Quantification of Epithelial Cell Differentiation in Mammary Glands and Carcinomas from DMBA- and MNU-Exposed Rats

Abstract

Rat mammary carcinogenesis models have been used extensively to study breast cancer initiation, progression, prevention, and intervention. Nevertheless, quantitative molecular data on epithelial cell differentiation in mammary glands of untreated and carcinogen-exposed rats is limited. Here, we describe the characterization of rat mammary epithelial cells (RMECs) by multicolor flow cytometry using antibodies against cell surface proteins CD24, CD29, CD31, CD45, CD49f, CD61, Peanut Lectin, and Thy-1, intracellular proteins CK14, CK19, and FAK, along with phalloidin and Hoechst staining. We identified the luminal and basal/myoepithelial populations and actively dividing RMECs. In inbred rats susceptible to mammary carcinoma development, we quantified the changes in differentiation of the RMEC populations at 1, 2, and 4 weeks after exposure to mammary carcinogens DMBA and MNU. DMBA exposure did not alter the percentage of basal or luminal cells, but upregulated CD49f (Integrin α6) expression and increased cell cycle activity. MNU exposure resulted in a temporary disruption of the luminal/basal ratio and no CD49f upregulation. When comparing DMBA- or MNU-induced mammary carcinomas, the RMEC differentiation profiles are indistinguishable. The carcinomas compared with mammary glands from untreated rats, showed upregulation of CD29 (Integrin β1) and CD49f expression, increased FAK (focal adhesion kinase) activation especially in the CD29hi population, and decreased CD61 (Integrin β3) expression. This study provides quantitative insight into the protein expression phenotypes underlying RMEC differentiation. The results highlight distinct RMEC differentiation etiologies of DMBA and MNU exposure, while the resulting carcinomas have similar RMEC differentiation profiles. The methodology and data will enhance rat mammary carcinogenesis models in the study of the role of epithelial cell differentiation in breast cancer.

Analysis of Immune Cells from Human Mammary Ductal Epithelial Organoids Reveals Vδ2+ T Cells That Efficiently Target Breast Carcinoma Cells in the Presence of Bisphosphonate

“…1C). EpCAM expressing cells within the organoid preparations also expressed other markers associated with mammary gland-derived epithelial cells, including CD49f low/+ (25, 26), CD10 low (25, 26), CD24 + (24), CD29 + (24), and Muc-1 int/high (25) (Supplementary Fig. 1A–E).…”

“…1B). Cells that were double negative for both the hematopoietic linage marker CD45 and the endothelial marker platelet/endothelial cell adhesion molecule-1 (PECAM-1; CD31) (24), were assessed for expression of the epithelial cell adhesion molecule (EpCAM) (25, 26) to delineate epithelial content (Fig. 1B).…”

Congenic and functional analysis of rat mammary cancer susceptibility.

“…The isolation of single cell suspensions enriched for MECs was optimized from the protocol found in [210]. old rats that were administered DMBA at 50-55 days of age.…”

“…MEC extract was prepared as previously described [210]. For each rat, both abdominal and inguinal mammary glands without lymph nodes were excised and kept separately in GIBCO DMEM/F12 media (Invitrogen).…”

“…MEC extract was stained as previously described [210]. Single cell suspensions were stained using antirat CD24, CD29, CD31 CD45 and live/dead stain (BD Pharmingen) antibodies.…”