Abstract
BackgroundThree-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.MethodsMCF-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.ResultsMCF-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.ConclusionsMCF-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.
Highlights
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
MCF7 cells were grown in DMEM supplemented with 5% fetal bovine serum (FBS)
The epithelial cells were mononuclear with a well-defined nucleolus and nuclear size varied between cells within the epithelial plaques
Summary
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. A number of immortalized mouse and tumorigenic humanderived mammary epithelial cell lines that express estrogen receptor alpha (ER) and display hormone sensitivity have been established and used for such a purpose; they include HC11 (mouse), MCF7, BT-474 ZR-75-B, MDAMB-361 and T47D [8, 9]. Cells of the latter established line have recently been shown to form normal-like structures in 3D culture in response to mammotropic hormones. While searching for a model that would closely resemble the human mammary tissue, we chose to test MCF-12A cells, a human mammary cell line allegedly reported to be non-tumorigenic and estrogen-responsive
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