mTOR as a mechanistic biomarker of FLASH radiotherapy: Implications for immune-modulated melanoma treatment

FLASH radiotherapy (FLASH-RT), which delivers ultra-high dose rate radiation, has emerged as a promising modality that reduces normal tissue toxicity without compromising tumor control. While preclinical data support its tissue-sparing properties, the underlying biological mechanisms remain incompletely defined. Understanding these pathways is critical for integrating FLASH into multimodal cancer therapy.Melanoma, a highly immunogenic and aggressive skin cancer, is increasingly treated with immune checkpoint inhibitors (e.g., anti-PD-1), yet many patients develop resistance. Radiation can enhance immunogenicity, but toxicity often limits dose escalation. The potential for FLASH to synergize with immunotherapy while minimizing side effects presents an exciting opportunity to optimize melanoma treatment.To investigate the immune contributions to the FLASH effect, female BALB/c mice received proton radiation delivered as FLASH (~80 Gy/s) or conventional (~0.5 Gy/s) at 0, 12, or 14 Gy. At 27 days post-treatment, skin tissues were analyzed using Data Independent Acquisition-Mass Spectrometry (DIA-MS). Machine learning-based feature selection identified tryptic peptides that distinguished FLASH from conventional treatment. mTOR (mammalian target of rapamycin)-a master regulator of immune signaling-and its binding partner Raptor were significantly upregulated in FLASH-treated skin, suggesting a distinct mTOR-driven pathway.Given mTOR's known role in modulating PD-L1 expression and immune suppression via interferon and TGF-beta signaling, we hypothesized that FLASH may potentiate the efficacy of immune checkpoint blockade. Using in vitro and in vivo melanoma models, we evaluated responses to FLASH or conventional radiation with or without anti-PD-1 therapy. Mice treated with FLASH plus anti-PD-1 demonstrated promising improvements in survival, supporting potential synergy. Immunoblotting and immunofluorescence confirmed increased phospho-mTOR (Ser2448) and downstream effector activation in both normal and tumor tissues following FLASH.These findings identify mTOR as a candidate biomarker and mechanistic driver of FLASH responses and support further development of mTOR-informed, immune-responsive FLASH strategies in melanoma and other cancers.