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Lentiviral shRNA Screen to Identify Epithelial Integrity Regulating Genes in MCF10A 3D Culture   

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Original research article

A brief version of this protocol appeared in:
Oncogene
Mar 2016

Abstract

MCF10A 3D culture system provides a reductionist model of glandular mammary epithelium which is widely used to study development of glandular architecture, the role of cell polarity and epithelial integrity in control of epithelial cell functions, and mechanisms of breast cancer. Here we describe how to use shRNA screening approach to identify critical cell pathways that couple epithelial structure to individual cell based responses such as cell cycle exit and apoptosis. These studies will help to interrogate genetic changes critical for early breast tumorigenesis. The protocol describes a library of lentiviral shRNA constructs designed to target epithelial integrity and a highly efficient method for lentiviral transduction of suspension MCF10A cultures. Furthermore, protocols are provided for setting up MCF10A 3D cultures in Matrigel for morphometric and cellular response studies via structured illumination and confocal microscopy analysis of immunostained 3D structures.

Keywords: 3D cultures, MCF10A, shRNA, Epithelial integrity, Immunofluorescence staining, 3D imaging, Morphometric analysis

Background

All epithelial cells form highly organized tissue structures, which provide physical support and a structured scaffold for coordinated cell signaling. Such coordinated signaling across the epithelial structures is fundamental for epithelial biology; enabling dynamic joint actions of epithelial cells in regulation of organ size, shape, function and individual cell based responses (Roignot et al., 2013; Shamir and Ewald, 2014). Joint command of epithelial signaling also presents a powerful tumor suppressor mechanism by gatekeeping extrinsic and intrinsic mitogenic signals to quiescent epithelial tissues (Partanen et al., 2013; Rejon et al., 2016). However, very little is still known about genetic mechanisms coupling the status of epithelial structure with individual epithelial cell functions. MCF10A 3D Matrigel culture system is a well-established genetically tractable model of mammary epithelial architecture that is widely used to explore epithelial context-dependent cell functions (Debnath and Brugge, 2005). However, individual structures in MCF10A 3D cultures are not fully uniform in size or symmetry, which makes high-throughput screens with shRNA or cDNA reagents challenging in this system. Here, we describe protocols that expand the use of MCF10A 3D culture system from single gene studies to cell pathway level perturbation studies. The protocols for medium-throughput 3D screen using validated lentiviral shRNAs were originally used in a screen designed to identify genes with epithelial integrity-linked proliferation functions (Marques et al., [2016], screen outlined in Figure 1). However, these protocols are suitable for any reverse genetic MCF10A 3D culture study within a range of about 50 perturbed genes of interest.


Figure 1. Overview of shRNA screen in MCF10A 3D culture designed to identify epithelial integrity regulating genes. The protocols described here were recently applied in a shRNA screen using 52 knockdown validated shRNAs, which were lentivirally transferred to MCF10A cells containing a switchable oncogenic form of Myc (MycERTM). This set up allowed two separate primary morphometric screens in MCF10A 3D cultures; one with and another without Myc oncogene challenge. These screens produced morphometric data from > 5,000 structures. The most interesting knockdown phenotypes were further analyzed with cell response markers (proliferation [i.e., Ki67], apoptosis [i.e., active caspase 3] and polarity change [i.e., α6-integrin, GM130]) and via 3D structures reconstructions obtained with confocal microscopy. The results from this screen for morphometric results have been published in Marques et al. (2016).

Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Marques, E. and Klefström, J. (2016). Lentiviral shRNA Screen to Identify Epithelial Integrity Regulating Genes in MCF10A 3D Culture. Bio-protocol 6(23): e2050. DOI: 10.21769/BioProtoc.2050.
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