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The Z-cad dual fluorescent sensor detects dynamic changes between the epithelial and mesenchymal cellular states

BACKGROUND: The epithelial to mesenchymal transition (EMT) has been implicated in metastasis and therapy resistance of carcinomas and can endow cancer cells with cancer stem cell (CSC) properties. The ability to detect cancer cells that are undergoing or have completed EMT has typically relied on th...

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Autores principales: Toneff, M. J., Sreekumar, A., Tinnirello, A., Hollander, P. Den, Habib, S., Li, S., Ellis, M. J., Xin, L., Mani, S. A., Rosen, J. M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4912796/
https://www.ncbi.nlm.nih.gov/pubmed/27317311
http://dx.doi.org/10.1186/s12915-016-0269-y
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author Toneff, M. J.
Sreekumar, A.
Tinnirello, A.
Hollander, P. Den
Habib, S.
Li, S.
Ellis, M. J.
Xin, L.
Mani, S. A.
Rosen, J. M.
author_facet Toneff, M. J.
Sreekumar, A.
Tinnirello, A.
Hollander, P. Den
Habib, S.
Li, S.
Ellis, M. J.
Xin, L.
Mani, S. A.
Rosen, J. M.
author_sort Toneff, M. J.
collection PubMed
description BACKGROUND: The epithelial to mesenchymal transition (EMT) has been implicated in metastasis and therapy resistance of carcinomas and can endow cancer cells with cancer stem cell (CSC) properties. The ability to detect cancer cells that are undergoing or have completed EMT has typically relied on the expression of cell surface antigens that correlate with an EMT/CSC phenotype. Alternatively these cells may be permanently marked through Cre-mediated recombination or through immunostaining of fixed cells. The EMT process is dynamic, and these existing methods cannot reveal such changes within live cells. The development of fluorescent sensors that mirror the dynamic EMT state by following the expression of bona fide EMT regulators in live cells would provide a valuable new tool for characterizing EMT. In addition, these sensors will allow direct observation of cellular plasticity with respect to the epithelial/mesenchymal state to enable more effective studies of EMT in cancer and development. RESULTS: We generated a lentiviral-based, dual fluorescent reporter system, designated as the Z-cad dual sensor, comprising destabilized green fluorescent protein containing the ZEB1 3′ UTR and red fluorescent protein driven by the E-cadherin (CDH1) promoter. Using this sensor, we robustly detected EMT and mesenchymal to epithelial transition (MET) in breast cancer cells by flow cytometry and fluorescence microscopy. Importantly, we observed dynamic changes in cellular populations undergoing MET. Additionally, we used the Z-cad sensor to identify and isolate minor subpopulations of cells displaying mesenchymal properties within a population comprising predominately epithelial-like cells. The Z-cad dual sensor identified cells with CSC-like properties more effectively than either the ZEB1 3′ UTR or E-cadherin sensor alone. CONCLUSIONS: The Z-cad dual sensor effectively reports the activities of two factors critical in determining the epithelial/mesenchymal state of carcinoma cells. The ability of this stably integrating dual sensor system to detect dynamic fluctuations between these two states through live cell imaging offers a significant improvement over existing methods and helps facilitate the study of EMT/MET plasticity in response to different stimuli and in cancer pathogenesis. Finally, the versatile Z-cad sensor can be adapted to a variety of in vitro or in vivo systems to elucidate whether EMT/MET contributes to normal and disease phenotypes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12915-016-0269-y) contains supplementary material, which is available to authorized users.
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spelling pubmed-49127962016-06-19 The Z-cad dual fluorescent sensor detects dynamic changes between the epithelial and mesenchymal cellular states Toneff, M. J. Sreekumar, A. Tinnirello, A. Hollander, P. Den Habib, S. Li, S. Ellis, M. J. Xin, L. Mani, S. A. Rosen, J. M. BMC Biol Methodology Article BACKGROUND: The epithelial to mesenchymal transition (EMT) has been implicated in metastasis and therapy resistance of carcinomas and can endow cancer cells with cancer stem cell (CSC) properties. The ability to detect cancer cells that are undergoing or have completed EMT has typically relied on the expression of cell surface antigens that correlate with an EMT/CSC phenotype. Alternatively these cells may be permanently marked through Cre-mediated recombination or through immunostaining of fixed cells. The EMT process is dynamic, and these existing methods cannot reveal such changes within live cells. The development of fluorescent sensors that mirror the dynamic EMT state by following the expression of bona fide EMT regulators in live cells would provide a valuable new tool for characterizing EMT. In addition, these sensors will allow direct observation of cellular plasticity with respect to the epithelial/mesenchymal state to enable more effective studies of EMT in cancer and development. RESULTS: We generated a lentiviral-based, dual fluorescent reporter system, designated as the Z-cad dual sensor, comprising destabilized green fluorescent protein containing the ZEB1 3′ UTR and red fluorescent protein driven by the E-cadherin (CDH1) promoter. Using this sensor, we robustly detected EMT and mesenchymal to epithelial transition (MET) in breast cancer cells by flow cytometry and fluorescence microscopy. Importantly, we observed dynamic changes in cellular populations undergoing MET. Additionally, we used the Z-cad sensor to identify and isolate minor subpopulations of cells displaying mesenchymal properties within a population comprising predominately epithelial-like cells. The Z-cad dual sensor identified cells with CSC-like properties more effectively than either the ZEB1 3′ UTR or E-cadherin sensor alone. CONCLUSIONS: The Z-cad dual sensor effectively reports the activities of two factors critical in determining the epithelial/mesenchymal state of carcinoma cells. The ability of this stably integrating dual sensor system to detect dynamic fluctuations between these two states through live cell imaging offers a significant improvement over existing methods and helps facilitate the study of EMT/MET plasticity in response to different stimuli and in cancer pathogenesis. Finally, the versatile Z-cad sensor can be adapted to a variety of in vitro or in vivo systems to elucidate whether EMT/MET contributes to normal and disease phenotypes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12915-016-0269-y) contains supplementary material, which is available to authorized users. BioMed Central 2016-06-17 /pmc/articles/PMC4912796/ /pubmed/27317311 http://dx.doi.org/10.1186/s12915-016-0269-y Text en © Toneff et al. 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Methodology Article
Toneff, M. J.
Sreekumar, A.
Tinnirello, A.
Hollander, P. Den
Habib, S.
Li, S.
Ellis, M. J.
Xin, L.
Mani, S. A.
Rosen, J. M.
The Z-cad dual fluorescent sensor detects dynamic changes between the epithelial and mesenchymal cellular states
title The Z-cad dual fluorescent sensor detects dynamic changes between the epithelial and mesenchymal cellular states
title_full The Z-cad dual fluorescent sensor detects dynamic changes between the epithelial and mesenchymal cellular states
title_fullStr The Z-cad dual fluorescent sensor detects dynamic changes between the epithelial and mesenchymal cellular states
title_full_unstemmed The Z-cad dual fluorescent sensor detects dynamic changes between the epithelial and mesenchymal cellular states
title_short The Z-cad dual fluorescent sensor detects dynamic changes between the epithelial and mesenchymal cellular states
title_sort z-cad dual fluorescent sensor detects dynamic changes between the epithelial and mesenchymal cellular states
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4912796/
https://www.ncbi.nlm.nih.gov/pubmed/27317311
http://dx.doi.org/10.1186/s12915-016-0269-y
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