0360-3016/92 $5.00 + .oO Copyright 0 1992 Pergamon Press plc
hr. J. Radianon Oncology Bid. Phys., Vol. 22, pp. 393-395 Pnnted I” the U.S A All rights reserved.
??Session A: Tissue Oxygenation Manipulation and Tumor Blood Flow
KEYNOTE ADDRESS: TISSUE OXYGEN MANIPULATION AND TUMOR BLOOD FLOW DIETMAR W. SIEMANN, PH.D. Tumor Biology Division and Department of Radiation Oncology, University of Rochester Cancer Center, 601 Elmwood Avenue, Box 704, Rochester, NY 14642, USA The classic view of tumor hypoxia was based on the chronic or diffusion-limited model of hypoxia as described by Thomlinson and Gray in 1953. In this model, large inter-capillary distances, resulting from rapid tumor cell proliferation, lead to hypoxic cells existing at the rim of the oxygen diffusion distance. In the 1970’s it was suggested by Brown as well as Sutherland and Frank0 that another type of hypoxia, namely acute or perfusion-limited hypoxia, might also exist in tumors. Such perfusion-limited hypoxic cells could arise as a consequence of a vascular collapse which renders areas in tumors, that were wellperfused only moments before, suddenly hypoxic. Using a dual fluorescent stain technique coupled with histological evaluations, Trotter and colleagues recently were able to provide eloquent experimental evidence for the existence of perfusion-limited hypoxic cells in SCCVII rodent tumors. In view of these findings it is currently commonly believed that both types of hypoxia may exist in tumors (Fig. 1) and that different therapeutic strategies may be required to effectively target these two populations. Our understanding of tumor hypoxia has increased significantly during the past few years. Novel developments and improvements in laboratory techniques have provided new and unique insights into tumor microenvironments. Techniques presently available to study tumor hypoxia include: (a) the use of nitroheterocyclics in labelling and imaging studies, (b) the development of fluorescent staining and sorting procedures to identify diffusion-limited and transient hypoxic cell populations, (c) the application of MRS and MRI techniques, (d) the initiation of ELISA assays of hypoxia marker binding, and (e) the improvement of invasive probes. A number of these approaches have been or will soon be applied to humans. Currently, two approaches in particular appear to hold great promise for the identification of hypoxic regions within tumors in patients: (a) the use of fluoromisonidazole, when labeled with the positron emitter fluorine-18, to image with PET and (b) the development of gamma-labeled nitroheterocyclics. Although the current model for hypoxia in tumors (Fig. 1) is attractive, it must be recognized that to date only a few rodent tumor models have been evaluated extensively
As a tumor grows, the vascular development often cannot keep pace with the rapid and uncontrolled proliferation of the malignant cell population. As a consequence, solid tumor masses typically exhibit abnormal blood vessel networks which, unlike vessels in normal tissues, fail to provide adequate and homogeneous nutritional support to the tumor cells. Solid tumors, therefore, can be comprised of cell subpopulations existing under a variety of microenvironmental conditions. Indeed, acidic regions and areas of low oxygen tensions have been well documented in both rodent and human tumors. This environmental heterogeneity within tumors has been implicated as a major contributing factor for failure to cure neoplastic disease by radiation or chemotherapy. As early as 1904 it was recognized that interference with the blood supply could reduce radiation-induced skin reactions. Subsequently it was demonstrated that oxygen-deficient or hypoxic cells were approximately 2-3 times more resistant to radiation than were oxic cells. However, it was not until 1953 that the full magnitude of the potential impact of hypoxia on clinical radiation therapy was realized. In what is now a milestone paper in radiobiology, Thomlinson and Gray utilized tumor histology measurements and oxygen diffusion distance calculations to predict that viable tumor cells might exist in regions of radiobiological hypoxia. Experimental confirmation of this hypothesis came in 1963, when it was demonstrated that hypoxic cells gave rise to the biphasic shape of the in situ tumor cell survival curve. Since these initial observations, it has now been well established that hypoxic cells exist in most rodent tumor models and that these cells lead to refractory radiation responses. Evidence for the presence of hypoxic cells in human tumors is less readily available. However, (a) the clinical gains observed with hyperbaric oxygen, (b) the recognition from both retrospective and prospective trials that anemia represents a poor prognostic factor often associated with local radiation failures, and (c) the recent success with the hypoxic cell sensitizer misonidazole reported in the DAHANCA study, all support the notion that hypoxic cells can affect the outcome of radiation therapy in patients with certain tumor types.
