth and a wide heterogeneity in individual measurements was noted (Vaupel, 1977). The reduction in vascular volume and surface area, and the increase in intercapillary distance have been attributed to differences in cell cycle times between neoplastic cells (22 hours) and vascular endothelial cells (50-60 hours) (Tannock, 1970). The concept that tumour cells "outgrow" their blood supply is not universally accepted (e.g. Rubin and Casarett, 1966; Shivas and Gillespie, 1969). Compression of vessels by elevated tumour interstitial pressure, vessel collapse due to stretching, or actual vessel destruction secondary to tumour cell invasion could also presumably contribute to reduction in vascularization observed.  Whatever the mechanism, reduction of tumour vasculature as tumours grow and  increased heterogeneity in vessel distribution have important implications for blood flow, oxygen delivery and the tumour microenvironment as will be examined later in more detail.  1.2.3.3 Vessel wall  The "tumour vasculature" is comprised of both incorporated host blood vessels and newly formed tumour vessels. Ultrastructural abnormalities of the vessel wall are confined to these newly formed vessels, most of which are capillaries with no evidence of arterial or venous differentiation (Grunt et al., 1986a).  The diameter of a normal, nontumourous capillary is 3-8 /vm. The vessel wall is composed of a unicellular layer of endothelial cells surrounded on the outside by a basement membrane.  Most  capillaries also have additional cells, called pericytes, which have contractile properties and are  22 thought to provide some mechanical support to the thin-walled vessels (Sims, 1986). The total wall thickness is approximately 0.5 //m. characteristics depending  The structure of the endothelial cell layer exhibits different  on the tissue supplied (Renkin and Michel, 1984; Jain, 1987). In  nonfenestrated (continuous) capillaries the endothelium is contiguous, without gaps between the endothelial cell processes. Such capillaries are found in skin, connective tissue, skeletal and cardiac muscle, lung alveoli, and brain.  In the intestine, kidney, and some endocrine glands,  capillaries are fenestrated, having transendothelial gaps about 50 nm lumen and the tissue interstitial space. diaphragm similar to plasma membrane.  The fenestrae may  in diameter between the  be open, or closed with a thin  Discontinuous or sinusoidal capillaries are large in  diameter and have wide openings between endothelial ceils. The basement membrane is either discontinuous or completely absent. Such capillaries are found in sinusoids of liver, spleen, and bone marrow.  Warren (1979) first proposed a classification of tumour blood vessels (modified by Jain, 1987) based  on  ultrastructural characteristics.  Nonfenestrated, fenestrated, and  discontinuous  capillaries have all been recognized in tumour tissue. In tumour vessels, however, both endothelial cells and tumour cells may form the lining of the vessel (Hammersen et al., 1985). Two other classes of vessels, found also in granulation tissue, are commonly present in tumours: blood channels and capillary sprouts (Warren, 1979). Blood channels are lined only by tumour cells (Grunt et al., 1986b). Capillary sprouts, blind-ending tapered or saccular tubes, are a result of angiogenesis.  These sprouts have a fragmentary endothelial lining and are fragile and leaky  (Grunt et al., 1986b). In tumour tissue, postcapillary venules and veins may also have structural abnormalities. Postcapillary venules, for example, may by very large in diameter, tortuous, and lacking basement membrane. Warren (1979) referred to these vessels as "giant capillaries" and identified them as a site for tumour cell intravasation into the blood stream (Warren et al., 1978).  23 The presence of contractile cells (vascular smooth muscle or pericytes) around tumour vessels is controversial, the prevailing view being that pericytes and smooth muscle cells are not present in the wall of tumour vessels.  Incorporated host arterioles, however, are essentially identical to  normal vessels and thus have contractile properties. Some recent studies have also demonstrated pericytes and smooth muscle cells in the wall of newly formed tumour vessels (Kaido and Uehara, 1987; Pawaletz and Knierim, 1989), the number of such cells increasing as the vessel "matures". Pericytes and smooth muscle cells appear to interact with endothelial cells in the regulation of vessel proliferation; in addition they serve as structural support for the new vessel and influence vessel permeability and perhaps, via contractile properties, vessel caliber (Pawaletz and Knierim, 1989). Adrenergic innervation of tumour vasculature is found only on incorporated host arterial vessels and not on newly formed vessels (Mattsson et al., 1977).  24  1.3 TUMOUR BLOOD FLOW  Abnormalities of tumour blood supply can be summarized under three  broad headings:  (1) inadequate tumour perfusion, (2) spatial and temporal heterogeneity, and (3) loss of intrinsic control mechanisms.  All three are interrelated  and can influence therapeutically  relevant  parameters such as tumour oxygen distribution, nutrient supply, metabolic microenvironment (eg. pH distribution and bioenergetic status), and response to treatment strategies, such as the use of vasoactive drugs, designed to manipulate tumour blood flow.  Measurements of overall tumour  blood flow may not reflect tumour perfusion or "nutritive flow" since, as will be described later, arteriovenous shunting may be significant in tumour vasculature.  At the single vessel level,  however, blood flow and perfusion can be equated.  1.3.1  PERFUSION RATE OF TUMOURS  Ribbert (1904) first proposed that overall tumour blood flow would be greater than that of normal tissues. Based on several lines of evidence, this proposal was generally accepted for many years. This evidence included: (1) the dense vascularity observed in histologic sections of many tumours, especially in the tumour periphery,  (2) the large vascular plexus surrounding the tumour mass,  and (3) the belief that a rapidly growing tumour needs a large blood flow to supply its metabolic requirements. It was not until the development of a direct method to measure tumour blood flow (Gullino and Grantham, 1961) that it was conclusively demonstrated that, in general, neoplastic tissues (especially carcinomas) have a lower blood flow than the normal tissues of origin.  In  human tumours in situ, however, blood flow may be higher or lower than the tissue of origin and the average perfusion rate of carcinomas does not differ substantially from that of sarcomas (Vaupel et al., 1989).  With the exception of two studies (Takacs et al., 1975; Slotman et al., 1980),  experiments in animal tumours reveal that the average blood flow rate decreases as the tumour grows (reviewed by Jain and Ward-Hartley, 1984) but such a relationship is not valid for many  25  human tumours (Vaupel et al., 1989). A reduction in blood flow with increasing tumour weight is presumably due to the rarefaction of tumour vascularity which occurs as the tumour enlarges.  The blood flow rate in a tumour vascular network is determined by three factors (Jain, 1988): (1) pressure difference between the arterial and venous sides,  (2) geometric resistance to flow,  and (3) viscous resistance to flow or the apparent viscosity of the blood. Flow is proportional to the arterio-venous pressure difference and inversely proportional to the geometric and viscous resistances. Tumour interstitial pressure can also greatly influence blood flow. Tumour perfusion is poor relative to many normal tissues because those factors which determine tumour blood flow are, in most cases, abnormal.  Reduction in vascular density is in large part responsible for the  decrease in overall blood flow which occurs with tumour growth (Vaupel, 1977) but aberrations in tumour microvascular pressure, geometric resistance, viscous resistance, and interstitial pressure result in inadequate, nonhomogeneous tumour perfusion (Jain, 1988).  1.3.1.1 Microvascular pressure  Using an indirect vascular occlusion method in two sarcomas and a mammary adenocarcinoma grown in observation chambers in mice, Algire and Legallais (1951) stated that "the blood pressure of tumor vessels approximates that of venous pressure". Hori et al. (1981) used a similar method and found that tumour microvascular pressure was lower than in normal subcutaneous tissue and that many tumour vessels had a pressure of <4 mm Hg. consistent  with  extravascular chambers.  indirect  evidence  for  decreased  intravascular  pressure in neoplastic tissue obtained  These observations are  pressure  and  using tumours grown in  increased transparent  As mentioned previously, loosening of the chamber can restore flow in previously  nonperfused tumour regions (Eddy and Casarett, 1973; Reinhold, 1979) microvascular pressure (Ide et al., 1939).  as can elevation of  Yamaura and Sato (1974) suggested that lowered  tumour intravascular pressure in rat hepatoma was responsible for instability of blood flow velocity in veins and capillaries as well as for changes in flow direction with the animal's movements.  26  Peters et al. (1980) reported the first direct measurements of tumour microvascular pressure using a servo-null method (Intaglietta et al., 1970) transparent chamber in the rat.  in a mammary adenocarcinoma grown in a  On the arterial side, the pressure did not differ significantly  between tumour and nontumour vessels. However, pressures in venous capillaries and venules in the tumour were significantly lower (8.5 preparations (12.3  cm H,,0).  cm H 0) 2  than in comparable vessels of control  Wiig (1982) also found a low perfusion pressure in tumour  microvessels; in central areas of large rat mammary tumours, perfusion pressure was calculated to be only 50% of that in skin.  The reasons for low microvascular pressures in tumours is not known but might include (Jain, 1988):  (1) tortuosity of tumour vessels,  (3) decreased vascular "tone".  (2) increased viscous resistance in tumours, and/or  A predominantly venous tumour circulation would also clearly  contribute to a lower mean intravascular pressure.  1.3.1.2 Viscous resistance  A major resistance to flow in blood vessels is blood viscosity. The rheological behaviour of blood in the microcirculation depends on many factors, including: plasma viscosity, shear rate (velocity gradient in the fluid), cell concentration (primarily red blood cells (RBCs), i.e. hematocrit), cell deformability, cell aggregation, protein concentration, temperature, and vessel diameter (Jain, 1988).  The most important of these factors in determining blood viscosity are hematocrit and  shear rate. As hematocrit increases, blood viscosity also increases. At low shear rates, RBCs aggregate to form rouleaux, causing an increase in blood viscosity.  Jain (1988) hypothesized that intratumour blood viscosity may be higher than the viscosity in normal tissues.  Sevick and Jain (1989b) have provided experimental evidence confirming an  increase in viscous resistance in solid tumours.  Within tumour vessels, opposing factors  contribute to both an increase and a decrease in viscosity. Possible mechanisms for an increased  27  viscosity in tumours include (Jain, 1988):  (1) the Fahraeus-Lindqvist effect (reduction in blood  viscosity in small vessels) may be less pronounced in tumour microvasculature since tumour vessels have relatively large diameters, (2) large vessel diameters and sluggish flow contribute to a reduced shear rate and an increase in blood viscosity, (3) reduced RBC deformability at the low pH environments found in some tumour regions (Cohen, 1979), (4) plasma losses of 5-7% occur as blood passes through the tumour vasculature (Butler et al., 1975; Vaupel et al., 1987; Sevick and Jain, 1988); the resulting increase in microvessel hematocrit contributes to increased viscosity, (5) presence of relatively nondeformable white blood cells (WBCs) and tumour cells in the tumour microcirculation; obstruction of vessels by platelet aggregates or WBCs/tumour cells can lead to micro- and even macro-thrombosis (Vaupel et al., 1989).  Reduction of systemic  hematocrit with tumour growth (Price and Greenfield, 1958; Dintenfass, 1982; Hirst, 1986) and the presence of RBC-free blood channels with plasma flow only (Jain, 1988) could contribute to a reduction in viscosity in tumours.  However, the abovementioned factors causing increased  viscosity are likely to dominate over those which reduce viscosity (Jain, 1988).  1.3.1.3 Geometric resistance  For a network of blood vessels, the geometric resistance to flow is a complex function of vascular architecture and morphology. According to the Hagen-Poiseuille relationship for nonturbulent flow through a rigid, cylindrical vessel, the geometric resistance is proportional to the vessel length and inversely proportional to the 4th power of the vessel diameter.  Thus, vessel diameter is the most  important determinant of geometric resistance to blood flow.  Within tumour tissue, many factors can contribute to changes in effective diameter of tumour vessels:  (1) presence or absence of vascular smooth muscle,  (2) swelling or destruction of  endothelial cells,  (3) adhesion of platelets, WBCs, or circulating tumour cells to the vascular  endothelium, and  (4) partial or total collapse of a vessel due to increased interstitial pressure  and/or decreased microvascular pressures.  28  Geometric resistance can be measured by perfusing a tissue or organ with a fluid of known, constant viscosity at various perfusion pressures.  The slope of the resulting pressure-flow  relationship allows determination of geometric resistance since viscosity is known (Zweifach and Lipowsky, 1984).  There are only three studies in the literature describing measurement of  geometric resistance in tumour tissue. In rat mammary tumours (Weiss et al., 1985) and in human renal carcinoma (Tveit et al., 1987) perfused ex vivo, tumour vascular resistance is higher than that of normal tissue, at least at low perfusion pressures. A more accurate determination of tumour geometric resistance was obtained using a tissue-isolated rat mammary adenocarcinoma preparation perfused ex vivo (Sevick and Jain, 1989a).  The results obtained showed that:  geometric resistance increased as arterial pressure was lowered below 20-40 mm Hg but reached a constant value at pressures greater than 40-50 mm Hg; and geometric resistance increased linearly with tumour weight.  A category of tumour vessels with very low resistance to flow is arteriovenous shunts. "Useful" tumour blood flow, that is perfusion through exchange vessels (capillaries), is thought to be reduced in tumours due to this large arteriovenous shunt flow, estimated at 30% of total tumour flow (Vaupel et al., 1987; 1989). For example, in patients receiving intra-arterial chemotherapy for head and neck cancer, shunt flow, measured using Tc-labelled macroaggregated albumin, was reported as 8-43% of total tumour blood flOw (Wheeler et al., 1986).  1.3.1.4 Tumour interstitial pressure  Microvascular pressure in tumour vessels is low but tumour interstitial pressure is elevated, with the result that microvessels are susceptible to compression and collapse (Ide et al., 1939; Eddy and Casarett, 1973; Yamaura and Sato, 1974; Reinhold, 1979; Wiig, 1982). Since the first direct measurements by Young et al. (1950), several investigators have shown that interstitial pressure in tumours is significantly higher than in normal tissues (Gullino et al., 1964; Butler et al., 1975;  29  Paskins-Hurlburt et al., 1982; Wiig et al., 1982; Tveit et al., 1985; Wiig and Gadeholt, 1985; Hori et al., 1986). These studies also showed that as tumours enlarge, interstitial fluid pressure rises, and that this increase correlates with tumour blood flow reduction and development of necrosis. Wiig et al. (1982) found that central tumour regions had higher interstitial pressures than peripheral regions and recorded a maximum pressure of 23 mm Hg in the central region of a large rat mammary tumour. Hori et al. (1986) measured pressures as high as 30 mm Hg. Such pressures could clearly  prevent  flow  in many tumour  microvessels, especially those with reduced  intravascular pressure (eg. 4 mm Hg (Hori et al., 1981); 9-13 mm Hg (Wiig, 1982)). It should be noted that in normal subcutaneous tissue, interstitial pressure is actually negative (-2 mm Hg) (Hori et al., 1986). Several factors are thought to contribute to elevated tumour interstitial pressure: (1) tumour cell proliferation in a relatively restricted area, vessels,  (2) absence of functioning lymphatic  (3) "leaky" blood vessels with increased permeability to macromolecules, and/or  (4) ischemic cell swelling (Wiig et al., 1982).  Elevated tumour interstitial pressure probably reduces perfusion in tumour microvessels by altering  the  extravascular  geometric  resistance to flow.  As long as microvascular pressure exceeds  pressure, blood vessels should remain  open and functional.  However,  if  microvascular pressure declines, partial occlusion or collapse of tumour vessels may occur with a resultant rise in geometric resistance and decrease in blood flow.  Evidence for this hypothesis  includes (Sevick and Jain, 1989a): (1) increasing geometric resistance at low perfusion pressures, (2) constant geometric resistance at moderately high perfusion pressures, and  (3) a non-zero  (16 mm Hg) pressure intercept of the pressure-flow relationship. Following administration of drugs which lower systemic blood pressure, direct observation of tumour vessel nonperfusion has been observed in transparent chambers (Algire and Legallais, 1951) and, as will be described in this thesis, in solid three-dimensional tumours (Trotter et al., 1989).  30  In addition to its adverse effects on tumour perfusion and its possible contribution to development of necrosis, elevated tumour interstitial pressure will also affect transport processes across the capillary wall.  Specifically, plasma filtration and thus convective transport will be reduced in  central tumour regions with high interstitial pressure.  The theoretical consequences for the  delivery of chemotherapeutic agents have been recently reviewed (Jain, 1989).  1.3.2 HETEROGENEITY  Tumour vascular architecture,  blood vessel morphology, and vessel density are  highly  heterogeneous, varying greatly from one tumour region to another, with the result that tumour blood flow also demonstrates a large degree of spatial nonuniformity. Several quantitative studies of tumour blood flow heterogeneity have been carried out, and for many experimental tumours at least, blood flow is often higher in the tumour periphery than in central regions (Endrich et al., 1979; Jirtle, 1981; Kaelin et al., 1984).  In a spontaneous canine lymphosarcoma (Straw et al.,  1974), tumour blood flow, measured using a thermal dilution technique, was as low as 0.6 ml/g/min in central regions and as high as 43 ml/g/min in the tumour periphery. Note that the absolute flow values in the tumour periphery were very high compared to normal tissue flow measured using the same method (e.g. renal cortex: 8.7 ml/g/min) and that even central tumour flow was comparable to that measured in normal brain (0.58 ml/g/min) (Straw et al., 1974). This is an example of an unusual tumour type which is highly vascularized (27%) and appears to have a blood flow rate far in excess of normal tissues. Endrich et al. (1979) measured blood flow in five tumour "zones" defined as necrotic, semi-necrotic, stabilized tumour circulation, advancing tumour front, and normal tissue. Blood flow in peripheral tumour regions (advancing front) was significantly higher than that of surrounding normal tissue.  Tumour blood flow is also characterized by temporal heterogeneity at a fixed location. Over the long term, as a tumour grows, the vascular pattern constantly changes due to variable rates of angiogenesis, vessel collapse and destruction, and formation of necrosis. A quantitative study of  31  these temporal changes was performed by Endrich et al. (1979) using tumours grown in transparent chambers.  In addition to flow changes which occur as a tumour grows, local, transient changes in blood flow have been shown to occur in tumours growing as thin two-dimensional sheets in observation chambers.  Such flow fluctuations may have important consequences for tumour oxygenation,  tumour microenvironment, and chemotherapeutic drug delivery. involves the  quantification  and  characterization  of  A major portion of this thesis  such temporal  heterogeneity  at  the  microregional level in solid three-dimensional tumours.  Based on observations made of tumours grown in transparent chambers (Goodall et al., 1965), Gullino (1975) proposed that "regurgitation and intermittent circulation (i.e. periods of stasis) followed by resumption of blood flow, sometimes in a direction opposite to the previous one, are probably the 'normal' features of the vascular transport system of tumors". Other similar studies supported this hypothesis: Yamaura and Sato (1974) noted that blood flow in tumour vessels was unstable and changed even with the animal's movement or respiration; Endrich et al. (1979) also described "regurgitation and intermittent flow"; and Reinhold (1971) observed differences in flow rates of a factor of 5 to 10, even in adjacent capillaries. Flow intermittency has been assessed quantitatively in two-dimensional tumours by measuring RBC velocity in single vessels (Intaglietta et al., 1977; Reinhold et al., 1977; Endrich et al., 1982). Arterioles 20-40/jm in diameter supplying the tumour microvasculature exhibited strong vasomotor activity in approximately 50-60% of the vessels observed and this resulted in flow fluctuations in downstream tumour vessels.  The  periodicity of this flow intermittency was 2-3 minutes (Intaglietta et al., 1977).  Apart from vasomotor activity in incorporated host arterioles, several other mechanisms might be responsible for intermittent flow in tumour microvasculature, for example, Theological factors such as transient plugging of vessels by WBCs or circulating tumour cells or vessel collapse occurring in  32  regions of low perfusion pressure/high interstitial pressure.  Possible mechanisms for tumour  blood flow intermittency will be discussed further in Section 4.  Much indirect evidence suggests that intermittent perfusion, at the single vessel level, might be responsible for impaired nutrient, and especially oxygen, delivery to tumour microregions (Urtasun and Merz, 1969; Jirtle and Clifton, 1978; Yamaura and Matsuzawa, 1979; Brown, 1979; Sutherland and Franko, 1980). Direct histological evidence for intermittent blood flow in three-dimensional tumours has been obtained using a double-staining technique employing Hoechst 33342, a fluorescent vascular marker, and fluorescent microspheres (Chaplin et al., 1987; Jirtle, 1988). However, microspheres are trapped in only a small fraction of tumour vessels, and the use of two agents capable of staining all functional tumour blood vessels would thus be more suitable for the quantitation of flow intermittency.  The work in this thesis addresses microregional changes in  tumour perfusion using two fluorescent stains with different excitation and emission spectra. Administration of the stains at different times prior to the preparation of histological sections provides temporal and spatial information about tumour blood flow at the microregional level.  1.3.3 INTRINSIC C O N T R O L OF TUMOUR BLOOD FLOW  In the circulatory system as a whole, several different mechanisms are provided (local, nervous, and humoral) to control blood flow to different parts of the body. At the microcirculatory level, control is almost entirely local, that is, flow is controlled by intrinsic mechanisms in proportion to the tissue's need for perfusion.  Local control of microcirculatory flow is mediated via vascular  smooth muscle, which dictates vessel "tone" at the arteriolar or pre-capillary sphincter level. Two important mechanisms are thought to be involved (Granger et al., 1983): (1) metabolic control, i.e. the capacity to adjust flow as tissue metabolic requirements change, and (2) myogenic control, i.e. local autoregulatory adjustments that stabilize flow and intravascular pressure, factors crucial in control of transcapillary fluid and solute exchange.  33  Abnormalities of tumour vascular architecture and vessel wall structure, specifically an apparent lack of smooth muscle in newly-formed tumour vessels, seriously disturb microcirculatory control mechanisms. Active vasomotor adjustments within the microcirculation occur only in vessels with smooth muscle in their walls, although the possibility that endothelial cells (Ragan et al., 1988) or pericytes (Sims, 1986) with contractile properties might also play a role cannot be discounted. Thus, incorporated host arterioles are likely to be the only tumour vessels involved directly in local control of blood flow, and such vessels are clearly in the minority among the predominantly capillary/venous tumour circulation.  