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

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.

Keywords: breast cancer; breast feeding; estrogen receptor; lactation; metabolomics; nuclear receptor; organoids; steroid.

Figure 1

Isolation and purification of mouse mammary epithelial cells. (A) Schematic representation of the isolation and purification protocols to obtain mouse mammary epithelial cells. (B) Number of mammary epithelial cells obtained before and after isolation and purification (between steps 5 and 6 in A). Results are shown as mean ± SEM of 11 independent experiments. (C) Distribution of mammary epithelial cells before (left) and after (right) purification according to their CD24 and CD49f expression. The mammary colony-forming cell (Ma-CFC) fraction was defined as the CD24^High; CD49f^Low and the mammary repopulating units (MRU) fraction as the CD24^Low; CD49f^High. Numbers are percentages. One representative experiment out of three independent experiments is shown.

Figure 2

High enrichment of mammary epithelial cell for primary culture in two-dimensions. (A) Brightfield images of mammary epithelial cells obtained after purification compared with the cells eliminated during the purification process. Scale bars = 300 μm. (B) Western blot analysis of purified cells normally discarded with differential plating (DP) compared to purified epithelial cells (Pur). Protein expression of the cytokeratin 8 and 18 (CK8/18), a marker of epithelial cells, and vimentin, a marker of fibroblasts, after three and six days in two-dimensional culture. S6 was used as the loading control. (C) Immunofluorescence showing the expression of CK8/18 (green) and vimentin (red) at three or six days in two-dimensional culture. Nuclei were stained with DAPI (blue). Scale bars = 300 μm and 75 μm. (D) Ratios of positive cells for CK8/18 or vimentin per the total number of cells (counts of nuclei) in percentage. Data are shown as mean ± SEM of one representative experiment (n = 6 images per condition). **p < 0.01; ***p < 0.001.

Figure 3

Ex vivo mammary epithelial cell organoids recapitulate the in vivo architecture of the mammary gland. (A) Brightfield visualization of organoids over 20 days in three-dimensional culture. At 10 days, spheroid-like organoids can be observed along with the beginning of lobular structures (black arrow). (B) Contractile structures are indicated by black arrows (see videos in Supplemental Figure S1 to observe contractions). (C) Diameter measurements of organoids over time (45 organoids measured/day). (D) Number of visible organoids through time in three-dimensional culture. (E) Brightfield visualization of lobular organoids after 10 and 20 days in three-dimensional culture. (F) Percentage of luminal, opaque, and lobular organoids over time. For (C, D, F), results are shown as mean ± SEM of one out of three independent experiments. Scale bars = 300 μm for (A, B, E).

Figure 4

Primary mammary epithelial cells in two- and three-dimensions express ERα. (A) Western blot analysis of ERα protein expression in mammary epithelial cells in two-dimensional culture after 3 and 6 days in culture with E₂ or vehicle. MCF7 human breast cancer cells and MCF10A human immortalized mammary epithelial cells were used as positive and negative controls for ERα protein expression, respectively. (B) qRT-PCR analysis of genes regulated by estrogens in two-dimensional culture following a 24 h treatment with E₂ or vehicle. (C) Western blot analysis of ERα protein expression in mammary organoids after 12 days in culture. MCF7 human breast cancer cells and MCF10A human immortalized mammary epithelial cells were used as positive and negative controls for ERα protein expression, respectively. (D) qRT-PCR analysis of genes regulated by estrogens in primary mammary gland organoids following 24 h or 10 d of treatment with E₂ or vehicle. For (B, D), results are shown as mean ± SEM of three independent experiments performed at least in duplicate. (E) Percentage of luminal, opaque, and lobular organoids over time, maintained in culture with and without E₂. Results are shown as the mean ± SEM of one out of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. (F) Number of visible organoids treated with E₂ or vehicle. Results are shown as the mean ± SEM of two independent experiments performed in triplicate.

Figure 5

Reprogramming of mammary epithelial cell metabolism in two-dimensions by estrogens. (A) Extracellular flux analysis of mammary epithelial cells using an XFe96 Seahorse apparatus. Oxygen consumption rate (OCR), an indicator of mitochondrial respiration, and the extracellular acidification rate (ECAR), an indicator of aerobic glycolysis, are shown normalized to their respective controls. (n= 3 biologically independent samples). Metabolic flexibility of OCR (B) and ECAR (C) after injections of mitochondrial modulators. Dashed lines indicate when in the assay the different drugs were injected and followed by the metabolic response. Oligo, oligomycin; R, rotenone; AA, antimycin. (A) Results are shown as the mean ± SEM of one out of four independent experiments, each with 4-5 biological replicates per group. All experiments were performed in primary mammary epithelial cells in two-dimensions after 3 days in culture and 48 h of treatment with estradiol or vehicle. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 6

Reprogramming of cell metabolism by estrogens in mouse mammary organoids. After 12 days in culture, with media changed every 72h, organoids and extracellular media were harvested for GC-MS targeted metabolomics to measure lactate (A), TCA cycle intermediates (B), secreted succinate (C), and other metabolite levels in organoids (D) or in the extracellular media (E). Organoids were either treated with E₂ or vehicle. All results are shown as the mean ± SEM of two independent experiments performed in triplicate. *p < 0.1; **p < 0.05; ***p < 0.001.