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. 2014 Jul 24;16(4):411.
doi: 10.1186/s13058-014-0411-0.

The Influence of Tamoxifen on Normal Mouse Mammary Gland Homeostasis

Free PMC article

The Influence of Tamoxifen on Normal Mouse Mammary Gland Homeostasis

Mona Shehata et al. Breast Cancer Res. .
Free PMC article

Abstract

Introduction: Lineage tracing using inducible genetic labeling has emerged to be a powerful method for interrogating the developmental fate of cells in intact tissues. A common induction mechanism is the use of tamoxifen-dependent Cre recombinase (CreER and CreERT2), but the effects of tamoxifen at doses normally used in lineage-tracing studies on normal adult mammary gland homeostasis are not known.

Methods: We used flow cytometry and immunostaining of intact glands to determine whether varying doses of tamoxifen skew the distribution and the apoptosis and proliferation status of different types of mammary epithelial cells in vivo. We also examined how tamoxifen influences the number of progenitor and mammary repopulating units (MRUs).

Results: Our results indicate that ≥5 mg/25 g body weight of tamoxifen induces a transient increase in cell proliferation and in the number of basal cells in the adult mammary epithelium up to 7 days after tamoxifen administration. However, in the medium term (3 weeks), all doses of tamoxifen≥1 mg/25 g body weight result in a decrease in the number of basal and EpCAM+CD49b- luminal cells and a decrease in progenitor cell function. Tamoxifen at doses≥5 mg/25 g body weight induced a transient increase in caspase-3-mediated apoptotic cell death within the mammary epithelium. However, mammary epithelial cell numbers in all subpopulations were restored to their original levels by 8 weeks. No long-lasting effects of tamoxifen on MRU numbers or on pubertal ductal development were observed.

Conclusion: Tamoxifen can skew the distribution of mammary cell types in a dose-dependent manner, and thus caution must be taken when interpreting lineage-tracing studies using high doses of tamoxifen, particularly when short-duration analyses of a quantitative nature are being performed.

Figures

Figure 1
Figure 1
Tamoxifen temporarily retards ductal elongation during pubertal mammary gland development. (A) Bar graph depicting the quantification of mammary gland outgrowth, showing relative elongation of the inguinal mammary gland between P23 and P43 in mice treated with sunflower oil (n = 4), 1 mg/25 g of tamoxifen (n = 4), or 4 mg/25 g of tamoxifen (n = 4). The position of the most distal tip of the lymph node is set at zero. (*P < 0.05, **P < 0.01). (B) Whole-mount preparations of carmine alum-stained mammary epithelium, showing representative images of the inguinal glands quantified in panel (A). Arrows indicate the relative outgrowth of the epithelium. Scale bar = 500 μm. (C) Close-up of the glands depicted in (B), showing loss of terminal-end bud structures in mice treated with 4 mg/25 g tamoxifen. Scale bar = 20 μm. (D) Whole-mount carmine alum staining of thoracic glands. Scale bar = 500 μm. (E) Close-up of thoracic glands depicted in (D). Scale bar = 20 μm.
Figure 2
Figure 2
High doses of tamoxifen can skew the relative distribution of mammary cell populations over time. Absolute number of different types of mammary cells per pair of inguinal glands treated with varying doses of tamoxifen and analyzed at (A) 1 and 2 or (B) 3, 7, 21, and 56 days after tamoxifen injection (*P < 0.05; **P < 0.01; ***P < 0.0001). (C) Representative flow-cytometry dot plots show the expression of EpCAM, CD49f, Sca1, and CD49b among mammary cells from control mice and mice treated with 3 × 5 mg tamoxifen and analyzed 21 (left panels) or 56 days (right panels) later.
Figure 3
Figure 3
Tamoxifen can decrease the number of CFCs in the mammary epithelium. (A) Cloning efficiency and (B) total number of CFCs per pair of inguinal mammary glands in mice treated with varying doses of tamoxifen and analyzed at 3, 7, and 21 days after treatment. (*P < 0.05; **P < 0.01; ***P < 0.0001); n = 5 to 11 for all experiments, except for 0.2-mg dose, where n = 3 to 4.
Figure 4
Figure 4
Tamoxifen can induce short-term proliferation of mammary epithelial cells. (A) Ki-67-immunostained sections of mammary glands from oil- and tamoxifen-treated mice at selected time points for 10–week- and 14-week-old treated mice. Scale bars = 50 μm. (B) Frequency of Ki-67+ cells in mammary glands of mice treated with oil or varying doses of tamoxifen, and analyzed at multiple time points. (C) Representative immunofluorescent sections of mammary glands from oil- and tamoxifen-treated glands at time points Days 3 and 7. Shown is the expression of keratin 5 (yellow), ER (red), and Ki-67 (green). Nuclei were visualized with DAPI. Scale bar = 25 μm. (D) Proportion of proliferating ER luminal cells, ER+ luminal cells, and basal cells in tamoxifen-treated glands. (*P < 0.05; **P < 0.01; ***P < 0.0001).
Figure 5
Figure 5
Tamoxifen can induce slight short-term apoptotic activity in mammary epithelial cells. (A) CC3 immunostained sections of mammary glands from oil- and tamoxifen-treated mice at selected time points. Arrows indicate CC3-positive cells. (B) Bar charts depicting the number of CC3+ cells per pair of oil- and tamoxifen-treated glands analyzed at 1, 2, 3, 7, and 21 days after treatment. (*P < 0.05; **P < 0.01; ***P < 0.0001).

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