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55 Relative stability of the tumor micromilieu during a fractionated radiotherapy in fadu human squamous cell carcinoma
S14
Monday, 11 June 2001
Symposia/Proffered Papers/Posters TUMOUR MICROENVIRONMENT
54
52
B. Wouters R.U. Limburg, Department of Radiotherapy, Maastricht, The Netherlands
invited
The tumour microenvironment and its importance for radiation therapy P. Vauoel Institute of Physiology & Pathophysiology, University of Mainz, Germany Tumor angiogenesis exhibits numerous abnormalities. Therefore newly formed microvessels in most solid tumors do not conform to the regular morphology and function of the normal tissue vasculature. These irregular, ities obligatorily lead to disturbances in the microcirculation and can induce severe rheological problems which in turn greatly participate in the development of a hostile metabolic microenvironment. This includes a poor and heterogeneous tissue oxygenation, nutrient supply, drainage of waste products and bioenergetic status. In addition, transmembraneous pH gradients develop which are the reverse of normal cells. The tumor cell microenvironment may often be a critical determinant of treatment response because it can play a pivotal role in acquired resistance to radiation therapy (and chemotherapy as well). Major mechanisms involved are as follows: 1. direct effects through decreased generation of free radicals and thereby less DNA damage, and/or "fixation" of DNA damage by molecular oxygen 2. indirect effects via genome changes and clonal selection 3. indirect effects via proteome changes 4. secondary indirect effects through an intensified glycolysis with extracellular acidosis 53
invited
Multiple parameter imaging of the tumour microenvironment A.J. van der Kooe/. J. Bussink, J.P.W. Peters, P. Rijken, A.S.E. Ljungkvist, J.H.A.M. Kaanders Department of Radiotherapy, University Medica/ Center Nijmegen, The Netherlands It is increasingly recognized that the response of tumors to radiation is not only determined by intrinsic sensitivity of cells, but also is greatly influenced by the microenvironment, consisting of stromal tissue, the vascular network and associated gradients of oxygen and nutrients. To characterize the various elements of the microenvironment and to analyse their interrelationships, we have developed a quantitative image analysis system. Tumor cell proliferation and hypoxia is detected by immunohistochemical methods, and mapped in relation to the vascular structures. This allows quantitative relationships to be measured in the context of tissue structure. Guided by e.g., gene expression profiles for hypoxia induced-genes, several molecular markers of tumor hypoxia were recently recognized and are immunohistochemically detectable with monoclonal antibodies. Most promising at present are the glucose transporters glut-1 and glut-3, as well as carbonic anhydrase IX. These genes are regulated via the von HippelLindau and HIF-1 pathway, and we and others have shown excellent coIocalizatioin with pimonidazole-stained hypoxic regions in clinical biopsy material and in human tumor xenografts. Other genes upregulated under hypoxic conditions are associated with proliferation, apoptosis, and anglogenesis. The extent and distribution of hypoxia can also be assessed by administering nitroimidazole-based markers such as pimonidazole, that can be detected immunohistochemically. The use of multiple hypoxia markers (CCI103F, NITP) has allowed us to study the elects of modifiers of pertusion or oxygenation on the distribution of hypoxic cells in the same tumor. Exampies of the use of these methods are the study of reoxygenation after radiotherapy or the investigation of the lifespan and dynamics of hypoxic cell populations over time. It is expected that the combination of well-time administration of external nitroimidazole-based markers of hypoxia with the detection of intrinsic molecular markers will yield a comprehensive view of the dynamics of hypoxic cells in tumors in relation e.g. their proliferation characteristics. We have started to apply this methodology in patients with squamous carcinomas of the head and neck area, with the ultimate goal to obtain a microenvironmental profile to guide in treatment selection.