Lack of a well defined system of local microcirculatory control in tumour tissue leads to several important  differences  between  tumour  vasculature  and that of  normal tissue.  Tumour  microcirculation can be characterized as a "passive" system, lacking the ability to autoregulate flow in response to changes in perfusion pressure.  Absence of autoregulation in tumours, as  evidenced by a linear pressure-flow relationship, has been demonstrated on several occasions (Vaupel, 1975; Suzuki et al., 1981; Sevick and Jain, 1989a).  The paucity of vascular smooth muscle may also dramatically alter the response of tumour vasculature to vasoactive influences, both pharmacological and nervous/humoral (eg. during animal stress). Indeed the observed effects of vasoactive drugs on tumour blood flow vary widely, are inconsistent, and are difficult to predict based on the known pharmacological properties of the agents (reviewed by Jain and Ward-Hartley, 1984).  For example, the drugs noradrenaline and  angiotensin II, both pressor agents, have opposite effects on tumour blood flow; noradrenaline decreases tumour blood flow (Mattsson et al., 1980; Hultborn et al., 1983) while angiotensin II increases it (Suzuki, 1981). The influence of vasoactive drugs on tumour circulation is likely to be mediated indirectly via effects on flow in surrounding normal tissue ("steal effects") (Jirtle, 1988) or on systemic blood pressure and thus tumour perfusion pressure (Sevick and Jain, 1989a).  34  Stress, too, has a variable influence on tumour circulation; animal restraint, for example, appears to have adverse effects on tumour blood flow (Zanelli and Lucas, 1976; Shibamoto et al., 1987).  Based on direct visual observation of the nonuniformity of red blood cell flow and distribution, Krogh (1922) proposed that in most normal tissues under basal conditions microcirculatory flow was intermittent. He also noted that in skeletal muscle only a fraction of the available vasculature was perfused at rest but that this number could be increased 4-5x in active, contracting muscle. Flow intermittency in capillary vessels is controlled primarily by the precapillary sphincters (Zweifach,  1977).  Zweifach (1974) proposed that the branching pattern of the  arteriolar-  precapillary functional complex is the most important parameter governing local flow regulation. Minor changes in entry conditions (narrowing of the terminal arteriole) or in size of the precapillary branch can stop capillary perfusion temporarily and lead to the flow intermittency observed in many tissues. In most tissues the final precapillary branch has vascular smooth muscle for only 20-50 //m and thus controls flow in 4-6 capillaries.  Abnormal vascular architecture in tumours  precludes such fine local changes in perfusion. Intermittent flow in experimental tumours, in those preparations in which it has been observed, appears to be mediated via arteriolar vasomotion (Intaglietta et al., 1977) and thus, presumably, will affect many downstream capillaries.  Loss of local microcirculatory control mechanisms in tumour tissue is based not only on structural derangements (lack of vascular smooth muscle) but also on functional considerations. As outlined by Intaglietta and Mirhashemi (1987) in reference to ischemic states, an inert microvascular network, with arterioles maximally dilated to serve a high metabolic demand, is characterized by: low arteriolar-venular pressure gradient, low flow velocity, higher blood viscosity, and non-selective capillary perfusion.  "In this situation capillary perfusion becomes heterogeneous and only the  microvessels with low hydraulic resistance maintain flow" (Intaglietta and Meyer, 1987).  The  similarities to tumour microcirculation are clear, and thus it may be possible to think of tumour tissue as representing a form of "ischemic" or "shock" state.  35  1.4 TUMOUR HYPOXIA Abnormal vascular structure and spatial/temporal heterogeneity in blood flow lead to a decline in nutritive blood flow per unit weight as a tumour grows.  As a result, hypoxia, acidosis, and  substrate depletion can occur in nonhomogeneously distributed tumour microregions (Vaupel et al., 1981; Kallinowski et al., 1989; Vaupel et al., 1989). Tumour cell oxygenation is of particular importance since the presence of viable, hypoxic cells in solid tumours is thought to influence the response to radiotherapy and, in some instances, to chemotherapy (Moulder and Rockwell, 1987; Teicher et al., 1981).  Oxygen sensitizes cells to the killing effects of ionizing radiation.  Approximately three times the radiation dose is required to kill hypoxic cells compared to that required for well-oxygenated cells (Dewey, 1960).  The potential importance of hypoxia in  radiotherapy has been recognized for many years (Crabtree and Cramer, 1933; Mottram, 1936). If hypoxic cells which survive a given dose of radiation subsequently reoxygenate they can provide a focus for tumour regrowth and local recurrence. Altered tumour cell microenvironment may also influence the effectiveness of chemotherapy.  Hypoxic cells, for example, if they have altered  metabolism and or if they move slowly through the cell cycle (Koch et al., 1973a; 1973b; Bedford and Mitchell, 1974; Born et al., 1976) may be resistant to antiproliferative agents (Teicher et al., 1981). There is evidence that hypoxia can lead to gene amplification and drug resistance (Rice et al., 1986). An understanding of the mechanisms underlying the development of tumour hypoxia and a knowledge of the proportion of hypoxic cells in a tumour would therefore have both therapeutic and prognostic significance.  1.4.1  EVIDENCE FOR TUMOUR HYPOXIA  Several radiobiological techniques have been employed to detect hypoxic cells and measure the hypoxic fraction of animal tumours (reviewed by Moulder and Rockwell, 1984).  Radioresistant  hypoxic cells exist in almost all experimental animal tumours and hypoxic fractions of 1% to more than 50% have been measured (Guichard et al., 1980; Moulder and Rockwell, 1984).  Other  36  methods  such  as  binding  cryospectrophotometric  of  radiolabeled  (Vaupel et al., 1978;  misonidazole  (Chapman  Mueller-Klieser et al., 1981)  et  al.,  1981),  and fluorescent  techniques (Olive and Chaplin, 1986), the use of oxygen-sensitive needle electrodes (Vaupel et al., 1981), and nuclear medicine, positron emission tomography, and magnetic resonance techniques (reviewed by Chapman, 1984) have also confirmed the presence of hypoxia in animal tumours.  The presence of hypoxic regions within solid human tumours can be inferred from both direct and indirect evidence. In squamous cell carcinoma of the lung, Thomlinson and Gray (1955) observed necrotic regions at the centre of cylindrical cords of tumour cells which had a peripheral blood supply.  The thickness of the rim of viable tumour cells surrounding a necrotic centre never  exceeded 180 /jm and had an average radius of 169 //m. This value was in reasonable agreement with theoretical calculations of oxygen diffusion distance in tumour tissue (145 //m).  Thus,  restrictions on the size of tumour cords were consistent with the theory of limited oxygen diffusion. Thomlinson and Gray proposed that cells adjacent to necrotic regions would be located in regions of low oxygen concentration and thus be relatively resistant to radiation therapy.  Local disease control and improved patient survival in response to treatment regimens designed to overcome the relative radioresistance of hypoxic cells provide indirect evidence for the existence of hypoxic cells in human tumours. Attempts to improve oxygen delivery using hyperbaric oxygen techniques (e.g. Henk and Smith, 1977; Dische, 1979; Dische et al., 1983)  have shown a  therapeutic benefit, although only in head and neck cancers and some cervix carcinomas. Pretherapy blood transfusion to correct anemic states (e.g. Evans and Bergsjo, 1965; Bush et al., 1978; Overgaard et al., 1986), and the use of hypoxic cell sensitizers (drugs which sensitize hypoxic cells to the killing effects of ionizing radiation) (e.g. Urtasun et al., 1976; Ash et al., 1979; Dische, 1984) tumours.  have also suggested that hypoxic cells are present, at least in some human  37  Measurements of oxygen tension in human tumours using oxygen electrodes also indicate the presence of tissue hypoxia (Cater and Silver, 1960; Evans and Naylor, 1963; Kolstad, 1968; Badib and Webster, 1969; Gatenby et al., 1985). In the human tumours examined, the mean values for oxygen partial pressure (pO^ are lower than in surrounding normal tissues and many tumour regions have p 0  2  values less than 5 mm Hg (reviewed by Vaupel et al., 1989).  In a study of  squamous cell carcinoma metastases, the oxygen distribution appears to influence the outcome of radiation therapy (Gatenby et al., 1988). Cryospectrophotometric measurement of oxyhemoglobin saturation of RBCs in human tumour microvessels also suggests the presence of tumour hypoxia (Mueller-Klieser et al., 1981) as does the technique of using misonidazole adducts as markers of hypoxic cells (Urtasun et al., 1986a; 1986b).  1.4.2 MECHANISMS OF IMPAIRED OXYGEN SUPPLY TO TUMOURS  The available evidence clearly demonstrates that hypoxic regions exist in almost all experimental rodent tumours and also in some solid human malignancies. Therapeutic regimens to improve tumour oxygenation or, conversely, to target treatment specifically towards hypoxic cells (hypoxiatargetted chemotherapy) must, if they are to prove successful, take into account the mechanisms underlying the development of tumour hypoxia.  Impaired oxygen supply to tumour tissue occurs by several mechanisms. As in normal tissue, oxygenation is governed by oxygen availability (nutritive blood flow x arterial oxygen concentration) and by the respiration rate (oxygen consumption rate) of the cells themselves.  Oxygen  consumption rates of tumours in vivo are intermediate between low and high metabolic rates measured in normal tissues, i.e. the respiratory function of tumour cells is within normal limits (Vaupel et al., 1989). Thus tumour hypoxia must arise as a result of impaired oxygen availability. Arterial oxygen concentration may be suboptimal within tumour microvasculature if the circulation is predominantly venous and in series with normal tissue. Shifts in the oxyhemoglobin dissociation curve (e.g. by altered pH) or modified RBC rheology could impair release of oxygen from  38  intratumour RBCs.  However, abnormalities of vascular structure and function are probably the  most important factors affecting tumour oxygenation. The resulting tumour cell hypoxia can be subdivided into two main types, chronic and acute.  1.4.2.1 Chronic hypoxia  In most normal tissues, the degree of vascularity is directly proportional to the metabolic requirements of the tissue, and blood vessels are distributed such that most cells are located no further than 20 to 30/jm from the nearest capillary (Weibel, 1984).  In neoplastic tissue, this  principle does not hold; vascularity in larger tumours, especially in central regions, is reduced relative to normal tissue and, consequently, some tumour cells are located distant from blood vessels, where the oxygen supply is significantly impaired.  The classic model of hypoxia in tumour tissue is that of "chronic" or "diffusion-limited" hypoxia. Based on histological study of human lung carcinoma, Thomlinson and Gray (1955) postulated that cells located beyond the diffusion distance of oxygen (calculated as 145/vm) would be anoxic and necrotic but that viable, radioresistant hypoxic cells would be located immediately adjacent to necrosis.  Such cells are subject to chronic or diffusion-limited hypoxia.  The oxygen diffusion  distance is of course determined by the consumption of oxygen by cells located nearer to the blood supply. Two main histological patterns are possible: oxygen diffusion may be outward from a single capillary or inward from blood vessels at the periphery of a cord of tumour cells (Tannock, 1972). Proliferation rates of tumour cells agree with this model: cells immediately adjacent to the blood supply, in an oxic microenvironment, proliferate more rapidly than those further away (Tannock, 1968). Whether or not chronically hypoxic cells can remain viable and clonogenic for extended periods and thereby influence the response of a tumour to treatment will be discussed in Section 4.  39  1.4.2,2 Acute hypoxia  Chronic, diffusion-limited hypoxia is a consequence of inadequate tumour vascularization. Perturbations in vascular function could also result in hypoxia if blood flow, and hence oxygen delivery, is significantly impaired.  Transient fluctuations in tumour blood flow could result in  "acute" or "perfusion-limited" hypoxia (Brown, 1979; Sutherland and Franko, 1980).  Several  lines of evidence suggest that experimental tumours may experience intermittent blood flow resulting in transiently hypoxic cells. Direct visualization of tumour blood flow using observation chambers indicates that periodic changes in flow occur (Intaglietta et al., 1977; Reinhold et al., 1977). Cell sorting experiments using the fluorescent perfusion probe Hoechst 33342 have shown that tumour cells close to blood vessels may be hypoxic in larger tumours, implying transient episodes of severely impaired oxygen delivery in those vessels (Chaplin et al., 1986; Chaplin et al., 1987). Also, tumour cell survival following misonidazole administration (a hypoxic cell cytotoxin) (Brown, 1979) or high dose irradiation (Urtasun and Merz, 1969; Jirtle and Clifton, 1978; Yamaura and Matsuzawa, 1979) has been shown to occur in peripheral, well vascularized tumour regions rather than in areas adjacent to necrosis (as would have been predicted by the chronic hypoxia model). Preliminary histological confirmation of intermittent blood flow has been obtained using Hoechst 33342 in combination with fluorescent particles in a transplantable murine tumour (Chaplin et al., 1987). However, only a small fraction of vessels can be visualized with this method and "opening" and "closing" of vessels cannot be quantified.  Acute hypoxia, especially if it results from transient but complete nonperfusion of some tumour vessels, is likely to be a major determinant of a tumour's response to radiation therapy (Sutherland and Franko, 1980; Chaplin et al., 1986; Jirtle, 1988). In addition, flow intermittency may provide a mechanism for the rapid postirradiation reoxygenation observed in some tumours (Kallman, 1972; Brown, 1979). Unfortunately, nothing is known about the mechanisms underlying transient tumour vessel nonperfusion, the duration of vessel "closure", or the importance of tumour site or histology.  A detailed characterization of such flow intermittency would allow a more accurate  40  prediction of its importance and, possibly, suggest therapeutic methods by which tumour perfusion can be improved at the microregional and single vessel level.  41  1.5 THESIS OBJECTIVES Dynamic changes in tumour microvascular function, spontaneous or drug-induced, can be studied in detail using tumours grown as thin two-dimensional sheets in transparent observation chambers (Reinhold, 1979).  No methods have been available for comparable investigations in solid,  unrestricted, three-dimensional tumours. Systemically administered markers of functional tumour vasculature have been used extensively to study vascular morphology in histological sections (Warren, 1979) but detection of temporal changes in tumour blood flow is more complex. Two or more unique vascular markers, with short circulation half-lives and stable staining characteristics, must be injected, separated by some interval, so that a "picture" of functional vasculature can be obtained at different instances in time.  Such a technique, employing fluorescent markers, was  first used to demonstrate intermittency of blood flow in a transplanted murine tumour (Chaplin, et al., 1987).  Fluorescent dyes offer high contrast and high light yield at very low concentrations  (Reinhold, 1979)  and, if binding to cells is stable, the stains can be used for sorting of  disaggregated tumour cell populations using flow cytometric methods (Chaplin, et al., 1985; Durand et al., 1990). The use of two fluorescent vascular markers with similar in vivo staining characteristics but differing fluoresence spectra (i.e distinguishable from each other in histological sections) would allow the localization and quantitation of vessels with changing perfusion patterns. Quantification of flow intermittency or of flow changes following vasoactive drug administration would then be possible at the microregional and single vessel level in solid, experimental tumours.  Therefore in this thesis, three major areas were studied:  1.  development of a double fluorescent staining technique capable of resolving changes in tumour perfusion at the level of individual blood vessels.  The methodology should be  capable of detecting infrequent, spontaneous fluctuations in tumour blood flow as well as more extensive drug-induced blood flow changes.  42  use of this double staining technique to detect, quantify, and characterize intermittent blood flow in a transplanted murine tumour and to predict the probable effect of these flow fluctuations on tumour oxygenation.  use of fluorescent vascular markers, in conjunction with blood flow measurements using laser Doppler flowmetry,  to quantify  drug-induced changes in tumour blood flow  (especially those resulting from manipulation microvascular level.  of systemic blood pressure)  at  the  43  2. MATERIALS AND METHODS  2.1 FLUORESCENT PERFUSION PROBES  2.1.1  H O E C H S T 33342  Hoechst 33342 (H33342) was obtained from Sigma Chemical Co. (St. Louis, MO, USA); H33342 is a DNA-binding fluorescent stain which emits blue fluorescence (470 nm) when excited by ultraviolet light (350 nm) (Loewe and Urbanietz, 1974). It has an anhydrous molecular weight of 562 Daltons; the structure is shown in Figure 1A. H33342 was dissolved in phosphate-buffered NaCl solution (PBS)  (120 mM  NaCl,  2.7 mM  KCI,  10 mM  phosphate buffer,  pH 7.4)  at  concentrations between 3 mg/ml and 9 mg/ml. For visualization of tumour vasculature, the stain was injected intravenously (i.v.) via the lateral tail vein at a dose of 15 mg/kg in a volume of 50//I.  2.1.2 CARBOCYANINE DYES  The fluorescent carbocyanine derivatives 3,3'-diheptyloxacarbocyanine  (DiOC (3)) and 7  3,3'-  diethyloxadicarbocyanine (DiOC (5)) were obtained from Molecular Probes Inc. (Eugene, OR, 2  USA). The structure of symmetric carbocyanine dyes is shown in Figure 1B and can be described using the abbreviated notation D i Y C ( 2 + 1) (Sims et al., 1974). The lipophilicity of cyanine dyes n  m  increases with n in the above formula and the wavelengths of maximum absorption and emission increase with m. DiOC (3) has a molecular weight of 600 Daltons and exhibits green fluorescence 7  (510 nm) when excited by blue light (480 nm).  DiOC (5) has a molecular weight of 486 Daltons 2  and exhibits red fluorescence when excited by green light. Carbocyanine dyes are poorly soluble in aqueous solution and the stains were dissolved in dimethyl sulphoxide (DMSO) and then diluted to 75% (vol/vol) with PBS before use (final concentration of 0.6 mg/ml). For in vivo use as a marker  Figure 1: Chemical structures of Hoechst 33342 (A) and of symmetric carbocyanine dyes (B)  45  of tumour vasculature, DiOC (3) was administered i.v. in 50 u\ via the lateral tail vein at a dose of 7  1 mg/kg. This dose provided optimal visualization of tumour blood vessels.  2.1.3 RHODAMINE 6G  Rhodamine 6G (R6G) (Sigma) is a fluorochrome which is positively charged at physiological pH, enters cells, and specifically stains mitochondria (Johnson et al., 1980).  The stain inhibits  oxidative phosphorylation (Gear, 1974). R6G was evaluated as a marker of tumour vasculature since its fluorescence spectrum differs considerably from that of H33342: R6G emits red fluorescence when excited by green light.  The stain was dissolved in sterile water at a  concentration of 3 mg/ml and injected i.v. at a dose of 10 mg/kg.  2.1.4 FITC-DEXTRANS  Fluoresceinated (fluorescein isothiocyanate, FITC) dextran of average molecular weight 156 kD was obtained from Sigma. The degree of substitution was 0.007 moles FITC per mole of glucose residue. The mean diameter of this dextran-150 was 17.4 nm (Dvorak et al., 1988). In the absence of leaky blood vessels, macromolecules such as dextran-150 are normally retained within the vascular space and thus can potentially identify tumour vasculature. FITC-dextran was dissolved in PBS at a concentration of 30 mg/ml and 100 fj\ of this solution was injected i.v. Animals were sacrificed by cervical dislocation 5 minutes after stain injection.  2.1.5 ZINC-CADMIUM SULPHIDE PARTICLES  Fluorescent zinc cadmium sulphide particles (Duke Scientific, Palo Alto, CA, USA) are trapped within the microcirculation following intravascular injection and were used to label perfused tumour vasculature.  The particles are 1-10 //m in size and are yellow fluorescing. They were  suspended in PBS and injected i.v. via the lateral tail vein at a dose of 4 g/kg in a volume of 250 The particles are distributed initially to the pulmonary vasculature and only a small percentage  46  reach the systemic circulation and are subsequently distributed to the tumour.  Mice die within  1 minute of particle injection, presumably as a consequence of extensive pulmonary embolization.  2.2 MODULATORS OF TUMOUR BLOOD FLOW  2.2.1 HYDRALAZINE  Hydralazine is a phthalazine derivative whose major action is the relaxation of vascular smooth muscle (Koch-Weser, 1976; Rudd and Blaschke, 1985). Hydralazine hydrochloride (Apresoline) was obtained from CIBA-Geigy Canada Ltd (Mississauga, Ont.). The drug was dissolved in PBS and administered i.v. via the lateral tail vein in a 50/J\ volume at doses of 2.5 to 10 mg/kg.  2.2.2 ANGIOTENSIN II  The vasoconstrictor octapeptide angiotensin II (Douglas, 1985) was obtained from Sigma. The drug was dissolved in PBS at a concentration of 10/yg/ml and administered i.v. via the lateral tail vein using a microliter infusion pump (Harvard Microliter Syringe Pump, Harvard Apparatus, South Natick, MA).  Angiotensin was infused at a rate of 6.4//l/min corresponding to a dose of  2//g/kg/min.  2.2.3 NICOTINAMIDE AND PYRAZINAMIDE  The niacin precursor nicotinamide (Marcus and Coulston, 1985) was dissolved in PBS and injected intraperitoneally (i.p.) at a dose of 1000 mg/kg in 2 5 0 P y r a z i n a m i d e , the pyrazine analogue of nicotinamide, and, like nicotinamide, also a radiosensitizer (Chaplin et al., 1990) was dissolved in PBS and administered i.p. at a dose of 500 mg/kg. Both drugs were purchased from Sigma.  47 2.3 MICE AND TUMOURS  Experiments were carried out using transplantable murine tumours or human tumour xenografts in laboratory mice. Mice were housed in an air-conditioned facility inspected and approved by the Canadian Council on Animal Care. All animal procedures were approved by a local animal care committee under guidelines comparable to those of the National Institute of Health.  The mice  were kept 6-8 animals to a cage on sawdust bedding. They were allowed free access to food (Laboratory Rodent Chow, Purina Mills Inc., St. Louis, MO, USA) and water.  C3H/He mice were bred in the animal facility and male animals 8-12 weeks old were used for all murine tumour experiments.  Human tumour xenografts were grown in 8-12 week old female  congenitally athymic nude mice, arising from the cross of heterozygous nu/+ homozygous nu/nu males. Dr. Marcelle  Guichard  females and  These mice were kindly supplied, with tumour lines implanted, by  (Institut  Gustave-Roussy, Villejuif,  France)  and  Dr.  Herman  Suit  (Massachussetts General Hospital, Boston, MA). Athymic nude mice, because they are not fully immunocompetent, were housed in a separate room in a laminar flow hood. Bedding, food, and water were sterilized before use.  For most experiments, the transplantable murine squamous cell carcinoma SCCVII was used. The SCCVII carcinoma originated spontaneously in the abdominal wall of a C3H mouse in the laboratory of Dr. Herman Suit (Massachussetts General Hospital). The tumour line used in these experiments, SCCVII/St, was obtained from Dr. Michael Horsman at Stanford University.  The  SCCVII is an extremely poorly differentiated malignant tumour composed of a mixture of spindle and epithelioid cells. The mitotic rate is high with many abnormal mitoses visible. There is no histologically detectable host response in the form of stromal proliferation or inflammatory cell infiltration (Dr. Jean Leriche, personal communication). However, host macrophages (identified in flow cytometry studies) are known to constitute 39+19% of SCCVII tumours (Olive, 1989). Grown  48  subcutaneously, the SCCVII tumour has a doubling time of 4.1 days (Dr. David Chaplin, personal communication).  