An innovative label-free approach for investigating epithelial-mesenchymal transition: pharmacological characterization of TGF-β1 effects in A549 cells.

Epithelial-mesenchymal transition (EMT) is a fundamental biological process involved in normal functions such as embryonic development and tissue repair, as well as in pathological conditions including cancer progression, metastasis, and fibrosis. TGF-β1 is a key inducer of EMT, activating pathways that alter cell morphology and gene expression (e.g., downregulation of E-cadherin, upregulation of α-smooth muscle actin (α-SMA)). EMT contributes to fibrotic tissue remodeling in idiopathic pulmonary fibrosis (IPF), a chronic and progressive lung disease characterized by excessive scarring of lung tissue. To achieve a comprehensive evaluation of EMT in respiratory epithelial cells (A549), we employed the standard Operetta CLS platform to assess morphological changes and protein expression of key biomarkers (E-cadherin, α-SMA), alongside an advanced approach that monitored cellular dynamics using the xCELLigence Real-Time Cell Analysis (RTCA) system and quantified biomarker gene expression via RT-qPCR. In Operetta experiments, TGF-β1 reduced cell roundness and E-cadherin protein levels, while it increased cell length and α-SMA protein levels. In xCELLigence RTCA experiments, TGF-β1 reduced the cellular index and E-cadherin gene expression while increasing α-SMA expression. SB-525334 blocked all effects of TGF-β1, whereas nintedanib was more effective in counteracting the stimulatory effects of TGF-β1 on cell length and α-SMA. Interestingly, nintedanib, per se, evoked small but consistent effects opposite to those of TGF-β1. In conclusion, integrating these experimental approaches provides a powerful platform for detailed investigation of EMT mechanisms and for the identification of novel drug candidates that counteract EMT.