The goal of this project is to explore the mechanisms by which Ad.Egr-TNF gene therapy enhances the response of tumors to radiotherapy. We have reported that gene therapy targeted by ionizing radiation (IR) enhances the anti-tumor effects of radiotherapy by activating the expression of a cDNA encoding tumor necrosis factor-alpha (TNF-alpha) under the control of a radiation-inducible promoter (Egr-1). Murine and human tumor models demonstrate activation of Ad.Egr-TNF following single dose or fractionated radiation and achieves therapeutic concentrations of intratumoral TNF-alpha protein. Combined treatment with Ad.Egr-TNF and IR significantly increased tumor regression and cures in these tumor models compared with either treatment alone with little or no increase in normal tissue toxicity. Ad.Egr-TNF is currently being investigated with radiation therapy in three Phase 2 clinical trials following demonstration of safety in two Phase 1 trials. Treatment efficacy in the phase trials was suggested by the fact that >20% patients with radioresistant (melanoma, sarcoma, rectal cancer) or very large tumors (up to 13,500 cm3) achieved complete responses and are without evidence of recurrence to date. Experiments outlined here will employ genetic and pharmacological models to explore the anti-tumor mechanisms of AD.Egr-TNF and IR to provide the experimental basis to rationally improve the efficacy of this new combined modality treatment. To achieve these goals we propose the following specific aims. Specific Aim 1. Explore whether the tumor microvascular endothelium mediates resistance to Ad.Egr-TNF and IR. Specific Aim 2. Explore whether the tumor secretion of vascular endothelial growth factor (VEGF) mediates resistance to Ad.Egr-TNF +IR by enhancing resistance of the tumor endothelium. Specific Aim 3. Explore the role of tumor cell resistance to Ad.Egr-TNF+IR.