Elsevier

Methods

Volume 30, Issue 3, July 2003, Pages 256-268
Methods

Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures

https://doi.org/10.1016/S1046-2023(03)00032-XGet rights and content

Abstract

The three-dimensional culture of MCF-10A mammary epithelial cells on a reconstituted basement membrane results in formation of polarized, growth-arrested acini-like spheroids that recapitulate several aspects of glandular architecture in vivo. Oncogenes introduced into MCF-10A cells disrupt this morphogenetic process, and elicit distinct morphological phenotypes. Recent studies analyzing the mechanistic basis for phenotypic heterogeneity observed among different oncogenes (e.g., ErbB2, cyclin D1) have illustrated the utility of this three-dimensional culture system in modeling the biological activities of cancer genes, particularly with regard to their ability to disrupt epithelial architecture during the early aspects of carcinoma formation. Here we provide a collection of protocols to culture MCF-10A cells, to establish stable pools expressing a gene of interest via retroviral infection, as well as to grow and analyze MCF-10A cells in three-dimensional basement membrane culture.

Introduction

Glandular epithelial cells, such as those in the mammary gland, have several distinguishing histological features including a polarized morphology, specialized cell–cell contacts, and attachment to an underlying basement membrane (Fig. 1A). The development and maintenance of this polarized structure are critical for the form and function of epithelial cells [1]. Moreover, the pathogenesis of epithelial tumors, termed carcinomas, requires the disruption of this intact, well-ordered architecture.

Studies of primary human tumor tissue and mouse models of epithelial tumors have provided information on the genetic events involved in carcinoma formation. Although these approaches have been critical for understanding epithelial tumors, they are relatively intractable for studying the biochemical and cell biological pathways involved in tumor formation, especially the mechanisms responsible for early oncogenesis. In contrast, the molecular underpinnings of the signaling machinery involved in oncogenic transformation have been studied primarily in fibroblasts and, to a lesser extent, epithelial cells cultured as monolayers on tissue culture plastic. However, because monolayer cells do not recapitulate the glandular structure of epithelium in vivo, they also do not provide the optimal system for fully understanding how the regulation of proliferation, cell death, and differentiation influence the form and function of glandular epithelium, both in the normal state and during early tumor formation [1], [2].

Two imminent questions that remain unanswered by traditional methods are: (1) What are the mechanisms and signaling pathways involved in organizing individual cells into a well-ordered polarized glandular architecture? (2) Does disruption of these pathways influence the architectural progression of cancer? Undoubtedly, these questions require unique approaches that can deal with the limitations imposed by monolayer cell culture and mouse modeling or human tissue studies.

One such experimental strategy involves the three-dimensional (3D) culture of epithelial cells. The value of studying the morphogenesis of glandular epithelium in vitro using three-dimensional culture systems has been recognized for many years [2], [3], [4]. Unlike monolayer cultures, mammary epithelial cells grown in three dimensions recapitulate numerous features of breast epithelium in vivo, including the formation of acini-like spheroids with a hollow lumen, apicobasal polarization of cells making up these acini, the basal deposition of basement membrane components (collagen IV and laminin V), and, in some cases, the production of milk proteins [5], [6], [7]. Thus, 3D epithelial culture provides the appropriate structural and functional context fundamental for examining the biological activities of cancer genes.

Recently, our laboratory has used the immortalized, nontransformed mammary epithelial cell line MCF-10A to characterize events associated with morphogenesis in vitro and to analyze the phenotypic effects of oncogenes on the morphogenesis of glandular structures. For example, overexpressing cyclin D1 or inactivating the retinoblastoma (Rb) protein with human papillomavirus E7 within mammary epithelial acini results in excessive proliferative activity within acinar structures cultured on basement membrane gels. However, despite this enhanced proliferation, the structures retain a hollow morphology because the proliferating cells lacking contact with the basement membrane undergo apoptosis [8]. In contrast, activation of the ErbB2 oncoprotein during mammary epithelial morphogenesis elicits a multiacinar phenotype, which is notable for excess proliferation, and filling of the luminal space; apparently, both protection from apoptosis and changes in apicobasal polarization contribute to this complex phenotype (Fig. 1B) [8], [9].

