PROJECT SUMMARY Modern X-ray–based radiation therapy, delivered at conventional dose rates (~0.1 Gy/s, 2-Gy fractions, once daily) (C-XRT) has led to improved outcomes for patients with head & neck squamous cell carcinoma (HNSCC) and prostate cancer (PC). However, acute and long-term treatment-related side effects in both cancers have prompted the search for more biologically sound novel therapeutic strategies. Conventional dose rate proton radiotherapy (C-PT) may provide better tumor control with less treatment-related toxicity than C- XRT in both HNSCC and PC because of its biological enhancement effects and unique physical properties, but use of PT has been limited by its high cost. Here we aim to test the effects of a novel form of RT delivery, in which electrons, X-rays, or protons are delivered at ultra-high dose rates (≥40 Gy/s). This so-called “FLASH” RT can deliver curative doses to tumors within milliseconds, while simultaneously minimizing damage to surrounding tissues. The apparent protective effect of FLASH on normal tissues may further allow the use of very large fractions, which would both shorten overall treatment time and reduce costs, especially for PT. However, among the many unknowns at this time include (i) how FLASH-PT affects HNSCC and PC tumors and surrounding normal tissues, and (ii) whether FLASH-PT can reduce treatment toxicity while preserving treatment outcomes. We propose to address these important unknowns by testing the effects of FLASH-PT and C-PT in a unique model system, established by us, on tumor response and normal tissue damage in vivo. Our long-term objective is to establish a foundation for the clinical use of FLASH radiation to improve outcomes for patients with HNSCC or PC. In our preliminary work, we have generated a FLASH-PT experimental platform and homogenous dose distributions for FLASH-PT and C-PT for both in vitro and in vivo experiments. Our novel in vitro findings are that: (i) FLASH-PT kills more HN5 HNSCC cells than C-PT; and (ii) FLASH-PT preserves or enhances viability of Hs680.Tg normal tongue cells versus C-PT. Our immediate goals for this R21 are reflected in our specific aims: (1) Determine the functional and mechanistic effects of FLASH-PT vs C-PT in vivo in high-α/β tumor models (HNSCC); and (2) Determine the functional and mechanistic effects of FLASH-PT vs C-PT in vivo in low-α/β tumor models (PC). We expect that this high- risk/high-reward project will provide preclinical evidence regarding the in vivo effects of FLASH-PT vs C-PT (delivered in a variety of fraction numbers and sizes) on HNSCC and PC tumors and surrounding normal tissues. This knowledge will serve as the basis for choosing FLASH-PT or C-PT for individual patients, with the ultimate goals of improving treatment efficacy, minimizing treatment-related toxicity, and reducing treatment costs.