Evidence that an early pregnancy causes a persistent decrease in the number of functional mammary epithelial stem cells--implications for pregnancy-induced protection against breast cancer

Abstract

A completed pregnancy at a young age reduces a woman's lifetime risk of breast cancer by up to 50%. A similar protective effect of an early pregnancy has been observed in rodent models using chemical carcinogens. However, the mechanisms responsible for this protective effect remain unclear. Stem cells have been proposed to be the cells of origin for breast cancer. We hypothesized that an early pregnancy reduces adult levels of either mammary stem cells or mammary multipotent progenitor cells. Unsorted mammary cells from adult mice that had undergone an early parity had the same mammosphere formation efficiency as cells from age-matched virgin mice. However, when we transplanted adult mammary cells in limiting dilutions into cleared fat pads of syngeneic mice, we found a significant reduction in the outgrowth potential of the cells from early parous mice compared with age-matched virgin mice. The extent of fat pad filling in successful outgrowths did not change, suggesting that although mammary stem cells in parous mice retained their functional competence, the number of mammary stem cells was reduced. Our results provide the first direct evidence that an early pregnancy has an effect on mammary stem cells.

Figure 1. An early pregnancy does not induce a persistent change in total mammary cells or epithelial content, and does not cause a persistent change in mammosphere forming cells

(A) Single cell suspensions were made from the pooled #2–4 mammary glands of each mouse, and the total number of viable cells recovered from each mouse was determined by FACS and hemocytometry. n=19 (virgin) or 20 (parous). (B) Single cell suspensions made from parous and age-matched virgin mouse mammary glands were spotted on slides and probed by immunofluorescence for luminal (keratin 8) and myoepithelial (keratin 5) markers. n=3. (C) Mammary cells from early parous or age-matched virgin mice were plated at 10,000 cells/well under ultra-low adherent culture conditions. Sample mammospheres that formed after 10 days in culture are shown. Scale bar = 50 μm. Primary mammospheres were fixed, sectioned, and stained for myoepithelial (keratin 5, left, arrows) and luminal (keratin 8, center) cell markers. All mammospheres contained keratin 8⁺ cells; approximately one in three also contained keratin 5⁺ cells. Scale bar = 10 μm. (D) Primary mammospheres larger than 50 μm were quantitated at 10 days, dissociated to single cells (by digestion in 0.05% trypsin EDTA followed by pipetting), and replated at 5000 cells/well to measure secondary mammosphere formation. The number of mammospheres formed as a percentage of cells plated is shown. n=9 (primary mammospheres) or 5 (secondary mammospheres).

Figure 2. An early pregnancy reduces the number of mammary stem cells

(A) The indicated number of mammary cells isolated from parous (n=11) or age-matched virgin mice (n=11) was injected into the cleared #4 fat pads of recipient mice. The number of successful outgrowths (>5% fat pad-filling) after 8 weeks and total number of transplants performed are shown. These data were analyzed by using Generalized Estimating Equations in a Generalized Linear Model with a logit link function (PROC GENMOD, SAS V9.1, Cary, NC). This ANOVA-like analysis, which tests for overall effects of parity while accounting for dilution and the paired nature of the outgrowth data, found that an early parity significantly reduced the take rate compared to virgin (p=0.017). Limiting dilution analysis was conducted to estimate the frequency of mammary stem cells per total mammary cells by fitting the single-hit Poisson model (SHPM) to the limiting-dilution data from (A) using a complementary log-log generalized linear model. The fit of the model was checked using the method proposed by Bonnefoix et al . (B) Regression lines of the estimated outgrowth frequency and 95% confidence intervals are graphed. The Wald confidence intervals were calculated via delta method for the frequency of regenerative stem cells. Limiting-dilution statistical analyses were performed using the limdil function in the Statmod package in the software R . (C) The extent of the cleared fat pad filled by outgrowths in (A) is shown.