Tumor peripheral stiffness modulates lenvatinib resistance in HCC preclinical models by regulating FIS1-dependent...

Hepatocellular carcinoma (HCC) displays heterogeneous responses to lenvatinib, with tumor microenvironment (TME) stiffness emerging as a key resistance modulator. This study investigates how tumor peripheral stiffness governs lenvatinib efficacy via mitochondrial fission/mitophagy and evaluates matrix-targeting combination therapies.

Clinical HCC tissues underwent stiffness measurement (atomic force microscopy [AFM]/rheometry) and survival correlation analyses. In vitro, cells grown on soft vs. stiff hydrogels (5 vs. 15 kPa) were assessed for their lenvatinib response, mitophagy, and mitochondrial fission 1 (FIS1)-trimethylation of histone H3 lysine 27 (H3K27me3) regulation. Subcutaneous xenografts received collagenase-lenvatinib combination therapy.

Elevated tumor peripheral stiffness, quantified by AFM and rotational rheometry, was significantly associated with HCC recurrence. Patients with stiff peripheries exhibited reduced recurrence-free survival (p <0.05), correlating with upregulated mitophagy markers (Parkin and FIS1) and diminished H3K27me3 in high-stiffness human HCC tissues (p <0.0001). In vitro, HCC cells on stiff matrices (15 kPa) showed attenuated lenvatinib-induced apoptosis (TUNEL: p = 0.0003 vs. soft 5 kPa) and preserved mitochondrial membrane potential (JC-1: p = 0.0004), concomitant with fragmented mitochondria driven by FIS1 upregulation via H3K27me3 depletion at its promoter (chromatin immunoprecipitation: p <0.0001). FIS1 knockdown reversed mitochondrial fragmentation (p <0.001) and resensitized cells to lenvatinib. Stiffness amplified cytoprotective mitophagy under lenvatinib stress, evidenced by enhanced LC3/TOM20 colocalization (p = 0.0008) and mitochondrial Parkin accumulation. In vivo, collagenase-mediated matrix softening synergized with lenvatinib, suppressing tumor growth (volume: p <0.001; weight: p <0.001) while reducing FIS1/Parkin expression and augmenting apoptosis.

Tumor peripheral stiffness drives lenvatinib resistance in HCC via H3K27me3-mediated FIS1 upregulation, triggering mitochondrial fission and cytoprotective mitophagy to evade drug-induced apoptosis. Targeting matrix stiffness (via collagenase-mediated softening) synergizes with lenvatinib to overcome microenvironment-driven resistance, providing a novel mechanoadjuvant strategy for HCC therapy.

This study shows that tumor peripheral matrix stiffness reduces lenvatinib sensitivity in HCC by enhancing FIS1-dependent mitophagy, explaining therapeutic response heterogeneity. These findings are clinically relevant, highlighting tumor stiffness as a potential biomarker for lenvatinib resistance and mitophagy as a targetable pathway. Clinically, stiffness assessments (e.g. imaging/biopsy) could be used to stratify patients for personalized treatment. Combining lenvatinib with matrix-softening agents or mitophagy inhibitors could improve efficacy. However, translational potential requires validation in larger cohorts and development of non-invasive stiffness measurement methods, given challenges associated with the clinical application of current invasive techniques or collagenase-based preclinical models.