Epithelial-mesenchymal transition (EMT) is a complex cellular program, involved in the progression of epithelial cancers to a metastatic stage. During this process characteristic epithelial marker proteins are repressed in favor of mesenchymal biomarkers, such as the intermediate filament protein vimentin. There is an ongoing need for reliable cell-based EMT model systems, which can be adapted to screening campaigns for novel anti-metastatic therapies. Imaging-based screening systems are restricted to endpoint immunofluorescence studies that provide only little information about the dynamics of the process, or to ectopic expression of EMT markers, fused to fluorescent proteins. However, overexpression of EMT marker proteins, particularly vimentin, have been shown to influence the EMT phenotype of the cells.
Here we introduce chromobodies as an innovative technology to monitor EMT in living non-small lung cancer cells. Chromobodies are fluorescently-labeled single-domain-antibodies that are functional within in living cells and directly target endogenous proteins. We generated chromobodies against the EMT biomarkers vimentin, actin and β-catenin and stably introduced them into the genome of A549 cells. Upon induction of EMT with transforming growth factor β (TGF- β), we could visualize the dynamic reorganization of endogenous vimentin, actin and β-catenin in the established EMT chromobody models in 2D as well as 3D cell culture. By means of these chromobody cell lines we further developed a 2D high content analysis system to visualize and quantify spatiotemporal changes of the endogenous biomarkers vimentin and actin during the EMT process. In a proof-of-principle screen, using known EMT-affecting reagents (e.g. TGF receptor I inhibitors LY364947 and SB505124) as well as EMT-unrelated agents, we were able to show that this high content analysis system can be applied to select compounds that inhibit TGF-β-induced EMT.
In summary, live cell imaging of EMT markers with chromobodies provides a novel versatile approach to study EMT in real time and allows the identification of EMT inhibitors as potential candidates for anti-metastatic therapies by high content screening.
Citation Format: Julia Maier, Theresa Plaga, Stefanie Egetemaier, Bjoern Traenkle, Ulrich Rothbauer. Tracing EMT with fluorescent biosensors (chromobodies) in living cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3054. doi:10.1158/1538-7445.AM2017-3054