IDENTIFICATION OF HYPOXIA IN CELLS AND TISSUES OF EPIGASTRIC 9L RAT GLIOMA USING EF5 -1H-IMIDAZOL-1-YL)-N-(2,2,3,3,3-PENTAFLUOROPROPYL) ACETAMIDE]

One of the most sensitive hypoxia detection methods is based on the ob servation that binding of nitroimidazoles to cellular macromolecules o ccurs as a result of hypoxia-dependent bioreduction by cellular nitror eductases. Nitroimidazole-binding techniques provide measurements of h ypoxia to virtually any degree of spatial resolution and with a multip licity of techniques. This paper demonstrates hypoxia imaging using in vivo EF5 binding with detection by a fluorochrome-conjugated monoclon al antibody. We investigated these techniques in the 9L glioma tumour, in part because the exact nature of the hypoxia in this tumour system is controversial. Our results demonstrate that following intravenous injection of EF5, binding and detection using a monoclonal antibody in 9L gliomas is specific and oxygen dependent. Detection of binding usi ng fluorescence microscopy can be performed on frozen tissues; tissue sections can be counterstained with haematoxylin and eosin for light m icroscopic analysis. Alternatively, the distribution of hypoxia in a t umour can be inferred by examining individual tumour cells using flow cytometric techniques. Based upon the results presented herein, the ra diation-resistant phenotype of 9L epigastric tumours grown in our labo ratories can be associated with the presence of hypoxic cells.