The tumour was maintained by serial passage in vivo:  tumour brei was  inoculated into the gastrocnemius muscle of inbred C3H/He mice. For tumour implantation prior to experimentation, a single cell suspension was prepared from an intramuscular maintenance tumour. agitation  The tumour was excised, chopped with scalpel blades, and disaggregated by gentle at  37°C  for  30  minutes  in an enzyme  cocktail consisting of trypsin  (0.2%),  deoxyribonuclease I (0.05%), and collagenase (0.05%). All enzymes were obtained from Sigma. The resulting cell suspension was filtered through a polyester mesh (50//m pore size), centrifuged, 5 and the pellet resuspended in medium.  5x10  cells in a volume of 20-50 //I were implanted  subcutaneously over the sacral region in methoxyflurane-anaesthetized  mice.  Intramuscular  gastrocnemius implants were also performed and for laser Doppler blood flow measurements in unanaesthetized mice, tumour cells were implanted subcutaneously in the hind foot dorsum. For all experiments, tumours of transplant generations 3-10 were used.  Three human tumour xenografts grown in athymic nude mice were used.  Na11 is a human  melanoma cell line maintained in monolayer culture (Guichard et al., 1977). HRT18 is a cell line obtained from a colonic adenocarcinoma biopsy (Guichard et al., 1983). Both of the above cell lines grow well as xenografts in nude mice. The FaDu line was established from a punch biopsy of a human pharyngeal squamous cell carcinoma; in the nude mouse, this cell line produces a welldifferentiated grade I epidermoid carcinoma (Hay et al., 1985).  2.4 PHARMACOKINETIC AND PHYSIOLOGIC PARAMETERS  2.4.1  PHARMACOKINETICS OF F L U O R E S C E N T VASCULAR MARKERS  Blood levels of H33342, DiOC (3), DiOC (5), and rhodamine-6G were measured at various times 7  2  following intravenous injection using a fluorometric method. Non-tumour-bearing, 8-10 week old  49  male C3H/He mice were used and fluorescent stains were injected via the lateral tail vein in a volume of 100/yl. H33342 was injected at a dose of 15 mg/kg, DiOC (3) and DiOC (5) at 1 mg/kg, 7  2  and rhodamine-6G at 10 mg/kg. Blood samples to determine stain levels were obtained from the orbital sinus at varying times post-injection.  44.7 /J\ of blood was collected in a heparinized  micropipette which was then emptied into 3.0 ml of 99% ethanol. The micropipette was rinsed several times with the ethanol to remove any retained blood or drug. Samples were stored in the dark prior to further processing.  Samples were centrifuged at 1500 rpm x 6 minutes and the supernatant analyzed for drug concentration using a Farrand spectrofluorometer (System 3 Scanning Spectrofluorometer, Farrand Optical Co., Inc., Valhalla, NY, USA). Appropriate excitation and emission wavelengths were chosen for each stain. A standard curve of known stain concentrations was obtained. Stock solutions of the stain were diluted in 99% ethanol and mouse serum.  Blood concentration of the stains was plotted against time after injection, and distribution and elimination half-lives were calculated based on a two compartment model of drug kinetics. The apparent volume of distribution was calculated as the amount of stain given divided by blood concentration obtained by extrapolation of the 1st order elimination curve to zero time (Benet and Sheiner, 1985).  2.4.2 BLOOD P R E S S U R E AND TEMPERATURE  Measurements of arterial blood pressure and heart rate were made in male C3H/He mice anaesthetized with halothane, administered continuously, via vaporizer, at a concentration of 0.5-2.5% in 100% oxygen.  Mice were allowed to breathe spontaneously.  Temperature was  monitored and maintained at 35-37°C using a heating pad. Microsurgical techniques were used for insertion of an arterial cannula (operating microscope, Olympus Optical Co., Tokyo, Japan). The left femoral artery was dissected free and the superficial circumflex iliac branch was ligated.  50 The femoral artery was then catheterized using saline filled PE10 tubing (O.D. 0.61 mm). Pressure measurements were recorded using a Statham P23D pressure transducer (Gould, Oxnard, CA, USA) connected to an amplifier and recorder (General Electric, Co., Liverpool, NY, USA). Heart rate could be read directly from the arterial pressure waveform.  Prior to intravenous injection of  H33342, mean arterial blood pressure was titrated to a baseline level of 80-90 mm Hg (near the maximum achievable using this anaesthetic) by adjusting the concentration of inhaled halothane.  The effect of the fluorescent vascular markers H33342 and DiOC (3) on mouse temperature was 7  measured using a Model 46 T U C Tele-thermometer and YSI Series 400 probes (Yellow Springs Instrument Co., Yellow Springs, OH, USA).  Rectal, skin, and intratumour temperature were  measured in restrained, unanaesthetized mice using the appropriate probes for a period of one hour after intravenous injection of H33342 (15 mg/kg) and DiOC (3) (1 mg/kg). Room temperature 7  was22°C.  2.4.3 METABOLIC PARAMETERS  The effect of H33342 (0-400/vg/ml) on relative oxygen utilization of SCCVII tumour cells in vitro was measured using the method described by Biaglow and Durand (1976). A single cell suspension was obtained from a subcutaneous SCCVII tumour by mechanical/enzymatic disaggregation as outlined previously. Cells were added to 9 ml media in a closed reaction vessel at a concentration of 5.0x10 cells/ml and stirred vigorously. H33342 was added at varying concentrations. As the cells consumed oxygen, the change in dissolved oxygen concentration was measured at 3 7 ° C with a Clark-type oxygen electrode (Yellow Springs Instrument Co., Yellow Springs, OH, USA). Oxygen utilization rate was calculated from the linear portion of the curve over the first 10 minutes of incubation.  Measurements of venous blood p 0 , C 0 , pH, hematocrit and hemoglobin saturation were made 2  2  before and after injection of H33342 (15 mg/kg).  Orbital sinus blood samples were collected in  51  microhematocrit tubes and blood was analyzed using an automated blood gas analyzer (ABL300) connected via a serial interface to an oximeter (OSM3) for measurement of hemoglobin saturation (Radiometer,  Copenhagen, Denmark).  The required sample volume was 35 /vi and all  measurements were performed at 3 7 ° C .  2.5 RADIOBIOLOGY STUDIES  To determine if H33342 influenced tumour oxygenation and thus the tumour response to radiation, the stain was administered at varying intervals before and after an x-ray dose of 10 Gy. Subcutaneous SCCVII tumours implanted over the sacral region were used. Mice were restrained in a lead-lined jig (no anaesthesia was used) and tumours were locally irradiated at a dose rate of 3.16 Gy/min using a 270 kVp X-ray source filtered with 0.5 mm Cu. H33342 was given i.v. in 50/t/l volume 20 minutes before, immediately before, or 20 minutes after irradiation. Some mice were killed 5 minutes after H33342 injection and the tumours then irradiated to obtain the response of a completely anoxic cell population. Following treatment, tumours were excised and enzymatically dissociated to a single cell suspension as described previously. Cells were then incubated for 1-2 minutes with fluorescein isothiocyanate (FITC)-labelled anti-mouse IgG (whole molecule; Sigma F 0257) to stain nontumour host cells, predominantly macrophages (Olive, 1989). Cells were analyzed on a fluorescence-activated cell sorter (FACS 440, Becton-Dickinson, Sunnyvale, CA, USA). Details of the cell sorting methodology have been described previously (Chaplin et al., 1985; Durand et al., in press). H33342 was excited using an argon laser operating at 350-360 nm. Fluorescence intensity was measured at 450+ 10 nm and the peripheral light scatter (proportional to cell size) was measured at 488+10 nm.  H33342 concentration was obtained by dividing  fluorescence intensity by peripheral light scatter. Sorting windows were established by assuming a cylindrical configuration of tumour cells around each H33342-stained tumour blood vessel. Equal numbers of FITC-negative (i.e. tumour) cells were sorted into 10 fractions ("equal volume sort") based on cellular H33342 concentration. The clonogenicity of each fraction was assessed  52  by plating cells, counted by the FACS, into 100 mm plastic cell culture plates (Becton Dickinson Labware, Lincoln Park, NJ, USA) and incubating in 5% 0 , 5% C 0 , and 95% N 2  2  2  at 3 7 ° C for  10 days. Colonies were then stained with malachite green and counted.  2.6 DOUBLE STAINING TECHNIQUE  Most experiments were performed in unrestrained, unanaesthetized tumour bearing mice. Mice were weighed, the tail was warmed in 4 5 - 5 0 ° C water and H33342 was administered by lateral tail vein injection. At varying time intervals after H33342 injection, DiOC (3) was given in an identical 7  fashion. Mice tolerated injection of both drugs without obvious ill effects. Animals were sacrificed by cervical dislocation 5 minutes after DiOC (3) injection and tumours were immediately excised 7  and frozen at - 2 0 ° C in O C T embedding compound (Tissue-Tek, Miles Laboratories, Inc., Elkhart IN, USA). Frozen sections 10 pm in thickness were rewarmed to room temperature, allowed to air dry, and examined using a Zeiss microscope (Carl Zeiss, Oberkochen, West Germany) with epifluorescence condenser and 100W mercury lamp illumination.  H33342 was visualized using  365 nm ultraviolet (UV) excitation, a 395 nm dichroic mirror, and a 397 nm long pass filter. DiOC-,(3) could be seen in the same tissue section using 450-490 nm excitation, a 510 nm dichroic mirror, and a 520 nm long pass filter.  The fluorescent stains identify perfused tumour vasculature allowing localization of tumour blood vessels within the histological section. All vessel counting was performed using a 25x Neofluor objective with a numerical aperature of 0.60; total magnification was 312x.  A frozen section  obtained from the tumour equatorial plane was examined for the absence of section artifacts and fluorescent-stained blood vessels were quantified.  Five hundred to 1000 vessels per tumour  section were counted and the percentage of vessels labelled with one fluorescent stain but not the other  ("mismatched vessels") was determined.  Perfusion mismatch was calculated for both  central and peripheral (< 500/ym from the tumour surface) regions.  53  2.7 QUANTITATIVE FLUORESCENCE MEASUREMENTS  The localization of intravenously administered fluorescent stains in relation to tumour blood vessels and the stability of stain binding to perivascular cells was assessed using a fluorescence image processing system (FIPS) (Jaggi et al., 1988). FIPS allows image acquisition, digitization and image processing of low-light level fluorescent images. Tissue sections were examined using a Zeiss microscope with epifluorescence condenser. The light source was a 100W mercury lamp equipped with a stabilized power supply. Fluorescent images from the microscope were acquired via an image intensified charge-injection device (CID) camera mounted on a microscope port (camera: CID Technologies, Liverpool, NY, USA; image intensifier: ITT, Fort Wayne, IN, USA). Using appropriate microscope objectives, the camera has a spatial resolution of 3/jm. Maximum sensitivity is 10" lux allowing detection of fluorescence at very low light levels (Jaggi et al., 1988).  The analog signal from the CID camera was digitized in real time using a digital imaging board (Digital Image Processor, Model FG-100-AT, Imaging Technology Inc., Woburn, MA, USA) controlled by an 80286/87-based IBM PC-AT computer.  Digitized images were held in a  512 x 512 pixel array frame memory and were either stored or simultaneously displayed on a high resolution RGB analog monitor.  An interactive program (IMGPRO) allowed acquisition and  storage of fluorescent images and quantitative measurement of fluoresence intensities in tissue sections.  For measurement  of fluorescence in perivascular regions stained with an intravenously-  administered fluorescent vascular marker, captured and stored.  128x120 pixel images of individual vessels were  Background images (regions containing no fluorescence) were also  obtained. Distributions of fluorescence intensity in each image were obtained using a software program written by Dr. Ralph Durand. The average fluorescence of the 10 brightest pixels (after subtraction of background fluorescence) was used as a measure of peak fluorescence intensity.  54  Vessel images were displayed on the RGB monitor and the distance of stain diffusion away from the vessel lumen was measured manually.  For quantitative measurement of perivascular fluorescence as an indicator of single vessel perfusion (stain delivery), both H33342 and DiOC (3) fluorescence were recorded for 100 vessels 7  per tumour. Experiments were performed on tumour-bearing mice in which both stains had been injected simultaneously and also in those in which the interval between stain injections was 20 minutes. Background fluorescence for each stain was obtained from 10 tumour fields in which (a) neither stain was detectable by eye (in the case of simultaneous injection) or (b) staining mismatch was observed (only one stain visible).  Perivascular fluorescence was expressed as a  ratio of DiOC (3) to H33342 fluorescence. Vessels in which only one stain was present were 7  arbitrarily assigned ratio values of 1000 (DiOC (3) only) or 0.001 (H33342 only). y  2.8 VASCULAR MORPHOLOGY  The morphology of functional (perfused) tumour vasculature was assessed using H33342 staining of perivascular tumour cells (Murray et al., 1987; Smith et al., 1988; Fallowfield, 1989). H33342 was injected i.v. in 50 /vl at a dose of 15 mg/kg and animals were sacrificed 5 minutes later. Frozen sections 10/vm in thickness obtained from the equatorial plane of the tumour were examined using fluorescence microscopy.  The functional vascular volume was measured using the point counting technique described by Chalkley (1943). The use of such methods and illustrations of the principle that "a tissue section is a quantitatively representative two-dimensional sample of a three-dimensional system of randomly distributed tissue structures" are reviewed in Weibel (1963).  The point counting method was  performed as follows. A grid consisting of a quadratic array of 25 points was placed over the digitized image of the tumour section displayed on the monitor of the fluorescent image  55  processing system. The number of times a point fell within a vessel lumen outlined by H33342 staining was recorded. This procedure was repeated over 100 randomly chosen fields each of dimensions 400x400 fjm (magnification 512x).  Counting was restricted to grossly viable (non-  necrotic) tumour regions. The vascular volume was calculated as the ratio of positive points (within a vessel lumen) to total points:  Vascular volume (%)  =  no. of positive points (n) x 100 total points  The total number of points necessary to estimate the vascular volume within a given error depends on the vascular volume itself. For an anticipated vascular volume of 5%, a total of 2500 points was counted (error = 9%).  Vessel lumen diameters were assessed directly from the image on the FIPS monitor; no correction was made for the minor asymmetry of the digitized image (240x256 pixels). Only vessels oriented at right angles to the plane of the tumour section (i.e. lumen outlined by H33342 was approximately circular) were measured. One hundred such vessels were randomly selected and the average of two perpendicular diameters was determined for each vessel. Since H33342 does not stain cell cytoplasm, this method may slightly overestimate vessel diameter.  Estimates of average vessel length and vascular surface area per tumour volume were determined using the following formulae (Vogel, 1965):  Vascular length  =  Vascular surface area  =  4V red  (mm/mm ) 3  2  4Vd d  (mm /mm ) 2  3  2  where  V = vascular volume, d = mean vessel diameter, d = mean of squared diameters 2  56  2.9 VASCULAR SMOOTH MUSCLE  Vascular smooth muscle was demonstrated in tumour sections using a muscle-actin-specific monoclonal antibody (Enzo Diagnostics, Inc., New York, NY, USA).  This antibody recognizes  smooth muscle cells of arteries and veins as well as pericytes surrounding smaller vessels (Tsukada et al., 1987a; 1987b). Tumours were excised and immersion-fixed overnight at room temperature in 99% ethanol, cleared with xylene, and paraffin-embedded.  Immunohistochemistry  to detect muscle-specific actin (MSA) was performed using an avidin-biotin immunoperoxidase method (Bourne, 1983). All incubations were carried out at room temperature. 5/vm sections were deparaffinized and endogenous peroxidase activity was blocked using 0.6% hydrogen peroxide in methanol for 30 minutes. Nonspecific staining was reduced by incubation with 5% goat serum for 30 minutes. The section was then incubated for 60 minutes with antibody against muscle-specific actin at a dilution of 1:8000 in PBS, followed by biotin-conjugated goat anti-mouse secondary antibody for 30 minutes. After a PBS rinse, peroxidase-conjugated biotin-avidin complex (equal parts of avidin and biotinylated peroxidase diluted 1:100 30 minutes prior to use) was added and the section incubated for 30 minutes. 3,3'-diaminobenzidine (DAB) was employed as the substrate solution.  Sections were counterstained very lightly for 1 minute in hematoxylin and a coverslip  applied.  2.10 OXYGEN DIFFUSION DISTANCE  In a technique analogous to that of Franko et al. (1987), AF-2, a fluorescent hypoxia probe (Olive and Chaplin, 1986), was used to label hypoxic cells in central regions of tumour tissue "cubes" incubated with the stain in vitro. The distance from the cube surface to the AF-2-stained region is an estimate of oxygen diffusion distance (Trotter and Olive, 1990). Cubes, 1-2 mm in size, were 3  cut from SCCVII tumours and incubated in 50 ml spinner flasks for 30-60 minutes in media containing 100/jg/ml AF-2. Incubations were carried out at temperatures between 2 2 ° C and  57  45°C.  Following removal from the flasks, cubes were washed twice in PBS and then frozen at  - 2 0 ° C in Tissue-Tek embedding compound. Sections 10/vm in thickness were cut and AF-2 was visualized using fluorescence microscopy. The distance from the cube surface to the region of maximum AF-2 intensity was measured.  2.11 THEORETICAL ESTIMATES OF TUMOUR HYPOXIA  Based on tumour vascular morphology and on measured oxygen diffusion distance, theoretical estimates were made of the predicted contribution of intermittent blood flow and acute hypoxia to overall tumour hypoxic fraction. Vessel diameters obtained from frequency histograms and the mean vascular volume of >500 mg SCCVII tumours were used to graphically plot tumour vessels (randomly distributed) together with their surrounding cord of tumour cells, the radius of which was determined by the oxygen diffusion distance. A simple computer program was written to calculate: (a) fraction of nonoxygenated tumour tissue (assumed to be nonviable);  (b) fraction of viable  tumour tissue with oxygen partial pressure between 0-1 mm Hg (chronic hypoxia); and (c) fraction of tumour tissue rendered acutely hypoxic/anoxic due to intermittent blood flow in (i) randomly distributed vessels or (ii) patches of vessels. The major unknown variable in this theoretical model was intravessel oxygen partial pressure (pO^. Based on a study by Vaupel et al. (1987) in which tumour arterial p 0 was 94 mm Hg and venous p 0 was 48 mm Hg, a value of 60 mm Hg (89% 2  saturation) was chosen as the mean p 0  2  2  in tumour vessels. This was the midpoint between  94 mm Hg (97% saturation) and 48 mm Hg (81% saturation).  An assumption was made that no  significant arteriovenous shunt perfusion was present. Individual microvessels were assumed to be either fully perfused or completely nonperfused; no consideration was given to graded flow reductions.  58  2.12  LASER DOPPLER FLOWMETRY  Relative changes in tumour blood flow were measured using a laser Doppler blood flowmeter (Laserflo Blood Perfusion Monitor, Model BPM 403, TSI Inc.,  St. Paul, MN, USA) which  incorporates a low-power, solid-state laser diode as the source of coherent light. The laser emits infrared light (wavelength = 780 ± 20 nm) which is delivered noninvasively to the tissue via a quartz optical fiber. The power at the end of the optical fiber is 2 mW. As light enters the tissue, photons are scattered randomly by stationary tissue cells and moving red blood cells (RBCs). Photons that interact with moving RBCs are Doppler (frequency) shifted.  Electrical signals from the laser  Doppler photodetector are proportional to the number of moving RBCs and to mean RBC velocity. RBC flow (expressed as ml/min/100g tissue) is the product of these two measurements.  This  instrument is capable of monitoring tissue microvascular flow continuously and noninvasively with a spatial resolution of approximately 1 mm (Haumschild, 1986; Shepherd et al., 1987). 3  For tumour blood flow measurements, mice bearing subcutaneously implanted tumours were anaesthetized with ketamine (50 mg/kg i.p.) and diazepam (10 mg/kg i.p.).  Ketamine has been  shown to be a useful anaesthetic in small animals (Weisbroth and Fudens, 1972; White and Holmes, 1976; Mulder, 1978) although the agent does cause some decrease in rodent blood pressure and in tumour blood flow (Cullen and Walker, 1985; Menke and Vaupel, 1988). A 1-2 mm incision was made in the thin skin directly overlying the tumour, and a 0.7 mm diameter laser Doppler needle probe was passed through this incision onto the tumour surface using a micromanipulator (Model 113, IITC Inc. Woodland Hills, CA, USA). Thus, flow measurements were recorded from a small, peripheral region of tumour with minimal contribution from skin or subcutaneous tissue.  Since the laser Doppler instrument is extremely sensitive to vibration  artifact, mice were placed on a vibration-damping platform consisting of a 2.5 cm thick slate block supported on a foam base and covered by a heated blanket (Vetko Thermal Barrier, Ealing Scientific Ltd., St. Laurent,  Quebec).  Fluorescent stains and/or vasoactive drugs were  59  administered via an indwelling 25G tail-vein catheter. In order to eliminate the undesirable effects of anaesthesia on the mouse cardiovascular system, measurement of tumour blood flow was also performed using unanaesthetized, restrained mice bearing subcutaneous tumours implanted in the hind foot dorsum. No skin incision was made and readings were therefore obtained from the tumour surface.  Analog output from the laser Doppler microprocessor-based signal analyzer was digitized via an analog-to-digital converter with 12-bit resolution and an acquisition speed of 10 KHz (Analog Connection Jr., Strawberry Tree Computers, Sunnyvale, CA, USA). Data were acquired, analyzed, and stored using an IBM-PC. Additional software for graphic display and data analysis was written by Bob Harrison, Department of Physics, C C A B C .  2.13 STATISTICAL ANALYSIS  In most instances, the significance of differences between two mean values was carried out using an unpaired (independent) f-test assuming a normal sample distribution. A pooled variance was used, the assumption being made that samples were drawn from populations with equal variance. P-values >0.05 were not considered statistically significant.  Where appropriate (for example,  comparison of staining mismatch values in central and peripheral tumour regions) a paired (dependent) f-test was used. In most instances, standard deviation was employed as a measure of dispersion. Significance of changes in blood pressure and tumour blood flow were determined using a one-way analysis of variance (ANOVA) performed using the SYSTAT statistical package. In post-hoc tests of multiple hypotheses, the overall error rate for comparison of pre-treatment and post-treatment blood flow (or blood pressure) was controlled using the Bonferroni procedure (Wilkinson, 1988).  60  3. DOUBLE FLUORESCENT STAINING TECHNIQUE  3.1  INTRODUCTION  Sequential systemic administration of two vascular markers, distinguishable from each other in tissue sections, allows the detection of blood flow changes at the microregional and single vessel level in solid three-dimensional experimental tumours. Such an approach was first employed by Chaplin et al. (1987) using the fluorescent stain Hoechst 33342 and fluorescent zinc-cadmium sulphide particles.  However, particles or microspheres (Jirtle, 1988), even when given in large  numbers, are trapped in only a small fraction of tumour blood vessels seen in thin tissue sections. Use of two stains capable of staining all functional vasculature would thus be more suitable for histologic quantification of changes in microvascular flow.  