Coaction of CCSCs/EPCs Promotes Development of CC Figure 7. Spheroid cells possess higher tumorigenic and metastatic potential in vivo and EPCs can increase these capacities. (A–F) The spheroid cells showed significantly higher tumorigenecity than adherent cells (A, B, E). And the volumes of subcutaneous tumors in spheroid cells plus EPCs groups were larger than those of spheroid cells groups (C, D, F). (G–J) Microvessel density (MVD) of tumor developed by HCT116 spheroid cells (H) was higher than that of HCT116 (G). The highest MVD among these groups was found in tumor tissue developed from HCT116 sphere plus 1/ 10 EPCs (I, J). And more blood vessel developed from human EPCs in HCT116 sphere plus EPCs group (L) were found than that of pure spheroid cells group (K). (M) The HCT116 spheroid cells developed more liver metastatic foci than HCT116. doi:10.1371/journal.pone.0039069.g007 by tumor [47]. Kyrezek et al. reported the expressions of VEGF and SDF-1 in tumors could be enhanced each other. And in the other hand, VEGF could up-regulate the expression of CXCR4 in vascular endothelial cell, thus promote the migration of EPCs and contribute to tumor neovascularization [48]. In our study, we isolated EPCs, which display a certain expression of CD34 and CD133, but high level expression of VEGFR2 in its later stage. And we found that supernatants of spheroid cells can promote proliferation, migration and tube formation of EPCs by up- regulating VEGF expression and bevacizumab can suppress the angiogenesis of EPCs by blocking the function of VEGF (Figure 5, 6). The results of xenograft model indicated that EPCs can promote the growth of subcutaneous tumor of spheroid cells by mediating neovascularization (Figure 7). And using the human specific CD31 antibody, we can detect the blood vessel developed from EPCs. The results indicated that EPCs may play an important role in the neovascularization. It can be seen that VEGF is relevant to the interaction between EPCs and CSCs. Since the EPCs may be responsible for turning on the ‘‘angiogenic switch’’, our results indicates a therapeutic strategy can be employed to keep this switch in the ‘‘off’’ position for colorectal cancer [47]. That is to say, if we block the interaction between EPCs and CSC, decreased tumor vascularization and suppressed tumor progression can be expected. Although no significant difference were found between spheroid cells plus EPCs groups and spheroid cells groups in liver metastasis, which may be due to the short observation period and limited sample size. To further confirm the correlation between colon cancer stem cells and the tumor neovascularization, we then performed immunoflu- orescence in tumor tissues. CD31 expression is associated to CD133 or Lgr5 expression in colon cancer tissues (Figure 8), which indicates robust angiogenesis is stimulated by cancer stem cells. In recent years, the evidence strongly indicates that CSCs not only promote self- renewal and tumor initiation, but also induce and stimulate angiogenesis. They seem to be adept at initiating a potent angiogenic response by inducing and secreting certain proangiogenic factors, such as VEGF, which constitute one of the CSC factor subsets [40,49]. These factors may promote the EPCs migration and neovascularization. Interestingly, in addition to the close link between angiogenesis and CSCs [50], certain stem-like cells can transdifferentiate into endothelial-like cells to form tumor blood vessels via a process termed vasculogenic mimicry [51,52,53]. This can be used to explain why human CD31 antigen exists in subcutaneous tumor developed from spheroid cells (Figure 7K). PLoS ONE | www.plosone.org 10 June 2012 | Volume 7 | Issue 6 | e39069