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Obesity is a known risk factor for the development and prognosis of breast cancer. Adipocytes have been identified as a source of exogenous lipids in other cancer types and may similarly provide energy to fuel malignant survival and growth in breast cancer. This relationship is of particular relevance to plastic surgery, because many reconstructions after oncologic mastectomy achieve optimal aesthetics and durability using adjunctive autologous fat transfer (AFT). Despite the increasing ubiquity and promise of AFT, many unanswered questions remain, including safety in the setting of breast cancer. Clinical studies to examine this question are underway, but an in vitro system is critical to elucidate the complex interplay between the cells that normally reside at the surgical recipient site. To study these interactions and characterize possible lipid transfer between adipocytes to breast cancer cells, we designed a 3-dimensional in vitro model using primary patient-derived tissues.Breast adipose tissue was acquired from patients undergoing breast reduction surgery. The tissue was enzymatically digested and sorted to retrieve adipocytes and adipose stromal cells. Polydimethylsiloxane wells were filled with type I collagen-encapsulated adipocytes labeled with the fluorescent lipid dye boron dipyrromethene, as well as unlabeled adipose stromal cells. A monolayer of red fluorescently labeled MDA-MB-231 and MDA-MB-468 breast cancer cells was seeded on the surface of the construct. Lipid transfer at the interface between adipocytes and breast cancer cells was analyzed.Confocal microscopy revealed a dense culture of native adipocytes containing fluorescent lipid droplets in the 3-dimensional collagen culture platform. RFP-positive breast cancer cells were found in close proximity to lipid-laden adipocytes. Lipid transfer from adipocytes to breast cancer cells was observed by the presence of boron dipyrromethene–positive lipid droplets within RFP-labeled breast cancer cells.We have established a 3-dimensional model to study complex breast cancer–adipose tissue interactions. Direct transfer of fluorescently labeled lipids from adipocytes to breast cancer cells may indicate aberrant metabolism to fuel malignant growth and adaptive survival. Our novel platform can untangle the complex interplay within the breast cancer tumor microenvironment for high-throughput analysis and better elucidate the safety of AFT in postoncologic mastectomy.