Abstract
Few in vitro models are used to study mammary epithelial cells (MECs), and most of these do not express the estrogen receptor α (ERα). 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 ERα 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 ERα 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.
Highlights
The mammary gland is known to be highly sensitive to sexsteroid hormones such as estradiol (E2), the most potent endogenous estrogen
We describe in detail a fluorescence-activated cell sorting (FACS)-free protocol to purify and culture epithelial cells from the mouse mammary gland
This protocol can be used to obtain cells for primary culture in both twoand three-dimensions, in which they can form complex organoid structures that recapitulate the mammary gland structure in vivo. We demonstrate that these mammary epithelial cells (MECs) retain estrogen receptor a (ERa) expression, allowing ex vivo studies of this critical hormonal signaling pathway in the mammary gland
Summary
The mammary gland is known to be highly sensitive to sexsteroid hormones such as estradiol (E2), the most potent endogenous estrogen. The mammary gland must sustain milk production and is a highly active metabolic tissue [1]. Mammary luminal epithelial cells produce and secrete milk, which consists of water, proteins, lipids, and carbohydrates (lactose) that are secreted mainly as secretory vesicles. These epithelial cells uptake nutrients from the blood and metabolize them in a specific manner to produce the milk constituents. All the major milk proteins, such as caseins, are synthesized by mammary epithelial cells (MECs) from amino acids, with some exceptions such as serum albumin and immunoglobulins. During lactation, the mammary gland exhibits high amino acid uptake and metabolism linked to protein synthesis [1]. A specific metabolic program must be regulated in the mammary gland to sustain its specific functions during lactation
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