Abstract Tumor extracellular matrix (ECM) stiffening is a defining biophysical alteration of solid cancers that amplifies mechanotransduction, reshapes gene expression, and fosters malignant behavior. In lung adenocarcinoma (LUAD), however, the extent to which matrix rigidity simultaneously tunes core phenotypes and maps onto patient survival remains incompletely resolved. We aim to determine how soft (2 kPa) versus stiff (32 kPa) substrates modulate proliferation, migration (scratch assay), and colony-forming efficiency (CFE) in A549 cells and to define the accompanying stiffness-driven transcriptome to evaluate the prognostic relevance of the resulting gene program using multivariate models across independent LUAD cohorts. Methods A549 cells were cultured on soft (2 kPa) and stiff (32 kPa) substrates. Proliferation was measured by MTT at days 3, 5, and 7. Cell migration was assessed by scratch assay, and clonogenic potential by colony-forming efficiency assay. In parallel, RNA-seq was performed. Differentially expressed genes at 7 days (padj 0.05, |log2FC| 1) were tested for survival association in GSE68465 (n = 439) using multivariate Cox regression. Genes both stiffness-regulated and survival-associated defined a 78-gene rigidity signature. Prognostic value was evaluated in Cox models and validated in an independent cohort (GSE72094, n = 393). Functional enrichment analysis identified related biological processes. Results Stiff matrices increased proliferation at day 7, accelerated scratch closure (enhanced migratory capacity), and elevated CFE (greater ability of single cells to form viable colonies) compared with soft substrates. Transcriptomic analysis identified 2,585 differentially expressed genes, 235 associated with survival resulting in a 78-gene rigidity signature. In the discovery cohort, the signature predicted worse survival (HR per SD = 1.7, 95% CI 1.45-1.90; concordance = 0.74; p 0.001). Validation confirmed its prognostic power (HR per SD = 1.7, 95% CI 1.4-2.0; concordance = 0.71; p 0.001). Enrichment analysis highlighted programs related to positive regulation of locomotion and cell migration, consistent with increased proliferation, migration and CFE observed on 32 kPa. Conclusions Substrate stiffness modulates cancer cell behavior enhancing proliferation, migration and colony-forming efficiency while inducing a transcriptional phenotype linked to increased mortality in lung adenocarcinoma. This abstract is funded by: CB17/06/00021/Centro de Investigación Biomedica en Red-Enfermedades respiratorias - IDE/2024/000797/Gobierno del Principado de Asturias
Chacón et al. (2026) studied this question.