Extracellular Matrix Stiffening Induces a Malignant Phenotypic Transition in Breast Epithelial Cells

Tumors are much stiffer than healthy tissue, and progressively stiffen as the cancer develops. Tumor stiffening is largely the result of extracellular matrix (ECM) remodeling, for example, deposition and crosslinking of collagen I. Well established in vitro models have demonstrated the influence of the microenvironment in regulating tissue homeostasis, with matrix stiffness being a particularly influential mediator. Non-malignant MCF10A mammary epithelial cells (MECs) lose their epithelial characteristics and become invasive when cultured in stiff microenvironments, leading to the hypothesis that tumor stiffening could contribute directly to disease progression. However, previous studies demonstrating MCF10A invasion have been performed in gels with constant mechanical properties, unlike the dynamically stiffening tumor microenvironment. Here, we employ a temporally stiffening hydrogel platform to demonstrate that matrix stiffening induces invasion from and proliferation in MCF10A mammary acini. After allowing MCF10A acini to form in soft hydrogels for 14 days, the gels were stiffened to the level of a malignant tumor, giving rise to a proliferative and invasive phenotype. Cells were observed to collectively migrate away from mammary acini while maintaining cell-cell contacts. Small molecule inhibition of PI3K and Rac1 pathways was sufficient to significantly reduce the number and size of invasive acini after stiffening. Our results demonstrate that temporal matrix stiffening can induce invasion from mammary acini and supports the notion that tumor stiffening could be implicated in disease progression and metastasis.