Inducibility of vascular endothelial growth factor (VEGF) in multicellular tumor spheroids of HT29 cells using a monoclonal antibody to a fluorinated bioreductive drug, EFS

We have investigated the hypoxia Inducibility of vascular endothelial growth factor (VEGF) in multicellular tumor spheroids of HT29 cells using a monoclonal antibody to a fluorinated bioreductive drug, EFS [2-(2-nitro-lJI-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamidel, a chemical probe for hypoxia. We have shown that VEGF expression is predominantly localized in interior spheroid cells that are sufficiently hypoxic to bioreductively activate the 2-nitrohnidazole and produce im munologically detectable adducts ofthe EF5 compound. Northern blotting analyses demonstrated that VEGF1@ is the predominant form of VEGF produced by HT29 cells and that the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate did not induce VEGF expression. This study demon st@rates that VEGF expression is up-regulated in response to hypoxia and In the microenvironments found in human multicellular twnor spheroids. This Investigation also Illustrates the utility of the EFS binding in multi cellular tumor spherolds as a means of studying the expression and regulation of hypoxia-Inducible genes.