Project Summary Radiation therapy delivered at ultra-high dose rates may be becoming a breakthrough treatment option for cancer patients. Targeting cancers with ultra-high radiation dose rates produces a FLASH effect, wherein control of tumor growth is maintained similarly to conventional (CONV) radiation dose rates, but normal tissue toxicity is significantly reduced. Although FLASH irradiation has been shown to evoke strong, reproducible responses across many different organ systems (e.g., brain, lungs, gastrointestinal [GI] tract, skin) across multiple species, some studies have shown that ultra-high-dose rate irradiation to have either no effect or detrimental effects on normal tissue. This discrepancy is not clear; however, it likely stems from inconsistency in the physical radiation beam and fractionation parameters. Furthermore, although previous studies have shown either no change in or improved tumor responses from FLASH irradiation as compared with CONV dose rate irradiation, no studies have looked beyond simple tumor growth delay when evaluating tumor responses. A more relevant analysis for preclinical tumor responses to radiotherapy is the Tumor Control (TCD50) assay, and to date, no comparisons between FLASH and CONV dose rate irradiation on the dose required to cure 50% of tumors (TCD50) have been performed. The lack of comparisons of radiation types, the lack of consistency between physical radiation beam parameters and fractionation, and the lack of accurate measurements of tumor control in previous FLASH irradiation studies provides impetus to conduct this rigorous, high throughput, multi-institutional study to provide confirmatory evidence of the reproducibility of FLASH effects. This proposed project will test the hypothesis that there is an optimal set of physical beam parameters that will maximize the FLASH effect, and that under the same dose parameters and the same physical dose, the FLASH effect dose response will be the same between different radiation types. In order to test the hypothesis, Aim 1 will focus on determining whether radiation type (e.g., electrons, photons, and photons) alters abdominal FLASH-mediated normal tissue-sparing effects, with the expectation of similar responses to the different radiation types. In order to optimize the physical beam and fractionation parameters to maximally reduce normal tissue toxicity, physical beam parameters (e.g., mean dose rate, dose per pulse, pulse duration, overall delivery time, priming dose, and oxygen tension) as well as fractionation will be systematically changed and tested (Aim 2). Aim 3 will focus on establishing the therapeutic effects of FLASH dose rate irradiation mediate similar control of syngeneic, heterotopic tumors of three different cancer cell lines using the more relevant TCD50 assay. The overarching goal of this project is to minimize side effects for all cancer patients receiving radiation therapy, which will inevitably improve quality of life. Preve...