Proton beam therapy induces protective immunity via HMGB1-dependent signaling

Proton beam therapy is widely regarded for its cost-effectiveness, precision, and protection of normal tissues. Emerging evidence shows that conventional radiotherapy can inhibit primary tumors and promote immunogenicity of distant tumors, possibly via damage-associated molecular patterns (DAMPs) like calreticulin (CRT) and high mobility group box 1 (HMGB1) released during immunogenic cell death (ICD). However, the role of proton beams in inducing DAMPs and enhancing immunogenicity remains unclear. This study aimed to investigate the effects of proton beam-induced DAMPs on the colonization of distal tumors. In this study, <i>in vitro</i> cell irradiation experiments were conducted to identify the optimal proton beam dose for enhancing DAMPs expression in mouse colon carcinoma Colon-26 cells. Based on the optimal proton dose determined <i>in vitro</i>, a tumor-bearing mouse model was employed to evaluate its efficacy in inhibiting distal tumor colonization. To explore the mechanisms behind the anti-tumor effects, shRNA targeting DAMPs-related immunogenic molecules was applied to assess the immune response. <i>In vitro</i> findings indicated high-dose proton irradiation markedly induces HMGB1 release yet exerts no significant effect on CRT membrane exposure. Following high-dose proton beam irradiation, tumor cells transfected with shRNA exhibited a significant reduction in CRT and HMGB1 expression compared with the con-shRNA-irradiated control group. <i>In vivo</i> experiments demonstrated that HMGB1 knockdown reduced distal-tumor rejection by 60%, whereas CRT knockdown reduced it by only 20%, indicating that HMGB1 release may dominate proton-induced ICD. Our research results indicated that high-dose proton irradiation trigger the rejection of distal tumor colonization through a signaling pathway that depends on HMGB1. This research advanced our understanding of proton beam therapy immunological mechanisms and offered insights for improving tumor treatment outcomes.