A FACS-Free Purification Method to Study Estrogen Signaling, Organoid Formation, and Metabolic Reprogramming in Mammary Epithelial Cells

Few in vitro models are used to study mammary epithelial cells (MECs), and most of these do not express the estrogen receptor alpha (ER alpha). Primary MECs can be used to overcome this issue, but methods to purify these cells generally require flow cytometry and fluorescence-activated cell sorting (FACS), which require specialized instruments and expertise. Herein, we present in detail a FACS-free protocol for purification and primary culture of mouse MECs. These MECs remain differentiated for up to six days with >85% luminal epithelial cells in two-dimensional culture. When seeded in Matrigel, they form organoids that recapitulate the mammary gland's morphology in vivo by developing lumens, contractile cells, and lobular structures. MECs express a functional ER alpha signaling pathway in both two- and three-dimensional cell culture, as shown at the mRNA and protein levels and by the phenotypic characterization. Extracellular metabolic flux analysis showed that estrogens induce a metabolic switch favoring aerobic glycolysis over mitochondrial respiration in MECs grown in two-dimensions, a phenomenon known as the Warburg effect. We also performed mass spectrometry (MS)-based metabolomics in organoids. Estrogens altered the levels of metabolites from various pathways, including aerobic glycolysis, citric acid cycle, urea cycle, and amino acid metabolism, demonstrating that ER alpha reprograms cell metabolism in mammary organoids. Overall, we have optimized mouse MEC isolation and purification for two- and three-dimensional cultures. This model represents a valuable tool to study how estrogens modulate mammary gland biology, and particularly how these hormones reprogram metabolism during lactation and breast carcinogenesis.

Automated summary

This study introduces a FACS-free protocol for purification and primary culture of mouse mammary epithelial cells (MECs), which maintain high purity and form organoids resembling mammary gland structures in vitro. The MECs express functional ER alpha signaling pathway and exhibit a metabolic switch favoring aerobic glycolysis in response to estrogens, providing insights into how these hormones reprogram metabolism during lactation and breast carcinogenesis.