Figure 4.

Spheroids formed by cholangiocarcinomas (CCAs) subpopulations. A: We determined the capacity of spheroid formation by cancer stem cell (CSC) subpopulations immunosorted from CCA primary cultures; the number of spheroids (see bar graph) formed in vitro being a self-renewal index. By immunofluorescence (IF) for the marker used for immunosorting, we tested the purity of the formed spheroids. Spheroids formed by CD90⁺ cells are enriched for CD90⁺ cells, whereas the negative counterpart CD90⁻ forms spheroids where CD90⁺ cells are <5%, indicating that these spheroids are formed by other CSC subpopulations. Each subpopulation forms spheroids efficiently, reaching a size of 100 to 500 μm after 7 days in culture. IF (nuclei were stained with DAPI) shows the enrichment in the spheroids by the immunosorted cell subpopulation. CD133, EpCAM, and LGR5 form a higher (P < 0.01) number of spheroids compared to cells expressing the mesenchymal cell marker, CD90, or CD13 (note that scales are different). CD13⁺ cells from mixed-IHCCA (IH-mixed) form a higher number of spheroids than CD13⁺ cells immunoselected from mucin-IHCCA (IH-mucin), whereas the opposite is observed for CD90⁺ or CD133⁺ cells (mucin > mixed.) B: Spheroids formed by CD133⁺ and CD90⁺ CSC subpopulations were analyzed by IF for markers of epithelial-mesenchymal transition (Twist, SNAIL, vimentin, P-cadherin). Spheroids show positive staining for Twist, SNAIL, vimentin, and P-cadherin without differences between CD133⁺ and CD90⁺ spheroids. ^∗P < 0.05, ^∗∗P < 0.01. Original magnification: ×30 (A); ×40 (B).