Extracellular Matrix Context Shapes Morphogenesis and Lactation-Associated Programs in Human Milk-Derived Mammary Organoids

The mammary gland relies on reciprocal interactions between epithelial cells and their surrounding extracellular matrix (ECM) to form and maintain milk-producing tissue structures. Yet these processes remain difficult to study in human model systems. Mammary epithelial cells (MECs) can be isolated noninvasively from breast milk, but whether they generate three-dimensional organoids and respond to matrix cues has been unclear. Here, human milk-derived MECs (milk MECs) spontaneously form complex organoids, including polarized acinar and terminal duct lobular unit-like structures after isolation. To investigate how matrix composition shapes these organoids, milk MECs were cultured in decellularized mammary ECM (dECM), Matrigel, and collagen I. Mammary dECM and collagen I both promote branching and actin alignment through collagen fibrils, but mammary dECM better preserves luminal-basal epithelial organization. Comparative proteomic analysis reveals that bovine and human dECM are rich not only in fibrillar collagens but retain additional ECM components such as basement membrane associated proteins and proteoglycans. Breast dECM is further supportive of estrogen receptor-related and cell-cycle pathways in branched milk MEC organoids. In contrast, Matrigel favors an acinar organoid morphology and a more secretory phenotype, with β-casein expression and enrichment of lactation-associated transcriptional programs. Together, these findings establish breast milk-derived MEC organoids as a human model to study how ECM architecture and composition regulate mammary morphogenesis as well as functional state, and demonstrate that ECM context instructs organoid geometry and secretory differentiation.