Figure 1.
Morphological character and molecular marker expression of adherent/ spheroid cells of HCT-116 and HT29.
Both HCT116 and HT29 can form large round, unattached floating colonsphere of 50–100 μm when cultured in SFM. FCM analysis showed that CD133 expression rise from ≤1.2% up to 60%–80% when cultured in SFM. And strongly expression of Lgr5 was detected by immunofluorescence staining. But as the marker of differentiated epithelial cell, CK20 expression presented adverse change.
Figure 2.
Cell adhesion and migratory/invasive capacity of adherent and spheroid cells.
Spheroid cells have lower capacities of both adhesions to FN (A) and to their homogeneous neighbors (B), as compared with their parental counterparts. HCT116/HT29 spheroid cells showed a 3.8-fold (p<0.01) and 4-fold (p<0.001) increase in chemotactic potential at 24 h (C). More spheroid cells were able to invade Matrigel as compared to their parental counterparts (p<0.001 and p<0.05, respectively, D).
Figure 3.
Spheroid cells possess higher tumorigenic and metastatic potential in vivo.
The volumes of subcutaneous tumor in HCT116 spheroid cells group were significantly higher than that in HCT116 cells group (A, B). HT29 spheroid cells showed the similar results (C). The HCT116 spheroid cells developed more liver metastatic foci than HCT116 (D, F). Metastatic tumors were confirmed as colon cancers with hematoxylin-eosin staining (E).
Figure 4.
EMT-phenotype detection of spheroid/adherent cells.
Representative photomicrographs depicting the higher expression of E-cadherin and the membrane localization of β-catenin in HCT116 and HT-29 adherent cells than the corresponding spheroid cells by immunofluorescence staining. And higher expression of Vimentin in spheroid cells was detected comparing to adherent cells.
Figure 5.
The comparative expression of different members of Wnt/â-catenin signaling pathway in parental cells and spheroid cells.
The Wnt/β-catenin signaling is constitutively activated in spheroid cells, including up-regulated expression of Vimentin, Slug, Snail, and nuclear β-catenin, but down-regulated expression of E-cadherin and GSK3β, comparing with the corresponding parental cells.