The bisbenzamide Hoechst 33342 (H33342) is a DNA-binding fluorescent stain used extensively in flow cytometry studies to quantify DNA content in live cells (Arndt-Jovin and Jovin, 1977) and to select cells from different locations within multicell spheroids (Durand, 1982) and experimental solid tumours (Chaplin et al., 1985, 1986, 1987; Loeffler et al., 1987; Siemann and Keng, 1988; Young and Hill, 1989; Durand et al., in press). Perivascular tumour cells avidly bind intravenously injected H33342 and are therefore more brightly stained than cells distant from the blood supply which are exposed to lower dye concentrations.  Thus, H33342 acts as a marker of perfused  tumour vasculature (Reinhold and Visser, 1983) and can be used to quantify vascular morphology (Smith et al., 1988; Fallowfield, 1989).  The stain has also been employed in double-labelling  techniques to identify tumour vessels subject to transient nonperfusion (Chaplin et al., 1987; Jirtle, 1988; Trotter et al., 1989a) and to isolate tumour cells which are rendered acutely hypoxic by these flow fluctuations (Chaplin et al., 1986, 1987; Minchinton et al., in press). H33342-staining methods  61  have also been used to examine the effects of vasoactive drugs (Trotter et al., 1989c) and chemotherapeutic agents (Murray et al., 1987; Murray and Randhawa, 1988; Zwi et al., 1989) on functional tumour vasculature.  Several possible candidates for a second fluorescent vascular marker, with excitation and emission spectra different from those of H33342, have been examined. A promising class of stains are the fluorescent carbocyanine dyes, for example 3,3'-diheptyloxacarbocyanine  (DiOC (3)) 7  (Olive and Durand, 1987). DiOC (3) has been employed previously in photographic processing 7  and as a membrane potential probe (Sims et al., 1974). The slow tissue penetration rate of this dye has been used to select cells from different depths within multicell spheroids (Olive & Durand, 1987).  DiOC (3) is cationic and has a long alkyl side chain, increasing its lipophilicity, and 7  facilitating rapid entry into cells. Rapid cell uptake and slow tissue penetration rate suggest that the dye might have in vivo staining characteristics similar to H33342; that is, the ability to stain cells immediately adjacent to blood vessels (Trotter et al., 1989b). Carbocyanine dyes are excited by visible light and can easily be discriminated from the UV-excited H33342 when both stains are present in tissue sections examined by fluorescence microscopy.  Thus, such dyes, used in  conjunction with H33342, could be employed in a double staining protocol to identify regions of transient vessel nonperfusion or drug-induced flow fluctuations in experimental tumours.  This section describes:  (1) evaluation of several fluorescent stains, in particular carbocyanine  dyes, as vascular markers for use in conjunction with H33342 in a double-labelling protocol designed to identify tumour blood flow intermittency or drug-induced microvascular flow changes; and  (2) determination of the effects of these vascular markers, especially H33342, on mouse  cardiovascular and tumour physiology.  62  3.2  RESULTS  3.2.1  PERIVASCULAR LOCALIZATION OF F L U O R E S C E N T STAINS  The ability of several fluorescent stains to act as markers of functional tumour vasculature was qualitatively assessed using fluorescence microscopy of tumour frozen sections. Fluorescence micrographs of SCCVII tumour sections stained  in vivo by H33342, DiOC (3), FITC-dextran, or 7  zinc-cadmium sulphide particles are shown in Figures 2 and 3. FITC-dextran and zinc-cadmium sulphide particles were restricted to the intravascular space.  The other stains examined were  localized, in varying degrees, to perivascular cells. Discrimination of perfused tumour vessels was qualitatively superior using H33342 or DiOC (3). 7  Rhodamine-6G and the carbocyanine dye  DiOC (5) diffused rapidly outward from tumour vessels and did not accurately delineate tumour 2  vasculature (data not shown).  The stability of H33342 or DiOC (3) fluorescence and the localization of dye in relation to blood 7  vessels were quantified using the fluorescence imaging system. In the first few minutes following intravenous injection, the stains were seen only in cells located a short distance from the vessel lumen.  With time, dye became detectable in cells more distant from the blood supply and  fluorescence of the perivascular cells declined; i.e. peak fluorescence intensity decreased and staining distance away from the vessel increased.  The peak-fluorescence/distance ratio is  therefore a measure of dye localization in relation to tumour blood vessels (Figure 4).  Below a  peak-fluorescence/distance ratio of approximately 2, counting blood vessels was not possible primarily because vessels near to each other could not be easily discriminated.  H33342 remained localized to perivascular cells to a greater degree than DiOC (3). 7  H33342  allowed discrimination of blood vessels even if tumours were removed and sectioned 1-4 hours after stain injection.  However, 30-60 min. following DiOC (3) administration, the difference in y  Photomicrographs of SCCVII tumour vessels marked with H33342 (A) and DiOC (3) (B) Stains were administered simultaneously. Section thickness is 10/vm and the scale bar represent' Fi  9  ure  2 :  100yum.  Figure 3: Photomicrographs of S C C V I I tumour vessels marked with H33342 (A), FITC-dextran-150 (B), or H33342 and zinc-cadmium sulphide particles (C). Section thickness is 10 /ym and the scale bars represent 100/vm.  65  <  CC LU  O  CO  UJ  o z  HI  o CO UJ GC  O 10  20  30  40  50  60  TIME A F T E R INJECTION (minutes)  Figure 4: Stability of H33342 (O) and DiOC (3) ( • ) staining of SCCVII tumour cells in vivo. Tumours were excised and perivascular fluorescence assessed at varying intervals after stain injection. Error bars represent SEM for 20 vessels in a tumour section. Peak fluorescence intensity declined and staining distance increased with time after dye injection. 7  66  fluorescence intensity between perivascular cells and cells more distant from the blood supply was essentially lost. Visualization of tumour vasculature was most distinct if tumours were removed 5 minutes after DiOC (3) injection.  Both DiOC (5) and rhodamine-6G fluorescence remained  7  2  poorly localized to perivascular cells; the stains appeared to transfer rapidly to adjacent cells and thus vasculature was not clearly delineated (data not shown).  Bleaching of the vascular markers H33342, DiOC (3), and DiOC (5) was also measured using the 7  fluorescence imaging system (Figure 5).  2  DiOC (3) was relatively resistant to bleaching by 7  continuous illumination of the tissue section with blue light (430-490 nm) using the optical system described.  Fluorescence declined with a half-life of 105 seconds.  In comparison, H33342  fluorescence faded rapidly when excited by UV light (376 nm) at least over the first 30 seconds of illumination (t1/2 = 20 seconds). DiOC (5) also bleached very quickly (t1/2 = 17 seconds) when 2  the tumour section was excited by green light (580 nm).  3.2.2 PHARMACOKINETICS  Systemically administered markers of tumour vasculature, if they are to be employed in a double staining regimen to detect changes in blood flow patterns, should have short distribution half-lives in blood.  Each marker will then provide a "picture" of tumour microregional flow, outlining only  those vessels which were perfused during the short period that the stain was present in the circulation.  The vascular markers H33342, DiOC (3), DiOC (5), and rhodamine-6G all fulfilled the criterium of 7  2  short half-life in blood (Figure 6). H33342 had a distribution half-life of 142 + 27 seconds and an elimination half-life of 42 + 15 minutes (based on a two-compartment model of drug kinetics). The carbocyanine dyes DiOC (3) and DiOC (5) had distribution half-lives of 160 ± 2 7 seconds and 7  2  elimination half-lives of 36 minutes and 42 minutes respectively. rhodamine-6G were 80 seconds and 52 minutes.  Corresponding values for  67  1  2 TIME  3 4 5 (minutes)  Figure 5: Rate of bleaching of H33342 (O) and DiOC (3) (#) under continuous 100W mercury lamp illumination using either 376 nm excitation (H33342) or 430-490 nm excitation (DiOC (3)). Peak fluorescence intensity surrounding SCCVII tumour blood vessels was measured as a function of illumination time in 3-4 tumours. The t1/2 for bleaching (time to decline to 50% of maximum fluorescence) was 105 seconds for DiOC (3). H33342 bleached rapidly with a t1/2 of 20 seconds and then, after approximately 30 seconds of illumination, bleaching occurred more slowly with a t1/2 of 90 seconds. Fluorescence intensity of the carbocyanine stain DiOC (5) (•) also decreased rapidly (t1/2 = 17 seconds) under continuous 550-580 nm excitation. 7  7  7  2  68  1 '  0.1I 0  20  40  TIME A F T E R  60  0  20  INJECTION  40  i 60  (minutes)  Figure 6: Blood levels of fluorescent vascular markers following intravenous bolus injection (100 /J\ volume) in 22-29 gram C3H mice. (A) H33342 (15 mg/kg); (B) rhodamine-6G (10 mg/kg); (C) DiOC (3) (1 mg/kg); and (D) DiOC (5) (1 mg/kg). The mean ± SD for 5-6 mice is shown. For those symbols without error bars the SD is less than the symbol size. 7  2  69  Both H33342 and DiOC (3) had a large apparent volume of distribution (Vd).  For H33342,  7  Vd = 216 ml, reflecting rapid cell uptake of the stain and binding to DNA. For DiOC (3), Vd = 59 ml 7  probably as a result of the lipophilic nature of the stain and binding to intracellular components such as mitochondria.  3.2.3 TOXICITY  In vivo and in vitro toxicity of H33342, in terms of effects on cell reproductive integrity, have been described previously (Durand and Olive, 1982; Olive et al., 1985). The  LD  rn  of H33342 in mice is  t>u >200 mg/kg (Chaplin et al., 1985). Toxicity of DiOC (3) in an in vitro spheroid system has also 7  been assessed (Olive and Durand, 1987). Mice tolerated a 1 mg/kg intravenous dose of DiOC (3) 7  without obvious ill effects and were alive and well 6 weeks after drug administration. An LD_„ 50 experiment was not performed but doses greater than 5 mg/kg killed the mice within several minutes; this might be due to extensive cell depolarization caused by influx of the cationic stain. All injection volumes were limited to 50 fjl, so that increasing doses of DMSO were not responsible for this toxicity. The in vitro survival of SCCVII tumour cells exposed to DiOC (3) (1 mg/kg i.v.) in 7  vivo was not significantly reduced.  Even cells located in the brightest H33342 sort fractions,  representing cells closest to tumour blood vessels and thus exposed to the highest concentration of DiOC (3), showed no reduction in clonogenicity (Figure 7). 7  3.2.4 CARDIOVASCULAR AND METABOLIC E F F E C T S O F H33342  3.2.4.1 Blood pressure/heart rate H33342 (15 mg/kg) caused a transient reduction in mean arterial blood pressure measured directly in anaesthetized C3H mice (Figure 8).  Blood pressure declined following i.v. H33342  injection in all 5 animals tested using this dose. Pretreatment blood pressure was 86 ± 4 mm Hg. The minimum pressure recorded after H33342 injection was 66 ± 9 mm Hg and thus the maximum  70  0.01  10 9 8 BRIGHT  7  6  5  4  3  2 1 DIM  SORT FRACTION  Figure 7: Effect of intravenous DiOC (3) (1 mg/kg bolus) on surviving fraction of SCCVII tumour cells plated in vitro. Tumours were excised 5 minutes after stain injection. Survival level is plotted as a function of H33342 sort fraction. The brightest cells are located immediately adjacent to the blood supply. Means + SD for 3 tumours are shown. 7  71  100 -  £  80  E E  60 600  Q.  coco0cococooco<>-^^  400  E a .a  200  *  ffl 40 20 -5  0 TIME  5  AFTER  10  15  HOECHST  20  33342  25  30  (minutes)  Figure 8: Effect of H33342 (15 mg/kg i.v. in 50 //I injection volume) on mean arterial blood pressure (MABP) ( # ) and heart rate (HR) (O) in 8 week old male C3H mice (n = 5) anaesthetized with halothane via vapourizer. Error bars represent SD. Blood pressure remained significantly lower than pretreatment values for 24 minutes after H33342 injection (p<0.05). No significant change in heart rate was observed.  72  decrease in blood pressure was 20+ 6 mm Hg (range 11-26 mm Hg; p< 0.005). Blood pressure remained slightly but significantly depressed up to 24 minutes after H33342 injection (p<0.05) although a return to near pretreatment values occurred by 14 minutes.  Heart rate was not  significantly changed: prior to H33342 injection heart rate was 431 ± 19 beats per minute and at times after H33342 injection corresponding to the minimum blood pressure, heart rate was 428 + 23 beats per minute.  3.2.4.2 Metabolic parameters  H33342 at concentrations as high as 400 /jg/ml (approximately 4-5x the peak blood levels achieved following in vivo administration of 15 mg/kg) had no significant effect on relative oxygen utilization by SCCVII cells in vitro.  Oxygen consumption (relative to untreated  cells) was  0.99 + 0.07 (n = 6) and 0.92 + 0.08 (n=10) for H33342 concentrations of 100/jg/ml and 400/jg/ml respectively. Venous blood pH, p 0 , and p C 0 2  2  were not significantly altered following H33342  administration (Table I). A slight drop in systemic hematocrit was noted but this was also seen in control animals and was likely the result of repeated orbital sinus blood sampling. No effect on the oxygen-hemoglobin dissociation curve was observed.  In five C3H/He mice bearing SCCVII  tumours, H33342 had no effect on rectal, skin, or tumour temperature (data not shown).  3.2.5 TUMOUR BLOOD FLOW  H33342 caused a dose-dependent reduction in tumour red blood cell (RBC) flow as measured by laser Doppler flowmetry.  In immobilized mice (ketamine/diazepam anaesthesia) bearing SCCVII  tumours implanted subcutaneously over the back, an H33342 dose of 15 mg/kg i.v. reduced RBC flow to 19+ 11% of normal (p< 0.001) with a return to pretreatment flow in <8 minutes (Figure 9). In foot tumours, the decline in RBC flow was less pronounced in both anaesthetized (50+ 15% of normal) and unanaesthetized mice (52+14% of normal).  In foot tumours, RBC flow was  significantly different from pretreatment values for only 2-3 minutes post-injection.  A dose of  5 mg/kg was not significantly vasoactive (Figure 2). No long term reductions in flow were observed  73  pH  PC0  2  PRETREATMENT  5 MINUTES  20 MINUTES  7.33 ±.01  7.33 ±.01  7.27 ± . 0 7  44 ± 3  37 ± 2  41 ± 3  34 ± 2  35 ± 3  34 ± 5  38 ± 3  40 ± 5  38 ± 1 0  46 ± 2  42 ± 1  40 ± 1  46 ± 2  43 ± 1  42 ± 3  (mm Hg) P0  2  (mm Hg) HB0  2  (%) HCt(%) (no drug) HCt(°/o) (H33342)  Table I: Effect of H33342 (15 mg/kg i.v.) on venous blood gases, pH, oxyhemoglobin saturation, and hematocrit in unanaesthetized C3H mice. Parameters were measured prior to, 5 minutes after, and 20 minutes after H33342 injection. Means ± SD for 5-7 mice are shown.  74  140 •  R  B  C  F  L  O  W  100  i  60  \  20  S  ' 140  '  '  i  N U M B E R O F MOVING R B C s  -i  < ^  I  u. O ^  100  60 20  140  RBC VELOCITY  100  60  20 0  5  TIME A F T E R H O E C H S T 33342  10  15  (minutes)  Figure 9: Effect of H33342 (15 mg/kg i.v.) on tumour red blood cell (RBC) flow, number of moving RBCs (indicative of functional microvascular volume), and mean RBC velocity as assessed by laser Doppler flowmetry. SCCVII tumours were either implanted subcutaneously over the sacral region and mice immobilized with ketamine/diazepam anaesthesia ( • ) (n = 8) or tumours were implanted subcutaneously in the hindfoot dorsum: ( • ) anaesthesia (n = 5), ( O ) no anaesthesia (n = 6). Error bars represent SEM. In back tumours, R B C flow, from times 1 to 7 minutes inclusive, is significantly different than pretreatment values (p<0.05). In foot tumours, RBC flow is only significantly different than pretreatment values up to 3 minutes post-H33342 injection (p<0.05).  75  HOECHST 33342 DOSE  (mg/kg)  Figure 10: Effect of i.v. H33342 dose on tumour RBC flow. Measurements were made in SCCVII tumours implanted subcutaneously over the sacral region. Mice were immobilized with ketamine/diazepam anaesthesia. (O) RBC flow. (•) Duration of flow reduction, i.e. time to return to pretreatment values. Error bars represent SD.  76  using this method. The number of moving RBCs (indicative of functional microvascular volume) also declined following H33342 administration (15 mg/kg) in back tumours (59% of normal, p< 0.001) but this effect was much less pronounced in foot tumours (78% and 89% of normal with and without anaesthesia respectively, p<0.05). Most of the tumour blood flow reduction induced by H33342 was a result of a decrease in mean RBC velocity: in back tumours RBC velocity declined to 31% of pretreatment values while in foot tumours velocity decreased to 62% in anaesthetized mice and to 58% in unanaesthetized mice (p< 0.001).  Blood flow reduction was  seen in tumours ranging in size from 50 to 1000 mg. Flow reductions in large back tumours >800 mg in size were not significantly different than that of small tumours < 200 mg in size (19 + 12% of pretreatment values vs 21 +7%)  H33342 had no significant effect on blood flow in skin of a non  tumour-bearing foot (Figure 11).  The tumour blood flow reduction observed following H33342  injection is unlikely to be an artifact of the measurement technique since the absorption spectrum of H33342 does not overlap with that of the infrared laser and the stain did not alter the DC voltage signal, i.e. H33342 staining did not affect the amount of light scattered by stationary tumour cells (data not shown).  DiOC (3) (1 mg/kg) also caused a reduction in RBC flow in SCCVII tumours as measured by laser 7  Doppler flowmetry (Figure 12).  DiOC-,(3) injection resulted in an initial transient reduction,  recovery, and then a gradual significant decline in flow to 26 + 9% of baseline values (p< 0.001). A significant reduction in the number of moving RBCs (34+ 13% of pretreatment values; p< 0.001) contributed to the flow decline following DiOC (3). 7  DMSO (75%) caused no change in tumour  blood flow (data not shown). Intravenous administration of DiOC (3) did not alter blood hematocrit 7  (48 + 2% pretreatment vs. 48+1%  60 minutes after DiOC (3)). 7  DiOC (3) caused a slight decrease (1.5°C) in rectal temperature. 7  In unanaesthetized animals  77  CD  g 140 CO  c  100  o # 60 O _J LL  O  20  CQ  CC  0  5  10  15  20  25  30  TIME A F T E R H33342 INJECTION (minutes)  Figure 11: Effect of H33342 (15 mg/kg) on RBC flow measured in foot skin of restrained, unanaesthetized C3H mice. Means ± SEM for 5 mice are shown. No statistically significant changes in skin R B C flow were observed.  78  0  TIME  AFTER  S  10  15  20  DiOC <3) ADMINISTRATION 7  25  (min)  Figure 12: Effect of DiOC (3) (1 mg/kg i.v.) on tumour red blood cell (RBC) flow, number of moving RBCs (indicative of functional microvascular volume), and mean RBC velocity as assessed by laser Doppler flowmetry. SCCVII tumours were implanted subcutaneously over the sacral region and mice immobilized with ketamine/diazepam anaesthesia. Error bars represent SEM (n = 5). 7  79  10  8  9  7  6  5  4  BRIGHT  SORT  1  2  i  1  D IM  FRACTION  Figure 13: Effect of H33342 bolus injection (15 mg/kg i.v.) on the response of subcutaneous SCCVII tumour to 10 Gy tumour localized x-irradiation in restrained, unanesthetized male C3H mice. Control plating efficiency (panel A; mean + SD ) is plotted as a function of H33342 sort fraction (based on H33342 concentration); brightly stained cells are those located immediately adjacent to the tumour blood supply. Panel B shows the surviving fraction of tumour cells after 10 Gy x-rays: H33342 was injected 20 minutes prior to X-rays (n = 5) (A); 20 minutes after X-rays (n = 5) (A): or immediately prior to X-rays (n = 7) (#). The responses of fully oxic SCCVII tumour cells irradiated in vitro (•) and of completely anoxic cells (killed animal; n = 5) (O) are also shown for comparison. Error bars represent S E M .  80  3.2.6 RADIOBIOLOGIC E F F E C T S  The overall radiation response of the tumour depended on the time of H33342 injection relative to radiation treatment. H33342 injection 20 minutes before or 20 minutes after irradiation resulted in essentially identical tumour cell survival in all sort fractions (Figure 13). If, however, tumours were irradiated during the period of reduced tumour blood flow following H33342 injection, ie. administration of H33342 immediately prior to irradiation, the average cell survival was elevated compared to the survival observed when H33342 injection and radiation were separated by 20 minutes although this increase is only statistically significant (p<0.01) in cells distant from the blood supply. These results are consistent with observations that the stain transiently reduces tumour blood flow ; reduction in nutritive flow will cause impaired oxygen delivery, tumour cell hypoxia, and a relative resistance to x-irradiation.  3.3 DISCUSSION Evaluation of fluorescent stains as  in vivo markers of functional tumour vasculature must address  several concerns: (1) fluorescence properties: spectra, efficiency, bleaching; (2)  in vivo staining  characteristics: dye localization relative to blood vessels; (3) stain toxicity; (4) pharmacokinetics; (5) physiologic and metabolic effects; and (6) effects on tumour blood flow and oxygenation.  In this section, the staining patterns and pharmacokinetics of several fluorescent probes are described  but,  based on qualitative  observations of  perivascular stain  localization and  fluorescence intensity, H33342 and DiOC (3) were chosen as vascular markers suitable for trial in 7  a double-staining protocol. H33342 and DiOC (3) were therefore investigated in more detail, with 7  emphasis on physiologic effects, especially effects on tumour blood flow.  H33342 and DiOC (3), despite differences in chemical structure and cellular binding sites, have 7  very similar  in vivo staining patterns in tumour tissue (Figure 2). With time, H33342 remains better  81  localized to perivascular cells than does DiOC (3) (Figure 4), suggesting that H33342 should be 7  used as the first marker in a double-staining protocol. DiOC (3) is more resistant to bleaching 7  than H33342 and thus might be superior for vessel counting experiments requiring prolonged illumination of the tissue section. The short blood half-lives of both stains makes them equally suited to investigation of transient changes in tumour microvascular flow. One clear disadvantage of DiOC (3) is its insolubility in aqueous solutions, requiring the use of DMSO as a solvent. 7  Based on stability of H33342 perivascular localization and the requirement that DiOC (3) must be 7  dissolved in 75% DMSO, H33342 was injected first in a double-staining protocol.  Effects of  H33342 on mouse cardiovascular function and on tumour blood flow thus became of primary concern.  Smith et al. (1988) suggested that H33342 may cause a dose-dependent reduction in tumour 86 blood flow.  Mice given H33342 concurrently with  RbCI (used to measure tumour blood flow) 86  showed a reduced tumour flow compared to animals receiving  RbCI alone. This effect was  more pronounced at an H33342 dose of 40 mg/kg than at a dose of 20 mg/kg. The time course of this flow perturbation is of critical importance since a transient, reversible change is not necessarily a contraindication to the use of H33342 as the first marker in a double staining regimen.  Using laser Doppler flowmetry, it has been confirmed that bolus injection of H33342 causes a dose-dependent reduction in blood flow in subcutaneous experimental tumours (Figures 9 and 10).  Several important observations require emphasis: (1) reductions in tumour RBC flow are  dose-dependent and transient;  (2) 5 mg/kg H33342 is not significantly vasoactive;  (3) flow  reductions are independent of tumour size; (4) foot tumours show a much smaller, <3 minute reduction in R B C flow; thus H33342-induced flow changes are tumour site dependent; (5) H33342 has no significant effect on skin RBC flow.  82  These results indicate that H33342 can be used as the first marker in a double-staining protocol provided several limitations  are  kept  in mind.  Firstly, high doses should be avoided.  Unfortunately, the nonvasoactive dose (in anaesthetized mice) of 5 mg/kg does not provide sufficient fluorescence in tumour tissue sections to allow accurate vessel counting.  However,  whenever unanaesthetized mice are used, a slightly higher dose may be employed without the expectation of significant tumour blood flow reduction.  Secondly, because H33342 reduces  tumour RBC flow for several minutes, the second marker in a double staining protocol should be injected at least 10 minutes after H33342, when tumour flow has returned to pretreatment levels (following doses <15 mg/kg).  The mechanism responsible for H33342-induced reductions in tumour blood flow are not clear, but, based on observations by Algire and Legallais (1951), it was hypothesized that small changes in mouse blood pressure might explain the apparently selective decrease in tumour flow. Indeed, H33342 causes a transient but significant decline in mouse blood pressure measured by direct arterial cannulation in anaesthetized animals. The time course of blood pressure changes is not, however, identical to that of tumour blood flow reductions, and therefore, perturbations in systemic pressure might not entirely explain H33342-induced effects on tumour flow.  