Autophagy-promoted immunogenic cell death elicited by tyrosine kinase inhibitor orchestrates a synergistic immunotherapeutic microenvironment in hepatocellular carcinoma

BACKGROUND: The combination of tyrosine kinase inhibitors (TKIs) like lenvatinib with immune checkpoint inhibitors (ICIs) has become a cornerstone of first-line therapy for advanced hepatocellular carcinoma (HCC). However, this approach faces a significant bottleneck, with objective response rates lingering around 20-36%, largely due to the non-immunogenic ("cold") nature of most HCC tumors. This highlights a critical need for strategies that can remodel the tumor immune microenvironment to enhance therapeutic efficacy. METHODS: T cell triple co-culture system was established for functional assays. In vivo efficacy was evaluated in subcutaneous and orthotopic mouse models. Tumor immune profiling was performed using Cytometry by Time-Of-Flight (CyTOF), multiplex immunofluorescence, and spatial proximity analysis. RESULTS: T cells and DCs. Genetic (shATG5) or pharmacological (3-MA) autophagy inhibition significantly attenuated anlotinib's antitumor efficacy, while autophagy enhancement through ATG5 overexpression or metformin potentiated it. The triple combination of anlotinib, anti-PD-1, and metformin achieved the highest levels of HMGB1 release and immune cell infiltration and maximal tumor suppression. CONCLUSION: This research pioneers the identification of anlotinib as an ER stress- and autophagy-dependent ICD inducer in HCC. Our comprehensive mechanistic dissection and robust preclinical evidence establish anlotinib's ability to convert immunologically "cold" tumors to "hot" ones through the FGFR-1/ER stress/autophagy/DAMP release axis. The synergy with anti-PD-1 and enhancement by metformin provide a rationale for novel combination strategies to overcome current limitations of targeted-immunotherapy, offering a promising approach to improve response rates in advanced HCC.