Musculoskeletal and Marrow Sparing with Proton FLASH Radiotherapy in Juvenile Mice.

Abstract

Radiation‑induced musculoskeletal toxicities are one of the major limitations in pediatric patients whose skeletons are still developing. FLASH radiotherapy, delivered at ultra-high dose rates (>40 Gy/s), has demonstrated normal tissue sparing in preclinical models, but its effects on the developing skeleton and marrow remain incompletely characterized. This study evaluated the normal-tissue effects 10 weeks after proton FLASH irradiation in juvenile mice, modeling the pediatric context. Juvenile C57BL/6 mice (3 to 4 weeks old) were randomized to receive 11 Gy protons with FLASH (≈200 Gy/s), conventional irradiation (0.2 Gy/s average), or sham treatment to the left hind leg using a synchrotron-based proton beamline. Mice were followed for 10 weeks post-treatment. Bone toxicity was assessed with microCT, including bone mineral density, bone volume fraction, trabecular indices, and limb length. Bone marrow cellularity was quantified on H&E-stained sections, and muscle fibrosis was assessed using Masson's trichrome staining. Proton FLASH irradiation preserved bone microarchitecture compared with conventional irradiation, with higher bone mineral density and bone volume fractions (p < 0.05). Trabecular number was maintained, while structure model index indicates a mechanically favorable trabecular structure in the FLASH group. Proton FLASH also partially preserved longitudinal bone growth, with tibial length ratios of 0.94 (FLASH) vs. 0.91 (CONV) vs. 1.00 (SHAM). Bone marrow cellularity was preserved in FLASH mice (5.3% reduction vs. sham) compared with conventional (11.3 % reduction, p < 0.05). Muscle fibrosis was significantly lower in FLASH group (fibrosis positivity 2.6 % vs. 3.0% for FLASH vs. conventional CONV, p < 0.05). No severe immobility or weight loss was observed across groups. Proton FLASH reduced musculoskeletal and marrow toxicities at 10 weeks post-irradiation in juvenile mice. These findings extend prior proton FLASH adult mice studies by demonstrating tissue sparing in a juvenile model and support further investigation of FLASH as a strategy to reduce normal‑tissue injury during radiotherapy of pediatric patients.