A direct effect of  H33342 on tumour blood vessels is possible, but no obvious reason for this selectivity is immediately apparent.  Transient tumour blood flow reductions induced by H33342 might result in decreased oxygen delivery and alteration of tumour oxygenation status. The stain does not however affect mouse temperature,  hematocrit, oxyhemoglobin dissociation curve, or blood pH/pC02 (Table I), all  factors which influence oxygen delivery to tissue. The response of tumour to x-irradiation is a sensitive assay of oxygenation since well-oxygenated cells are approximately 3x more sensitive to radiation than hypoxic cells (Hall, 1988).  H33342-induced reduction in tumour blood flow and  83  hence tumour cell oxygenation is clearly demonstrated if the stain is given immediately prior to radiation (Figure 13) but no such effect is observed if H33342 injection and radiation are separated by 20 minutes.  In fact, injection 20 minutes before and 20 minutes after irradiation result in  identical tumour cell survival levels in all H33342 sort fractions. Thus, in support of the results obtained with laser Doppler flowmetry, H33342 reductions in tumour oxygenation are short-lived and no evidence of radiobiological hypoxia (hypoxia sufficient to cause resistance to radiation) is observed 20 minutes after stain injection.  The radioresistance induced by H33342 injection  immediately prior to x-rays is unlikely to be due to a direct radioprotective effect of the stain (Smith and Anderson, 1984; Young and Hill, 1989) since no tumour cell radioprotection has been observed even after in vivo administration of 400/jg/g (Young and Hill, 1989), a dose 25x that used in the radiobiologic experiments described above.  An ideal control experiment for the double staining method would involve injection of DiOC (3) at 7  some interval before H33342, i.e. the sequence of stain injection would be reversed. Therefore, the effect DiOC (3) on tumour blood flow was also measured using laser Doppler techniques. 7  DiOC-,(3), like H33342, causes an abrupt transient decline in tumour R B C flow, but this is followed, after a brief period of recovery, by a significant prolonged decline in flow. This effect precludes the use of DiOC (3) as a first marker in a double staining regimen. 7  If animals are  sacrificed, for example, 5 minutes after DiOC (3) injection (as a second marker), these vasoactive 7  properties are minimized.  84  4. INTERMITTENT BLOOD FLOW  4.1 INTRODUCTION  Tumour hypoxia may result from a reduced functional vascular density (chronic, diffusion-limited hypoxia) (Thomlinson and Gray, 1955) or from temporal fluctuations in oxygen delivery caused by intermittent blood flow  (acute,  Sutherland and Franko, 1980).  perfusion-limited  hypoxia)  (Reinhold,  1977;  Brown,  1979;  Other disturbances in oxygen supply conditions (e.g. arterial  hypoxemia, anemia) can also contribute to inadequate oxygenation of tumour tissue. Acutely hypoxic tumour cells have been demonstrated using flow cytometry and cell sorting techniques in two experimental murine tumours, the SCCVII squamous cell carcinoma (Chaplin et al., 1986; 1987) and the KHT sarcoma (Young and Hill, 1989; Minchinton et al., 1990). Preliminary histologic evidence for transient tumour vessel nonperfusion in the SCCVII carcinoma was obtained by Chaplin et al. (1987) using a double-staining technique employing H33342 and fluorescent zinccadmium sulphide particles. 12.7% of SCCVII vessels exhibited staining "mismatch" indicative of intermittent blood flow.  Jirtle (1988) obtained a comparable result in rat SMT-2A mammary  adenocarcinoma (8 + 2%) using a similar method (H33342 and fluorescent microspheres).  This section describes the characterization of intermittent blood flow in a transplantable murine tumour using the double fluorescent staining technique described in Section 3.  The main  questions addressed were: (1) Which vessels are subject to transient nonperfusion, what amount of tumour tissue do they supply, and where are they located within the tumour mass (central vs. peripheral)? (2) What are the effects of tumour size, tumour implantation site, and animal restraint or anaesthesia?  (3) What is the time course of flow intermittency; for what period do vessels  remain nonperfused? (4) Do some tumour vessels experience reductions in flow but not complete  85  flow stasis or collapse? (5) Are vessels capable of vasomotion found in SCCVII tumours (i.e. is vascular smooth muscle present)? and (6) Does intermittent flow occur in human tumours grown as xenografts in nude mice?  A further aim was to predict the importance of intermittent blood flow on tumour oxygen supply. Previous theoretical investigations of tumour hypoxia have estimated the impact of perfusion heterogeneity on tumour oxygenation (Degner and Sutherland, 1986; 1988; Groebe and Vaupel, 1988) but no quantitative measurements of intermittent blood flow were then available. In order to predict the consequences of transient vessel nonperfusion in terms of tumour cell oxygenation, measurements of tumour vascular morphology (specifically vascular density and vessel diameters) and oxygen diffusion distance are required.  Measurements of tumour vascular morphology can be performed relatively easily using tumour sections stained in vivo with the vascular marker H33342 (Smith etal., 1988; Fallowfield, 1989). In order to estimate oxygen diffusion distance, oxygen consumption rates can be obtained from historical in vitro data and applied to theoretical calculations (Thomlinson and Gray,  1955;  Tannock, 1968; Vaupel, 1979) or consumption can be measured in vivo using tissue-isolated tumour preparations (Gullino, 1970; Vaupel et al., 1987; Kallinowski, 1989). technique,  performed  on the  same  tumour  specimen from  A simple in vitro  which vascular  morphology  measurements are made, would allow an alternative method for estimating tumour-specific oxygen diffusion distance.  Such a method, using AF-2, a fluorescent nitroheterocycle employed  previously to quantify hypoxia in single cells, spheroids, and murine tumours (Olive, 1982; 1984; Olive and Durand, 1983; Olive and Chaplin, 1986), is described in this section. In a technique analogous to that of Franko et al. (1987) using radiolabeled misonidazole, AF-2 has been used to label hypoxic cells in central regions of tumour tissue "cubes" incubated with the stain in vitro. The distance from the cube surface to the AF-2-stained region is an estimate of oxygen diffusion distance. Oxygen consumption rates can be calculated from these measurements and used to  86  estimate diffusion distance radially outward from a cylindrical tumour blood vessel (Tannock, 1968; Boag, 1969).  Based on the above tumour-specific measurements and on quantitative  determinations of  intermittent blood flow, a theoretical estimate of the contribution of flow intermittency to tumour hypoxia can be made.  4.2 RESULTS  4.2.1 TUMOUR SIZE  Using the double fluorescent staining technique, in which H33342 and then DiOC (3) were 7  administered intravenously 20 minutes apart to unrestrained mice, staining mismatch (indicative of transient vessel nonperfusion) was regularly observed in murine SCCVII carcinoma implanted subcutaneously (s.c.) over the sacral region. In s.c. tumours >100 mg weight, overall staining mismatch was 7.2 + 2.8% (mean + SD, n = 42) which is significantly different from the background levels obtained when both stains were injected simultaneously ( 1 . 3 ± 0 . 5 % , n = H) (p<0.001). Vessels stained with DiOC (3) 7  only (Figure 14) or with H33342 only (Figure 15) were both  observed, although in a given tumour, vessel "closing" could predominate over "opening" or wee  versa.  The amount of intermittent vessel nonperfusion was a function of SCCVII tumour size (Figure 16). For any given size >100  mg, large variability in the percentage of staining mismatch was  observed. Tumours <100 mg did not exhibit statistically significant amounts of mismatch. At sizes >100 mg, overall staining mismatch was significantly increased over background levels (p< 0.005) and maximum mismatch was observed at tumour sizes between 400-600 mg (8.6 ± 2 . 9 % ) .  Figure 14: Evidence for " o p e n i n g " (reperfusion) of previously nonperfused S C C V I I c a r c i n o m a microvessels during the 20 minute interval between intravenous injection of the fluorescent vascular markers H33342 (A) and D i O C ( 3 ) (B). Scale bar represents 1 0 0 / j m . 7  88  Figure 15: Evidence for " c l o s i n g " (nonperfusion) of previously perfused S C C V I I c a r c i n o m a microvessels during the 20 minute interval between intravenous injection of the fluorescent vascular markers H33342 (A) and D i O C ( 3 ) (B). S c a l e bar represents 100/vm. 7  89  Figure 16: Influence of tumour size on overall perfusion mismatch in subcutaneous SCCVII carcinoma. Fluorescent vascular markers were injected i.v. into unanaesthetized mice; animals were not restrained during the 20 minute interval between stain injections. For each weight group the m e a n ± S D ([^]) for 5-11 tumours is shown. The dashed line represents one SD above the mean for control tumours (interval = 0 minutes). Staining mismatch is significantly different from controls for tumours > 100 mg (p < 0.005).  90  In most tumours, transient vessel nonperfusion was more pronounced in central tumour regions (>500 /vm from tumour surface) than in the tumour periphery. A comparison of mean mismatch values revealed that central mismatch was significantly larger than that in peripheral regions for tumour sizes >600mg (p<0.005) (Table II). When a paired statistical analysis was employed, this difference was significant at all tumour weights (p < 0.001).  4.2.2 PATCHES O F NONPERFUSION  Tumour blood vessels exhibiting staining mismatch were not distributed homogeneously throughout the tumour section.  In addition to mismatch observed in individual vessels, large  "patches" of unequal staining were also seen.  Patches of mismatch ranged in area from  0.006 mm to 0.520 mm , the frequency distribution was very broad (Figure 17), and mean patch 2  2  size was 0.096 ± 0 . 1 0 8 mm . 2  This mean value corresponds to a region of nonperfusion  approximately 350 /vm in diameter (range 88-814/vm) consisting of 7-8 blood vessels.  A given  patch consisted of either vessel opening (DiOC (3), no H33342) or vessel closing (H33342, no 7  DiOC (3)) but never both. 7  4.2.3 IMPLANTATION SITE  SCCVII tumours implanted into gastrocnemius muscle also exhibited intermittent blood flow as demonstrated by the double staining technique (Figure 18).  In unrestrained mice, intramuscular  tumour mismatch was 6 . 5 ± 6 . 0 % (range 1.7-25.0%, n = 14).  This value, despite the large  dispersion, is significantly different from background levels of 1.2 ± 0 . 4 % (range 0.7-1.7%, n = 7) obtained when both stains were injected simultaneously (p< 0.025).  91  STAINING MISMATCH GROUP  WEIGHT  N=  (mg)  PERIPHERAL  CENTRAL  OVERALL  (%)  (%)  (%)  2.3 ±1.2  1.7 ±0.3  <100  67 ±19  5  1.1 ±0.4  <200  144 ± 2 8  5  3.1 ± 1 . 8  <400  303 ± 53  6  4.6±3.6  <600  520 ± 60  11  7.3 ±3.2°  10.2±4.3  <800  690 ± 50  9  6.6±2.0  C  12.1 ± 4 . 6  >800  900 ± 80  11  5.6±1.7  C  9.6±3.3  a  b  5.6±1.8  C  4.2±1.7  6.2±2.6  C  5.1 ± 3 . 1  C  C  C  C  8.6±2.9  C  8.4±2.8  C  7.3±1.9  C  b  Table II: Staining mismatch indicative of intermittent tumour vessel nonperfusion in peripheral (<500 /jm from tumour surface) and central regions of subcutaneous SCCVII carcinoma. Overall levels (plotted in Figure 16) are also shown. Values represent mean + SD. Corresponding mismatch values following simultaneous stain injection are: peripheral 1.1+0.5%, central 1.6 + 0.5%. overall 1.3 ± 0.5%. Levels of statistical significance for a 20 minute interval vs. controls are as follows: a = p<0.010; b = p<0.005; c = p<0.001. Mean values for peripheral and central staining mismatch are significantly different at tumour weights >600 mg (p<0.005). When a paired statistical analysis is employed, this difference is significant at all tumour weights (p<0.001).  92  Figure 17: Frequency histogram of staining mismatch "patch" size for patches (n = 103) measured in subcutaneous SCCVII tumours (n = 8). Mean ( ± S D ) patch size of 0.096 + 0.108 mm represents a region of nonperfusion approximately 350 //m in diameter.  2  93  Figure 18: Staining mismatch in SCCVII tumours implanted in gastrocnemius muscle. Mean values ( ± S D ) are shown for simultaneous stain administration ( • ) ( n = 7) and for a 20 minute injection interval (<B )(n= 14). Mice were not restrained during the interval between injections. The corresponding staining mismatch values for subcutaneous SCCVII tumours (400-600 mg) are also shown.  94  4.2.4 RESTRAINT/ANAESTHESIA  If animal stress or movement are involved in the genesis of tumour blood flow intermittency, immobilization by restraint or anaesthesia might have an effect on the amount of staining mismatch observed. Subcutaneous SCCVII tumours in unrestrained, unanaesthetized mice (n = 5, tumour weight: 450 ± 1 1 0 mg))  exhibited 7.9 ± 3 . 6 %  6.9 ± 4 . 3 % , central: 9.0 ± 3 . 2 % ) (Figure 19).  overall staining mismatch (peripheral:  In unanaesthetized but physically restrained mice  bearing subcutaneous tumours from the same transplant generation (n = 6, tumour weight: 5 8 0 ± 9 8 mg), overall mismatch was 6 . 0 ± 2 . 2 % (peripheral: 4 . 3 ± 2 . 4 % , central: 8 . 8 ± 4 . 8 % ) . This value, although less than in mice allowed to move freely in the interval between stain injections, was not significantly decreased. Similarly, immobilization using ketamine/diazepam anaesthesia had no significant effect on transient tumour vessel nonperfusion: in anaesthetized mice (n = 5, tumour weight: 4 4 0 ± 6 0 mg), overall staining mismatch was 9 . 0 ± 3 . 4 % (peripheral: 6.0 + 4.7%, central 1 2 . 5 ± 4 . 2 % ) . Thus, animal movement  perse  was not a precondition for the observation of  intermittent blood flow in SCCVII microvasculature.  4.2.5 TIME C O U R S E  The double staining protocol used to study intermittent blood flow is based, in part, on the short circulation half-lives of the fluorescent vascular markers employed.  Using the optical system  described, no fluorescence was detectable in SCCVII tumours following an H33342 dose of 1 mg/kg i.v. (data not shown). Such a dose would be expected to achieve peak blood levels of about  20/JM.  After 15 mg/kg H33342, blood levels declined to 20/uM within approximately  4 minutes (Section 3, Figure 6). Since mice are sacrificed and tumours removed 5 minutes after DiOC (3) injection, the period of DiOC (3) circulation (t1/2 7  7  = 3 min) was artificially limited.  Therefore, cells immediately adjacent to vessels alternating between "open" and "closed" states with a duration of nonperfusion <4-5 minutes would always be marked with H33342 and DiOC (3) 7  95  Figure 19: Influence of animal restraint (n = 6) or ketamine/diazepam anaesthesia (n = 5) on transient perfusion in 400-600 mg subcutaneous SCCVII tumours. Values for tumours in unrestrained mice (n = 5) and control mismatch levels (interval = 0; n = 11) are also presented. Staining mismatch (mean + SD) is shown for the entire tumour ( | ) as well as for peripheral (| |) and central ( ^ ) tumour regions. Mismatch in restrained or anaesthetized mice is not significantly different than that in unrestrained animals.  96  and thus would not be scored as "mismatched" using the method described. The corollary is that SCCVII tumour vessels scored as mismatched must be nonperfused for >4-5 minutes.  In order to better understand the time course of transient tumour vessel nonperfusion and to further validate the use of H33342 as the first vascular marker in a double staining regimen, the interval between stain injections was varied between 5 and 60 minutes (Table III).  For all intervals,  the overall mismatch was significantly greater than when stains were injected simultaneously (p<0.01). An interval of 5 minutes gave artifactually elevated staining mismatch (11.8 + 11.7%) with a disproportionate amount of vessel closure (H33342, no DiOC (3)) (Figure 20) due to the 7  vasoactive effect of H33342 (Section 3, Figure 9).  Mismatch after an injection interval of  10 minutes was 2.8 + 1.0% (n = 7) a value significantly lower (p<0.01) than that obtained using intervals >15 minutes.  This suggests an average duration of vessel nonperfusion of between  5-10 minutes. Note that the overall staining mismatch observed using a 20 minute interval in this series of experiments (performed as a group over a 2 month period) was 5.4 + 1.8% (n = 15, tumour weight: 560 + 210 mg) a value less than the 8.6 + 2.9% (n = 11, tumour weight: 520 + 60 mg) found in earlier equivalent experiments.  If H33342-induced transient reductions in tumour blood flow affect the staining mismatch levels obtained then mismatch should decline with increasing injection interval.  In addition, vessel  closing would presumably predominate relative to vessel opening. No significant difference in mismatch levels was noted for injection intervals of 15 to 60 minutes. Apart from the 5 minute injection interval, no significant increase in vessel closing relative to vessel opening was observed (Figure 20).  97  STAINING MISMATCH INT  N=  (min)  WEIGHT  OPEN  CLOSED  TOTAL  (mg)  (%)  (%)  (%)  1.3 ±0.5  0  11  0.70 ±0.53  1.0 ±0.4  0.3 ±0.4  5  8  0.50 ±0.11  1.1 ±0.7  10.7 ± 11.9  10  7  0.51 ±0.07  1.7±0.7  b  1.1±0.7  b  2.8±1.0  15  9  0.66 ±0.11  3.4±2.5  b  2.3±2.9  a  5.7±3.9  20  15  0.56 ±0.21  2.7±1.4  C  2.7±2.0  C  5.4 ±1.8°  30  8  0.70 ±0.22  3.1 ±1.6°  2.0±1.2  C  5.0 ± 1.9°  45  8  0.74 ±0.23  3.2±2.8  3.3 ±2.2°  6.5±3.1  C  60  9  0.60±0.17  1.7 ±1.4  3.3±2.7  5.0±2.7  C  a  b  b  11.8 ± 11.7  b  b  C  Table III: Effect of the interval between stain injections (INT) on perfusion mismatch in subcutaneous SCCVII carcinoma. The percentage of total vessels exhibiting staining mismatch is shown (mean + SD); this value is further subdivided into vessels which "opened" (DiOC (3), no H33342) and those which "closed" (H33342, no DiOC (3)) during the interval between stain injections. Levels of statistical significance for non-simultaneous injections vs. controls (interval = 0) are as follows: a = p<0.05; b = p<0.01; c = p<0.001. 7  7  98  CO  100r  UJ  80 -  10  20  30  40  50  60  INTERVAL B E T W E E N STAIN INJECTIONS (minutes)  Figure 20: Effect of the interval between stain injections on vessel "opening" ( ^ ) and vessel "closing" ( | ) in subcutaneous SCCVII carcinoma. Values are expressed as a percent ( + SEM, n = 7-15) of all mismatched vessels. Differences between the amount of vessel opening and the amount of closing are not statistically significant with the exception of interval = 5 minutes where closing (10.7+11.9%, mean + SD) exceeded opening (1.1+0.7%. mean + SD) (p< 0.025) due to the transient effects of H33342 on tumour blood flow.  99  4.2.6 QUANTITATIVE F L U O R E S C E N C E MEASUREMENTS  Since fluorescence intensity in H33342- and DiOC (3)-stained tumour cells is related to stain 7  delivery and therefore blood flow, quantitative measurement of perivascular fluorescence after sequential dye administration should give information regarding relative changes in blood flow at the single vessel level.  A reduced stain intensity, as opposed to complete absence of  fluorescence, implies a decreased average vessel perfusion during the period of stain circulation. Following simultaneous stain injection, the mean DiOC (3)/H33342 ratio for 100 individual vessels 7  in each of two tumours was 1.18 ± 0 . 5 7 (range 0.29-3.80) and 1.10 ± 0 . 6 8 (range 0.19-3.90) (Figure 21).  In contrast, when stain injections were separated by a 20 minute interval, fluorescence ratio  values for individual vessels deviated widely from those obtained after simultaneous stain injection. In Figure 21, vessels with staining mismatch, around which either H33342 or DiOC (3) was entirely 7  absent, have been arbitrarily assigned ratio values of 1000 (DiOC (3), no H33342) or 0.001 7  (H33342, no DiOC (3)). 7  Interestingly, between these two extremes, a large proportion of vessels  marked with both stains nevertheless exhibited fluorescence ratios markedly different from unity. Mismatch scored by eye in these tumours was 5.8 + 2.4% (n = 5) but a further 15.8 ± 3 . 0 % of vessels had fluorescence ratios outside the range observed following simultaneous stain administration. While such vessels would not be considered mismatched using the scoring criteria adopted (essentially a "yes" or "no" decision) they presumably experienced a period or periods of transient flow reduction and decreased stain delivery.  4.2.7 HUMAN TUMOUR XENOGRAFTS  In order to characterize intermittent blood flow as completely as possible, most experiments were restricted to a single tumour type, the murine SCCVII carcinoma. However, the generality of the phenomenon is clearly of great interest especially if such flow fluctuations occur in human  100  1000  0.001  Figure 21: Quantitative analysis of intermittent blood flow using the fluorescence image processing system (FIPS). The ratio of DiOC (3) to H33342 fluorescence is an estimate of relative single vessel perfusion at time = 0 minutes (H33342 fluorescence) and at time = 20 minutes (DiOC (3) fluorescence). Ratios for 100 individual blood vessels per tumour are shown. Vessels exhibiting staining mismatch have been assigned extreme ratio values of 1000 (DiOC (3) staining only) or 0.001 (H33342 staining only). Shaded region represents the range of ratio values found in tumour vessels when the two stains are administered simultaneously (A.B). For tumours C-G, the interval between stain injections was 20 minutes. 7  7  7  101  cancers.  For this reason, a small number of experiments were performed in human tumours  grown as xenografts in nude mice. It should be pointed out that, although the tumour cells in these systems are of human origin, the stromal component, including the vascular system, is derived from the mouse (Rofstad, 1985).  Using the fluorescent double staining technique, perfusion mismatch was observed in both the FaDu squamous cell carcinoma and in the Na11 melanoma (Table IV).  Mean staining mismatch  levels in xenografts were significantly different from control values obtained following simultaneous stain injection in SCCVII tumours (p<0.025).  No simultaneous injections were performed in  xenograft tumours. Variability in the amount of mismatch was increased in xenografts relative to SCCVII tumours grown in C3H mice. In addition, SCCVII tumours implanted subcutaneously in nude mice had staining mismatch of 7.7 + 5.7% , a mean value not significantly different than that in SCCVII implanted in C3H mice, but with a much larger dispersion.  When neoplastic tissue is transplanted into previously irradiated host tissue, tumours grow more slowly in comparison to tumours growing in unirradiated sites.  This "tumour bed effect" is  believed to be caused by radiation damage to vascular endothelium (Reinhold and Buisman, 1973) resulting in impaired tumour neovascularization (Summers et al., 1964; Hewitt and Blake, 1968; Urano and Suit, 1971). Thus, prior irradiation of the tumour implantation site could have an effect on tumour blood flow intermittency. Nude mice are often irradiated with 5 Gy whole body prior to xenograft implantation (to suppress tumour rejection), and therefore, although this dose is usually not large enough to cause a significant tumour bed effect, the influence of prior radiation on transient tumour vessel nonperfusion was assessed.  No significant difference in staining  mismatch was found in Na11 tumours grown either in irradiated or non-irradiated host mice.  No control experiments were performed in nude mice bearing human tumour xenografts to explore the effects of H33342 on cardiovascular parameters and on tumour blood flow.  Indeed, the  102  STAINING MISMATCH TUMOUR  PERIPHERAL (%)  CENTRAL (%)  OVERALL (%)  SCCVII (n = 18)  6.0±2.1  11.7±4.1  8.1 ±2.5°  SCCVII CONTROLS (n = 11)  1.1 ±0.5  SCCVII NUDE MICE (n = 5)  3.9±2.5  b  18.0 ± 19.8  FaDu (n = 6)  2.8±1.8  a  5.3±3.8  Na11 (n = 8)  6.4±2.9  C  Na11 (WBI) (n = 9)  4.3±4.3  a  C  C  1.6 ±0.5  1.3 ±0.5 7.7±5.7  b  b  3.7±2.7  a  6.7±4.5  b  6.8±4.4  C  6.0±4.8  b  4.8±3.1  b  a  Table IV: Intermittent blood flow in human tumours grown as xenografts in athymic, nude mice. FaDu = head and neck squamous cell carcinoma; Na11 = malignant melanoma. WBI= 5 Gy whole body irradiation prior to tumour implantation. Overall staining mismatch values (mean ± SD) and those for peripheral and central tumour regions are shown. Values for SCCVII tumours grown in both C3H and nude mice are presented for comparison. Levels of statistical significance for a 20 minute injection interval vs. SCCVII controls (interval = 0) are as follows: a = p<0.05: b = p<0.0i: c = p<0.001.  