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. 2018 Mar 20:18:43.
doi: 10.1186/s12935-018-0534-y. eCollection 2018.

Characterization of MCF-12A cell phenotype, response to estrogens, and growth in 3D

Affiliations

Characterization of MCF-12A cell phenotype, response to estrogens, and growth in 3D

Michael F Sweeney et al. Cancer Cell Int. .

Abstract

Background: Three-dimensional cultures of mammary epithelial cells allow for biologically-relevant studies of the development of the mammary gland in rodents and humans under normal and pathological conditions, like carcinogenesis. Under these conditions, mammotropic hormones play significant roles in tissue morphogenesis. Therefore, a system that recreates the normal, hormonally responsive epithelium would be a valuable tool to study the normal state and its transition to carcinogenesis. MCF-12A cells have been claimed to be non-tumorigenic mammary epithelial cells with reported sensitivity to estrogens. In this study, we aimed at characterizing MCF-12A cells for use in a hormone-responsive 3D culture system to determine their usefulness as a tool to identify normal and abnormal microenvironmental cues.

Methods: MCF-12A cells were single-cell cloned in order to investigate their heterogeneous makeup. The parental cells were then treated with estradiol to investigate proliferative and transcriptional responses through the estrogen receptor alpha. Finally, parental cells and epithelial-like cell-derived clones were seeded in rat-tail collagen I to profile the morphogenesis of multicellular 3D structures. The resultant structures were then analyzed using unsupervised morphometric analysis.

Results: MCF-12A cells consist of epithelial-like colonies which shed elongated, freely growing cells on the colony's edges. The cells express E-cadherin as well as mesenchymal vimentin but do not express markers associated with myoepithelial cells or fibroblasts. Treatment with estradiol does not affect either the proliferation rate or the induction of gene expression in MCF-12A cells. Parental MCF-12A cells form acini, solid spheres and elongated branching ducts when grown in rat-tail collagen type I matrix, the geometries and distribution of which are altered following the removal of fibroblast-like cells.

Conclusions: MCF-12A cells are a heterogeneous pseudo-epithelial cell line capable of forming a variety of multicellular structures in 3D culture. We found no indication that the cells display estrogen-responsive characteristics, thus refuting previous studies which reported estrogen responsiveness. We report that MCF-12A cells are not suited for use in studies in which differential behaviors of "normal" and "cancerous" estrogen-responsive cells are to be compared.

Keywords: 3D culture; Estrogen response; Tissue morphogenesis.

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Figures

Fig. 1
Fig. 1
MCF-12A cells grow as a heterogeneous population. Parental cells grow as epithelial plaques surrounded by single fibroblast-like and spherical cells (a). Single cell cloning lead to the isolation of epithelial-like colonies (b) and a mixed population consisting of both fibroblast-like and spheroid cells (c). (Scale bar = 250 µm)
Fig. 2
Fig. 2
MCF-12A cells express epithelial and mesenchymal cell markers. E-cadherin is expressed throughout plaque-associated cells and highly localized to cell–cell junctions in the center of epithelial plaques. Localization at cell–cell junctions decreases at plaque edges. Cytokeratins are expressed in all MCF-12A subtypes. Beta-catenin is expressed by epithelial-like MCF-12A colonies with increased localization at their cell–cell junctions and dispersed throughout the cytoplasm of fibroblast-like and spheroid cells. Vimentin is expressed in the cytoplasm of all cells but concentrated in fan-like projections from peripheral cells. P63 is expressed in dividing cells but absent from others. MCF-10A cells used as a control for epithelial marker expression. Scale bar = 100 µm
Fig. 3
Fig. 3
MCF-12A cells do not respond to stimulation by estradiol. MCF-12A cells proliferate maximally under all experimental conditions while MCF7 cell proliferation is inhibited in the absence of estradiol and stimulated at increasing concentrations (a). Relative to MCF7 cells, MCF-12A cells do not express estrogen receptor alpha (b). The induction of estrogen responsive genes amphiregulin (AREG) and progesterone receptor (PGR) is not seen in MCF-12A (c) cells while estrogen receptor alpha-positive MCF7 cells show expression following estradiol exposure (d). Note differences in y-axis scales to accommodate variation in expression differences
Fig. 4
Fig. 4
3D growth of MCF-12A cells in rat-tail collagen after 14 days. Confocal projections of select structures (top row) and E-cadherin stained cross sections of corresponding structures (bottom row). MCF-12A cells form hollow (a) and solid acini (b) as well as solid bifurcating ducts (c) and lumenized branching ducts (d). Spherical clusters of concentric cells are often seen within elongated ductal structures (black arrow). Scale bar = 100 µm
Fig. 5
Fig. 5
Contraction of collagen gels after 14 days in culture. The addition of MCF-12A cells to rat-tail collagen gels results in a decreased gel diameter compared to acellular gels. MCF-12A-derived BF9 cells cause a similar extent of contraction. Asterisks signify gels that are significantly more contracted (p-value < 0.05) than acellular gels
Fig. 6
Fig. 6
Non-epithelial-like subpopulations alter the morphology of epithelial-like MCF-12A cells. Structures formed by the heterogeneous parental MCF-12A cells are more spherical (a), have shorter major radii (b), and are flatter (d) than those formed by primarily epithelial subclone BF9. Elongation (c) is not significantly different between the parental cells and BF9 cells. Structures were pooled from all technical and biological replicates for analysis (MCF-12A n = 1537, BF9 n = 1099). Error bars show SD. Asterisks denote p values < 0.05

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