Targeting Plasmacytoid Dendritic Cell-Invariant Natural Killer T Axis through TLR7 Agonist to Enhance Anti-Tumor Immune Therapy in Residual Cancer after Incomplete Radiofrequency Ablation for Hepatocellular Carcinoma

Introduction: The immunosuppressive tumor microenvironment (TME) contributes to poor outcomes in hepatocellular carcinoma (HCC) after incomplete radiofrequency ablation (iRFA). Plasmacytoid dendritic cells (pDCs) and invariant natural killer T (iNKT) cells play crucial roles in modulating the TME, but their functional contributions following iRFA remain to be elucidated. In this study, we investigate the role of the pDC-iNKT axis in the TME after iRFA, as well as the potential of Toll-like receptor 7 agonist CAN1012 as a viable therapeutic candidate in combination with anti-PD-1 in patients with residual HCC. Methods: Immune cell changes following iRFA were evaluated in clinical HCC samples and mouse models using immunohistochemistry, multiplex immunofluorescence, transcriptomic profiling, and flow cytometry. The activation and functional responses of pDCs and iNKT cells to CAN1012 were examined using quantitative PCR, flow cytometry, ELISA, Western blotting, and co-culture assays. Anti-tumor efficacy, immune activation, and systemic (abscopal) responses were evaluated in subcutaneous and orthotopic HCC mouse models treated with intratumoral CAN1012, with or without PD-1 antibody therapy. The role of the pDC-iNKT axis was further evaluated using antibody blockade. Results: Importantly, our results demonstrated that in both HCC patient samples and mouse models, the infiltration of pDCs and iNKT cells within the TME after iRFA remained diminished, and these cells exhibited functionally inhibitory characteristics. CAN1012-activated pDCs via the TLR7-MyD88-IRF7/NF-κB signaling pathway, upregulated CXCL16 expression, promoted iNKT-cell recruitment, and enhanced their IFNγ secretion. Furthermore, intratumoral CAN1012 inhibited residual tumor growth and induced systemic abscopal effects through the pDC-iNKT axis. In bilateral tumor models, the enhanced efficacy of CAN1012 combined with anti-PD-1 was markedly reduced by pDC depletion or iNKT blockade, indicating that an intact pDC-iNKT axis is required. Conclusion: CAN1012 restores anti-tumor immunity after iRFA by reactivating the pDC-iNKT axis. These findings underscore the potential of the pDC-iNKT axis as a novel therapeutic target in residual HCC and support intratumoral CAN1012, particularly in combination with anti-PD-1, as a potential post-ablation immunotherapeutic strategy.