Combined Anti-Pd-1 with Irreversible Electroporation Relieves Immunosuppression and Reduces Hepatocellular Carcinoma Regrowth

BACKGROUND & AIMS:Local ablation triggers anti-tumor response and is regarded as an encouraging treatment combined with immunotherapy for hepatocellular carcinoma (HCC). Irreversible electroporation (IRE) is a novel ablative technique eliminating tumor cells by electroporation, however, the characteristics of IRE-induced immune microenvironment and underlying mechanism remain unclear. METHODS:We developed an orthotopic immunocompetent HCC mouse model and performed incomplete IRE-ablation. The post-IRE immune microenvironment was characterized by RNA sequencing, single-cell RNA sequencing, flow cytometry, and multiplex immunofluorescence. Cytokine-chemokine array and organoid-immune cell co-culture were used to investigate the functions and underlying mechanism. The combination therapy of IRE and anti-PD-1 was examined for HCC treatment in mice. RESULTS:IRE initially inhibited HCC growth, but tumors rapidly regrew by day 14 after ablation, with increased Ly6G+ Polymorphonuclear (PMN) Myeloid-Derived Suppressor Cells (MDSCs) infiltration and CD8+ T cell exhaustion. In patient-derived HCC organoids, IRE-induced CD8+ T cell cytotoxicity was suppressed by MDSCs. Ly6G antibody-mediated PMN-MDSC depletion repressed HCC regrowth after IRE-ablation in vivo (1.29±0.12 vs 0.49±0.09 g, p<0.001). Mechanistically, HCC cells degraded CXCL10 mRNA via YTH N6-methyladenosine RNA binding protein F2 (YTHDF2), which activated MDSCs that led to CD8+ T cells exhaustion. Combined anti-PD-1 and IRE reduced tumor burden by 84.3% compared with IRE alone (0.22±0.08 vs 1.43±0.13 g, p<0.01), prolonged survival (day-60 survival: 93.33% vs 40%, p<0.001), and induced long-term anti-tumor immunity in mice. Four unresectable recurrent HCC patients treated with IRE plus anti-PD-1 therapy showed improved recurrence-free time than patients receiving IRE alone. CONCLUSIONS:PMN-MDSCs mediated immunosuppression that promoted HCC regrowth after incomplete IRE ablation. Combining anti-PD-1 therapy is a promising approach to eliminate post-ablation residual HCC, supporting evaluation in prospecitve clinical trials. IMPACT AND IMPLICATIONS:Post-ablation recurrence remains a major challenge in HCC. Here, we show that although IRE initially activates CD8+ T cells but subsequently promotes PMN-MDSCs activation and CD8+ T-cell exhaustion through YTHDF2-mediated CXCL10 mRNA degradation, resulting in a suppressive tumor immune microenvironment (TIME). These findings identify a previously unrecognized mechanism of post-IRE tumor regrowth and provide a rationale for early combination with anti-PD-1 therapy. Although further prospective clinical validation is required, this work suggests that monitoring post-ablation immune changes and applying rational immunotherapy combinations may help optimize treatment strategies for patients with HCC undergoing local ablation.