THE RELATIONSHIP BETWEEN PROLIFERATIVE AND OXYGENATION STATUS IN SPONTANEOUS CANINE TUMORS

Purpose: Immunocytochemical markers have been applied to biopsy specim ens from spontaneous canine tumors to assess the prevalence and spatia l distribution of proliferating and hypoxic cells, and their ''geograp hic'' relationship to each other. Both types of cells have been implic ated in the failure to locally control human tumors treated with radia tion and chemotherapy. Methods and Materials: For the detection of hyp oxic cells, a rabbit polyclonal antibody raised against a protein-boun d, hexafluorinated, 2-nitroimidazole, designated CCI-103F, was used. T he unmetabolized drug must first be injected into the dog to allow tim e for hypoxic metabolism and cellular binding to occur. For the detect ion of proliferating cells, a mouse monoclonal antibody raised against an endogenous nuclear protein, the ''proliferating cell nuclear antig en,'' or PCNA, was used. This protein is expressed in most actively pr oliferating cells, but not in quiescent ones. An indirect immunostaini ng technique was used to visualize these markers in the tissue section s, and image analysis was used to estimate the area fraction of positi ve staining in representative, low magnification microscope fields. Re sults: Tumors with both high and low hypoxic and proliferative area fr actions have been identified. No systematic relationship between the p revalence of the two markers, nor of the relationship between tumor gr ade and proliferative fraction, could be established. Staining with th e proliferation marker was more commonly found near blood vessels, but some ''nests'' of tumor cells apparently distant from vasculature con tained many proliferating cells. Staining with the hypoxia marker tend ed to be distant from the vasculature and/or bordering regions of tumo r necrosis, but some labeled cells appeared near blood vessels, and in the absence of necrosis. Staining of sequential sections, one with th e proliferation marker and one with the hypoxia marker, indicated that the two cell populations overlapped to varying extents. Some incident al staining of canine normal tissues with both the proliferative and h ypoxia markers was observed as well. Conclusion: The immunochemical ma rker approach promises to be a useful tool to increase both our basic understanding of tumor physiology and the complex nature of tumor hete rogeneity.