(A) Flow cytometric analysis of the colon cancer cell lines HCT116 (left panel) and SW480 (right panel) with antibodies directed against CD44 and EpCAM. EpCAM/CD44-positive and -negative regions (gray quadrants) were defined as in Figure supplement 1 using multiple isotype controls and are shown by the quadrants in the plots. Notably, both HCT116 and SW480 revealed a continuum of different EpCAM and CD44 expression levels with a large CD44highEpCAMhigh (EpCAMhi) cluster followed by a tail of gradually decreasing EpCAM and increasing CD44 levels. By applying specific gates, cells were divided in a large EpCAMhi cluster, together with a considerably smaller CD44highEpCAMlow (EpCAMlo) subpopulation. To ensure good separation from the large EpCAMhi cluster and maximal sorting purity, EpCAMlo cells were gated as CD44hi events ≤ 60% of the EpCAM fluorescence intensity of the left border of the EpCAMhi gate and sorted from ≤50% of that value. Variable percentages of EpCAMlo cells were found to feature the HCT116 (5.0% ± 2.5%) and SW480 (16.7% ± 13%) cell lines, respectively. For the sake of simplicity, gates are shown in the figure only if they encompass sizeable percentages of cells. Graphs show representative analysis of one experiment. (B) Phase-contrast microscopy images of sorted EpCAMhi and EpCAMlo cells from HCT116 (upper images) and SW480 (lower images) cells. While EpCAMhi cells formed compact colonies with characteristic epithelial morphology, EpCAMlo cells showed a more spindle- and mesenchymal-like appearance. Scale bar: 100 µm. (C) Intrasplenic injection of bulk, EpCAMhi, and EpCAMlo cells from HCT116 (left panel) and SW480 (right panel). For each transplantation experiment, 2 × 104 cells were injected in the spleen of a recipient NSG mouse. 4 (HCT116) and 8 (SW480) weeks after injection, mice were sacrificed and individual tumors counted. Single and double asterisks indicate significant differences (p<0.05 and p<0.01, respectively). HCT116: bulk (n = 8), EpCAMhi (n = 9), and EpCAMlo (n = 7). SW480: bulk (n = 4), EpCAMhi (n = 4), and EpCAMlo (n = 4). (D) Images of mouse livers 4 (HCT116) and 8 (SW480) weeks after orthotopic injection with 104 cells. Scale bar: 5 mm.