MAPPING OF THE VASCULAR ENDOTHELIAL GROWTH FACTOR-PRODUCING HYPOXIC CELLS IN MULTICELLULAR TUMOR SPHEROIDS USING A HYPOXIA-SPECIFIC MARKER

We have investigated the hypoxia inducibility of vascular endothelial growth factor (VEGF) in multicellular tumor spheroids of HT29 cells us ing a monoclonal antibody to a fluorinated bioreductive drug, EF5 l-1- yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide], a chemical probe for hy poxia. We have shown that VEGF expression is predominantly localized i n interior spheroid cells that are sufficiently hypoxic to bioreductiv ely activate the 2-nitroimidazole and produce immunologically detectab le adducts of the EF5 compound, Northern blotting analyses demonstrate d that VEGF(165) is the predominant form of VEGF produced by HT29 cell s and that the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate did not induce VEGF expression, This study demonstrates that VEGF express ion is up-regulated in response to hypoxia and in the microenvironment s found in human multicellular tumor spheroids, This investigation als o illustrates the utility of the EF5 binding in multicellular tumor sp heroids as a means of studying the expression and regulation of hypoxi a-inducible genes.