103  staining mismatch observed in these tumours consisted almost entirely of vessel "closure", implying that, despite the 20 minute interval between injections, H33342 may have had a prolonged effect on xenograft blood flow which interfered with the double staining technique.  4.2.8 VASCULAR SMOOTH MUSCLE  Vascular smooth muscle was demonstrated in tumour sections immunohistochemically using a muscle-actin-specific monoclonal antibody.  This antibody recognizes smooth muscle cells of  arteries and veins as well as pericytes surrounding smaller vessels (Tsukada et al., 1987a; 1987b). Contractile elements were clearly present in the wall of some blood vessels in murine SCCVII carcinoma; such vessels were confined exclusively to the tumour periphery (<500/jm from the tumour surface) (Figure 22). Less than 10 vessels containing vascular smooth muscle were found in tumour sections in which > 1000 vessels could be demonstrated by in vivo staining with H33342 or DiOC (3). 7  4.2.9 VASCULAR MORPHOLOGY  In subcutaneous SCCVII tumours >500 mg in size (n = 6), the mean vessel diameter, as measured in vessels outlined by H33342, was 1 4 . 5 ± 3 . 2 / j m , a value significantly larger than the mean diameter of 11.0+1.8/jm in tumours <500mg (n = 12) (p<0.01).  The frequency histogram of  vessel diameters was shifted slightly towards larger values in the >500 mg tumours (Figures 23 and 24). Other morphology parameters are shown in Figure 25. Vascular volume, vessel length, and vessel surface area were all significantly decreased in large tumours (>500 mg) relative to smaller tumours (< 500 mg) (p<0.01).  4.2.10 OXYGEN DIFFUSION DISTANCE  Measurement of oxygen consumption in SCCVII tumours was accomplished using binding of the fluorescent hypoxia probe, AF-2, to tumour "cubes" incubated with the probe in vitro.  The  104  Figure 22: Photomicrograph of SCCVII tumour section stained with monoclonal antibody against muscle-specific-actin. Vessels with vascular smooth muscle in their wall ( ^ ) are rare and are always located near the tumour periphery ( • • ) . Such vessels presumably represent host vasculature incorporated by the advancing tumour margins. Section thickness is 5 /um and the scale bar represents 100/jm.  105  VESSEL  DIAMETER  (um)  Figure 23: Frequency histogram of H33342-defined vessel diameters for subcutaneous SCCVII tumours <500mg. Total number of vessels = 1194.  106  VESSEL  DIAMETER  (Mm)  Figure 24: Frequency histogram of H33342-defined vessel diameters for subcutaneous SCCVII tumours >500mg. Total number of vessels = 600.  107  20 E a  i  •  i  10  • i  •• •  16  Ui  2 UJ  t-  1  —l  2  UJ  O >  <  CC  < 3  o  < ui  2  CO  4  < > •  800 eo E E g E  20  ^ E  600  CM  15  E  X  <  z  10 CC  5 -»oo  UJ  UJ ui  (0  8 200  ' 5  CO UJ  CC 3 CO  >  •  200  600  1 000  600  200  TUMOUR WEIGHT  1000  (mg)  Figure 25: H33342-defined vascular morphology parameters in subcutaneous SCCVII tumours (n = 18) as a function of tumour weight. Diameter measurements are mean values for 100 vessels per tumour. Increases in mean diameter (A) and decreases in vascular volume (B), vessel l e n g t h (C), and vessel surface area (D) are statistically significant (p<0.01).  108  Figure 26: Digitized grey-scale image of a "cube" of SCCVII tumour tissue incubated in vitro with the hypoxia probe AF-2. Thickness of the unstained (oxic) rim is an estimate of oxygen diffusion distance. Section thickness is 10/jm and the scale bar represents 100/jm.  109  distance from the cube surface to the region of maximum AF-2 binding is an estimate of oxygen diffusion distance (Figure 26).  When tumour cubes were incubated at 3 7 ° C in air, the oxygen  diffusion distance was 105 + 3/vm (mean + SEM; 20 experiments).  At temperatures below 3 7 ° C  the oxygen diffusion distance was increased as a consequence of impaired cell respiration. The results indicate that, at temperatures < 4 0 ° C , oxygen diffusion distance was linearly related to temperature  (r  =  -0.963); diffusion distance increased 7.8>c/m for every  temperature  (Figure 27).  The temperature  1 ° C decline in  of 500 mg subcutaneous SCCVII tumours was  32.0 + 0 . 9 ° C (n = 8, room temperature = 2 2 . 5 ° C , rectal temperature = 35.2°C) and therefore the measured diffusion distance was corrected accordingly; at 3 2 ° C , oxygen diffusion distance was estimated to be 144yum.  The above method gives an oxygen diffusion distance measured in plane geometry, i.e. using a tumour cube. To obtain an estimate of in vivo diffusion distance radially outward from a cylindrical blood vessel, the oxygen consumption, K, was first calculated from the results obtained in plane geometry using the following equation (Boag, 1969):  K  2DC (distance)  2  .5  where  oxygen diffusion coefficient, i.e 1.6x10 cm /sec at 3 2 ° C (Grote etal., 1977) concentration of oxygen in media around tumour cube oxygen diffusion distance (plane geometry) 2  C distance  The value for K obtained above is the oxygen consumption at 3 2 ° C in moles/litre/sec. diffusion distance in radial geometry is then calculated as follows (Boag, 1969):  Diffusion distance (radial outward)  where  a = vessel radius C = intravessel oxygen concentration  0.90a  (2CD  )  Ua  J  2  0.442  The  110  250  |  i  i  •  i i i i i ii i i i ii i i i | i  i  t i i  200  LU  O  z <  1 50  co 1 00 CO LU  50 -  *  20  •  •  •  1  25  •  •  •  *  1  30  •  •  *  •  1  •  -  •  35  TEMPERATURE  •  1  40  45  50  (°C)  Figure 27: Influence of temperature on oxygen diffusion distance in SCCVII tumour cubes. Below 4 0 ° C , diffusion distance increases 7.8/vm for every 1 ° C decline in temperature. At 3 2 ° C (temperature of subcutaneous SCCVII tumours in vivo), diffusion distance is approximately 145 /vm.  111  In >500 mg subcutaneous SCCVII tumours, mean vessel diameter was 14.5/um (radius = 7.25/jm) and therefore, at the arterial end of vessels, mean oxygen diffusion distance outward from blood vessels was 72//m, assuming an arterial p 0 of 94 mm Hg (Vaupel et al., 1987). At the venous 2  end of vessels ( p 0 = 48 mm Hg), oxygen diffusion distance was 52/ym. 2  In order to estimate  diffusion distance for the tumour microvasculature as a whole, the intravessel p 0 was assumed to 2  be 60 mm Hg, the midpoint between arterial and venous oxyhemoglobin saturations.  4.2.11 INTERMITTENT BLOOD FLOW AND A C U T E HYPOXIA  Many investigators have carried out extensive modelling, involving a myriad of variables, of oxygen diffusion in tumour tissue (Tannock, 1968, 1972; Boag, 1969, 1977; Vaupel, 1979; Degner and Sutherland, 1986; 1988; Jirtle, 1988; Groebe and Vaupel, 1988). Tumour microvasculature is so highly irregular that such mathematical models must be used with caution (Boag, 1977). The purpose of this section is simply to demonstrate the likely consequences of intermittent flow on tumour oxygenation and many simplifications and assumptions have been made.  Cells in environments in which p 0 is < 1 mm Hg exhibit only 10% of the maximum sensitivity to 2  ionizing radiation shown by cells under oxic conditions (Gerweck et al., 1981; Denekamp, 1983). Such poorly oxygenated, radioresistant cells, when they are located immediately adjacent to anoxic regions (regions beyond the oxygen diffusion distance) are under conditions of chronic hypoxia.  Using the oxygen diffusion distance estimated for >500 mg SCCVII tumours, and  measurements of vascular volume and vessel diameter made in the same tumours, one can estimate the percentage of tumour tissue which will located in regions in which p 0 is between 02  1 mm Hg, i.e. percentage of chronically hypoxic cells.  To estimate the amount of chronic hypoxia in SCCVII tumours >500 mg, a random distribution of blood vessels was assumed (Baez, 1977).  Mean vascular volume for such tumours was 3.4%  (Figure 25); vessel diameters were used corresponding to the frequency histogram in Figure 24.  112  Intravessel p C ^ was assumed to be 60 mm Hg. Based on these parameters, the fraction of anoxic tumour tissue (located beyond the oxygen diffusion distance) was estimated to be 20%. amount of tumour tissue located in regions in which pO^  = 0-1 mm Hg was 2.2%.  The  If one  considers the anoxic tissue to be necrotic and nonviable, then chronically hypoxic regions made up 2.8% of viable tumour tissue.  If 8% of SCCVII vessels are subject to intermittent blood flow of an "on-off" nature, then at any one time, 4% of tumour vessels are nonperfused. For example, in a tumour region containing 1000 blood vessels, 40 vessels will be nonperfused. Since intermittent flow affects patches of vasculature of mean diameter 350/jm, each containing approximately 8 vessels, it is estimated that 5 such patches will be present in this hypothetical tumour region.  When intermittent flow resulting in complete nonperfusion of 4% of vessels is superimposed on the model of chronic hypoxia described above, one finds that a further 2.2% of viable tumour tissue will become hypoxic. Thus, the total hypoxic fraction in SCCVII tumours >500 mg is calculated to be 5% of viable tissue. Of this total, 55% arises from chronic hypoxia and 45% is caused by transient nonperfusion of a small percentage of tumour blood vessels. Nonperfusion of randomly distributed individual tumour vessels (as opposed to patches) is predicted to contribute less to total hypoxia (18%) since vascularity in the SCCVII tumour is dense enough to allow nearby vessels to supply oxygen to the cord of cells normally supplied by a single nonperfused vessel.  As described previously (Section 4.2.6), approximately 15% of SCCVII tumour vessels experience transient reductions in flow without complete nonperfusion.  These vessels would clearly  contribute to acute hypoxia at least in cells distant from the blood supply; however, vessels subject to such flow reductions have not been included in the model used.  113  4.3 DISCUSSION  4.3.1 MECHANISMS FOR TUMOUR BLOOD FLOW INTERMITTENCY  In the murine SCCVII carcinoma, implanted subcutaneously or intramuscularly, 5-10% of blood vessels exhibit staining mismatch indicative of transient nonperfusion lasting at least 4-5 minutes. The mechanisms responsible for such intermittent flow have not been directly investigated but some possible causative factors include: (1) collapse of tumour vessels as a consequence of reduced  intravascular  and  elevated  extravascular  pressures; (2)  spontaneous  arteriolar  vasomotion; (3) vessel plugging by WBCs, platelet thrombi, RBC rouleaux, or circulating tumour cells; and (4) endothelial cell contractility or swelling resulting in vessel lumen occlusion.  Large subcutaneous SCCVII tumours exhibit more intermittent flow than small tumours; acute hypoxia is also more pronounced in larger tumours (Chaplin et al., 1986, 1987).  In addition,  staining mismatch is significantly higher in the tumour centre than in peripheral regions. The interrelationship between vascular morphology, microvascular pressure, and interstitial pressure seems likely to be of importance in the susceptibility of tumour vessels to periods of nonperfusion. First, incomplete development of tumour vasculature results in thin, distensible vessels which can easily be compressed or collapsed by elevated extravascular pressures. Second, intravascular pressure is considered to be lower in tumours than in normal tissue (Peters et al., 1980; Wiig, 1982). Finally, tumour interstitial pressure is elevated and appears to be higher in central tumour regions (Wiig et al., 1982). All these factors suggest that central vessels in large rapidly growing tumours should be subject to collapse and/or nonperfusion. Results obtained using the double staining method in SCCVII tumours support this hypothesis. However, a mechanism responsible for reversal of nonperfusion (i.e. vessel "opening") is less apparent.  Fluctuations in tumour  interstitial pressure have not been reported in the literature; therefore, changes in microvascular pressure are presumably responsible for vessel reperfusion. However, Wiig (1982) postulated that  114  an abrupt increase in interstitial pressure (by an unknown mechanism) could arrest microvessel flow. As a result of flow cessation, transcapillary filtration would be reduced and local interstitial pressure would decline.  At the same time, increasing intravascular pressure in the supplying  arteriole could cause a resumption of flow.  This brings us to the view that intermittent blood flow in tumours is, like its counterpart in normal tissue, a consequence of arteriolar vasomotion (Intaglietta et al., 1977; Reinhold et al., 1977). Partial constriction of smooth muscle in the wall of an arteriole (or other precapillary vessel) might result in a decreased downstream intravascular pressure and subsequent collapse of central capillaries supplied by the feeding vessel. The observation in SCCVII tumours that large patches of vasculature may be nonperfused is evidence for this mechanism. Interestingly, actual closure of a feeding arteriole would not be required to produce complete nonperfusion in supplied capillaries since in tumour tissue, the critical closing pressure (intravascular pressure required to prevent capillary collapse; Nichol et al., 1951; Burton, 1951) may be elevated, not due to excessive vasomotor tone as might occur in normal tissues, but as a consequence of increased interstitial pressure (Sevick and Jain, 1989a). Occasional vessels with contractile components in their walls are present in the periphery of SCCVII tumours.  Regardless of their origin (neovasculature or  incorporated host-arterioles), such vessels clearly provide a possible locus for vasomotor activity in this tumour model.  Periods of microvessel nonperfusion could originate not only from vasomotion in vessels feeding tumour regions of high interstitial pressure but also by plugging of capillaries by WBCs, platelet thrombi, RBC rouleaux, circulating tumours cells, or contractile endothelial cells. Vessel occlusion by poorly deformable cells has been postulated by Jain (1988) to be a cause of intermittent blood flow in tumours. Capillary plugging by granulocytes has also been considered a likely mechanism for the microcirculatory no-reflow phenomenon following low flow conditions as occur, for  115  example, in circulatory shock (Schmid-Schonbein, 1987); WBCs can apparently block capillary RBC flow transiently for periods of minutes to hours.  The intratumour  microenvironment  exaggerates the rheological problems that WBCs have in negotiating the microcirculation, i.e. low perfusion pressure, low pH (resulting in increased W B C rigidity), and increased adhesion to abnormal tumour vessel endothelium. Aggregation of RBCs with rouleaux formation is also likely to occur in the low flow tumour microcirculation (Jain, 1988; Sevick and Jain, 1989b). As perfusion pressure declines, intratumour blood viscosity increases as a result of rouleaux formation at low shear rates. Thus, transient declines in perfusion pressure, mediated via increases in interstitial pressure or by arteriolar vasomotion could cause vessel plugging by RBC rouleaux.  However,  vessel plugging by WBCs does not seem to be a likely explanation for transient nonperfusion in the SCCVII tumour. The average W B C diameter (7.5 /ym) is small relative to the caliber of SCCVII vessels and thus blockage of larger vessels supplying patches of vasculature is doubtful. A similar argument holds for circulating tumour cells (approximately 10/um diameter).  Nevertheless, cell  entrapment (including RBC aggregates) could conceivably occur within vessels supplied by a constricted arteriole since the decline in perfusion pressure would predispose such small vessels to blockage by nondeformable cells. After short periods of nonperfusion, trapped granulocytes which are plugging capillaries can be gradually pushed through the vessel lumen by restoring the perfusion pressure (Schmid-Schonbein, 1987). Thus, it is not possible to rule out the contribution of vessel plugging to reversible tumour vessel nonperfusion.  Finally, spontaneous cyclic contractions of capillary endothelial cells have been shown to block RBC flow for periods up to 1 minute (Ragan et al., 1988), but no longer periods of nonperfusion have been measured and the phenomenon does not explain flow fluctuation in large patches of vasculature.  In tumours grown in transparent chambers, animal movement appears to contribute to temporary flow stasis and flow reversal (Yamaura and Sato, 1974).  However, animal immobilization, by  116  chemical or physical restraint, does not inhibit intermittent flow in subcutaneous SCCVII tumours. Anaesthesia could, of course, have effects on tumour blood flow independent from those arising as a consequence of immobilization (Zanelli et al., 1975; Zanelli and Fowler, 1977; Cullen and Walker, 1985; Menke and Vaupel, 1988). Of particular interest is the observation that anaesthesia can reduce spontaneous arteriolar vasomotion (Funck and Intaglietta, 1983; Colantuoni et al., 1984).  If vasomotion is in part responsible for tumour flow intermittency, one would predict a  reduction in staining mismatch when mice are anaesthetized; this was not observed in the SCCVII tumour.  However, arteriolar vasomotion in a vessel supplying tumour tissue has been observed  using transparent chambers in rats even under barbiturate anaesthesia (Intaglietta et al., 1977). Physical restraint can also have a variable influence on tumour perfusion and oxygenation (Zanelli and Lucas, 1976; Shibamoto et al., 1987); the usual effect is an increase in tumour ischemia. Therefore, the observation that restraint causes a very small decrease in staining mismatch is difficult to explain.  Using either anaesthesia or physical restraint, no large decrease in tumour  microvascular flow was qualitatively observed using the double staining technique.  4.3.2 DURATION OF TUMOUR V E S S E L NONPERFUSION  Based on the pharmacokinetics of the fluorescent vascular markers used, the duration of transient nonperfusion in SCCVII vessels is at least 4 to 5 minutes. This is a very long period of time relative to vasomotion in normal tissues and in tumours grown in observation chambers.  In a bat wing  microvascular preparation, Nicoll and Webb (1955) reported a frequency of active vasomotion of 415 cycles/minute.  Even in capillaries in cat mesentery which have an "on-off" flow pattern, the  period of nonperfusion is only 25-30seconds (Johnson and Wayland, 1967).  Thus, transient  nonperfusion of >5 minutes duration is quite unusual in normal tissue microvasculature. In a rat rhabdomyosarcoma grown in a transparent chamber, Intaglietta et al. (1977) measured a flow periodicity in a supplying arteriole of only 2-3 minutes (i.e. decreased flow for <1  minute).  Extended periods of nonperfusion in tumour vessels likely arises as a consequence of an increased critical closing pressure. As a result of increased tumour interstitial pressure, a minor  117  intravascular pressure reduction caused by precapillary vasomotion (which in normal tissue would not cause complete cessation of capillary flow) could cause prolonged nonperfusion of tumour vessels. Alternatively, vessel plugging by RBC rouleaux, WBCs, or tumour cells could cause an extended but potentially reversible period of vessel nonperfusion.  Interestingly, the double  staining method cannot detect perfusion fluctuations of <5 minutes duration, although such short periods of nonperfusion could also occur and cause impaired oxygen delivery and substantial acute tumour cell hypoxia.  While the minimum duration of intermittent nonperfusion detectable by the double staining method is 4-5 minutes, no upper limit is easily identified. The results suggest that vessel nonperfusion of very long duration is unlikely: if vessels receive no flow for periods substantially longer than the interval between dye injections then large tumour regions would be present in which neither fluorescent stain would be visible.  Such unstained regions are seldom seen, even when the  interval between injections is reduced to 10 minutes, although clearly, lack of vessel staining by either marker is difficult to quantitate, since at least one stain is required for vessel identification. The reduced amount of staining mismatch observed using a 10 minute interval could be explained by a small percentage of nonperfused vessels receiving neither stain; such vessels, although transiently nonperfused, would not be detectable using the double staining method. Nevertheless, the results suggest that intermittent blood flow in the SCCVII carcinoma results in vessel nonperfusion, of 5-15 minutes duration, affecting 5-10% of tumour vessels.  Results obtained  3 using the hypoxic cell marker  H-misonidazole support a duration of vessel "closure" of  < 30 minutes (Olive and Durand, 1989).  Binding of misonidazole requires sufficient time under  hypoxic conditions; no heavy misonidazole labelling was observed in SCCVII cells immediately adjacent to the blood supply, suggesting that vessel nonperfusion and acute hypoxia were of relatively short duration. The use of H33342 infusions (as opposed to bolus injections) to alter the effective circulating half-life of the stain would be an alternative approach to studying the duration of nonperfusion.  118  4.3.3 TUMOUR OXYGENATION: SIGNIFICANCE OF INTERMITTENT FLOW  The measurements of SCCVII vascular morphology reveal a gradual rarefaction of the vasculature in larger tumours and an increase in the mean vessel diameter.  These results are in agreement  with most other work on tumour vascular morphology (reviewed by Jain, 1988). An understanding of vascular volume and the distribution of vessel diameters is necessary if one wishes to predict, using a theoretical model, the proportion of hypoxic cells expected in a tumour mass. Of further importance is the measurement of a tumour-specific oxygen consumption rate so that the oxygen diffusion distance in the tumour of interest can be obtained.  The calculated in vivo oxygen diffusion distance for subcutaneous SCCVII carcinoma (52-72/vm) is less than that estimated by Thomlinson and Gray (1955) for human bronchial carcinoma (145 /jm), but it is in good agreement with values calculated by Tannock (1968, 1972) in a mouse tumour model (55-80/jm). A theoretical estimate of the hypoxic fraction in SCCVII tumours was made using the calculated oxygen diffusion distance in subcutaneous SCCVII tumours and the measured vascular volume and vessel diameter distributions.  The predicted overall hypoxic  fraction of 5% is less than values obtained radiobiological^ in the same tumour system (10-20%; D.J. Chaplin, personal communication; approximately 20% calculated from Figure 13), although this comparison does not take into account differences in tumour size, an important determinant of hypoxic fraction. In addition, the model employed considers only severe hypoxia (0-1 mm Hg) and assumes that anoxic cells (beyond the oxygen diffusion distance) are nonviable when in fact they may have the ability to survive such conditions for several hours (see below). Of major significance is the conclusion that acutely hypoxic regions, arising as a result of intermittent blood flow, make up 45% of all hypoxia in the tumour. Jirtle (1988) has previously calculated the relative importance of acute and chronic hypoxia in a rat mammary adenocarcinoma. The model employed predicted that in order for 45% of the total hypoxic fraction to be due to acutely hypoxic cells, 15% of tumour vessels would have to be nonperfused.  However, no tumour-specific vascular morphology or  oxygen consumption rates were used and, more importantly, no information was then available  119  regarding patches of nonperfusion.  At the vessel density observed in the SCCVII,  4%  nonperfusion of randomly distributed individual vessels contributes only 18% to the total hypoxic fraction, a less dramatic effect than when nonperfusion occurs in patches of vessels. This value is in good agreement with the 16% predicted by Jirtle (1988) under the same conditions.  The size of the clinically relevant (i.e. radiation resistant) tumour cell hypoxic fraction is determined not only by oxygen delivery but also by the ability of cells to survive prolonged periods of chronic hypoxia.  The half-time for cell death of hypoxic cells in the innermost regions of multicell  spheroids (regions of chronic hypoxia) is estimated to be 3-4 hours (Franko and Sutherland, 1978). The combination of prolonged hypoxia (6 hour exposure) and an acidic environment (pH 6.0) is -4 extremely cytotoxic in vitro, reducing the surviving fraction of cells to 10  (Rotin et al., 1986).  When tumours are clamped, preventing oxygen delivery, significant cell death occurs after a period of 4-8 hours (Denekamp et al., 1983). deprived cells have a limited lifespan.  Thus, in the absence of reoxygenation, oxygen  If chronically hypoxic cells have reduced clonogenic  capacity, then acute hypoxia, presumably causing minimal cytotoxicity, may be of even more importance in terms of tumour cell resistance to radiation.  