Coaction of CCSCs/EPCs Promotes Development of CC Figure 1. Morphological change of adherent, spheres, and differentiated HCT-116 and HT29. Both HCT116 and HT29 can form large round, unattached floating colonsphere of 50–100 mm when cultured in SFM. When spheroid cells were cultured in 10% FBS containing medium, the floating spheroid cells attached to the plastic, gradually migrated from colonspheres and transformed to adherent cells. doi:10.1371/journal.pone.0039069.g001 Spheroid Cells Display Lower Adhesion but Higher in vitro Migratory/invasive Capacity To investigate the malignant profile of spheroid cells, we conducted in vitro assays to evaluate the adhesion and migratory/ invasive capacity of spheroid cells in comparison with their adherent counterparts. The results of cell adhesion assay demonstrated that spheroid cells have lower capacities of both adhesion to FN (one of the key ECM components) and adhesion to their homogeneous neighbors, as compared with their parental cells (Figure 4A and 4B). Then CXCR4 and ICAM-1 expression in HCT116/HT29 spheroid cells was examined and compared with their parental cells. The ICAM-1 level was modestly down- regulated, while CXCR4 expression was up-regulated in HCT116/HT29 spheroid cells when compared with HCT116/ HT29 monolayer cells respectively (Figure 4C). Using Transwell migration chambers, we found that spheroid cells display a significant increase in cell motility. Compared with HCT116/HT29 adherent cells, 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, respectively (Figure 4D). These cells also demonstrated higher capacity to invade Matrigel-coated inserts in Transwell migration chambers. More HCT116/HT29 spheroid cells were obviously observed to invade Matrigel as compared with corresponding adherent cells (p,0.001 and p,0.05, respectively) (Figure 4E). These results indicate that spheroid cells are endowed with lower adhesion, but higher migratory/invasive capacity, a functional phenotype associated with tumor aggressiveness. Spheroid Cells Promotes Proliferation, Migration and Tube Formation of EPCs Through Up-regulating VEGF Expression Purified EPCs were obtained from human umbilical cord blood. Flow cytometry analysis showed that EPCs in later stage displayed a certain expression of CD34 and CD133, but high level expression of VEGFR2 (Figure 5A–5C). Cell proliferation and migration of EPCs induced by supernatants of spheroid cells were higher than their counterparts (Figure 5D and 5E). The tube formation assay showed that more tubes formed in spheroid cells group than adherent cells group. Additionally, when 0.25 mg/ml bevacizumab (avastin, a VEGF monoclonal antibody) was added in supernatant of spheroid cells, the tube formation capacity of EPCs was suppressed (Figure 6A). Then the VEGF secretions in supernatants of spheroid/adherent cells were detected using ELISA. The results showed that concentration of VEGF in supernatant of adherent cells was lower than spheroid cells (Figure 6B). The expression of spheroid/adherent cells were evaluated by Western blot (Figure 6C). The results illustrated that VEGF may play an important role in the effect of spheroid cells on EPCs, which suggested that spheroid cells may promote angio- genesis of EPCs through up- regulating VEGF expression. Spheroid Cells Possess Higher Tumorigenic and Metastatic Potential in vivo and EPCs Can Increase these Capacities The spheroid cells showed significantly higher tumorigencity than adherent cells (Figure 7A, 7B and 7E). When 26106 cells were inoculated into nude mice, all mice developed tumors. Transplanted tumors were confirmed as colon cancers with hematoxylin-eosin staining. And the volumes of subcutaneous tumor in spheroid cells groups were significantly higher than that of adherent cells groups. Moreover, the volumes of subcutaneous tumor in spheroid cells plus 1/10 EPCs groups were higher than that of pure spheroid cells groups (Figure 7C, 7D and 7F). But no significant difference was found between pure spheroid cells and spheroid cells plus 1/100 EPCs (data not shown). Then blood vessels density in tumor tissues were evaluated using CD31 immunofluorescence staining. More blood vessels were found in PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e39069