Soft ECM Environments Prime Central Contractility and Stiffness via Myosin II–Mediated

Abstract

Cells are constantly exposed to a dynamic mechanical environment, and their ability to sense and adapt to extracellular matrix (ECM) stiffness plays a critical role in cancer progression. While it is known that ECM stiffness influences cancer cell invasion, morphology, and cytoskeletal organization, how past mechanical experience leaves a persistent intracellular signature remains unclear. In this study, we investigated whether breast cancer cells encode a mechanical memory of prior ECM stiffness through spatially compartmentalized cytoskeletal remodeling and local stiffness adaptation. Two luminal A breast cancer cell lines, MCF-7 and T47D, were cultured on soft (0.5 kPa) or stiff (8 kPa) polyacrylamide hydrogels for 48 hours. Following re-plating on uniform substrates, we performed AFM-based nanoindentation and quantitative immunofluorescence to assess regional stiffness and myosin II (pMLC) intensity in central (perinuclear) and peripheral zones. Our results revealed that preconditioning on soft ECM increased myosin II intensity and mechanical stiffness specifically in the perinuclear region, while peripheral regions remained largely unaffected. Mediation analysis showed that myosin II activity partially accounted for the effect of ECM condition on central-region stiffness. This pattern was conserved in both MCF-7 and T47D cells, suggesting a generalizable mechanism within ER-positive breast cancer phenotypes. These effects were consistently observed across three independent experiments, reinforcing their biological robustness. Together, these findings support a model in which ECM stiffness history imprints spatial mechanical memory within individual cells through region-specific actomyosin remodeling. By linking past extracellular mechanical inputs to persistent intracellular mechanics, this memory may prime cancer cells for context-adapted migration or invasiveness. Our work identifies intracellular stiffness not only as a passive biophysical trait but as an active, regionally tuned effector of mechanoadaptive cell states.