Background <p>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.</p> Methods <p>We systematically screened existing TKIs for their ability to induce immunogenic cell death (ICD). ICD hallmarks were validated through multiple assays in HCC cell lines and patient-derived organotypic tumor spheroids (PDOTS) from 20 primary and 15 neoadjuvant-treated HCC samples. Mechanistic studies employed RNA-seq, pharmacological inhibitors, and genetic knockdown approaches. A tumor cell-Bone Marrow-Derived Dendritic Cells (BMDC)-CD8⁺ 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.</p> Results <p>Among screened TKIs, anlotinib emerged as the most potent ICD inducer, triggering extracellular ATP release, HMGB1 secretion, and surface calreticulin exposure. RNA-seq and GSEA identified ER stress and autophagy as top enriched pathways. Mechanistically, anlotinib inhibited FGFR-1 phosphorylation, activating ER stress (upregulation of CHOP/ATF4 with ATF4 nuclear translocation) and secondary autophagy (LC3-II conversion). Pharmacological inhibition of ER stress or autophagy, as well as ATG5/LC3B knockdown, abrogated damage-associated molecular patterns (DAMPs) release, confirming autophagy's essential role in ICD execution. Luminex cytokine assay and mass spectrometry identified the key secretome (CCL19, CXCL12) and DAMPs essential for dendritic cell recruitment. In BMDC co-culture experiments, anlotinib-conditioned media enhanced DC migration (blocked by anti-HMGB1), DC activation (CD86/CD40 upregulation), and subsequent CD8⁺ T cell priming (CD25/CD69/IFN-γ expression). In vivo, anlotinib inhibited tumor growth, enhanced CD8⁺ T cell and dendritic cell infiltration, and increased spatial interactions between ICD⁺ tumor cells and T cells. Anlotinib synergized with anti-PD-1 therapy, an effect mediated by CD8⁺ 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.</p> Conclusion <p>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.</p>