PROJECT SUMMARY Over 40% of adult women in the U.S. are obese and obesity will soon overtake smoking as the leading risk factor for cancer. For breast cancer, obese women demonstrate both a higher incidence and higher rate of cancer- related mortality compared to normal weight women. While many studies focus on potential systemic connections behind this observation, the breast is rich in white adipose tissue (WAT), which is highly remodeled in the context of obesity, and thus local tissue-resident cues must also be considered. At the cellular level, white adipocytes are the functional units of WAT and secrete extracellular vesicles (EVs) that promote tumor progression. Recent studies indicate that adipocyte-derived EVs contain lipids and other metabolites for fatty acid oxidation that modulate tumor cell metabolism to increase migration, proliferation, and chemoresistance. In obese individuals, adipocytes become hypertrophic with known consequences for metabolic disease. Whether adipocyte hypertrophy similarly impacts breast cancer risk and prognosis is less clear. Preliminary data in this proposal indicate that hypertrophic adipocytes promote the proliferation and migration of co-cultured tumor cells to a greater extent than donor-matched, non-hypertrophic control adipocytes. Moreover, I found that hypertrophic adipocytes secrete more EVs and exhibit remodeled cortical actin. Given that the actin cytoskeleton mediates the biogenesis of EVs by other cell types, this proposal aims to investigate if hypertrophy, remodeled cortical actin, and increased EV secretion are interconnected. Moreover, the proposed research also aims to discern if these differences impact breast cancer progression by altering tumor cell metabolism. In Specific Aim 1, I will characterize the concentration, size distribution, and cargo of EVs released by hypertrophic vs. control adipocytes via nanoparticle tracking analysis and mass spectrometry. In Specific Aim 2, I will expose breast cancer cells to EVs secreted by hypertrophic vs. control adipocytes to assess how treatment impacts tumor cell behavior in vitro and in vivo. Moreover, I will perform pharmacological and genetic inhibitor studies to determine if altered fatty acid oxidation underpins any observed differences in tumor cell phenotypes. Collectively, this work will help discern if hypertrophic adipocytes constitute a distinct subpopulation of cells conducive to tumor progression and thus contribute to the poor prognosis of obesity-associated breast cancer. In the clinic, identified molecular mechanisms between adipocytes and tumor cells could be targeted therapeutically and the degree of mammary adipocyte hypertrophy could serve as a prognostic biomarker for patient outcomes. Beyond research, I will develop skills around experimental design, data analysis, mentorship, and science communication through my training goals and team of mentors outlined in this proposal. These skills will be essential to achieve my long...