Accepted for publication 9 July 1991.
This work was supported by NIH grant CA 36858. 393
394
I. J. Radiation Oncology 0 Biology 0 Physics
Vessel Open,
hronically Hypoxic
Fig. 1. Model for hypoxia in solid tumors indicating the presence of chronic or diffusion-limited hypoxic cells. This figure was
modified from that of Coleman.
for the manifestation of diffusion- and/or perfusion-limited hypoxia. Little is known about the universality of these findings and to what degree the various types of hypoxia exist in different rodent tumor models. More critically, no definitive evidence exists concerning their presence in human tumors. Thus, despite significant research progress, many questions remain. 0 Do both diffusion- and perfusion-limited hypoxic cells exist in all tumors? 0 If both types are present, will one dominate the therapeutic response and is this therapy dependent? 0 What role does the host play in the evolution of the hypoxia? Several strategies are available for attacking treatment refractory hypoxic cell populations. The two approaches to be discussed in this presentation involve altering oxygen transport or manipulating tumor blood flow. Studies aimed at improving tissue oxygenation through increased oxygen transport have historically focused primarily on either high oxygen content gas inhalation or correcting the detrimental effects of anemia in the tumorbearing hosts. Both techniques have led to (a) reductions in the fraction of hypoxic tumor cells in preclinical investigations and (b) a few prospective clinical trials with blood transfusions or hyperbaric oxygen given prior to or during the course of radiation therapy. While certainly not definitive, positive results in some of these clinical trials support the notion that hypoxic cells may influence radiotherapy outcome. Given the technical difficulties of high oxygen content gas administration as well as the potential problems associated with blood transfusions, other manipulations of the oxygen supply have received considerable attention. For example, artifical blood substitutes, particularly the perfluorochemical emulsions, have been studied extensively for possible cancer treatment application. Unfortunately, recent results from Phase I/II investigations of the lead perfluorochemical, Fluosol-DA, as an adjuvant to radiation therapy have not been overly encouraging.
Volume 22, Number 3, 1992
An alternate approach to improving blood oxygen transport is the shifting of the hemoglobin-oxygen dissociation curve to favor oxygen off-loading at the tissues. A shift to the right, corresponding to a reduction in hemoglobin affinity for oxygen, enhances the oxygen release capacity such that tissue oxygenation is improved. Successful right shifting of the hemoglobin-oxygen dissociation curve has been shown to have therapeutic implications for ischemia and hypoxemia. Pre-clinical oncology investigations have demonstrated that the in viva oxygenation of solid tumors could be improved by reducing the hemoglobin affinity for oxygen through (a) the manipulation of allosteric factors normally controlling the position of the hemoglobin-oxygen dissociation curve (increasing 2,3 diphosphoglycerate levels) and (b) the application of chemicals known to bind the hemoglobin molecule and altering oxygen release (antilipidemics such as clofibrate). Therapeutical approaches such as these not only reduced the hemoglobin affinity for oxygen in vivo but also led to radiosensitization of hypoxic cells in several animal tumor models. The manipulations described above are directed at increasing the quantity of oxygen carried in the blood. Under these circumstances, tissue oxygenation is improved because the higher blood oxygen tensions result in an increase in the oxygen diffusion distance. As such, it would appear that manipulations of this sort exert their influence primarily on the chronic or oxygen-diffusion limited hypoxic cells. Furthermore, since such manipulations have been demonstrated to improve tumor response to therapy, these results also imply that diffusion-limited hypoxia can critically impact therapy outcome. Another approach to the hypoxic cell problem is the use of vasoactive agents directed at improving tissue oxygenation b;, increasing blood flow. However, attempts to improve tumor oxygenation with such compounds have been largely unsuccessful. Indeed, vasodilators like hydralazine often decrease tumor blood flow and increase tumor hypoxia. It now is believed that such agents, rather than directly affecting tumor blood vessels, reduce tumor blood flow by inducing changes in systemic blood pressure and/or resistance of the normal tissue microvasculature. While reductions in tumor blood flow may at first glance appear to be counter indicative to the strategy of applying vasoactive agents, this result may be exploited by applying therapies specifically targeted to hypoxic tumor cells. For example, there is currently considerable interest in reducing tumor blood flow as a means of enhancing the efficacy of bioreductive anticancer agents. This topic is the subject of another session of these proceedings. Classes of vasoactive compounds that can increase tumor blood flow do, however, also exist. Foremost among these are the calcium antagonists, particularly verapamil, flunarizine, and cinnarazine. In several animal models these agents have been shown to be capable of effecting the micro-circulation sufficiently to increase tumor blood flow and enhance the hypoxic cell radiation response. Another interesting agent that has received considerable atten-
Keynote address 0 D. W.
