PROJECT SUMMARY We recently discovered an unexpected and intriguing role for white adipose tissue (WAT) in breast cancer. Our past studies identified that the mechanical properties of WAT extracellular matrix (ECM) regulate tumor cell invasion, a key rate-limiting step of metastasis, and that these properties are altered in obesity, contributing to the increased prevalence and worse prognosis of breast cancer in obese patients. Now, recent preliminary data from our labs additionally suggest that adipocyte mechanical properties may be similarly important. However, how adipocyte mechanics change with obesity and which effect these changes have on ECM remodeling and tumor invasion remains largely unclear. Understanding these connections is important for several reasons: First, while the biochemical functions of WAT are widely known to contribute to the pathogenesis of breast cancer, the influence of WAT mechanical properties on breast cancer invasion is largely unexplored. Second, our preliminary data suggest that aberrant remodeling of WAT in obese individuals promotes breast cancer invasion due to adipocyte lipid loss, transdifferentiation into myofibroblasts, and consequential changes in ECM deposition all of which affect WAT mechanics. Last, our preliminary results also indicate that tumor-induced lipid loss may synergistically promote invasion by changing WAT mechanical properties and tumor cell metabolism. Elucidating how these parameters are interconnected will be critical to decrease breast cancer burden and requires computational methods to uncover how single-cell properties of adipocytes and tumor cells affect WAT mechanics and tumor cell invasion. Through three focused and complementary Specific Aims, the proposed work iteratively couples computational models of tumor cell invasion into WAT, materials characterization of adipocytes and ECM, engineered cell culture models, and transgenic mouse models that allow visualization and manipulation of WAT in the mammary gland. Furthermore, single cell and spatial RNA transcriptomics, coupled with advanced bioinformatics approaches and human specimens, will determine the associated molecular mechanisms and potential value to patient prognosis. In particular, we will (1) define WAT physical properties in the breast as a function of obesity and determine their effect on tumor invasion, (2) determine the synergistic effect of tumor-induced lipid loss on WAT physical properties and tumor cell metabolism, and (3) establish the molecular basis of tumor-induced lipid loss in lean versus obese adipocytes and determine their effect on WAT physical properties and tumor invasion. These studies will identify specific obesity-dependent changes in WAT mechanical properties and their associated molecular mechanisms that will help predict the risk of breast cancer invasion for a given patient based on histological analysis.