Ductal carcinoma is the most common form of breast cancer and progresses to invasive ductal carcinoma (IDC) when the carcinoma invades through the basement membrane (BM) into the stromal tissue. Invasion is a key step in ductal carcinoma progression that is associated with an increased likelihood for metastasis, the most deadly aspect of breast cancer. During metastasis, cancer cells must also invade BM during intravasation and extravasation. The overall goal of our work is to determine how matrix mechanical plasticity (malleability) regulates breast cancer invasion and migration. In the initial 5-year phase of this R37 award, we found that breast cancer tissue is mechanically plastic, and that individual breast cancer cells can migrate through nanoporous matrices independent of proteases using invadopodia if the matrix exhibits sufficient matrix mechanical plasticity. We also found that increased covalent crosslinking of the matrix or increased stiffness inhibits invadopodia formation, and that cancer cells can utilize filopodia to migrate along soft basement-membrane-like substrates if the substrate is sufficiently viscoelastic or malleable. We have also pursued related lines on inquiry, finding that extracellular matrix viscoelasticity regulates cell-cycle progression, that cancer cells generate force in order to undergo mitotic elongation and divide in confining type-1 collagen rich matrices, that increased stiffness regulates breast cancer progression through a YAP-independent mechanism, and that increased stiffness induces broad changes in the epigenome that functionally mediate a stiffness-induced malignant phenotype in mammary epithelium. While our initial studies on the role of malleability in invasion focused on single cell invasion, initial invasion of the BM during cancer progression is thought to be collective in nature, involving the coordinated activity of multiple cells. The specific hypothesis to be tested in this R37 extension application is that matrix mechanical plasticity (malleability) is a key physical parameter mediates collective invasion of the BM and subsequent migration through the type-1 collagen rich stromal matrix during cancer progression. This hypothesis will be tested by pursuing the following two specific aims: (1) Determine how basement membrane mechanical plasticity (malleability) mediates collective cell invasion; (2) Determine how mechanical plasticity (malleability) of type-1 collagen rich matrices mediates collective migration of breast cancer cells. This approach is innovative because of its focus on understanding the role of malleability in mediating protease-independent and -dependent invasion and migration, as malleability is a physical characteristic of ECM, related to matrix viscosity but distinct from elasticity or density, which has been largely ignored in studies to date. This work is also innovative in its focus on collective invasion of the basement membrane instead of single cell invasion. The ...