Project Summary Radiotherapy (RT) is used in the treatment of soft tissue sarcomas (STS), frequently in conjunction with surgical removal of gross disease. For pre-op RT, 50 Gy equivalent in 2 Gy fractions (EQD2) is commonly used, while post-op RT requires 60–66 Gy (EQD2). For definitive (curative with RT alone) schedules, doses from 75– 100 Gy (EQD2) are needed. Paralleling changes in the RT field, STS are being treated with increasingly hypofractionated schedules, but the combination of high dose and/or high dose per fraction RT to larger tumor volumes can increase normal tissue toxicity. Indeed, patients with STS who receive RT risk major bone and soft tissue problems including skin toxicity, non-healing ulcers, necrosis, lymphedema, bone fractures and second malignant neoplasms (SMNs). This project will test the overall hypothesis that FLASH proton radiotherapy (F- PRT) spares normal soft tissues and bone from early/late toxicities compared with standard PRT (S-PRT), whereas the two modalities will be isoeffective in controlling sarcoma growth. Our preliminary data show reduced skin damage, normal tissue inflammation, lymphedema, and vascular damage with F-PRT vs S-PRT. In addition to modeling typical side effects of RT, as a malignancy that requires high dose RT to achieve local control, sarcoma represents a good proving ground to evaluate whether F-PRT sufficiently modulates RT therapeutic index to be clinically useful in other cancers. In Aim 1, the ability of F-PRT to abrogate tissue effects that pose barriers to the treatment of sarcomas with RT will be tested using the following hypotheses in in vivo mouse models: (i) the inflammatory component of normal tissue toxicity to F-PRT will be attenuated relative to S-PRT, leading to less severe fibrosis and lymphedema after F-PRT compared to S-PRT by reducing the production of pivotal drivers, such as TFG-b and VEGF-C (ii) F-PRT will produce less injury to tissue vasculature and preserve its associated matrix, providing for faster and more complete recovery of skin and bone than is feasible with S- PRT. In Aim 2, we will assess clinical outcome in murine sarcomas treated with F-PRT. Among the long-term survivors of mice treated with whole body irradiation, we find fewer tumors in the F-PRT-treated group than in the group treated with S-PRT. In Aim 2.1, we will employ a mouse model with transient p53 knockdown in conjunction with wildtype controls, to test the ability of F-PRT to reduce the incidence of SMNs compared with S-PRT. In Aim 2.2 we will perform dose escalation studies in mice bearing sarcomas to define the therapeutic window of F-PRT when added in the pre-op setting as either one or three fractions. In Aim 3, we will conduct a phase 1 dose escalation study using pre-op (amputation) doses from 21-30 Gy (4 dose levels) in one fraction to determine safety and tolerability of F-PRT and provide pathologic evidence of efficacy. For a phase 2 definitive trial, we will determine feasibilit...