ITGB1 Contributes to Matrix Stiffness–Induced Lipid Raft Organization and Promotes Hepatocellular Carcinoma Cell Migration

Matrix stiffening is a characteristic pathological feature of the hepatocellular carcinoma (HCC) microenvironment and promotes HCC cell migration. Lipid rafts are cholesterol–enriched plasma membrane microdomains involved in cell adhesion and migration. However, whether matrix stiffness promotes lipid raft organization and whether lipid rafts contribute to stiffness–promoted HCC cell migration remain unclear. Using two HCC cell lines cultured on polyacrylamide hydrogels with distinct stiffnesses, we found that stiff matrix enhanced cell migration, increased Area-normalized GM1 fluorescence, and upregulated Caveolin-1 and Flotillin-1 protein expression, consistent with enhanced lipid raft–associated membrane organization. Cholesterol depletion with methyl-β–cyclodextrin (MβCD) reduced GM1 fluorescence and attenuated migration on the Stiff matrix. Cholesterol replenishment partially restored GM1 fluorescence and wound closure after MβCD treatment. To explore how extracellular matrix stiffness is coupled to lipid raft organization, we next examined integrin β1 (ITGB1), a major extracellular matrix mechanoreceptor. The Stiff matrix increased ITGB1 protein expression, ITGB1–GM1 colocalization, and ITGB1 levels in raft-enriched fractions. ITGB1 knockdown reduced GM1 fluorescence, decreased Caveolin-1 and Flotillin-1 levels in raft-enriched fractions, and suppressed migration under Stiff–matrix conditions. These findings support a contributory role for ITGB1 in stiffness-associated lipid raft organization and HCC cell migration.