Figure 1. Identification and characterization of EpCAM^lo cells in colon cancer cell lines.

(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 CD44^highEpCAM^high (EpCAM^hi) cluster followed by a tail of gradually decreasing EpCAM and increasing CD44 levels. By applying specific gates, cells were divided in a large EpCAM^hi cluster, together with a considerably smaller CD44^highEpCAM^low (EpCAM^lo) subpopulation. To ensure good separation from the large EpCAM^hi cluster and maximal sorting purity, EpCAM^lo cells were gated as CD44^hi events ≤ 60% of the EpCAM fluorescence intensity of the left border of the EpCAM^hi gate and sorted from ≤50% of that value. Variable percentages of EpCAM^lo 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 EpCAM^hi and EpCAM^lo cells from HCT116 (upper images) and SW480 (lower images) cells. While EpCAM^hi cells formed compact colonies with characteristic epithelial morphology, EpCAM^lo cells showed a more spindle- and mesenchymal-like appearance. Scale bar: 100 µm. (C) Intrasplenic injection of bulk, EpCAM^hi, and EpCAM^lo 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), EpCAM^hi (n = 9), and EpCAM^lo (n = 7). SW480: bulk (n = 4), EpCAM^hi (n = 4), and EpCAM^lo (n = 4). (D) Images of mouse livers 4 (HCT116) and 8 (SW480) weeks after orthotopic injection with 10⁴ cells. Scale bar: 5 mm.

Figure 1—figure supplement 1. Further characterization of EpCAM^lo cells in colon cancer cell lines: FACS analysis.

(A) FSC-A/SSC-A, FSC-W/FSC-A, and SSC-W/SSC-A single-cell gates (confirmed by gating on FSC-A/FSC-H). Purity of sorted single cells was confirmed by microscopy. (B) Acquisition parameters used for FACS analysis. (C) Gating strategy employed to select for live cells by DAPI staining (top: unstained cells; bottom: after DAPI staining). (D) Top: unstained and isotype controls, showing absence of relevant unspecific antibody binding. Bottom: fluorescence minus one (FMO) samples, showing the absence of uncompensated spillover between fluorescence channels. For the sake of clarity, a quadrant is placed to show the relative position of unstained and single stains in both cells lines. For the EpCAM-FITC antibody, a mouse IgG1-FITC isotype control S. Cruz sc-2855 was used; for the CD44-APC antibody, a Rat IgG2a-APC isotype control S. Cruz sc-2895 was used. Similar results were obtained with SW480 cells (not shown). (E) Full EpCAM/CD44 staining, also showing the rationale behind the definition of high and low referred to EpCAM and CD44 levels. Despite the differences in CD44 levels between EpCAM^hi and EpCAM^lo cells, for the sake of simplicity the populations were defined ‘low’ for a defined marker if they were mainly covering a region within the 1st log above the negative gate, high if they were above the 1st log. FSC-H: forward-scatterheight; FSC-W: forward-scatter width.

Figure 1—figure supplement 2. Further characterization of EpCAM^lo cells in colon cancer cell lines: migration/invasion and EMT analysis.

(A) Top panel: transwell migration assay of EpCAM^hi (black bar) and EpCAM^lo (gray bar) cells from the HCT116 and SW480 lines. 10⁵ cells were plated on TC-coated membrane in triplicate and left overnight before counting the number of migrated cells on the bottom side of the membrane. Each bar represents the mean ± SD of two independent experiments. Asterisks indicate significant differences (p<0.05). Bottom panel: invasion assay of EpCAM^hi (black bar) and EpCAM^lo (gray bar) cells from the HCT116 and SW480 lines. 10⁵ cells were plated in triplicate on top of an extracellular matrix-coated membrane and left overnight before counting the number of cells migrated to other side of the membrane. Each bar represents the mean ± SD of two independent experiments. Asterisks indicate significant differences (p<0.05). (B) RT-qPCR expression analysis of epithelial (EPCAM and CDH1) and mesenchymal (VIM) markers in sorted EpCAM^hi (black bars) and EpCAM^lo (gray bars) from the HCT116 and SW480 lines. GAPDH was employed for normalization purposes. Each bar represents the mean ± SD of three independent experiments. Asterisks indicate significant differences (p<0.05). (C) RT-qPCR expression analysis of epithelial to mesenchymal transition transcription factors (ZEB1, ZEB2, TWIST, FOXC2, SLUG, and SNAIL) in EpCAM^hi (black bars) and EpCAM^lo (gray bars) cells. Left panel: HCT116. Right panel: SW480. GAPDH was employed for normalization. Each bar represents the mean ± SD of three independent experiments. Asterisks indicate significant differences (p<0.05). (D) Immunofluorescence (IF) analysis of EpCAM^hi and EpCAM^lo cells. Cells were sorted and directed plated on cover slips. After 4 days, cells were fixed with 4% paraformaldehyde and stained with antibodies against EpCAM (green) and ZEB1 (red). Nuclei were visualized by DAPI staining of DNA (blue). Scale bar: 50 µm.

Figure 1—figure supplement 3. Further characterization of EpCAM^lo cells in colon cancer cell lines: EMT and cell cycle analysis.

