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. Cancer cells are thought to utilize proteases to degrade the BM during invasion of the BM using specialized structures known as invadopodia. Known modes of protease-independent invasion and migration, involving cells squeezing through pores in the ECM, would be inhibited by the nanoporous nature of the BM. However, physiological ECM is viscoelastic, exhibiting some characteristics of viscous fluids, and cellular forces can induce flow and permanent deformation of the matrix. In other words, viscoelastic ECM is malleable, and cell generated forces may expand pores, providing a mechanism for cells to mechanically remodel the ECM and physically clear a path for migration, independent of proteases. While malleability is related to matrix viscosity, it is distinct from matrix elasticity. Interestingly, malignant breast lesions have been found to exhibit a greater degree of viscosity than benign lesions. Importantly, the concept of malleability might be relevant to protease-dependent migration as well, as the action of proteases may be to make the matrix more malleable. The specific hypothesis to be tested in this application is that malleability is a key physical parameter of the BM that mediates protease-dependent and protease-independent cancer cell invasion and migration. This hypothesis is supported by preliminary studies finding that cancer cells can invade and migrate through nanoporous matrices that contain BM ligands with intermediate or high-malleability in a protease- independent manner, utilizing invadopodial like protrusions to initiate invasion, but are unable to invade and migrate through matrices with low malleability. This hypothesis will be tested by pursuing the following three specific aims: (1) Fabricate materials for 3D cell culture with independently tunable malleability that present ligands and stiffness relevant to the BM of mammary epithelium; (2) Determine how ECM malleability regulates invadopodial protrusions; and (3)! Identify molecular and biophysical mechanisms underlying protease- independent migration through ECMs with different levels of malleability. 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. The proposed research is significant because it will reveal the role of ECM malleability in mediating both protease-dependent and protease-independent invasion and migration by breast cancer cells, potentially uncovering previously un- described modes of invasion or migration.

PROJECT NARRATIVE The proposed research is relevant to public health because invasion and metastasis during breast cancer progression are associated with increased mortality, yet the biophysical mechanisms underlying these two processes are not well understood. Therefore, the proposed research on understanding how extracellular matrix malleability regulates invasion and migration of breast cancer cells is relevant to the part of NIH's mission that seeks to develop fundamental knowledge that will help reduce the burdens of human disability and disease. Ultimately, it is anticipated that this study will uncover previously un-described modes of invasion and migration, thereby leading to new diagnostic measures of pre-invasive breast cancer and new therapeutic strategies to block invasion and metastasis.