Figure 1

Sphere-forming primary colon tumour cells under stem cell culture conditions retain CD133 expression and tumourigenic potential. (A) Formation of non-adherent, three-dimensional (3D) spheroids was observed within 4 weeks in the cultures of primary tumour cells derived from the majority of tumour samples tested (left; bar, 150 μm). In extended culture over a month, some tumour spheroids may become adherent (arrows) and partially differentiated into adherent cell populations (middle; bar, 300 μm). A closeup of the boxed area in the middle panel shows the morphology of epithelial lineages differentiated from a tumour sphere (right; bar, 75 μm). (B) Morphology of adherent cells and non-adherent tumour spheres derived from a sorted CD133⁺/EpCAM⁺ population in serum-containing and serum-free media, respectively (top panel). Cell surface staining of CD133 expression (green) was detected by immunofluorescent staining in spheroid populations, whereas their serum-driven adherent counterparts lost the expression of CD133 (bottom panel). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Bar, 50 μm. (C) Flow cytometry analysis shows that 89.20% of spheroid cells are positive for CD133/EpCAM. (D) A fraction of clonally derived spheroid cells reform spheroids in self-renewal assays (mean±s.d., n=5). (E) Histology of representative xenografted tumours derived from whole-tumour spheres (haematoxylin and eosin (H&E) staining; × 10 objective).