Abstract

The tumor microenvironment (TME) critically regulates cancer progression by providing biochemical and biophysical cues that shape cellular behavior. However, how defined physical microenvironments govern cancer stemness and chemoresistance through mechanotransduction remains poorly understood. Here, we systematically engineered eight tumor-mimetic microenvironments by integrating serum, oxygen, and 3D compacted culture to investigate their effects on A549 non-small cell lung cancer cells. Among all conditions, cells cultured under 3D culture (PM4C) exhibited reduced cellular stiffness, enhanced expression of cancer stemness markers (EpCAM and CD44), and significantly increased resistance to cisplatin in both in vitro and nude mouse xenograft models. Transcriptomic analysis revealed that differentially expressed genes in the PM4C group were predominantly enriched in cell adhesion, mechanotransduction, stemness, and cisplatin resistance pathways. Metabolomic profiling further revealed a substantial accumulation of anaerobic metabolites associated with the maintenance of stemness. Mechanistically, the PM4C microenvironment remodeled matrix production, cell-ECM interactions, and cytoskeletal organization while inducing epigenetic reprogramming (reduced H3K9 acetylation), collectively promoting a stem-like and chemoresistant phenotype. These findings establish a direct mechanistic link between TME and cancer cell stemness, demonstrating that TME can reprogram stemness and drug responsiveness through mechano-epigenetic regulation. This work provides a mechanobiological framework for engineering physiologically relevant tumor organoids and offers new strategies for developing TME-targeted drugs and therapies.