Coaction of CCSCs/EPCs Promotes Development of CC Figure 6. Supernatants of spheroid cells promote tube formation of EPCs through VEGF up-regulation. (A) Tube formation of EPCs treated by supernatants of spheroid cells was higher than their counterparts. And tube formation of EPCs was suppressed when we block the function of VEGF by bevacizumab. (B) The VEGF concentrations in supernatants of spheroid cells were higher than those of adherent cells. (C) Higher VEGF expression was observed in the spheroid cells compared with their counterparts. doi:10.1371/journal.pone.0039069.g006 [37]. Furthermore, SP and non-SP cells of colon cancer cell lines demonstrate equivalent multipotential differentiation capacity and similar tumorigenicity in xenograft model [38]. In the present study, we obtained tumor spheres from HCT116 and HT29 colon cancer cell lines by culturing these cells in serum- free medium [39,40]. And the following analysis indicated that CD133 and Lgr5 expression of these spheroid cells was higher than their parental counterpart, while CK20 expression was lower which represents the differentiated endothelial cell. The contra- dictory phenotypes were detected when the spheroid cells were induced to differentiate in FBS containing medium. Moreover, the spheroid cells can form the same spheres, indicating that they have the self-renew capacity. From these results, we can draw the conclusion that cancer stem cells were enriched in these spheroid cells. In order to take a further insight into the biological behavior of these spheroid cells, we performed a series of functional experiments. The results showed that spheroid cells display higher capacity of proliferation and clone formation, which represent self- renewal potential, as compared to their adherent counterparts. And also, spheroid cells demonstrate lower capacities of both adhesions to FN (one of the key ECM components) and adhesion to their homogeneous neighbors. That was to say, this kind of cells are easier to release from the bulk tumor. ICAM-1 is an important cell surface molecular mediating the intercellular adhesion and its downregulation can explain the lower homogenic adhesion of spheroid cells. Our results indicated that spheroid cells have enhanced in vitro migration and invasion potential. CXC chemokine receptor 4 (CXCR4) interacts specifically with the stromal cell-derived factor-1 (SDF-1), which is expressed in stromal cells, including fibroblasts and endothelial cells [41,42]. Recent studies showed that chemotaxis effect of CXCR4/SDF-1 axis is related with lymph node and liver metastasis of CRC [43,44,45,46]. And in our study, we found expression of CXCR4 of spheroid cells were upregulated. The circulating, bone-marrow-derived EPCs contribute to angiogenesis- mediated pathological neovascularization, and recent studies have begun to recognize the biological significance of this contribution, especially to the growth and metastasis of tumors. Generally speaking, circulating EPCs recruit to sites of tumors following gradients of growth factors, such as vascular endothelial growth factor (VEGF), SDF-1 etc, which are released PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e39069