(A). qRT-PCR expression analysis of ZEB1 in HCT116 and SW480 transduced with an inducible control (shCT) or ZEB1-shRNA (shZEB1) construct. shRNA expression was induced with 1 µg/mL of doxycycline. Each bar represents the mean ± SD of three independent experiments. (B) Bar graph of flow cytometric analysis (see B). Each bar represents the relative mean ± SD of three independent experiments. (C) Representative analysis of the flow cytometric analysis of the shCT- and shZEB1-transfected HCT116 and SW480 cell lines using antibodies against CD44 and EpCAM. Cells were induced with 1 µg/mL doxycycline for 72 hr before analysis. (D) RT-qPCR expression analysis of the members of the miRNA 200 family (miR-200a, miR-200b, miR-200c, miR-141, and miR-429) in EpCAM^hi (black bars) and EpCAM^lo (gray bars) cells. Upper panel: HCT116. Bottom panel: SW480. U6 was employed for normalization. Each bar represents the mean ± SD of three independent experiments. Single asterisks indicate significant differences of p<0.05, double asterisks of p<0.01, and triple asterisks of p<0.001. (E) Cell proliferation assay. Sorted bulk, EpCAM^hi and EpCAM^lo cells were seeded in triplicate in plates and cultured in conventional medium. HCT116 and SW480 cells were harvested and number of cells was counted at 4 and 11 days, respectively. Each bar represents the mean ± SD three independent experiments. (F) Cell cycle analysis of EpCAM^hi and EpCAM^lo cells in HCT116 (upper panel) and SW480 (lower panel). Cell fractions were sorted and plated in culture. After 72 hr, cells were fixed and stained with propidium iodide. Cell cycle distribution was assayed by flow cytometry. Graphs show representative analysis of one experiment. Tables demonstrate average and standard deviation of three independent experiments. White graph: EpCAM^hi; gray graph: EpCAM^lo. Asterisks show the significant (p<0.05) differences between EpCAM^hi and EpCAM^lo cells in G₁ and G₂M-phases.

Figure 1—figure supplement 4. Further characterization of EpCAM^lo cells in colon cancer cell lines: chemoresistance.

Bulk, EpCAM^hi, and EpCAM^lo HCT116 and SW480 cells were sorted and plated to recover and expand for 4 days.10⁵ cells per well were plated in triplicate in a 96-well plate and left to attach. After 24 hr, standard cell culture medium was changed for medium containing chemotherapeutic drug oxaliplatin or 5-fluorouracil (5-FU). HCT116 cells were left with 5-FU and oxaliplatin for respectively 3 and 5 days. For each drug, SW480 cells were treated for 7 days. After removal of the chemotherapeutic drug, cells were washed with PBS and left to regrow in standard culture medium. Cell viability was assessed using the MTT upon removal of the drug, and after regrowth (ranging 13–30 days). O.D. reading was performed at 595 nm with a Microplate Reader. Background measurements were subtracted from each data point. All experiments were performed at least in duplicate for each individual cell line and drug. (A, B) Chemo-sensitivity assays using oxaliplatin (A) or 5-FU (B) in bulk (black bars), EpCAM^hi (dark gray bars) and EpCAM^lo (light gray bars) HCT116 cells. Bars represent the average ± SD of the O.D. corrected for the background measurements. (C, D) Chemo-sensitivity assays using oxaliplatin (C) or 5-FU (D) in bulk (black bars), EpCAM^hi (dark gray bars) and EpCAM^lo (light gray bars) SW480 cells. Bars represent the average ± SD of the O.D. corrected for the background measurements.

Figure 1—figure supplement 5. Further characterization of EpCAM^lo cells in colon cancer cell lines: invasive and metastatic behaviour.

(A) Hematoxylin and eosin (H&E; first two panels) and immunohistochemistry (IHC) with antibody directed against β-catenin (third and fourth panels) in liver metastasis obtained 4 or 8 weeks after intrasplenic injection with HCT116 (upper panels) and SW480 (lower panels) cells, respectively. Second and fourth panels show zoom of the marked area in respectively first and third panels. Scale bar first and third panels: 100 µm. Scale bar second and fourth panels: 50 µm. (B) FACS analysis of liver metastases obtained by spleen injection of HCT116 bulk, EpCAM^hi, and EpCAM^lo cells. Upper panel: representative FACS plots. (C) Microscopic (top, left panel) analysis of AKP-Z organoids tagged with GFP and click beetle luciferase. The graph (bottom, left panel) shows the RT-qPCR expression analysis of Zeb1 in AKP (left) and AKP-Z (right) organoids upon in vitro doxycycline treatment for 48 hr. Black bars: no doxycycline treatment; gray bars: 1 μg/mL doxycycline. Each bar represents the mean ± SD of three independent experiments. (D) Upon establishment of a primary tumor in the caecum, transplanted mice were administered doxycycline in the drinking water to induce Zeb1 expression. FACS analysis of the primary tumor was performed 1 week after the start of the doxycycline. The panels show representative FACS plots of a control and two dox-treated primary tumors. (E) Quantification of the number of lung and liver metastases in uninduced (black; n = 4) and dox-induced (gray; n = 5) AKP-Z transplanted mice. Liver tissue was cut into 500 µm slices, processed for IHC, stained for β-catenin to visualize tumor cells, scanned using a NanoZoomer, and counted using NDP view software. The area of tissue analyzed was used to normalize the data.