Matrix Stiffness Drives Aggressive Phenotype in Tongue Squamous Cell Carcinoma via Mechanotransduction-Stromal Signalling

Objectives: Tongue squamous cell carcinoma (TSCC) is a highly aggressive malignancy where extracellular matrix (ECM) stiffening drives epithelial-mesenchymal transition (EMT). However, the specific mechanotransduction pathways and the distinction between primary and metastatic cell responses remain insufficiently defined. This study investigated how substrate stiffness regulates TSCC progression via a dual-regulatory mechanism: direct cell-intrinsic mechanotransduction and indirect stromal paracrine signalling. Methods: Two human TSCC cell lines with distinct origins, HSC-4 (metastatic) and HSC-7 (primary), were cultured on tunable collagen-coated polydimethylsiloxane (PDMS) substrates of varying stiffness (soft and stiff). Cell morphology, migration, proliferation, EMT marker expression, integrin and YAP expressions were assessed using wound healing assays, qRT-PCR and immunofluorescence staining. The involvement of actin cytoskeleton was examined using cytochalasin D. Additionally, the paracrine effects were evaluated by culturing TSCC cells with conditioned media from gingival fibroblasts (HGF-CM) cultured on different substrate stiffness. Results: Stiff substrates induced elongated, mesenchymal-like morphology and significantly enhanced migration in TSCC cells. Increased stiffness also upregulated EMT-associated markers (CDH2, VIM, MMP2), while induced YAP nuclear translocation and increased mechanosensitive integrin expression. Disruption of the actin cytoskeleton with cytochalasin D suppressed this stiffness-induced EMT marker expressions, indicating that cytoskeletal tension mediates mechanotransduction. Furthermore, HGF-CM derived from stiff substrates significantly upregulated EMT-related expression in HSC cells. Conclusions: Matrix stiffness drives TSCC progression through a dual mechanism: direct actin-mediated and YAP-associated mechanotransduction and indirect stiffness-modulated fibroblast signalling. These findings highlight that mechanical cues in the tumour microenvironment differentially regulate primary and metastatic phenotypes in TSCC. Clinical significance: Mechanical properties of the tumour microenvironment drive TSCC progression, suggesting that ECM stiffness is likely to be associated with altered TSCC phenotypes, providing a basis for future mechanobiology-focused studies on TSCC progression and management.