A ZEB1-Neon knock-in uncovers traceable dynamics of epithelial-mesenchymal transition in tumors in vivo

BACKGROUND: Progression and metastasis of solid cancers are orchestrated by activation of epithelial-mesenchymal transition (EMT) in the primary tumor. This process is typically restricted to a limited number of cells that acquire partial or hybrid EMT states to unleash cellular plasticity. Capturing such dynamic and often reversible events in vivo on the single cell level is hampered by the lack of proper labeling tools that yet often induce permanent staining persisting beyond transient EMT activation. METHODS: To enable live-tracking of EMT in vivo, we utilized CRISPaint and homologous recombination to endogenously tag ZEB1, one key transcription factor to activate EMT during tumorigenesis. Using the bright fluorescent protein mNeonGreen, we generated ZEB1-Neon fusion knock-in alleles in MDA-MB-231 and MCF10A cells, as well as in mice. RESULTS: We demonstrate that mNeonGreen fluorescence is suitable to faithfully report on ZEB1 expression in vitro over time, becomes properly upregulated by TGFβ, and allows separation of ZEB1hi and ZEB1lo cells to capture different cellular properties, e.g., handling of DNA damage. The fusion does not affect ZEB1 function as evident by proper EMT induction, embryogenesis, and tissue homeostasis when present homozygously. Moreover, introducing Zeb1-Neon into the KPC mouse model of pancreatic cancer permits tracking of ZEB1+ cells in precision-cut slices and time-lapse imaging of isolated tumor cells. CONCLUSIONS: In summary, we provide a versatile tool that allows precise detection and live cell imaging of EMT, which will help to more accurately decipher the role of EMT in tumor progression and to identify therapeutic agents that can specifically manipulate EMT for novel combination therapies.