Abstract

Background: Mechanical forces, particularly tensile stress, influence tumor progression by modulating cancer-associated fibroblast (CAF) behavior and extracellular matrix remodeling, yet their role in oral cancer remains insufficiently defined. To our knowledge, this is the first study investigating in vitro tensile stress responses in CAF and normal fibroblasts (NF) derived from oral squamous cell carcinoma (OSCC). Objective: To assess how tensile stress affects fibroblast gap closure, proliferation, metabolic activity, and paracrine signaling relevant to tumor-stroma interactions. Methods: CAFs and NFs were isolated from OSCC tissue and matched healthy mucosa and exposed to cyclic tensile strain (3%, 0.02 Hz, 96 h) using the FX-6000T system. Gap closure was assessed by wound-healing assay, proliferation by cell counting, metabolic activity by AlamarBlue, and paracrine effects on A549 tumor cell gap closure using fibroblast-conditioned supernatants. Results: Tensile loading significantly increased CAF gap closure capacity compared with both stimulated NFs (p<0.0001) and unstimulated CAF controls (p=0.0167). Metabolic activity showed a non-significant trend toward higher values in CAFs. Proliferation did not differ between groups up to 48 h, arguing against a major early contribution of proliferation to the observed group differences in the fibroblast scratch assays; however, because proliferation was not inhibited and was not quantified beyond 48 h, later time points should be interpreted conservatively as composite gap closure. Supernatants from mechanically stimulated CAFs increased A549 gap closure by ~40% compared with non-stimulated controls (p=0.0485), whereas stimulated NFs did not enhance the tumor cell's capacity to close the cell-free gap. Conclusions: Tensile strain was associated with increased fibroblast gap closure and enhanced gap-closure-promoting paracrine effects of oral CAFs under the conditions tested. These exploratory findings support the concept that biomechanical cues can modulate CAF functional behavior in OSCC. Given the limited cohort size and inter-patient variability, these results should be interpreted as hypothesis-generating and require confirmation in larger studies. Mechanistic pathways were not interrogated in this study and should be addressed in future work using more complex tumor-stroma models.