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



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.



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.
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA.
M J
MJ
Toneff
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA.
A
A
Sreekumar
Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
A
A
Tinnirello
Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
P Den
PD
Hollander
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA.
S
S
Habib
Washington University Institute of Clinical and Translational Sciences, St. Louis, MO, USA.
S
S
Li
Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX, USA.
M J
MJ
Ellis
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA.
L
L
Xin
Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
S A
SA
Mani
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA. jrosen@bcm.edu.
J M
JM
Rosen
D000818:Animals; D015703:Antigens, CD; D015374:Biosensing Techniques; D015820:Cadherins / Q000235:genetics; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000166:cytology*; D058750:Epithelial-Mesenchymal Transition; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D051379:Mice; D011401:Promoter Regions, Genetic; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology; D000071799:Zinc Finger E-box-Binding Homeobox 1 / Q000235:genetics; D000097573:Red Fluorescent Protein
D015703:Antigens, CD; D015820:Cadherins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D053773:Transforming Growth Factor beta1; D000071799:Zinc Finger E-box-Binding Homeobox 1; D000097573:Red Fluorescent Protein; C513384:CDH1 protein, human; C486307:ZEB1 protein, human
Cancer stem cell; EMT; MET; Molecular sensors; Plasticity