Fig. 1: A subpopulation of quasi-mesenchymal cells co-exists with epithelial cells in HGS ovarian cancer cell lines.
A FACS analysis of the ovarian cancer cell lines OV90, CAOV3, SKOV3, and COV504 with antibodies directed against CD44 and EpCAM. EpCAM/CD44 positive and negative areas were defined as previously described [17, 21] using multiple isotype controls and are shown by the quadrants in the plots. Using specific gates, cells were separated in CD44hiEpCAMhi and CD44hiEpCAMlo subpopulations. The percentages of CD44hiEpCAMlo and CD44hiEpCAMhi cells within each cell line are depicted in each quadrant. Notably, as previously observed for SW480 and HCT116, the ovarian cancer cell lines revealed a continuum of different EpCAM and CD44 expression levels with a large EpCAMhi (or EpCAMlo as in the case of SKOV3) cluster followed by a tail of gradually decreasing (increasing for SKOV3) EpCAM levels. By applying the indicated gates, cells were sorted into EpCAMhi and EpCAMlo subpopulations. Graphs show representative analysis from an individual experiment. B Phase contrast microscopy images of sorted EpCAMhi and EpCAMlo cells from EpCAMhi and EpCAMlo OV90 and CAOV3 sorted cells. Scale bar: 100 μm. While EpCAMhi cells show characteristic epithelial morphology, EpCAMlo cells showed a more spindle- and mesenchymal-like appearance. Scale bar: 100 µm. C Transwell migration assay (upper graph) and invasion assay (lower graph) of EpCAMhi (blue bar) and EpCAMlo (red bar) OV90 and CAOV3 cells. Each bar symbolizes the mean ± SD. D Principal component analysis (PCA) of the RNAseq profiles of EpCAMhi and EpCAMlo cells from the OV90 and CAOV3 lines. E Heatmap of common differentially expressed gene among EpCAMhi/lo and bulk subpopulations from the OV90 and CAOV3 cell lines (abs LFC > 1.5, P value < 0.01). Complete-linkage hierarchical clustering with split by k-means (k = 2) clustering was used. F Hallmark pathways based on the Gene Set Enrichment Analysis (GSEA) of OV90 and CAOV3 EpCAMlo expression profiles compared with EpCAMhi. Plots show only significantly expressed pathways, with a normalized enrichment score (NES) > 1 and P value < 0.05. G RT-qPCR expression analysis of ZEB1 in OV90, SKOV3 and COV504 transduced with an inducible control (shCT) and with a ZEB1-shRNA (shZEB1) construct. shRNA expression was induced with 1 μg/mL of doxycycline. GAPDH expression was used as control. Each bar represents the mean ± SD. P value is indicated. H FACS analysis of the OV90, SKOV3 and COV504 cell lines transfected with the shZEB1 and control constructs using antibodies against CD44 and EpCAM. Cells were induced with 1 μg/mL doxycycline for 72 h prior to the FACS analysis. The percentages of EpCAMlo and EpCAMhi cells within each cell line are depicted in each quadrant.
