PROJECT ABSTRACT Radiation therapy is a core treatment modality that benefits patients with many types of cancer, and recent studies show that RT can enhance the efficacy of immune checkpoint blocking antibodies by inducing immunogenic tumor cell death and “in situ vaccination”. We recently discovered that higher doses of RT given over a smaller number of fractions ("accelerated fractionated RT") can cure single tumors in mouse models and that such cures rely on enhanced anti-tumor immune responses. Although the dose of RT was limited by damage to surrounding tissues, these findings raised the prospect of more robust responses and even cures of metastatic disease provided that we can understand how to optimize RT for maximum synergy with immunotherapy and minimize collateral damage. Based on this premise, we began developing a next-generation clinical radiation therapy platform that can deliver ultra-rapid radiation (FLASH) and complete treatment in less than a second for extremely precise RT, addressing the challenge of hitting moving targets like tumors and enabling safe delivery of higher RT doses. We have already developed a unique preclinical FLASH irradiator for mice and demonstrated enhanced tumor control and increased immune cell infiltration with FLASH vs. conventional dose rate irradiation (i.e., sub-second vs. 5-minute delivery of the same radiation doses) in a syngeneic subcutaneous tumor model. Prior studies in our lab and others also demonstrated dramatically decreased normal organ injury with FLASH in multiple systems, all of which have an inflammatory basis, including lung (fibrosis), brain (cognition and neuroinflammation) and GI tract (intestinal crypt ablation and GI syndrome). This R01 diversity supplement will support a new project, to be conducted by Dr. Soto, that is related to Aims 1 and 3 of the parent R01. One aim will examine the curative potential of FLASH radiation by identifying a radiation dose at which local tumor cure is achieved in subcutaneous tumor mouse models. This will build on Aim 1 of the parent R01 by further studying the therapeutic effect of FLASH radiation, not in the context of tumor growth delay, but rather in the context of tumor cure. The second aim will investigate the levels of activated TGF after FLASH radiation and compare them to levels of activated TGF following CONV radiation. Activated TGF has been shown to support tumor survival by enhancing DNA repair, suppressing the immune response, and promoting a growth-favorable tumor microenvironment (6). This will build on Aim 3 of the parent R01 by helping to understand a potential cellular mechanism for the efficacy of FLASH radiation.