Clinical methods to overcome hypoxic cell radioresistance, (e.g. hyperbaric oxygen therapy, preradiotherapy blood transfusion, perfluorochemical emulsions) have been designed based on the assumption that tumour hypoxia is of the "chronic" type, occurring in cells adjacent to regions of necrosis.  These methods attempt to increase the oxygen diffusion distance outward from  perfused blood vessels (reviewed by Hirst, 1986a).  If acute hypoxia resulting from vessel  nonperfusion contributes to radioresistance in human tumours then such techniques, which increase the oxygen carrying capacity of blood, would not be expected to eliminate tumour hypoxia. An alternative approach to the problem of tumour hypoxia involves the use of drugs which are selectively toxic to hypoxic cells or which sensitize such cells to radiation (reviewed by Fowler,  120  1985). Hypoxic cell cytotoxins, if they require relatively long exposure under hypoxic conditions or if they are not freely diffusable throughout the tumour mass, would not be expected to be particularly efficacious against transiently hypoxic cells. Methods to overcome acute hypoxia have been briefly reviewed by Coleman (1988); pharmacologic reduction of tumour flow heterogeneity is an approach which has been largely overlooked. In Section 5, manipulation of tumour blood flow, with emphasis on the role of systemic blood pressure and its effect on microvascular flow heterogeneity, is discussed in detail.  121  5. MODULATION OF TUMOUR BLOOD FLOW  5.1  INTRODUCTION  Selective manipulation of tumour blood flow using vasoactive drugs has been extensively investigated as a method to improve conventional cancer treatment modalities (reviewed by Jain and Ward-Hartley, 1984; Jirtle, 1988).  Perfusion heterogeneity is perceived as detrimental to  successful radiation therapy, chemotherapeutic drug delivery, and tumour heating in hyperthermia treatment.  Thus, modulation of tumour blood flow is undertaken with the goal of reducing or  eliminating such heterogeneity.  Investigation of the effects of vasoactive drugs on blood flow in  three-dimensional tumours has been limited, in most cases, to macroscopic observations, that is, measurement of changes in overall or regional tumour blood flow.  Methods have not been  available to measure drug-induced flow changes at the microregional or single vessel level in solid experimental tumours. The double fluorescent staining technique described in this thesis provides such a method. Effects of vasoactive agents on both spatial and temporal perfusion heterogeneity can be examined at the microvascular level. Two hypotheses were tested using this approach:  (1)  Pharmacologic manipulation of systemic blood pressure will cause large changes in tumour perfusion since the tumour microcirculation is essentially passive and lacks autoregulatory mechanisms (Algire and Legallais, 1951; Vaupel, 1975; Zanelli and Fowler, 1977; Sevick and Jain, 1989a).  (2)  Elevation of systemic blood pressure will reduce microregional perfusion heterogeneity by preventing transient tumour vessel nonperfusion.  122  Three classes of drugs were examined: hydralazine, a vasodilator; angiotensin II, a potent pressor agent; and nicotinamide, a drug which improves tumour oxygenation by an unknown mechanism.  5.1.1 HYDRALAZINE  The vasodilator hydralazine has been shown to selectively reduce blood flow in experimental solid tumours (Brown, 1987; Chaplin, 1988; Okunieff et al., 1988a; Vorhees and Babbs, 1982) thus decreasing tumour oxygenation (Brown, 1987; Stratford et al., 1988), energy metabolism, and pH (Okunieff et al., 1988b). Hydralazine enhances the action of chemotherapeutic agents targetted against poorly oxygenated or nutrient-deprived cells (Brown, 1987; Chaplin and Acker, 1987; Chaplin, 1988; Stratford et al., 1988; Chaplin, 1989) and allows selective heating of tumour tissue during hyperthermia treatment (Vorhees and Babbs, 1982).  The mechanism by which hydralazine reduces tumour blood flow is not clear. Hydralazine causes systemic vasodilation by direct relaxation of arteriolar smooth muscle (Koch-Weser, 1976). Tumour blood vessels, however, are apparently maximally dilated, have minimal smooth muscle (Warren, 1979) and are incapable of blood flow autoregulation in response to changes in perfusion pressure (Vaupel, 1975; Sevick and Jain, 1989a).  Hydralazine-induced vasodilation in normal  tissue could result in shunting or "steal" of blood flow away from the tumour (Chan et al., 1984; Kruuv et al., 1967; Vorhees and Babbs, 1982; Jirtle, 1988). Alternatively, or in addition, tumour microvascular pressure, already lower than in normal tissue (Peters et al., 1980; Wiig, 1982), could further decline as a result of systemic hypotension. Elevated tumour interstitial pressure might exceed intravascular pressure in some tumour regions (Wiig et al., 1982) with resultant vascular collapse and flow stasis (Sevick and Jain, 1989a).  Most determinations of hydralazine-induced blood flow reduction have been undertaken at the macroscopic level, that is, by measuring changes in overall tumour blood flow. To understand the mechanism by which hydralazine reduces tumour blood flow, quantitative microscopic information  123  is desirable, preferably at the level of individual tumour vessels. Flow reductions in single tumour vessels have been observed after hydralazine injection using transparent chamber techniques (Jain and Ward-Hartley, 1984).  Laser Doppler flowmetry has also recently been employed to  monitor, in small tumour regions, hydralazine-induced blood flow changes (Okunieff et al., 1988a).  In this section, the double fluorescent staining technique and laser Doppler methods have been used to investigate the hypothesis that vascular collapse and complete flow stasis, occurring as a consequence of systemic hypotension, are components of tumour blood flow reduction induced by hydralazine. The heterogeneity of the flow changes at the single vessel level has been examined.  5.1.2 ANGIOTENSIN II  Tumour blood flow is very sensitive to changes in systemic blood pressure (Algire and Legallais, 1951; Zanelli and Fowler, 1977). Flow autoregulation is apparently absent; at perfusion pressures >40 mm Hg, tumour blood flow is linearly related to mean arterial blood pressure (Vaupel, 1975; Seveck and Jain, 1989a).  This passive pressure-flow relationship, together with evidence that  vascular smooth muscle and adrenergic innervation are often poorly developed or absent in tumour neovasculature (Warren, 1979; Mattsson et al., 1977), suggests that elevation of systemic blood pressure by vasoactive drugs might selectively increase tumour blood flow.  The vasoconstrictor angiotensin II, a potent pressor agent, has been shown, with the exception of one study (Jirtle et al., 1978a), to increase absolute and relative blood flow in solid experimental tumours (Suzuki etal., 1981; Burton et al., 1985; 1988; Ackerman and Jacobs, 1987; Yanagibashi et al., 1987; Maeta et al., 1989). Administration of angiotensin II can enhance tumour visualization during angiography  (Ekelund  and  Lunderquist,  1974),  improve  the  efficacy  of systemic  chemotherapy (Suzuki etal., 1981; Takematsu et al., 1985; Kuroiwa etal., 1987; Kobayashi et al., 1989), and selectively increase tumour blood flow under conditions of applied local hyperthermia (Maeta et al., 1989).  124  The mechanism of angiotensin-induced increases in tumour blood flow is assumed to be indirect, secondary to an elevation of systemic blood pressure (Suzuki et al., 1981). Since angiotensin II causes vasoconstriction via specific receptors on vascular smooth muscle (Grega and Adamski, 1987) the contribution of indirect effects and direct effects on tumour vasculature will obviously depend on the number of tumour vessels (likely incorporated host arterioles) which possess such musculature. Angiotensin II has also been shown to increase tumour vascular area (Hori et al., 1985) and to reduce the macroscopic heterogeneity in tumour blood flow distribution (Burton et al., 1985). An anticipated improvement in blood flow distribution at the microvascular level has not, to date, been investigated.  Angiotensin-induced increases in tumour blood flow, if they reduce  microvascular perfusion heterogeneity, especially intermittent tumour blood flow, might potentially enhance tumour oxygenation (by eliminating acute hypoxia) and thereby improve the tumour response to radiation therapy.  5.1.3  NICOTINAMIDE  Nicotinamide, the amide form of vitamin B3 (Marcus and Coulston, 1985), is a relatively non-toxic, tumour-specific radiosensitizer in vivo (Horsman et al., 1986; 1989a).  Nicotinamide does not  enhance radiation damage under hypoxic conditions in vitro or in clamped, nonperfused tumours and it does not appear to reduce hypoxia by altering cellular oxygen consumption (Horsman et al., 1989a).  The mechanism by which tumours are sensitized to radiation is thought to be via a  reduction in the amount of tumour hypoxia as a consequence of increased tumour blood flow (Horsman et al., 1988; 1989a; 1989b). However, nicotinamide is not vasoactive, although nicotinic acid, for which nicotinamide is a precursor, does have some weak vasodilator effects (Needleman et al., 1985). A reduction in acute hypoxia by nicotinamide could account for its radiosensitizing properties.  The hypothesis that nicotinamide can improve microregional perfusion and tumour  oxygenation by reducing intermittent blood flow can be tested by applying double fluorescent staining techniques (Chaplin et al., 1990).  125  5.2 RESULTS  5.2.1 HYDRALAZINE  Bolus intravenous hydralazine administration caused a reduction in tumour blood flow as measured using laser Doppler flowmetry.  Red blood cell (RBC) flow in subcutaneous back  tumours declined to 33 ± 1 4 % (mean + SD, n = 9) of pretreatment  levels 15 minutes after i.v.  injection of hydralazine (10 mg/kg) in anaesthetized animals (Figure 28). The mean number of moving RBCs decreased to 65 ± 2 4 % of pretreatment values suggesting a reduction in functional microvascular volume. Hydralazine doses of 5 mg/kg and 2.5 mg/kg resulted in flow reductions to 32 ± 5 % (n = 5) and 36 ± 1 2 % (n = 5) of pretreatment levels respectively. These reductions were essentially identical to those achieved using a 10 mg/kg dose (Figure 29).  In subcutaneous foot tumours, hydralazine-induced R B C flow reductions were similar to those observed in back tumours (26 ± 12% of pretreatment values, n = 5). However, if mice were simply restrained, without the use of anaesthesia, the decrease in flow was significantly larger (8.7 + 6.4% of pretreatment values, n = 5, p< 0.025) (Figure 30). In foot tumours, the decrease in number of moving RBCs was significantly larger than in back tumours for both anaesthetized ( 2 7 ± 1 1 % , p< 0.010) and unanaesthetized (33 ± 18%, p< 0.025) animals.  The influence of hydralazine on blood flow in human tumour xenografts grown in nude mice was also examined using laser Doppler flowmetry.  All tumours were located subcutaneously in the  flank and mice were immobilized using ketamine/diazepam anaesthesia.  Hydralazine (5 mg/kg)  caused a reduction in RBC flow to 67 ± 1 9 % of pretreatment values in the Na11 melanoma (n = 5) (Figure 31).  This change was less than that observed in SCCVII carcinoma also grown  subcutaneously in nude mice (40 ± 1 8 % , n = 5, p<0.05). Whole body irradiation (5 Gy) prior to tumour implantation had no significant effect on the blood flow change induced by hydralazine in  126  Figure 28: Effect of hydralazine (10 mg/kg i.v.) on red blood cell (RBC) flow, number of moving RBCs (indicative of functional microvascular volume), and mean RBC velocity as assessed by laser Doppler flowmetry in subcutaneous SCCVII carcinoma. Measurements were made in mice immobilized with ketamine/diazepam anaesthesia. Error bars represent SEM (n = 9).  127  1 20 1 00 80 60 40  r  20  >  1 20 1 00  c o  80  f4  60 40 20  O  CQ 0C  0  5  TIME  10  15  20  25  30  (minutes)  Figure 29: Effect of hydralazine dose on SCCVII tumour RBC flow measured using laser Doppler flowmetry in anaesthetized mice. No significant differences in flow reductions were observed using the doses shown: 10 mg/kg, n = 9 (#): 5 mg/kg ( O ) and 2.5 mg/kg ( • ) . n = 5. Error bars represent S E M .  128  (U  I  i  i  i  -2 1 2 0 •  TIME  (minutes)  Figure 30: Influence of ketamine/diazepam anaesthesia on hydralazine-induced RBC flow reductions in SCCVII carcinoma grown subcutaneously in the hindfoot dorsum. Mean + SD for 5 tumours is shown. The tumour flow reduction in anaesthetized animals ( • ) was not as pronounced as that observed in mice which were unanaesthetized but restrained ( O ) (p<0.025).  129  60 40 • 20 • 0  5  TIME  10  15  20  25  30  (minutes)  Figure 31: Hydralazine-induced reductions in blood flow in human tumour xenografts grown in athymic nude mice. (A) Effect of host mouse on hydralazine (5 mg/kg)-induced RBC flow changes in SCCVII tumours. ( • ) C3H mice, (O) nude mice. (B) Flow reductions in Na11 melanoma (hydralazine 5 mg/kg). ( • ) whole-body irradiation prior to tumour implantation: ( • ) no preirradiation. (C) Flow reductions in HRT18 colonic adenocarcinoma (hydralazine 10 mg/kg). For all panels, error bars represent SEM for 5 tumours. Animals were immobilized with ketamine/diazepam anaesthesia.  130  Na11 melanoma (69 + 17%, n = 5). A dose of 10 mg/kg caused a slight reduction in R B C flow in the HRT18 adenocarcinoma (to 73 ± 2 8 % of pretreatment  values, n = 5) but this was not  statistically significant.  When tumour microregional perfusion was assessed using the double staining technique, intravenous hydralazine administration caused a reduction in functional tumour vasculature in the murine SCCVII carcinoma implying complete flow stasis and/or vascular collapse in affected vessels.  Regions of nonperfusion extended throughout the tumour mass and were especially  prominent in central and apical tumour regions (Figure 32).  Involvement of a single vessel is  shown in Figure 33. DiOC (3) (injected 15 minutes after hydralazine) stained only cells adjacent to 7  the proximal portion of an apparently contiguous vessel, suggesting collapse of the distal vessel segment or blockage of the vessel lumen.  Following hydralazine injection large patches of  vasculature were outlined only by H33342. These areas were distributed non-uniformly throughout the tumour mass but were more pronounced in central as compared to peripheral tumour regions (Figure 34).  Fifteen minutes after hydralazine doses of 5 mg/kg or 10 mg/kg, perfusion was  completely abolished in 12 ± 7 % ( m e a n ± S D , n = 6) and 36 ± 1 6 % (n = 10) of tumour vessels respectively (Figure 34). These values are significantly different (p<0.001) than those observed in saline treated controls in which 3.1 ± 1 . 9 % (n = 10) of vessels closed in the interval between injection of the fluorescent stains.  Nonperfusion was greater in central tumour regions; this  difference was only statistically significant when a dose of 10 mg/kg was used (p<0.05). Small SCCVII tumours (<120mg) did not exhibit vessel nonperfusion following 10 mg/kg hydralazine (1.1 ± 0 . 3 % , n = 5) (data not shown).  5.2.2 ANGIOTENSIN II  Intravenous infusion of angiotensin II (2/jg/kg/min) in halothane anaesthetized mice caused an increase in arterial blood pressure from a pretreatment value of 8 5 ± 2 m m H g to a maximum pressure of 112 ± 7 mm Hg (p< 0.001). An elevated blood pressure was sustained for the duration  131  Figure 32: Staining mismatch in a subcutaneous SCCVII carcinoma (700 mg) following administration of hydralazine (5 mg/kg i.v.). Regions of nonperfusion, that is, areas having no DiOC (3) staining of perivascular cells, are indicated by ( 0 ) . Matched staining patterns are depicted by ( • ) and are located predominantly in the tumour periphery. Serial sections (actual size) 400 /jm apart are shown. 7  132  Figure 33: Evidence for vessel closure in murine SCCVII carcinoma following hydralazine administration (10 mg/kg i.v.). Grey-scale representation of digitized fluorescence microscope images are shown. An apparently contiguous vessel stained with H33342 (A) exhibited lack of DiOC (3) staining 15 minutes after hydralazine injection (B). Section thickness is 10 um and the scale bar represents 100/jm. ?  133  60 CO _J LU CO CO HI  >  Q HI CO U, QC HI  0_  50 40 30  p  T  20 10  T>  I  11 10  HYDRALAZINE DOSE  (mg/kg)  Figure 34: Effect of hydralazine dose on functional vasculature in murine S C C V I I carcinoma implanted subcutaneously over the sacral region. Blood vessels outlined by H33342 but not DiOC (3) represent nonperfused vasculature. Staining mismatch is shown for peripheral (| |) and central ( t u m o u r regions as well as overall ( | )< Error bars represent SD (n = 10). 7  134  of drug infusion (Figure 35). Heart rate also increased, although with a delayed time course, from 446 ± 5 beats per minute (bpm) to 518 + 60 bpm (p< 0.005).  Angiotensin II infusion resulted in a 2-3 fold increase in SCCVII tumour RBC flow as measured by laser Doppler flowmetry (Figure 36).  The maximum tumour flow increase occurred 2-7 minutes  after starting angiotensin II infusion and, in ketamine/diazepam anaesthetized mice bearing s.c. back tumours, this maximum flow was 305 ± 9 0 % (range 169-386%) of pretreatment values (p< 0.001). In unanaesthetized, restrained mice bearing foot tumours, RBC flow increased to a maximum value of 2 0 6 ± 5 0 % (range 150-270%) (data not shown).  The elevation in RBC flow  observed was due both to an increase in the number of moving RBCs ( 1 5 7 ± 3 5 % of normal) and an increase in mean RBC velocity ( 1 9 9 ± 4 4 % ) .  Interestingly, the number of moving RBCs,  indicative of functional microvascular volume, remained slightly elevated relative to pretreatment values even after cessation of angiotensin II infusion (117 + 4%, p<0.001). Infusion of saline had no effect on tumour RBC flow and angiotensin II had no significant effect on blood flow in normal skin.  An estimation of the pressure-flow relationship for tumour vasculature during angiotensin II infusion was obtained by plotting relative tumour RBC flow changes measured in anaesthetized mice (n = 9) using laser Doppler flowmetry against mean arterial blood pressure measured in a separate experiment (n = 6).  Pressure-flow curves for conditions of increasing blood pressure at  the start of infusion and decreasing pressure after infusion ceases are shown in Figure 37. The linearity of this relationship suggests absence of autoregulation. Some hysteresis was noted; that is, for a given blood pressure, flow is higher after angiotensin II infusion than when pressure is increasing at the start of infusion.  In unrestrained, unanaesthetized C3H mice bearing s.c. SCCVII carcinoma, staining mismatch (indicative of intermittent tumour vessel nonperfusion) is found in approximately 8% of tumour  135  Figure 35: Effect of angiotensin II infusion (2 /jg/kg/min) on mean arterial blood pressure (MABP) (A) and heart rate ( A ) in 8week old male C3H mice (mean SD, n = 6) anaesthetized wiih inrrlT^ ^ ° , ^ angiotensin II infusion the blood pressure was significantly increased over pretreatment levels (p<0.001). +  v  a  p  r  i  z  e  r  D  u  r  y  136  Figure 36: Effect of angiotensin II infusion (shaded area) on RBC flow, number of moving RBCs (indicative of functional microvascular volume), and mean RBC velocity as assessed by laser Doppler flowmetry. Results are shown for tumour (#) and for normal skin adjacent to the tumour site ( • ) . The effect of saline infusion on tumour RBC flow is also indicated ( O ) Error bars represent SEM for 5 mice per treatment.  137  80  90  MABP  1 0 0 1 1 0 1 2 0 (mm  Hg)  Figure 37: Relationship of tumour RBC flow to mean arterial blood pressure changes induced by angiotensin II infusion. (#) increasing blood pressure; (O) decreasing blood pressure (following cessation of drug infusion). Linearity of pressure-flow relationship suggests lack of flow autoregulation in tumour tissue.  138  STAINING MISMATCH (%) PERIPHERAL  CENTRAL  OVERALL  NO RESTRAINT (n = 18)  6.0±2.1  C  11.7±4.r  8.1 ± 2 . 5 '  SALINE INFUSION (n = 10)  2.4±1.5  J  5.5±3.8  3.6±1.8  ANGIOTENSIN II INFUSION (n = 9)  1.6 ±0.9  2.7 ±2.2  2.0 ±1.3  SIMULTANEOUS INJECTION (n = 11)  1.1 ±0.5  1.6 + 0.5  1.3 + 0.5  b  C  Table V: Effect of angiotensin II or saline infusion on percentage ( ± SD) of SCCVII tumour vessels with staining mismatch indicative of transient nonperfusion. Mismatch values significantly different from background levels obtained when H33342 and DiOC (3) are injected simultaneously are indicated (a = p<0.025; b = p<0.005; c = p<0.001). 7  139  vessels. Angiotensin II causes a reduction in intermittent blood flow (Table V); during angiotensin II infusion, vessels exhibiting mismatch comprised only 2.0 ± 1.3% of total tumour vessels, a value not significantly different than the background levels obtained when both vascular markers are injected  simultaneously  (1.3±0.5%).  The  reduction in vessel nonperfusion induced by  angiotensin II occurred in both peripheral and central tumour regions. Restraint of the mice and infusion of saline also resulted in a reduction in staining mismatch compared to unrestrained, noninfused animals.  This decrease to 3.6 ± 1 . 8 % remained however, significantly different than  background levels (p< 0.001).  5.2.3  NICOTINAMIDE  Nicotinamide has been shown to reduce tumour hypoxia, apparently as a consequence of increased tumour blood flow (Horsman et al., 1988; 1989a; 1989b). To study the influence of this drug on tumour blood flow intermittency,  nicotinamide was administered (1000 mg/kg  i.p.)  60 minutes prior to double staining of tumour vasculature with H33342 and DiOC (3) (using a 7  20 minute interval between stain injections). Apart from brief periods of handling for injections, animals were allowed to move freely on a warm heating pad. In mice pretreated with PBS injected i.p. (10/yl/g) staining mismatch in subcutaneous SCCVII tumours was 1 0 . 3 ± 2 . 9 % (n = 5; tumour weight 6 5 0 ± 4 0 mg).  Nicotinamide pretreatment significantly reduced the amount of staining  mismatch to 2 . 0 ± 0 . 7 % (n = 5; tumour weight 7 0 0 ± 1 3 0 mg) (p<0.001) (Figure 38), a level not significantly different  from control values when both fluorescent stains were administered  simultaneously (1.3 ± 0 . 5 % ) .  Pyrazinamide,  the  pyrazine  analogue of nicotinamide, also  decreased staining mismatch when given 45 minutes prior to double staining (500 mg/kg i.p.). The mismatch level following pyrazinamide was 3.3 ± 1 . 5 % (n = 8); this value remains higher than that observed in control (interval = 0) tumours (p< 0.001).  Nicotinamide might conceivably reduce transient vessel nonperfusion in the SCCVII tumour by interfering with the double staining method, either by altering the circulation half-life of H33342 or  140  \  Figure 3 8 : Effect of nicotinamide (1000 mg/kg i.p., n = 5) or pyrazinamide (500 mg/kg i.p., n = 8) on staining mismatch (indicative of transient vessel nonperfusion) in SCCVII carcinoma. Values for tumours in mice pretreated with PBS (10/jl/g i.p.) prior to double staining are also shown (n = 5) as are the background staining mismatch levels when the stains are administered simultaneously (n = 11). ( • ) overall mismatch, (P]) peripheral tumour regions (<500 /jm from surface), (fs^) centraTtumour regions. Error oars represent SD. Mismatch levels following nicotinamide pretreatment are not significantly different than background values.  141  by prolonging the normally short-lived vasoactive effects of the stain. H33342 pharmacokinetics were repeated after pretreatment with nicotinamide (1000 mg/kg i.p) one hour before H33342 injection. The distribution t1/2 of H33342 was not affected by prior nicotinamide administration (no pretreatment t1/2 = 142 + 27 seconds; nicotinamide pretreatment t1/2 = 158 + 13 seconds) (Figure 39).  Nicotinamide also did not prolong the transient reduction in tumour blood flow  induced by H33342 (Figure 40).  Thus, pretreatment with nicotinamide does not appear to  preclude the use of the fluorescent double staining method to detect changes in tumour microvascular flow.  5.3 DISCUSSION This section describes the effects of two vasoactive drugs, hydralazine and angiotensin II, on tumour blood flow, with emphasis on red blood cell flow at the microregional level and on fluorescent stain delivery to individual tumour vessels. In addition, the influence of nicotinamide (a drug without known vasoactive properties) on intermittent blood flow is examined.  In the  discussion which follows, the results obtained will be evaluated within the context of factors which influence tumour microcirculatory function and temporal flow heterogeneity. Originally, the drugs were chosen for study in order to assess their possible relevance to cancer therapy, that is, as modulators of tumour blood flow, but clearly, at least in the cases of hydralazine and angiotensin II, the physiological effects of the drugs provide a means to investigate factors which influence microvascular flow in solid experimental tumours.  