These studies have illustrated that three-dimensional culture systems provide an important tool to interrogate the how cancer genes influence glandular architecture as well as model early events involved in carcinoma formation. Hence, these culture systems may serve as a valuable addition to the technical repertoire used by cancer cell biologists. This article provides a detailed primer of the methods used in our laboratory to culture MCF-10A cells, establish stable pools expressing a gene of interest, as well as grow and analyze MCF-10A cells in three-dimensional basement membrane culture.

Section snippets

Use of MCF-10A mammary epithelial cells in acinar morphogenesis

Several methods have been developed to culture epithelial cells as three-dimensional spheroids. Certain tumor-derived cell lines, such as LIM 1863 colon carcinoma cells and DU4475 mammary carcinoma cells, spontaneously form gland-like organoids when grown in suspension culture [10], [11]. Although spheroids derived from tumors may be useful for studies of tissue organization, cell–cell adhesion, and, in some cases, apicobasal polarization, the structures derived from these cell lines do not

Establishment of stable pools in MCF-10A cells by retroviral infection

Retroviral expression vectors can be used for the stable expression of genes in MCF-10A cells. We have found that vectors that use the long terminal repeat regions of Moloney leukemia virus are very effective for stable expression in MCF-10A cells (e.g., pBABE, pMSCV, pLNCX, pLXSN). Because MCF-10A cells are of human origin, it is necessary to use virus that either possesses an amphotropic envelope or is pseudotyped with the envelope of vesicular stomatitis virus (VSV). Established retroviral

Three-dimensional culture of MCF-10A cells on reconstituted basement membrane

In most previous studies, mammary cells have been cultured in three dimensions by totally embedding in a gel of reconstituted basement membrane. Although MCF-10A cells can be cultured using a total embedment method, our laboratory has used primarily an overlay method in which single cells are seeded on a solidified layer of Growth Factor Reduced Matrigel measuring approximately 1–2 mm in thickness (Fig. 3). The cells are grown in an Assay Medium (see Table 1) containing 5 ng/ml epidermal growth

Analysis of morphogenetic parameters within 3D cultures by fluorescent and immunofluorescent techniques

The utility of MCF-10A mammary acini grown in three-dimensional culture as an experimental tool is due largely to the ability to analyze the spatial and temporal aspects of key biological processes (e.g., proliferation, apoptosis) and signal transduction events during morphogenesis. Recently, we have delineated a temporal progression of biological processes that contribute to the development and maintenance of a hollow glandular architecture within MCF-10A acini, which is schematically

Guidelines for preparation of protein lysates from 3D cultures

In addition to indirect immunofluorescence, three-dimensional cultures can be examined by immunoblotting to assess protein expression levels, the presence of protein complexes, as well as phosphorylation events. Protein lysates for immunoblot analysis can be prepared directly from three-dimensional cultures using detergent based-lysis buffer such as RIPA buffer (1% NonidetP-40, 0.2% SDS, 0.5% sodium deoxycholate, 150 mM sodium chloride, 50 mM Tris–HCl, pH 7.4, 10 mM sodium fluoride, 1 mM sodium

Concluding remarks

The disruption of an intact glandular structure, including loss of apicobasal polarity and filling of the luminal space, is a hallmark of epithelial cancer, even at its earliest stages; yet, very little is known about the mechanisms that elicit these changes during carcinoma formation. The morphogenesis of MCF-10A mammary acini in three-dimensional basement membrane culture provides a biologically rich system and the appropriate structural context in which to assay the effects of oncogenes on

Acknowledgements

We thank Dr. Mina Bissell (Lawrence Berkeley Laboratories), as well as members of the Brugge and Bissell laboratories, for assistance in developing and optimizing the protocols presented in this review. This work is supported by grants from National Cancer Institute (CA80111, CA89393), Aventis Pharmaceuticals, and American Cancer Society (to J.S.B.); an HHMI Physician Postdoctoral Fellowship (to J.D.); a Massachusetts Public Health Breast Cancer Research Award; and The V Foundation Scholar

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