Background: Hepatocellular carcinoma (HCC) remains a major global health challenge, with limited efficacy of current immunotherapeutic strategies. Immunogenic cell death (ICD), characterized by the release of damage-associated molecular patterns (DAMPs), offers a promising approach to enhance antitumor immunity. Arsenic trioxide (ATO), an ICD inducer, may synergize with PD-1 inhibitors to overcome therapeutic resistance, though the underlying mechanisms remain unclear.

Methods: The cytotoxicity of ATO was evaluated via MTT, clonogenic, and apoptosis assays. ROS levels were quantified using ROS fluorescent probes. ERS activation was confirmed by Western blot detection of Calnexin, PDI, ATF-4, p-elF2α, and Caspase-12. ICD induction was assessed by measuring DAMPs (CRT exposure, HMGB1/ATP/IFN-β release). The roles of ROS/ERS pathways were dissected using NAC (ROS inhibitor) or 4-PBA (ERS inhibitor) pre-treatment. Ex vivo dendritic cell maturation assays analyzed ATO-treated HCC cells' immunostimulatory capacity, while In Vivo models evaluated immune microenvironment modulation via flow cytometry. Prophylactic/therapeutic tumor vaccine experiments assessed antitumor immunity using ATO-treated HCC cells as vaccines. Synergy between ATO and PD-1 blockade was tested in tumor-bearing mice by combining ATO with anti-PD-1 antibodies, monitoring tumor growth kinetics and survival outcomes.

Results: ATO dose-dependently reduced HCC cell viability while elevating intracellular ROS levels and activating ERS. These processes triggered the release/surface exposure of ICD-related DAMPs, including CRT, HMGB1, ATP, and IFN-β, leading to dendritic cells maturation and tumor immune microenvironment remodeling. ATO-treated HCC cells exhibited enhanced immunogenicity, functioning as prophylactic and therapeutic vaccines to stimulate antitumor immunity. Notably, ATO significantly potentiated the therapeutic efficacy of PD-1 inhibitors In Vivo.

Conclusion: ATO induces ICD in HCC via a ROS/ERS signaling axis, thereby amplifying antitumor immune responses and synergizing with PD-1 blockade. These findings support the clinical evaluation of ATO-PD-1 inhibitor combinations to improve outcomes in HCC patients.

Keywords: arsenic trioxide; endoplasmic reticulum stress; hepatocellular carcinoma; immunogenic cell death; oxidative stress.

© 2025 The Author(s). Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.

背景： 肝细胞癌（HCC）仍然是一个重大的全球健康挑战，目前的免疫治疗策略效果有限。具有释放损伤相关分子模式（DAMPs）特征的免疫原性细胞死亡（ICD）为增强抗肿瘤免疫力提供了有前景的方法。三氧化二砷（ATO），一种ICD诱导剂，可能与PD-1抑制剂协同作用以克服治疗耐药性，尽管其潜在机制尚不清楚。

方法： 通过MTT、克隆生成和凋亡实验评估ATO的细胞毒性。使用ROS荧光探针量化ROS水平。通过检测Calnexin、PDI、ATF-4、p-elF2α 和Caspase-12确认ERS激活。通过测量DAMPs（CRT暴露，HMGB1/ATP/IFN-β释放）评估ICD诱导。使用NAC（ROS抑制剂）或4-PBA（ERS抑制剂）预处理来解析ROS/ERS途径的作用。体外树突状细胞成熟实验分析ATO处理的HCC细胞的免疫刺激能力，而体内模型通过流式细胞术评估免疫微环境调节。预防性/治疗性肿瘤疫苗实验使用ATO处理的HCC细胞作为疫苗来评估抗肿瘤免疫力。通过将ATO与抗PD-1抗体结合在携带肿瘤的小鼠中测试ATO和PD-1阻断之间的协同作用，监测肿瘤生长动力学和生存结果。

结果： ATO剂量依赖性地降低HCC细胞的活力，并升高胞内ROS水平及激活ERS。这些过程触发ICD相关DAMPs（包括CRT、HMGB1、ATP 和IFN-β）的释放/表面暴露，导致树突状细胞成熟和肿瘤免疫微环境重构。ATO处理的HCC细胞表现出增强的免疫原性，可以作为预防性和治疗性疫苗来刺激抗肿瘤免疫力。值得注意的是，ATO在体内显著增强了PD-1抑制剂的疗效。

结论： ATO通过ROS/ERS信号轴诱导HCC中的ICD，从而放大抗肿瘤免疫反应并与PD-1阻断协同作用。这些发现支持对ATO-PD-1抑制剂组合进行临床评估以改善HCC患者的预后。

关键词： 三氧化二砷；内质网应激；肝细胞癌；免疫原性细胞死亡；氧化应激。