Most breast cancer (BC) deaths are caused by metastatic estrogen receptor positive (ER+) BC, which currently remains incurable. Cancer cell invasion in the primary tumor is a crucial step in metastatic dissemination of BC and is influenced by biochemical and biophysical properties of the tumor microenvironment (TME). The overall goal of this project is to investigate the distinct role of matrix stiffness (mechanical tumor microenvironment) in regulating durotactic invasion of breast tumors in vivo in the context of ER status and EVL expression. Our prelimary data show the following: Stiffness-associated gene expression predicts poor outcomes for ER+, but not ER-, BC patients; matrix stiffness gradients increase durotactic migration of ER+, but not ER-, BC cells; and ER transcriptional target, EVL, is critical for adhesion strengthening on stiff matrix and for mechanosensing of stiffness gradients. Based on these data we hypothesize that matrix stiffness in primary breast tumors promotes durotactic invasion of ER+ breast cancer via EVL-mediated adhesion. We will test this hypothesis in two Specific Aims (SAs): SA1) Determine whether ER promotes durotactic invasion via EVL in ER+ breast tumors; and SA2) determine that targeting matrix stiffness suppresses invasion of ER+ breast tumors. In our approach, we are combining the following experimental systems: a) Mammary INtra-Ductal (MIND) xenograft mouse models, which allows development of human-like tumors and examination of invasion progression; b) advanced two-photon Bessel Light Sheet (2pBLS) instrument, which allows live-cell deep tissue imaging at single-cell resolution; and c) Mammary dome-shaped window chamber, which is optimized for 2pBLS intravital imaging of the MIND model and allows manipulation of the mechanical tumor microenvironment. We are using multiple culture model systems and one patient-derived xenograft model. This work will contribute to our understanding of how the mechanical properties of the microenvironment impact ER+ BC invasion progression and establish the importance of considering BC subtype when designing therapies that target the microenvironment. Further, this work will provide an evidence-based foundation for future studies focused on novel therapies for ER+ BC that target the tumor microenvironment, using anti-fibrotic drugs in combination with anti-estrogenic therapies.