Atomic force microscopy reveals the influence of substrate collagen concentration and TGF-β on lung fibroblast mechanics

Understanding how extracellular matrix (ECM) stiffness and biochemical factors such as TGF-β affect cell behaviour is critical for elucidating mechanisms underlying several pathologic conditions such as tissue fibrosis and cancer metastasis. This study investigates the effects of varying collagen substrate concentration and consequently varying stiffness conditions along with TGF-β treatment on the morphology, nanomechanical properties, and gene expression of normal human lung fibroblasts (NHLF). Our results reveal that increased substrate stiffness leads to more elongated cell morphology, decreased cellular stiffness, and significant alterations in gene expression related to cytoskeletal organization and myofibroblast activation genes. TGF-β treatment further induces myofibroblast differentiation, as evidenced by increased α-SMA and collagen expression, while also reducing cellular stiffness and promoting a more elongated, invasive phenotype. These findings highlight the critical role of both mechanical and biochemical cues in modulating fibroblast behaviour, with significant implications in fibrosis development and cancer progression.