Title : TLR7 agonist reactivates the pDC–iNKT axis to enhance anti-tumor immunity in residual HCC after iRFA
Abstract:
Background & Aims: Incomplete radiofrequency ablation (iRFA) in hepatocellular carcinoma (HCC) leaves residual tumors within an immunosuppressive microenvironment. Plasmacytoid dendritic cells (pDCs) and invariant natural killer T (iNKT) cells are critical for anti-tumor immunity, but their coordinated role after iRFA remains unclear. We investigated whether the pDC–iNKT axis is impaired after iRFA and whether intratumoral delivery of the TLR7 agonist CAN1012 could restore this axis and enhance anti-PD-1 therapy.
Methods: Immune changes after iRFA were assessed in clinical HCC samples and mouse models via immunohistochemistry, multiplex immunofluorescence, single-cell RNA sequencing, and flow cytometry. CAN1012 effects on pDCs and iNKT cells were examined using qPCR, ELISA, Western blotting, and co-culture assays. Anti-tumor efficacy and abscopal responses were evaluated in orthotopic and bilateral subcutaneous HCC models treated with intratumoral CAN1012 ± anti-PD-1, with pathway blockade using anti-Bst2 (pDC depletion) or anti-CD1d (iNKT blockade).
Results: iRFA led to progressive loss of pDCs and iNKT cells, along with reduced IFNγ+ CD8+ T cells. CAN1012 activated pDCs via TLR7–MyD88–NF-κB/IRF7, upregulated CXCL16, promoted iNKT chemotaxis, and enhanced IFNγ production. In orthotopic models, intratumoral CAN1012 (0.3 μg/μL) significantly inhibited residual tumor growth, with necrotic-region injection offering comparable efficacy, less leakage, and prolonged local retention. In bilateral models, local CAN1012 suppressed both treated and contralateral untreated tumors, indicating abscopal effects. Combining CAN1012 with anti-PD-1 achieved near-complete tumor regression and prolonged survival. Blockade of pDCs or iNKT cells markedly attenuated these effects, confirming the pDC–iNKT axis as the mechanistic driver.
Conclusion: Intratumoral CAN1012 reactivates the pDC–iNKT axis, reverses post-iRFA immunosuppression, and synergizes with anti-PD-1. This strategy represents a promising post-ablation immunotherapy for residual HCC.
