Description of a spheroid model for the study of radiation and chemotherapy effects on hypoxic tumor cell populations.

The presence of poorly oxygenated cells in solid tumors may account for clinical resistance to ionizing radiation and some chemotherapy in many cancers. Studies of the presence and spatial distribution, sensitivity to cancer therapies, and other physiological characteristics of hypoxic cells are hindered by the lack of markers specific for hypoxia and a relevant yet easily manipulated model system. We have chosen to use multicellular spheroids composed of murine EMT6 (fibrosarcoma) tumor cells as a model system and have applied an immunohistochemical marker specific for hypoxic cells with the ultimate goal of determining how cell populations change in response to radiation and/or chemotherapy. Large spheroids (500-700 microns in diameter) were selected and incubated in the presence of a hexafluorinated 2-nitroimidazole derivative, designated CCI-103F, which undergoes reductive metabolism and irreversibly binds to cellular macromolecules only under low oxygen tensions. A rabbit polyclonal antibody raised against a CCI-103F/protein adduct was used to visualize hypoxic cells using standard streptavidin-biotin-peroxidase immunohistochemical methods. Investigations using this spheroid model system promise to further our understanding of hypoxic cell resistance to cytotoxic therapies and of hypoxic cell biology in general.