tion in preclinical tumor radiobiology investigations is nicotinamide. This compound has been shown to improve tumor blood flow and to enhance tumor radiation sensitivity. Perhaps most interestingly, Horsman and Chaplin have shown that the ability of nicotinamide to increase the radiation response of tumors is at least in part a consequence of a reduction in the proportion of perfusion-limited or acutely hypoxic cells. In conclusion, our current experience related to the hypoxia problem in cancer therapy suggests that oxygen-deficient cell populations may arise in tumors from both oxygen diffusion limitations and intermittent fluctuations
SIEMANN
395
in blood flow. While increasing the oxygen carrying capacity of the blood would serve to reduce diffusion-limited hypoxic cells, such therapeutic manipulations would have little impact on eliminating hypoxia resulting from transient variations in microregional blood flow. However, strategies that improve blood flow could conceivably impact the latter hypoxic cell type. Obviously, if both perfusion- and diffusion-limited hypoxic cells exist in tumors, one approach might be to combine different therapies such that each is aimed at eliminating a particular hypoxic cell subpopulation. This hypothesis is currently under investigation in a number of laboratories.
hr. J. Radianon Oncology Bid. Phys., Vol. 22, pp. 393-395 Pnnted I” the U.S A All rights reserved.
??Session A: Tissue Oxygenation Manipulation and Tumor Blood Flow
KEYNOTE ADDRESS: TISSUE OXYGEN MANIPULATION AND TUMOR BLOOD FLOW DIETMAR W. SIEMANN, PH.D. Tumor Biology Division and Department of Radiation Oncology, University of Rochester Cancer Center, 601 Elmwood Avenue, Box 704, Rochester, NY 14642, USA The classic view of tumor hypoxia was based on the chronic or diffusion-limited model of hypoxia as described by Thomlinson and Gray in 1953. In this model, large inter-capillary distances, resulting from rapid tumor cell proliferation, lead to hypoxic cells existing at the rim of the oxygen diffusion distance. In the 1970’s it was suggested by Brown as well as Sutherland and Frank0 that another type of hypoxia, namely acute or perfusion-limited hypoxia, might also exist in tumors. Such perfusion-limited hypoxic cells could arise as a consequence of a vascular collapse which renders areas in tumors, that were wellperfused only moments before, suddenly hypoxic. Using a dual fluorescent stain technique coupled with histological evaluations, Trotter and colleagues recently were able to provide eloquent experimental evidence for the existence of perfusion-limited hypoxic cells in SCCVII rodent tumors. In view of these findings it is currently commonly believed that both types of hypoxia may exist in tumors (Fig. 1) and that different therapeutic strategies may be required to effectively target these two populations. Our understanding of tumor hypoxia has increased significantly during the past few years. Novel developments and improvements in laboratory techniques have provided new and unique insights into tumor microenvironments. Techniques presently available to study tumor hypoxia include: (a) the use of nitroheterocyclics in labelling and imaging studies, (b) the development of fluorescent staining and sorting procedures to identify diffusion-limited and transient hypoxic cell populations, (c) the application of MRS and MRI techniques, (d) the initiation of ELISA assays of hypoxia marker binding, and (e) the improvement of invasive probes. A number of these approaches have been or will soon be applied to humans. Currently, two approaches in particular appear to hold great promise for the identification of hypoxic regions within tumors in patients: (a) the use of fluoromisonidazole, when labeled with the positron emitter fluorine-18, to image with PET and (b) the development of gamma-labeled nitroheterocyclics. Although the current model for hypoxia in tumors (Fig. 1) is attractive, it must be recognized that to date only a few rodent tumor models have been evaluated extensively
