Dissociation of estrogen receptor expression and in vivo stem cell activity in the mammary gland

Abstract

The role of estrogen in promoting mammary stem cell proliferation remains controversial. It is unclear if estrogen receptor (ER)-expressing cells have stem/progenitor activity themselves or if they act in a paracrine fashion to stimulate stem cell proliferation. We have used flow cytometry to prospectively isolate mouse mammary ER-expressing epithelial cells and shown, using analysis of gene expression patterns and cell type-specific markers, that they form a distinct luminal epithelial cell subpopulation that expresses not only the ER but also the progesterone and prolactin receptors. Furthermore, we have used an in vivo functional transplantation assay to directly demonstrate that the ER-expressing luminal epithelial subpopulation contains little in vivo stem cell activity. Rather, the mammary stem cell activity is found within the basal mammary epithelial cell population. Therefore, ER-expressing cells of the mammary epithelium are distinct from the mammary stem cell population, and the effects of estrogen on mammary stem cells are likely to be mediated indirectly. These results are important for our understanding of cellular responses to hormonal stimulation in the normal breast and in breast cancer.

Figure 1.

CD24, Sca-1, and prominin-1 expression define a distinct mammary epithelial cell compartment. (A) Flow cytometric staining patterns of freshly isolated mouse mammary cells stained for CD24 expression (left) and CD24 and Sca-1 expression (right). Only live, single CD45⁻cells were included in the analysis. (B) Flow cytometric staining patterns of freshly isolated mouse mammary cells triple stained for CD24, prominin-1, and Sca-1 expression. The CD24 and prominin-1 staining pattern is shown on the left, and the Sca-1 and prominin-1 staining pattern of the CD24^High fraction is shown only on the right. (C) Analysis of cytoskeletal antigen expression in cells separated by CD24 and prominin-1 expression. Results of three independent cell isolations in which freshly isolated cells were sorted onto slides and single stained for CK14 or CK18. Numbers represent the percentage of positive cells ± the SD for three experiments. The total numbers of cells observed are given below the percentages. Intensity of CK18 staining is indicated by CK18⁺ (weak) and CK18⁺⁺ (strong).

Figure 2.

qPCR analysis identifies a hormone receptor–expressing cell population. (A) Cytoskeletal genes. ¹A P value could not be determined for CD24^High/prominin-1⁺ with CK14, as all three replicates gave a value of 0. ²A P value could not be determined for CD24^High/prominin-1⁻ or CD24^High/prominin-1⁺ with CK5, as in both cases two of the three samples gave a value of 0. (B) Hormone response genes. (C) Milk component genes. (D) Miscellaneous genes. Each data point on each graph is a mean ± the SD of fold-relative expression of the gene in three independently isolated samples of the population of interest compared with total mammary epithelial cells. The fold-relative expression for each sample is itself a mean of two independent cDNA syntheses performed on that sample. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3.

Prominin-1⁺ cells are ERα⁺. (A) Frozen section of adult mouse mammary gland stained with anti–prominin-1, anti-ERα, and a nuclear counterstain (TO-PRO-3). The majority of cells with nuclear ERα staining (red; arrows) also show apical prominin-1 staining (green; arrowheads). Heavy background staining in the stroma is caused by the Alexa Fluor 555 antibody used to detect the ERα. (B) To quantify prominin-1/ERα double staining, CD24^Low, CD24^High/prominin-1⁻, and CD24^High/prominin-1⁺ cells were sorted onto slides and stained for nuclear ERα expression. The histogram indicates the percentage of ERα⁺ cells in each population from analysis of three independent sorts and the total numbers of cells counted (n). Examples of cells from the three populations are shown below the corresponding populations. Nuclear ERα staining (red) can only be seen in the CD24^High/prominin-1⁺ population. Significant differences between the populations were determined using a t test of log10-transformed data. NS, not significant. Bars, 30 μm.

Figure 4.

CD24^High/prominin-1⁻ mammary epithelial cells are enriched for in vitro CFCs. (A) Bar chart indicating colony-forming efficiencies of single cells sorted into individual wells of 96-well plates from CD24^Low (fifteen 96-well plates from four sorts), CD24^High/prominin-1⁻ (17 96-well plates from 4 sorts), and CD24^High/prominin-1⁺ (17 96-well plates from 4 sorts) populations. Data are the mean percentage cloning efficiencies ± the SD. CFCs, mammary colony forming cells. (B–D) Immunophenotyping of 8–10-d-old colonies cultured on glass coverslips derived from CD24^Low (B), CD24^High/prominin-1⁻ (C), and CD24^High/prominin-1⁺ (D) cells. Stained for CK14 (red) and CK18 (green) expression and with DAPI (white) to distinguish nuclei. Bar, 150 μm.

Figure 5.

Functional assays identify in vivo stem/progenitor activity in CD24^Low epithelial cells. (A) Results of cleared fat pad transplantation of CD24^Low, CD24^High/Sca-1⁻, CD24^High/Sca-1⁺, CD24^High/prominin-1⁻, and CD24^High/prominin-1⁺ cells. Freshly isolated sorted cells were transplanted at dilutions ranging from 20,000 to 1,000 cells per fat pad. Results from transplants of double-sorted populations are indicated by “DS.” (B) Results of transplantation of 200 mammary colony forming cells (MaCFCs), myoepithelial cells (MYOs), or mammary repopulating cells (MRUs) isolated by CD24 and CD49f staining. For both datasets, the number of successful outgrowths and the number of fat pads transplanted for each population are indicated. Also shown are graphic indications of the extent to which each transplant filled the fat pad. ND, not determined. (C–J) Wholemounts and sections through representative 100% (C–F) and <25% (G–J) transplants. (C, D, G, and H) Carmine-stained wholemounts. (E, F, I, and J) Sections through transplant outgrowths stained for SMA to detect myoepithelial cells (E and I; arrows) or for ERα to detect ER⁺ cells (F and J; arrowheads). Bars: (C and G) 6 mm; (D and H) 2 mm; (E, F, I, and J) 40 μm.