Figure 5: Characterization of an EMT-like phenotype in NICD/p53−/− primary tumours. | Nature Communications

Figure 5: Characterization of an EMT-like phenotype in NICD/p53−/− primary tumours.

From: Concomitant Notch activation and p53 deletion trigger epithelial-to-mesenchymal transition and metastasis in mouse gut

Figure 5

(a) Staining of the mesenchymal marker N-cadherin and the EMT transcription factors, SNAIL, SLUG and TWIST in NICD/p53−/− primary tumours. Left panel: N-cadherin is overexpressed in tumour cells invading the desmoplastic area compared with those from the tumour bulk. Scale bars, 40 μm. Middle and right panels: a nuclear overexpression of EMT transcription factors is observed in cancer cells from the desmoplastic area compared with differentiated tubular structures. Scale bars, 100 μm. (b,c) Expression of the EMT-inducer ZEB1 in primary tumours of NICD/p53−/−. (b) Left panel: triple immunofluorescence on NICD/p53−/− primary tumours using 4,6-diamidino-2-phenylindole (DAPI; grey) and GFP staining (green), ECAD (blue) and ZEB1 (red). Scale bars, 40 μm. Right panel: magnification of different tumour cell features coming from the same slide showing that cells undergoing EMT-like processes express the EMT-inducer ZEB1. *Zeb1 negative epithelial tumour cells; ** and *** Zeb1 and GFP-positive, ECAD-negative cells, independently of their morphology. Scale bars, 4 μm. The white arrow labels individual ZEB1-negative cells with a non-epithelial origin. (c) The left bar graph shows the frequency of vimentin, SMA or Zeb1-positive cells among GFP-positive cells. Cells were separated in two groups presenting either an epithelial phenotype described by ECAD-positive cells (E+) or cytokeratin-positive cells (CK+), or an EMT-like phenotype characterized by ECAD negative (E−) or CK negative (CK−). The associated tables of contingency (Supplementary Table 4) depict a strong negative correlation (***) between ZEB1 and ECAD expressions (P<0.001), vimentin and ECAD expressions (χ2 test, P<0.001) or SMA and CK expressions (χ2 test, P<0.001). The right bar graph shows the distribution of Zeb1 and SMA among GFP positive cells. ZEB1 is expressed in SMA-negative cells in 27% of GFP-positive cells and in SMA-positive cells for 22.4% GFP-positive cells. Only 3% of GFP-positive are ZEB1-negative cells and SMA positive, suggesting that a small fraction of cells have fully completed the EMT process. (d) Triple immunofluorescence on NICD/p53−/− metastatic tissues using DAPI (grey), GFP staining (green), ECAD (blue) and ZEB1 (red; scale bars, 20 μm) with magnifications of one area showing the presence of GFP-intestinal cancer cells with mesenchymal features in the secondary organs (scale bars, 4 μm).

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