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March 28, 2026Open Access

Mechanoregulation of cell proliferation and migration of human oral squamous cell carcinoma by substrate stiffness

Key Points

Investigate how substrate stiffness affects cell proliferation, migration, and epithelial-mesenchymal transition in oral squamous cell carcinoma.
Cultured HSC-4 and HSC-7 cells on PDMS substrates with varying stiffness for 24 and 72 hours.
Evaluated cell morphology using phase-contrast microscopy.
Assessed cell proliferation via MTT assay.
Conducted migration assays and analyzed gene expression using quantitative real-time polymerase chain reaction (qRT-PCR).
HSC-4 cells on stiff substrates showed significantly higher proliferation after 24 hours compared to soft substrates (p < 0.05).
Cell morphology changed significantly, with an increase in cell area and perimeter on stiff substrates.
Gene expression analysis indicated upregulation of vimentin, N-cadherin, and MMP-2 on stiff substrates (p < 0.05).

Abstract

Objectives: Oral squamous cell carcinoma (OSCC) is the most prevalent and lethal oral malignancy, often presenting with abnormally stiff tissues caused by extracellular matrix (ECM) remodeling. ECM stiffness has been implicated in cancer progression through mechanotransduction pathways. This study aimed to investigate how substrate stiffness influences cell proliferation, migration, and epithelial–mesenchymal transition (EMT) in human tongue squamous cell carcinoma cell lines (HSC-4 and HSC-7). Methods: HSC cells were cultured on type I collagen-coated polydimethylsiloxane (PDMS) substrates with tunable stiffness representing normal oral mucosa (4.4 kPa) and OSCC-stiffened tissue (17.6 kPa) for 24 and 72 hours. Cell morphology was evaluated using phase-contrast microscopy to determine cell area, perimeter, and shape index. Cell proliferation was assessed via MTT assay. Cell migration assays and expression analyses of migration- and EMT-related markers were performed using quantitative real-time polymerase chain reaction (qRT-PCR). Results: HSC-4 cells cultured on the stiff substrate demonstrated significantly higher proliferation after 24 hours compared to those on the soft substrate (One-way ANOVA, p < 0.05). Increased stiffness induced morphological changes from polygonal to spindle-like shapes, with significant enlargement of cell area and perimeter. Gene expression analysis revealed that EMT- and migration-associated markers, including vimentin, N-cadherin , and matrix metalloproteinase-2 (MMP-2) , were significantly upregulated on stiff substrates (p < 0.05), while cells on soft substrates exhibited reduced expression of these genes. Conclusions: Substrate stiffness promotes cell proliferation, migration, and EMT in OSCC cell lines, accompanied by notable cytomorphometric alterations. These findings underscore the pivotal role of ECM mechanical properties in OSCC pathogenesis and highlight the mechanobiological regulation of tumor progression as a potential therapeutic target.

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