# Proton beam therapy induces protective immunity via HMGB1-dependent signaling

## TL;DR

Proton beam therapy can trigger immune responses against distant tumors, mainly through the release of HMGB1, a molecule linked to cell death and immunity.

## Contribution

This study reveals that HMGB1, not CRT, is the key driver of proton beam therapy's immunogenic effects in tumor rejection.

## Key findings

- High-dose proton irradiation significantly increases HMGB1 release but not CRT membrane exposure in tumor cells.
- HMGB1 knockdown reduces distal tumor rejection by 60%, compared to only 20% for CRT knockdown.
- Proton beam therapy's anti-tumor effects are primarily mediated through HMGB1-dependent signaling pathways.

## Abstract

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, in vitro 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 in vitro, 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. In vitro 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. In vivo 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.