5.3.1 HYDRALAZINE  The critical importance of perfusion pressure in determining tumour blood flow has been appreciated since the work of Algire and Legallais (1951) in which histamine-induced hypotension was shown to cause nonperfusion of vessels in tumours grown in transparent chambers.  "The  reduction in tumor circulation was directly correlated with the duration and degree of the  142  Figure 39: Effect of nicotinamide (1000 mg/kg i.p. 60 minutes prior to H33342 injection) on H33342 pharmacokinetics in male C3H mice. ( O ) no pretreatment; (#) nicotinamide pretreatment. Means + SD for 5 mice are shown.  143  140 • RBC  FLOW T  4  '•••9  100  I H  or  60  20  in D  < > -J <  tl z  NUMBER OF MOVING RBCs  140  tooogeSSieiifi^  100 •  60  y  LL  O  2  0  140 100  RBC  VELOCITY  ,  0  60 ' 20 0 TIME  5  10  15  (minutes)  Figure 40: Effect of nicotinamide pretreatment (1000 mg/kg i.p.) on H33342-induced reductions in tumour RBC flow, number of moving RBCs, and mean RBC velocity as measured by laser Doppler flowmetry. H33342 (15 mg/kg i.v.) was administered 60 minutes after nicotinamide. All measurements were made in SCCVII foot tumours. Mice were restrained but not anaesthetized. (O) H33342 alone; (#) H33342 following nicotinamide pretreatment. Error bars represent SEM (n = 5).  144  peripheral hypotension" (Algire and Legallais, 1951). In isolated, perfused experimental tumour preparations, blood flow is linearly related to perfusion pressure (Vaupel, 1975; Sevick and Jain, 1989a); flow autoregulation is apparently absent. An increased non-zero pressure intercept of the pressure-flow relationship in a rat mammary tumour perfused ex vivo (Sevick and Jain, 1989a) supports the concept of an elevated "critical closing pressure" (Nichol et al., 1951; Burton, 1951) in tumour microvasculature.  This means that in tumour microvasculature, the intravascular  pressure required to maintain flow is increased relative to normal tissue. Sevick and Jain (1989a) reported a critical closing pressure of 16 mm Hg in the microvasculature of a rat mammary adenocarcinoma. This probably occurs as a consequence of elevated tumour interstitial pressure (reviewed by Jain, 1987; 1988). Thus, reduction of systemic pressure should cause actual vessel collapse and cessation of flow within the microvasculature of solid tumours (Kruuv et al., 1967) especially in regions of low intravascular and high interstitial pressure. This hypothesis was tested in the murine SCCVII tumour using the vasodilator hydralazine.  Hydralazine has been shown to  cause a large decrease in blood pressure in C3H mice; mean arterial blood pressure declined by 30 mm Hg in anaesthetized mice (Okunieff et al., 1988b) and by 60 mm Hg in unanaesthetized, restrained mice (Horsman et al., 1989c).  The data presented provide evidence that a proportion of blood vessels in subcutaneouslyimplanted murine SCCVII carcinoma experience complete cessation of flow following intravenous administration of the vasodilator hydralazine (2.5-10 mg/kg).  The results obtained from laser  Doppler flowmetry studies suggest that the flow reduction observed after hydralazine has two components: a decrease in functional microvascular volume and a decline in velocity of flow. The fluorescent staining technique allows localization of these reductions in functional vasculature within tumour frozen sections. Blood vessels outlined with H33342 and not DiOC (3) (mismatched 7  vessels) suggest the presence of vascular collapse and/or complete flow stasis. response of SCCVII vasculature to hydralazine is highly heterogeneous.  Clearly, the  Not all vessels are  rendered nonfunctional and those that do exhibit lack of fluorescent staining after hydralazine are  145  distributed nonuniformly throughout the tumour mass. Collapse or flow stasis in large patches of vasculature contributes to the staining mismatch observed.  The histological method used does not differentiate between complete flow stasis and actual collapse of the vessel.  In both cases, no fluorescent dye would reach the cells adjacent to the  nonfunctional vessels and staining mismatch would result.  However, careful analysis of tumour  sections reveals many blood vessels in which the transition between bright DiOC (3) stained 7  perivascular cells and more distally-located unstained cells is very abrupt.  This supports the  postulate that vessel collapse, or perhaps lumen occlusion, by WBCs, RBC aggregates, or circulating tumour cells, occurred at this point. Gradual slowing of flow with resultant stasis would likely result in a progressive decline in DiOC (3) staining intensity along the affected vessel. 7  Vessel collapse must involve complete lumen occlusion since plasma flow past a partial obstruction would still allow some fluorescent stain delivery to distal perivascular cells.  The amount of hydralazine-induced vessel nonperfusion appears to be dose-related although it is not correlated with reductions in tumour RBC flow or with changes in mean arterial blood pressure, both of which are dose-independent in the range of 2.5 to 10 mg/kg (Okunieff et al., 1988b). In addition, relative flow reduction is more pronounced in unanaesthetized animals, perhaps because in mice which are immobilized with ketamine and diazepam, systemic blood pressure is, even prior to hydralazine injection, slightly depressed (Menke and Vaupel, 1988).  Many investigators have postulated that tumour blood flow reductions induced by vasodilator drugs are secondary to a "steal phenomenon"; that is, increased flow to surrounding normal tissue "steals" flow from the tumour circulation which is less responsive to the drug (reviewed by Jirtle, 1988). Two assumptions are inherent in this hypothesis: (1) tumour circulation is in parallel with normal tissue circulation, and (2) tumour vessels are less responsive to vasoactive drugs than normal vessels. However, Sevick and Jain (1989a) have clearly demonstrated that at low perfusion  146  pressures tumour geometric resistance increases, presumably as a result of vascular collapse. These authors conclude that "a parallel deployment of normal and tumor vasculature is not a requirement for the reduction of tumor blood flow after hydralazine administration" and that "any agent which lowers the tumor arterial pressure below 20-40 mm Hg increases the geometric resistance to tumor blood flow and results in blood flow reductions and even complete stasis". The presence or absence of vascular smooth muscle in tumour tissue is also of major importance in predicting the impact of vasoactive drugs on tumour blood flow.  A small percentage of  peripheral vessels in SCCVII carcinoma possess contractile elements and probably represent host arterioles incorporated by the advancing tumour mass. The tumour capillaries supplied by these arterioles would be expected to exhibit less flow reduction after hydralazine than would the majority of tumour vasculature. This is a possible explanation for the "patchy", heterogeneous response observed in SCCVII tumours.  Hydralazine-induced dilation of such arterioles could  conceivably result in an intratumour steal phenomenon; increased flow to tumour regions supplied by incorporated host arterioles might "steal" flow from other tumour regions causing patches of nonperfusion.  Hydralazine has less effect on RBC flow in the human tumour xenografts studied and this does not appear to depend on the host mouse or on preimplantation whole body irradiation (which could alter tumour vascularization as a result of the tumour bed effect).  Jirtle et al. (1978b) also  observed that tumours grown in preirradiated or unirradiated tissue showed a similar response to vasoactive drugs. Since xenograft vasculature arises from the murine host (Rofstad, 1985), the differences observed between SCCVII  carcinoma and xenografts presumably arise as a  consequence of a different tumour-host interrelationship. For example, if xenografts incorporate a greater number of functional host arterioles, then hydralazine, via its vasodilator effect, may actually increase overall tumour blood flow.  147  5.3.2 ANGIOTENSIN II  Based on the observations that drug-induced lowering of systemic pressure decreases tumour blood flow and causes vessel collapse, it is expected that blood pressure elevation should have an opposite effect, increasing tumour blood flow and preventing or reversing microvascular collapse. Angiotensin II was chosen as the pressor agent for investigation since this vasoconstrictor has been shown in almost all studies to increase tumour blood flow.  Tumour selectivity of an  angiotensin ll-induced blood flow increase is likely a result of abnormal tumour vascular morphology and function, specifically, a scarcity of intratumour vascular smooth muscle and an inability to autoregulate  tumour  blood flow in response to changing perfusion pressure.  Angiotensin II, because it acts primarily at the level of pre-capillary sphincters (Grega and Adamski, 1987), is unlikely to have any major direct effect on tumour neovasculature, which, at least in the SCCVII tumour model, has few contractile elements.  If angiotensin ll-induced hypertension raises microvascular pressure in the SCCVII carcinoma then an increased functional microvascular volume should be observed. The observation that angiotensin II increases functional microvascular volume by a factor of 1.6 (as measured by laser Doppler flowmetry) supports the results obtained by Hori et al. (1985) using tumours grown in transparent chambers in which perfused vascular area during angiotensin II infusion was 2.1x pretreatment levels.  The slight increase in microvascular volume noted even 10 minutes after  cessation of drug infusion is consistent with the hysteresis-like response of the pressure-flow relationship, an effect described previously by Suzuki et al. (1981). The linearity of the pressureflow curve obtained in SCCVII tumours during angiotensin II infusion is evidence for the lack of autoregulation in this tumour model.  As described in Section 4, intermittent blood flow in the SCCVII tumour may result from spontaneous vasomotion in a few incorporated host arterioles.  Prolonged nonperfusion of  downstream microvessels probably occurs as a consequence of increased tumour interstitial  148  pressure. Elevation of perfusion pressure by angiotensin II could prevent such extended periods of nonperfusion if intravascular pressures now exceed the critical closing pressure. Clearly, the pressor  effect  of angiotensin II  (resulting  in increased tumour  perfusion pressure)  must  predominate over any vasoconstrictor influence on intratumour vascular smooth muscle, otherwise angiotensin II would only exacerbate nonperfusion of capillaries supplied by host arterioles. Furthermore, in normal tissue, elevated intravascular pressure has been found to increase active vasomotion (Johnson and Wayland, 1967); again the lack of intrinsic control mechanisms in tumour vasculature must explain how angiotensin II infusion can result in a reduced heterogeneity in tumour microvasculature flow.  Elevated tumour microvascular pressure could also prevent  vessel plugging by relatively non-deformable circulating cells (Sevick and Jain, 1989b), an alternate mechanism for tumour blood flow intermittency.  One other explanation must be  considered; since H33342 and DiOC (3) were injected during angiotensin II infusion it is possible 7  that  neither  stain  vasoconstriction.  reached  vessels  nonperfused  as  a  result  of  angiotensin ll-induced  Thus, staining mismatch would be reduced, but no real reduction in  microregional flow heterogeneity would have been achieved. As mentioned previously, vessels receiving neither stain cannot be quantified directly but no "patches" of reduced functional vascular density were observed in tumour sections stained during angiotensin II infusion. Future experiments using the Chalkley point counting technique might provide a quantitative approach to this problem.  In the experiments described here, restraint of the mice and infusion of saline also results in a significant reduction in staining mismatch although tumour RBC flow is not increased. Restraintinduced hypertension might account for the reduction in mismatch; this hypothesis predicts an increase in tumour flow, an effect that was not observed during saline infusion. However, the use of anaesthesia in the laser Doppler studies would clearly prevent blood pressure elevation induced by stress/restraint. Alternatively, hemodilution caused by saline infusion (the total infusion volume of 190 fj\ represents approximately 10% of mouse blood volume) could, by lowering intratumour  149  hematocrit, improve tumour blood flow at the microvascular level (Sevick and Jain, 1989b). Finally, restraint of small rodents reduces body temperature, an effect likely to inhibit spontaneous vasomotion (Nicoll and Webb, 1955).  Angiotensin II infusion has also been shown to reduce but not completely eliminate radiobiological acute hypoxia occurring in SCCVII tumours as a result of transient of vessel nonperfusion (Trotter, Chaplin, and Olive, submitted for publication).  In this study, the observed survival level of  perivascular tumour cells is consistent with a residual hypoxic fraction of 2% (0.2% of total tumour cells).  Angiotensin ll-induced vasoconstriction of the rare tumour vessel containing vascular  smooth muscle might explain the existence of this small population of acutely hypoxic cells. Flow of RBC-free plasma through tumour vessels which remain partially collapsed or occluded despite elevated systemic pressure could also account for a lack of staining mismatch but retention of an acutely hypoxic radiation response.  If acutely hypoxic cells influence the response of human tumours to radiation, a reduction in intermittent blood flow by angiotensin II infusion (or by other methods designed to increase tumour perfusion pressure) would have important implications for radiotherapy.  Angiotensin ll-induced  changes in tumour microregional flow are certainly consistent with improvements in tumour visualization (Ekelund and Lunderquist, 1974) and in chemotherapeutic drug delivery (Suzuki et al., 1981; Takematsu et al., 1985; Kuroiwa et al., 1987; Kobayashi et al., 1989).  There is no  obvious reason to assume that angiotensin II is unique among pressor agents in its ability to selectively improve tumour blood flow distribution.  However, the effects of vasoactive drugs on  tumour blood flow are notoriously variable, being highly dependent on tumour type and experimental technique (see reviews by Jain and Ward-Hartley, 1984; Jirtle, 1988). Ideally, the response of experimental tumours to vasoactive drugs should be evaluated using several complimentary  methods for  measuring  blood flow  (preferably  techniques which  monitor  microvascular flow), and should be correlated with vascular structure. Subsequent measurements  150  in human tumours of vascular morphology, host-arteriole incorporation, and perhaps even the amount of functional as opposed to collapsed vasculature, would then allow a prediction of how such tumours might respond to pharmacological manipulation of blood flow.  5.3.3  NICOTINAMIDE  Nicotinamide is another drug which appears to have profound effects on intermittent blood flow and acute hypoxia in the SCCVII carcinoma (Chaplin et al., 1990).  Prior administration of  nicotinamide clearly reduces staining mismatch in this tumour model but the mechanism responsible for this effect is unknown. Nicotinamide does not increase blood pressure in mice; indeed, the drug appears to lower mean arterial pressure (Horsman et al., 1989b). The drug has no influence on H33342 pharmacokinetics or vasoactivity and thus does not appear to interfere with the double staining technique. The effect of nicotinamide on tumour blood flow has been examined previously (Horsman et al., 1988, 1989a; 1989b) and results obtained depend on the measurement technique used. measured by H33342 uptake or clearance.  Nicotinamide causes an increase in tumour blood flow as RbCI extraction but no such effect is observed using  Xe  However, the drug does improve tumour oxygenation as evidenced by a decreased 14  tumour cell binding of  C-misonidazole (Horsman et al., 1988); misonidazole binding is  considered a good indicator of tumour hypoxia (Chapman, 1984; Hirst et al., 1985).  From the  double staining data presented here, and from cell-sorting experiments (Chaplin et al., 1990) it can be concluded that nicotinamide improves tumour oxygen delivery by eliminating transient vessel nonperfusion and acute hypoxia, that is, the drug reduces microregional flow heterogeneity.  Nicotinamide has no known vasoactive effects (Needleman et al., 1985).  Several alternative  mechanisms, all speculative, can be suggested for the reduction in flow heterogeneity induced by nicotinamide.  Nicotinamide, at the high doses necessary to improve tumour oxygenation and  produce increased radiosensitivity, has been shown to reduce animal temperature (Horsman et al., 1989a).  Tumour cooling might, for example, inhibit spontaneous vasomotion in vessels  151  supplying tumour tissue; the frequency of active vasomotion in normal tissue is known to vary directly with temperature within the physiological range (Nicoll and Webb, 1955).  However,  nicotinamide has recently been shown to reduce tumour hypoxia even when animal temperature is maintained at 3 7 ° C (Horsman et al., 1989a), and therefore, tumour cooling does not explain the reduction in intermittent flow observed after treatment with this drug. Nicotinamide may also have an influence on tumour metabolic pathways via its contribution to increased levels of nicotinamide adenine dinucleotide (NAD).  Nicotinamide at a dose of 500 mg/kg (used in combination with  adenosine triphosphate) has been reported to reduce mortality in endotoxin-induced shock in rats (Fulton, 1974), a condition in which lethality is due in part to tissue ischemia.  However, no  improvement in survival was observed when nicotinamide, NAD, or nicotinic acid were used in the treatment of hemorrhagic shock (Chaudry et al., 1976). Nevertheless, nicotinamide, via its effects on cell metabolism, could possibly prevent dysfunction of tumour microcirculation caused by, for example, tissue hypoxia. Increased NAD levels in endothelial cells subject to acute hypoxia might reduce or prevent the cell swelling which is considered an important component of the "no-reflow" phenomenon occurring after transient periods of ischemia (Ames et al., 1968; Kloner et al., 1974). In general terms,  nicotinamide might act by improving microvascular perfusion in shock-like or  post-ischemic states, although a recent review on the pharmacologic modification of reperfusion injury makes no mention of nicotinamide or related compounds (Opie, 1989).  This leads to a brief discussion of tumour microcirculation as a paradigm of a low flow or shock state.  During prolonged low flow conditions, the terminal vascular bed becomes incapable of  compensatory adjustments and is therefore essentially passive (Lefer and Williams, 1986). As summarized by Intaglietta and Mirhashemi (1987): an "inert microvascular network,  where  arterioles are maximally dilated by the metabolic demand stimulus provides a low resistance to flow, and consequently low arteriolar-venular pressure gradient, low flow velocity, higher blood viscosity, and non-selective capillary perfusion". The similarities to tumour microcirculation are  152  clear, especially with regard to loss of intrinsic control mechanisms and increased flow heterogeneity.  Tumour microcirculation behaves like its counterpart in a shock state, not due to  low systemic pressure but rather as a consequence of dysfunctional vasculature, vasculature incapable, due to structural abnormalities, of carrying out the normal functions of microcirculation, namely flow adjustments as tissue metabolic requirements change and local autoregulatory adjustments that stabilize flow and intravascular pressure. An approach to the study of tumour vascular physiology based on the concept of "tumour as a shock state" could potentially reveal further similarities between the two conditions and thus suggest new approaches to cancer treatment.  153  6. SUMMARY AND CONCLUSIONS  This thesis describes several areas of experimental work: (1) the development of a histological technique useful for quantitation of microvascular blood flow changes in solid experimental tumours; (2) the characterization of intermittent blood flow in the murine SCCVII carcinoma and the prediction of the influence of transient vessel nonperfusion on tumour cell oxygenation; and (3) the study of the effects of several vasoactive drugs on tumour blood flow in an attempt to elucidate mechanisms involved in the control of tumour microcirculation.  Use of the double fluorescent staining technique is a feasible approach to the study of microvascular flow in solid experimental tumours growing in three dimensions. Spontaneous or drug-induced flow fluctuations can be visualized at the level of individual microvessels. Clearly, the "ideal" vascular markers have not been found; vasoactivity of the stains is an undesirable limitation and neither marker is suitable for use in human patients.  Nevertheless, in the murine  tumour model described, the double staining technique gives quantitative information available using no other method.  In the SCCVII carcinoma, 5-10% of tumour vessels are subject to periods of nonperfusion lasting at least 5 minutes.  The mechanism(s) responsible for these flow fluctuations are unclear.  postulate that arteriolar  vasomotion in a few  intratumour  arterioles  The  results in decreased  downstream perfusion pressure and vessel collapse or flow stasis as a consequence of elevated tumour interstitial pressure is supported by two main observations: increased staining mismatch in central regions of large tumours and patches of vessel nonperfusion. Nevertheless, alternative mechanisms such as vessel plugging by RBC rouleaux, WBCs, or circulating tumour cells, fluctuations in tumour interstitial pressure, or endothelial cell contractility/swelling have not been addressed experimentally and could clearly play a role. Indeed, the hypothesis that vasomotion is  154  a cause of tumour flow intermittency is weakened somewhat by the results obtained with angiotensin II and nicotinamide. Both drugs essentially eliminate tumour intermittent blood flow but angiotensin II would be expected to increase vessel tone (and thereby exacerbate flow reductions in central tumour regions) and nicotinamide has no known vasoactive properties. Thus, the mechanisms responsible for intermittent blood flow in tumour vessels, while obviously of great interest, remain unclear.  Regardless of mechanism, transient vessel nonperfusion is predicted to have a significant effect on tumour oxygenation and radiosensitivity. In large SCCVII tumours, intermittent flow is expected to contribute as much as 45% to the total fraction of hypoxic cells within a tumour mass.  It is  therefore essential to explore the possibility that intermittent blood flow might occur in human malignancies and that it may be an important factor limiting tumour cure or local control by radiotherapy or perhaps even chemotherapy.  The importance of perfusion pressure to tumour blood flow has been confirmed in this thesis. Blood pressure reduction by hydralazine has been shown, for the first time, to cause actual collapse or complete flow stasis of microvessels in three-dimensional experimental tumours. Elevation of pressure by angiotensin II causes a linear increase in tumour blood flow and prevents transient episodes of vessel nonperfusion.  The results support the concept that tumour  vasculature is both structurally and functionally immature and that tumours lack intrinsic mechanisms to control blood flow.  These abnormalities of tumour vascular physiology deserve  continued research attention since they clearly present aspects of malignancy which can be exploited therapeutically.  155  Intermittent blood flow was postulated to occur in tumours over ten years ago, but was not confirmed to exist in solid experimental malignancies until the late 1980's.  In this thesis, tumour  flow fluctuations have been characterized in the murine SCCVII carcinoma-the quantitative results obtained suggest that abnormalities in tumour vascular physiology can contribute to periods of vessel nonperfusion which significantly compromise oxygen delivery.  Continued work should  focus on three main areas: (1) precise determination of the duration of vessel nonperfusion, (2) mechanisms responsible for flow intermittency, and (3) generality of the phenomenon, i.e. does intermittent flow occur in human tumours? 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