invited
Exploiting the tumour microenvironment There is overwhelming evidence that solid h~man tumours grow within a unique microenvironment. This environment is characterized by an abnormal, vasculature, which leads to an insuff~cier~t supp~ of oxygen and nutrients to the turnout cells. These characteristics of the environment limit the effectiveness of both radiotherapy and chemotherapy. Measurement of the oxygenation status of human tumours has unequivocally demonstrated the importance of this parameter on patient prognosis. Tumour hypoxJa has been. shown to be an independent prognostic indicator of poor outcome in prostate, head and neck, and cervix cancers. Recent laboratory and clinical data have shown that hypoxia is also associated with a more malignant phenotype, affecting genomic stability, apoptosis, angi,ogenesis and metastasis~ Several years ago scientists realized that the unique properties within the tumour microenvironment could provide the basis for tumour speci,fic therapies. Efforts that are underway to develop therapies that exploit the tu,mour microenvironment can be categorized into 3 groups. The first includes agents that exploit the environmental changes that occur within the microenvironment such as hypoxia and reduced pH. This includes bioreductive drugs that are specifically toxic to hypoxic cells, as well as hypoxiaspecific gene defivery systems. The second category includes therapies designed to exploit the unique properties of the tumour vasculature and include both angiogenesis inhibitors and vascular targeting agents. The final category includes agents that exploit the molecular and cellular responses to hypoxia. For example, many genes are induced by hypoxia and promotor elements from these genes can be used for the selective expression of therapeutic proteins in hypoxic tumour cells. An overview of the properties unique to the microenvironment of tumours and the attempts of scientists to exploit these unique properties will be discussed. 55
Relative stability of the tumor micromilieu during a fractionated radiotherapy in fadu human squamous cell carcinoma xenografts W. Ei~heler 1, D. Huebner 1, A. Jiresova 1, D. Zips 1, A.J. van der Kogel3, J.A. Raleigh 4, M. Baumann 1,2 1Medical Faculty C.G. Carus, Radiation Oncology, Dresden, Germany 2Medical Faculty C.G. Carus, Experimental Center, Dresden, Germany 3University of Nijmegen, Institute of Radiotherapy, Nijmegen, The Netherlands 4UNC School of Medicine, Radiation Oncology and Toxicology, Chapel Hill, NC, USA The tumor microenvironment has a major impact on the radiosensitivity and the growth characteristics of human tumors and is therefore a target for therapeutic intervention. We analyzed the vascularization, perfusion, and the extent of cellular hypoxia in the human squamous cell carcinoma FaDu, xenografted in nude mice, during a fractionated radiotherapy up to a total dose of 60 Gy within 6 weeks. The vascular network as visualized using the 9F1 antibody, the distribution of the perfusion marker HOECHST, and the accumulation of the hypoxic marker pimonidazole was measured by image analysis using a multiparametric assay on immunofluorescence scans of total tumors sections. A significant decrease of the vascular space was accompanied by a decrease of vital tumor cells. No significant differences in the vascular density, perfused area, cellular hypoxia, or the perfused fraction was observed within the vital tumor areas, suggesting a relative constant microenvironment during radiation therapy. The perfused fraction did not change, indicating functional blood vessels during the observation time. We conclude that the adaptation of the tumor to the vascular network results in a relative constant microenvironment of the vital tumor cells during radiation therapy. Supported by the Deutsche Forschungsgemeinschaft (Grant BA1433/2).
Monday, 11 June 2001
Symposia/Proffered Papers/Posters TUMOUR MICROENVIRONMENT
54
52
B. Wouters R.U. Limburg, Department of Radiotherapy, Maastricht, The Netherlands
invited
The tumour microenvironment and its importance for radiation therapy P. Vauoel Institute of Physiology & Pathophysiology, University of Mainz, Germany Tumor angiogenesis exhibits numerous abnormalities. Therefore newly formed microvessels in most solid tumors do not conform to the regular morphology and function of the normal tissue vasculature. These irregular, ities obligatorily lead to disturbances in the microcirculation and can induce severe rheological problems which in turn greatly participate in the development of a hostile metabolic microenvironment. This includes a poor and heterogeneous tissue oxygenation, nutrient supply, drainage of waste products and bioenergetic status. In addition, transmembraneous pH gradients develop which are the reverse of normal cells. The tumor cell microenvironment may often be a critical determinant of treatment response because it can play a pivotal role in acquired resistance to radiation therapy (and chemotherapy as well). Major mechanisms involved are as follows: 1. direct effects through decreased generation of free radicals and thereby less DNA damage, and/or "fixation" of DNA damage by molecular oxygen 2. indirect effects via genome changes and clonal selection 3. indirect effects via proteome changes 4. secondary indirect effects through an intensified glycolysis with extracellular acidosis 53
invited
Multiple parameter imaging of the tumour microenvironment A.J. van der Kooe/. J. Bussink, J.P.W. Peters, P. Rijken, A.S.E. Ljungkvist, J.H.A.M. Kaanders Department of Radiotherapy, University Medica/ Center Nijmegen, The Netherlands It is increasingly recognized that the response of tumors to radiation is not only determined by intrinsic sensitivity of cells, but also is greatly influenced by the microenvironment, consisting of stromal tissue, the vascular network and associated gradients of oxygen and nutrients. To characterize the various elements of the microenvironment and to analyse their interrelationships, we have developed a quantitative image analysis system. Tumor cell proliferation and hypoxia is detected by immunohistochemical methods, and mapped in relation to the vascular structures. This allows quantitative relationships to be measured in the context of tissue structure. Guided by e.g., gene expression profiles for hypoxia induced-genes, several molecular markers of tumor hypoxia were recently recognized and are immunohistochemically detectable with monoclonal antibodies. Most promising at present are the glucose transporters glut-1 and glut-3, as well as carbonic anhydrase IX. These genes are regulated via the von HippelLindau and HIF-1 pathway, and we and others have shown excellent coIocalizatioin with pimonidazole-stained hypoxic regions in clinical biopsy material and in human tumor xenografts. Other genes upregulated under hypoxic conditions are associated with proliferation, apoptosis, and anglogenesis. The extent and distribution of hypoxia can also be assessed by administering nitroimidazole-based markers such as pimonidazole, that can be detected immunohistochemically. The use of multiple hypoxia markers (CCI103F, NITP) has allowed us to study the elects of modifiers of pertusion or oxygenation on the distribution of hypoxic cells in the same tumor. Exampies of the use of these methods are the study of reoxygenation after radiotherapy or the investigation of the lifespan and dynamics of hypoxic cell populations over time. It is expected that the combination of well-time administration of external nitroimidazole-based markers of hypoxia with the detection of intrinsic molecular markers will yield a comprehensive view of the dynamics of hypoxic cells in tumors in relation e.g. their proliferation characteristics. We have started to apply this methodology in patients with squamous carcinomas of the head and neck area, with the ultimate goal to obtain a microenvironmental profile to guide in treatment selection.
