Modeling Mammary Tumor Invasion in Response to Changes in Matrix Stiffness

The progression of mammary carcinoma involves a variety of cellular and environmental factors that facilitate invasion of the stroma. Stiffening of the extracellular matrix is associated with increased proliferation and migration of mammary epithelial cells (MECs). Adding further complexity, the MEC microenvironment is dynamic, stiffening nearly 40-fold gradually as the stroma remodels from normal stiffness of ~150 Pascals to ~5700 Pascals (Pa). These environmental changes occur alongside the emergence of aggressive cellular subpopulations within the tumor mass whose sensitivity to ECM stiffness may vary dramatically. To mimic stiffening of the tumor microenvironment, we utilized a methacrylated hyaluronic acid hydrogel that could be stiffened in the presence of cells. MCF10A organoids cultured on this substrate showed partial loss of their ductal phenotype in response to stiffening. Activation of TGF-β and YAP signaling was observed and dual inhibition of these pathways significantly reduced the number of invasive cells per organoid. We then sought to determine whether malignant subpopulations within a tumor could alter the mass’ stiffness sensitivity. MECs were cultured on polyacrylamide gels ranging from normal mammary stiffness, ~150 Pa, to malignant and stiff, ~5700 Pa. H-Ras transformed cells spread at 150 Pa. with their invasive fraction adopting mesenchymal-like morphology, disruption of E-Cadherin, reduced basement membrane, and nuclear localization of the EMT transcription factor Twist1. Stiffness-mediated transformation was blocked by treatment with R-Roscovitine, suggesting an important link between stiffness sensitivity and intercellular CDK activity. These data demonstrate a complex relationship between ECM stiffness, mutational status, and invasive potential in MECs