As a tumor grows, the vascular development often cannot keep pace with the rapid and uncontrolled proliferation of the malignant cell population. As a consequence, solid tumor masses typically exhibit abnormal blood vessel networks which, unlike vessels in normal tissues, fail to provide adequate and homogeneous nutritional support to the tumor cells. Solid tumors, therefore, can be comprised of cell subpopulations existing under a variety of microenvironmental conditions. Indeed, acidic regions and areas of low oxygen tensions have been well documented in both rodent and human tumors. This environmental heterogeneity within tumors has been implicated as a major contributing factor for failure to cure neoplastic disease by radiation or chemotherapy. As early as 1904 it was recognized that interference with the blood supply could reduce radiation-induced skin reactions. Subsequently it was demonstrated that oxygen-deficient or hypoxic cells were approximately 2-3 times more resistant to radiation than were oxic cells. However, it was not until 1953 that the full magnitude of the potential impact of hypoxia on clinical radiation therapy was realized. In what is now a milestone paper in radiobiology, Thomlinson and Gray utilized tumor histology measurements and oxygen diffusion distance calculations to predict that viable tumor cells might exist in regions of radiobiological hypoxia. Experimental confirmation of this hypothesis came in 1963, when it was demonstrated that hypoxic cells gave rise to the biphasic shape of the in situ tumor cell survival curve. Since these initial observations, it has now been well established that hypoxic cells exist in most rodent tumor models and that these cells lead to refractory radiation responses. Evidence for the presence of hypoxic cells in human tumors is less readily available. However, (a) the clinical gains observed with hyperbaric oxygen, (b) the recognition from both retrospective and prospective trials that anemia represents a poor prognostic factor often associated with local radiation failures, and (c) the recent success with the hypoxic cell sensitizer misonidazole reported in the DAHANCA study, all support the notion that hypoxic cells can affect the outcome of radiation therapy in patients with certain tumor types.
Accepted for publication 9 July 1991.
This work was supported by NIH grant CA 36858. 393
394
I. J. Radiation Oncology 0 Biology 0 Physics
Vessel Open,
hronically Hypoxic
Fig. 1. Model for hypoxia in solid tumors indicating the presence of chronic or diffusion-limited hypoxic cells. This figure was
modified from that of Coleman.
for the manifestation of diffusion- and/or perfusion-limited hypoxia. Little is known about the universality of these findings and to what degree the various types of hypoxia exist in different rodent tumor models. More critically, no definitive evidence exists concerning their presence in human tumors. Thus, despite significant research progress, many questions remain. 0 Do both diffusion- and perfusion-limited hypoxic cells exist in all tumors? 0 If both types are present, will one dominate the therapeutic response and is this therapy dependent? 0 What role does the host play in the evolution of the hypoxia? Several strategies are available for attacking treatment refractory hypoxic cell populations. The two approaches to be discussed in this presentation involve altering oxygen transport or manipulating tumor blood flow. Studies aimed at improving tissue oxygenation through increased oxygen transport have historically focused primarily on either high oxygen content gas inhalation or correcting the detrimental effects of anemia in the tumorbearing hosts. Both techniques have led to (a) reductions in the fraction of hypoxic tumor cells in preclinical investigations and (b) a few prospective clinical trials with blood transfusions or hyperbaric oxygen given prior to or during the course of radiation therapy. While certainly not definitive, positive results in some of these clinical trials support the notion that hypoxic cells may influence radiotherapy outcome. Given the technical difficulties of high oxygen content gas administration as well as the potential problems associated with blood transfusions, other manipulations of the oxygen supply have received considerable attention. For example, artifical blood substitutes, particularly the perfluorochemical emulsions, have been studied extensively for possible cancer treatment application. Unfortunately, recent results from Phase I/II investigations of the lead perfluorochemical, Fluosol-DA, as an adjuvant to radiation therapy have not been overly encouraging.