invited
Exploiting the tumour microenvironment There is overwhelming evidence that solid h~man tumours grow within a unique microenvironment. This environment is characterized by an abnormal, vasculature, which leads to an insuff~cier~t supp~ of oxygen and nutrients to the turnout cells. These characteristics of the environment limit the effectiveness of both radiotherapy and chemotherapy. Measurement of the oxygenation status of human tumours has unequivocally demonstrated the importance of this parameter on patient prognosis. Tumour hypoxJa has been. shown to be an independent prognostic indicator of poor outcome in prostate, head and neck, and cervix cancers. Recent laboratory and clinical data have shown that hypoxia is also associated with a more malignant phenotype, affecting genomic stability, apoptosis, angi,ogenesis and metastasis~ Several years ago scientists realized that the unique properties within the tumour microenvironment could provide the basis for tumour speci,fic therapies. Efforts that are underway to develop therapies that exploit the tu,mour microenvironment can be categorized into 3 groups. The first includes agents that exploit the environmental changes that occur within the microenvironment such as hypoxia and reduced pH. This includes bioreductive drugs that are specifically toxic to hypoxic cells, as well as hypoxiaspecific gene defivery systems. The second category includes therapies designed to exploit the unique properties of the tumour vasculature and include both angiogenesis inhibitors and vascular targeting agents. The final category includes agents that exploit the molecular and cellular responses to hypoxia. For example, many genes are induced by hypoxia and promotor elements from these genes can be used for the selective expression of therapeutic proteins in hypoxic tumour cells. An overview of the properties unique to the microenvironment of tumours and the attempts of scientists to exploit these unique properties will be discussed. 55
Relative stability of the tumor micromilieu during a fractionated radiotherapy in fadu human squamous cell carcinoma xenografts W. Ei~heler 1, D. Huebner 1, A. Jiresova 1, D. Zips 1, A.J. van der Kogel3, J.A. Raleigh 4, M. Baumann 1,2 1Medical Faculty C.G. Carus, Radiation Oncology, Dresden, Germany 2Medical Faculty C.G. Carus, Experimental Center, Dresden, Germany 3University of Nijmegen, Institute of Radiotherapy, Nijmegen, The Netherlands 4UNC School of Medicine, Radiation Oncology and Toxicology, Chapel Hill, NC, USA The tumor microenvironment has a major impact on the radiosensitivity and the growth characteristics of human tumors and is therefore a target for therapeutic intervention. We analyzed the vascularization, perfusion, and the extent of cellular hypoxia in the human squamous cell carcinoma FaDu, xenografted in nude mice, during a fractionated radiotherapy up to a total dose of 60 Gy within 6 weeks. The vascular network as visualized using the 9F1 antibody, the distribution of the perfusion marker HOECHST, and the accumulation of the hypoxic marker pimonidazole was measured by image analysis using a multiparametric assay on immunofluorescence scans of total tumors sections. A significant decrease of the vascular space was accompanied by a decrease of vital tumor cells. No significant differences in the vascular density, perfused area, cellular hypoxia, or the perfused fraction was observed within the vital tumor areas, suggesting a relative constant microenvironment during radiation therapy. The perfused fraction did not change, indicating functional blood vessels during the observation time. We conclude that the adaptation of the tumor to the vascular network results in a relative constant microenvironment of the vital tumor cells during radiation therapy. Supported by the Deutsche Forschungsgemeinschaft (Grant BA1433/2).