Arsenic Trioxide Enhances the Efficacy of PD-1 Inhibitors in Hepatocellular Carcinoma by Inducing Immunogenic Cell Death via the ROS/ERS Pathway

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.