ABSTRACT Although many women with estrogen receptor positive (ER+) primary cancers are successfully treated with surgery and adjuvant anti-estrogen therapies, metastatic therapy-resistant ER+ cancers account for the majority of breast cancer related deaths. New understanding of the biology underlying disease processes is required to develop new approaches. In this renewal application, we build on our previous findings demonstrating dynamic reciprocity between estrogen and features of the extracellular matrix (ECM) in the microenvironments of both the primary tumor and the metastatic niche which fuel the pulmonary metastatic burden. We showed that estrogen remodels the ECM architecture of the primary tumor, aligning collagen fibers and increasing synthesis of ECM components associated with more aggressive cancers, and alters ECM components in the lung metastatic niche. Moreover, we showed that this collagen alignment signature in patient tumors correlates with inflammatory markers, including COX-2 and CD163+ macrophages, and poor prognosis. In the current proposal, we hypothesize that estrogen orchestrates synergy among ER+ tumor cells, the extracellular matrix (ECM)/ cancer associated fibroblasts (CAFs), and macrophages/ inflammation, to fuel primary and metastatic tumor aggression. We will utilize our robust immunocompetent in vivo model of metastatic ER+ breast cancer, in vitro systems, PDX models and clinical patient samples to test this hypothesis in the following aims. Aim 1: Identify the steps in metastatic progression of ER+ breast cancer which are altered by estrogen activity in tumor cells as well as other estrogen targets. Aim 2: Determine how estrogen action on CAFs modifies the stromal ECM to mediate tumor progression, determine the receptors that mediate this action, and identify ECM signatures from native stromal matrix from ER+ invasive ductal carcinoma biopsies that instruct estrogen action on tumor cell growth and migration. Aim 3: Determine how estrogen impacts immune metastatic mediators, including macrophage activity and the inflammatory tumor microenvironment. Aim 4: Evaluate the ability of standard of care therapeutic approaches to reverse E2-promoted steps in metastasis and post- treatment estrogen re-exposure to promote growth of residual pulmonary lesions, and interrogate the interrelationships among E2-altered ECM structure, and macrophage activity/inflammation. Our studies will illuminate the role of estrogen in dissemination and pulmonary metastatic colonization, with implications for therapy, metastatic dormancy and recurrence of ER+ breast cancer, and reveal potential sites for intervention.