Volume 22, Number 3, 1992
An alternate approach to improving blood oxygen transport is the shifting of the hemoglobin-oxygen dissociation curve to favor oxygen off-loading at the tissues. A shift to the right, corresponding to a reduction in hemoglobin affinity for oxygen, enhances the oxygen release capacity such that tissue oxygenation is improved. Successful right shifting of the hemoglobin-oxygen dissociation curve has been shown to have therapeutic implications for ischemia and hypoxemia. Pre-clinical oncology investigations have demonstrated that the in viva oxygenation of solid tumors could be improved by reducing the hemoglobin affinity for oxygen through (a) the manipulation of allosteric factors normally controlling the position of the hemoglobin-oxygen dissociation curve (increasing 2,3 diphosphoglycerate levels) and (b) the application of chemicals known to bind the hemoglobin molecule and altering oxygen release (antilipidemics such as clofibrate). Therapeutical approaches such as these not only reduced the hemoglobin affinity for oxygen in vivo but also led to radiosensitization of hypoxic cells in several animal tumor models. The manipulations described above are directed at increasing the quantity of oxygen carried in the blood. Under these circumstances, tissue oxygenation is improved because the higher blood oxygen tensions result in an increase in the oxygen diffusion distance. As such, it would appear that manipulations of this sort exert their influence primarily on the chronic or oxygen-diffusion limited hypoxic cells. Furthermore, since such manipulations have been demonstrated to improve tumor response to therapy, these results also imply that diffusion-limited hypoxia can critically impact therapy outcome. Another approach to the hypoxic cell problem is the use of vasoactive agents directed at improving tissue oxygenation b;, increasing blood flow. However, attempts to improve tumor oxygenation with such compounds have been largely unsuccessful. Indeed, vasodilators like hydralazine often decrease tumor blood flow and increase tumor hypoxia. It now is believed that such agents, rather than directly affecting tumor blood vessels, reduce tumor blood flow by inducing changes in systemic blood pressure and/or resistance of the normal tissue microvasculature. While reductions in tumor blood flow may at first glance appear to be counter indicative to the strategy of applying vasoactive agents, this result may be exploited by applying therapies specifically targeted to hypoxic tumor cells. For example, there is currently considerable interest in reducing tumor blood flow as a means of enhancing the efficacy of bioreductive anticancer agents. This topic is the subject of another session of these proceedings. Classes of vasoactive compounds that can increase tumor blood flow do, however, also exist. Foremost among these are the calcium antagonists, particularly verapamil, flunarizine, and cinnarazine. In several animal models these agents have been shown to be capable of effecting the micro-circulation sufficiently to increase tumor blood flow and enhance the hypoxic cell radiation response. Another interesting agent that has received considerable atten-
Keynote address 0 D. W.
tion in preclinical tumor radiobiology investigations is nicotinamide. This compound has been shown to improve tumor blood flow and to enhance tumor radiation sensitivity. Perhaps most interestingly, Horsman and Chaplin have shown that the ability of nicotinamide to increase the radiation response of tumors is at least in part a consequence of a reduction in the proportion of perfusion-limited or acutely hypoxic cells. In conclusion, our current experience related to the hypoxia problem in cancer therapy suggests that oxygen-deficient cell populations may arise in tumors from both oxygen diffusion limitations and intermittent fluctuations
SIEMANN
395
in blood flow. While increasing the oxygen carrying capacity of the blood would serve to reduce diffusion-limited hypoxic cells, such therapeutic manipulations would have little impact on eliminating hypoxia resulting from transient variations in microregional blood flow. However, strategies that improve blood flow could conceivably impact the latter hypoxic cell type. Obviously, if both perfusion- and diffusion-limited hypoxic cells exist in tumors, one approach might be to combine different therapies such that each is aimed at eliminating a particular hypoxic cell subpopulation. This hypothesis is currently under investigation in a number of laboratories.
