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The morphological and molecular features of the epithelial-to-mesenchymal transition

Nature Protocols volume 4, pages 1591–1613 (2009)Cite this article

Abstract

Here we describe several methods for the characterization of epithelial–mesenchymal transition (EMT) at the cellular, molecular and behavioral level. This protocol describes both in vitro and in vivo approaches designed to analyze different features that when taken together permit the characterization of cells undergoing transient or stable EMT. We define straightforward methods for phenotypical, cellular and transcriptional characterization of EMT in vitro in monolayer cultures. The procedure also presents technical details for the generation of in vitro three-dimensional (3D) cultures analyzing cell phenotype and behavior during the EMT process. In addition, we describe xenotransplantation techniques to graft 3D cell cultures into mice to study in vivo invasion in a physiological-like environment. Finally, the protocol describes the analysis of selected EMT markers from experimental and human tumor samples. This series of methods can be applied to the study of EMT under various experimental and biological situations. Once the methodology is established, the time required to complete the protocol may vary from 3 to 4 weeks (monolayer cultures) and up to 6–8 weeks if including 3D cultures.

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Figure 1: Flow chart of the procedure.
Figure 2: Schematic diagram of the reporter vectors used in promoter assays.
Figure 3: Immunophenotypical characterization of EMT in two-dimensional cultures.
Figure 4: Analyses of the expression of EMT-related markers at the protein, mRNA and promoter levels.
Figure 5: Wound healing assay.
Figure 6: Implantation of three-dimensional cell cultures into nude mice.
Figure 7: Analysis of cell growth and behavior in 3D cultures.
Figure 8: High-throughput analysis and validation of EMT markers in tumors.

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Acknowledgements

We are grateful to A. Montes and R. Díaz-Uriarte for technical assistance in generation of stable transfectants and in microarray data analysis, respectively, and to S. Lavotshkin for his friendly help with editorial assistance. This study has been supported by grant nos. SAF2004-00361, SAF2007-63051, SAF2007-63075 and Consolider-Ingenio 2010 CSD2007-00017 (MICIN, Spain), by MRTN 2004-005428 (European Commission) and by FMM07 (Fundación Mutua Madrileña).

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  1. Héctor Peinado & David Olmeda

    Present address: Present addresses: Departments of Pediatrics, Cell & Developmental Biology, Weill Medical College of Cornell University, New York, New York, USA (H.P.); Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain (D.O.).,

  2. Gema Moreno-Bueno, Héctor Peinado and Patricia Molina: These authors contributed equally to this work.

Authors and Affiliations

  1. Departamento de Bioquímica, Universidad Autónoma de Madrid (UAM), Instituto de Investigaciones Biomédicas 'Alberto Sols' CSIC-UAM, Madrid, Spain

    Gema Moreno-Bueno, Héctor Peinado, Patricia Molina, David Olmeda, Eva Cubillo, Vanesa Santos, Francisco Portillo & Amparo Cano

  2. Servicio de Anatomía Patológica, Hospital Virgen del Rocío, Sevilla, Spain

    José Palacios

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Contributions

G.M.-B. was responsible for design and performance of microarray profiling studies including the statistical analysis, performed IHC analysis on tissue arrays and promoter assays, designed and carried out the Supplementary Methods, Figures 2, 4 and 8, and Tables 1, 2 and 7 and drafted this paper; H.P. was responsible for the design and analysis of 3D cultures (in vitro and in vivo), performed immunofluorescence analysis in monolayer and 3D cultures, designed and drew Box 2, Figures 6 and 7, and Table 3, collaborated in Figure 3 and drafted this paper; P.M. was responsible for the wound healing assays and collaborated in the IF analysis in monolayer cultures, designed and drew Box 1, Figures 1, 2 and 5, and Table 4 and drafted this paper; D.O. was responsible for the RT-PCR and qPCR analysis and for the design of species-specific primers (Table 5) and specific shRNAs probes; E.C. participated in the characterization of MDCK-E47 cell system at protein, mRNA and promoter level, collaborated in microarray analysis and drew Figures 3 and 4; V.S. collaborated with HP in the optimization of 3D cultures (in vitro and in vivo) and performed experiments described in Figure 7; J.P. performed, analyzed and interpreted the tissue arrays, and collaborated in drawing Figure 8; F.P. drew Tables 1, 2, 4, 5 and 6 and drafted this paper; A.C. coordinated the writing of this paper and together with F.P. was responsible for the final draft of this paper.

Corresponding author

Correspondence to Amparo Cano.

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Supplementary Methods

Selection of EMT genes after gene profiling analysis (PDF 121 kb)

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Moreno-Bueno, G., Peinado, H., Molina, P. et al. The morphological and molecular features of the epithelial-to-mesenchymal transition. Nat Protoc 4, 1591–1613 (2009). https://doi.org/10.1038/nprot.2009.152

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