Fig 2: Phenotypic plasticity maintains EpCAMlo and EpCAMhi cells in a stochastic equilibrium.(A) Analysis of plasticity of EpCAMhi and EpCAMlo cells from HCT116 (upper panel) and SW480 (lower panel). EpCAMhi and EpCAMlo cell fractions were sorted and plated in culture. At different time points, as indicated, cells were reanalyzed by flow cytometry for their levels of CD44 (y-axis) and EpCAM (x-axis) expression. (B) Flow cytometric analysis of single CD44hiEpCAMhi and CD44hiEpCAMlo HCT116 cells sorted by FACS and cultured for 72 days. Three representative individual single-cell clones per cell fraction are shown. (C) Dynamics of the EpCAMhi and EpCAMlo subpopulations from the HCT and SW480 cell lines as measured by FACS (% of total) over time. Under each graph, a schematic shows the estimated transition probabilities from the fitted two-state Markov model. Figure 2—source data 1.Phenotypic plasticity maintains EpCAMlo and EpCAMhi cells in a stochastic equilibrium: clonal analysis.The table encompasses a selection of individual single-cell sorted EpCAMhi and EpCAMlo HCT116 and SW480 clones. Single cells were sorted, grown up, and analyzed at two indicated times points by flow cytometry for their CD44 and EpCAM expression. Percentage of cells present in the set EpCAMhi and EpCAMlo gates is presented in the bar graph. Total success rate of clones grown out of single cells: HCT116 EpCAMhi 15 clones out of 24 sorted cells, HCT116 EpCAMlo: 31/48; SW480 EpCAMhi: 5/36; SW480 EpCAMlo: 11/36.

Fig 3: In vitro analysis of tumorigenic capacity of BXPC3 CSCs after PRP treatment. (A) Antiproliferative activity of PRP against CSCs from pancreatic BXPC3 cell line. (B) Decreased ALDH1 activity and CSCs markers expression: CD44, CD326 and CXCR4 in BXPC3 CSCs. (C) Representative confocal images of CD44, CxCR-4 and CD326 expression in BxPC3 treated and control CSCs. CD44, CD326 and CxCR-4 expression were detected in red and nuclei were counterstained with DAPI (blue). Scale bar represents 40 µm. (D) Western blot analysis of CD44, CD326 and CxCR-4 in BXPC3 CSCs treated with PRP versus non-treated. ?-actin and GAPDH were used as a internal controls. (E) Representative images of BXPC-3 primary and secondary spheres treated with PRP (T/C 0.07/0.42 mg/mL) at 48 and 72 hours after treatment. Scale bar represents 50 µm. (F) In vitro proliferation assay on BXPC-3 CSCs after treatment with PRP and gemcitabine. BXPC-3 primary and secondary spheres were incubated with PRP, gemcitabine (0,01 µM treatment) or with a combination of PRP/gemcitabine 72 h. The PRP, gemcitabine and combination of PRP and gemcitabine treatment resulted in a statistically significant decrease in primary and secondary CSCs spheres compared to control. Statistical significance indicated **p < 0.01. vs. samples not treated.

Fig 4: RNAseq analysis reveals enhanced Wnt signaling in EpCAMlo cells.(A) Multidimensional scaling analysis of RNAseq profiles of EpCAMhi and EpCAMlo cells from the HCT116 and SW480 lines. Red: HCT116, black: SW480, circle: EpCAMhi, triangle: EpCAMlo. (B) Ingenuity Pathway Analysis (IPA) of the HCT116 and SW480 expression profiles from the multicell line analysis (p adjusted value <0.01; log2 fold change <–1.5 and >1.5). Red marked pathways highlight the enhanced involvement of pathways involved in epithelial to mesenchymal transition, Wnt signaling, and the formation of colon cancer metastasis in the EpCAMlo subpopulation compared to EpCAMhi cells. (C) TOP-Flash luciferase reporter analysis of Wnt signaling activity in colon cancer cell lines HCT116 and SW480 upon treatment with 4 µM Chiron for 3 days. Each bar represents the mean ± SD of two independent experiments. (D) Flow cytometric analysis using antibodies directed against CD44 and EpCAM of control and 4 µM Chiron—figure supplemented HCT116 (A) and SW480 (B) cultures. Graphs show percentage of cells within the CD44hiEpCAMhi and CD44hiEpCAMlo gates relative to the control. Each bar represents the mean ± SD of two independent experiments. (E) Immunofluorescence analysis of control and Chiron-treated HCT116 (left panel) and SW480 (right panel) cells. After 3 days of treatment, 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: 100 µm. (F) Flow cytometric analysis of three HCT116 cell lines with differential ß-catenin mutation status, a parental HCT116 (HCT116-P) cell line harboring one WT and one mutant allele (Ser45 del), and two HCT116-WT and HCT116-MT cell lines harboring one WT or one mutant allele, respectively, generated by disruption of the other allele in the parental cell line (Kim et al., 2019). Figure 3—source data 1.List of differentially expressed genes in EpCAMlo vs. EpCAMhi cells in HCT116 and SW480.A total of 152 and 353 differentially regulated genes were identified between the CD44hiEpCAMhi and CD44hiEpCAMlo cells in HCT116 and SW480, respectively (p adjusted<0.01, when applying a log2 fold change of <–1.5 and > 1.5).

Fig 5: Gallbladder organoids express a biliary marker profile. (A) Technical outline: organoids were isolated from the gallbladders of adult mice, expanded in Matrigel, and genetically modified using CRISPR/Cas9 or by retroviral introduction of cDNAs. Genetically altered organoids were transplanted into recipient mice, either s.c. or orthotopically into the gallbladder. (B) Immunohistochemistry (IHC) confirms EpCAM expression within the epithelial layer of adult murine gallbladders. (C) Brightfield image of gallbladder organoids. (D) Flow cytometry analysis for EpCAM on single cell suspensions from adult mouse liver (left column), adult mouse gallbladder (middle column) and gallbladder organoids (right column). (E) Immunofluorescence on gallbladder organoids confirms expression of ß-catenin (left), CK19 (middle), and SOX9 (right).