Quantitation of hypoxia in multicellular spheroids by video image analysis.

Polyclonal antibodies to a series of radiation chemically produced 2-nitroimidazole-protein adducts have been used in the indirect immunofluorescent detection of hypoxic cells in EMT6/Ed spheroids. The spheroids were incubated with the radioactive 2-nitrioimidazoles under atmospheres of air, several intermediate oxygen concentrations, and nitrogen. The fluorescence intensity of the marker across radii of spheroids labeled at various oxygen tensions, as measured by video image analysis, paralleled the autoradiographic grain density. Thus, we conclude that the immunohistochemical approach provides a quantitative measure of the distribution of 2-nitroimidazole adducts in spheroids. The advantages of speed, technical simplicity, economy, and independence from the requirement of radiolabeled precursor render the fluorescent marker of potential use in the clinical detection of cellular hypoxia and tissue ischemia.

Dynamic heterogeneity: metastatic variants to liver are generated spontaneously in mouse embryonal carcinoma cells.

Mouse embryonal carcinoma (EC) cells derived from F9 cells form predominantly liver tumors following the intravenous injection (i.e. experimental metastasis assay) of EC cells into syngeneic 129/J male mice. In this study, EC cells (OTF9) expressing stage-specific embryonic antigen-1 (SSEA-1) are compared with cells (SOTF9) lacking SSEA-1 antigen in the experimental liver metastasis assay. When parallel clones of EC cells were grown to a measured cell number and tested in the experimental metastasis assay, it was observed that the frequency of experimental liver metastases increases with the population size. When the clonal population size is less than the critical number of cells (approximately 2 x 10(5) cells), the frequency of liver tumors is reduced relative to that of the parent EC population. The metastatic ability of clones derived from individual liver metastases did not differ from that of the parental cells. An analysis of the recessive biochemical and immunochemical markers of parental cells and of independent liver metastases suggests that somatic hybridization to host cells by the EC cells is not involved. These results are consistent with predictions from our dynamic heterogeneity model that was formulated by examining the experimental lung metastasis of KHT fibrosarcoma and B16 melanoma cells. Mathematical analysis of the results indicates that the effective rate of generation of the liver metastasizing variant cells is (7 +/- 3) x 10(-6) per cell per generation for both OTF9 and SOTF9 cells.