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

backgroundThe 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.

methodsWe 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

resultsAmong 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

conclusionThis 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.