Coaction of CCSCs/EPCs Promotes Development of CC Figure 2. CD133/CD44, Lgr5, and CK20 expression of adherent, spheres, and differentiated HCT-116 and HT29. CD133 (known as cancer stem cell marker) expression rises from #1.2% up to 60%–80% when cultured in SFM, but reduced to 5%–10% when the colonsphere changed back into adherent cells. The same change of Lgr5 (known as crypt stem cell marker) expression can be found while CK20 expression, the marker of differentiated epithelial cell, showed the adverse change. doi:10.1371/journal.pone.0039069.g002 spheroid cells group compared with adherent cells group, and even more angiogenesis in tumor tissue was observed in the combined group of HCT116 sphere plus 1/10 EPCs (Figure 7G– 7J). And the MVD were counted according to the non-specific polyclonal CD31 antibody. In order to determine the EPCs contribution, we performed IF staining using human-specific monoclonal CD31 antibody. The result showed more blood vessels in the combined group of HCT116 sphere plus EPCs than pure spheroid cells (Figure 7K–L). As the liver metastasis was concerned, four mice (4/5) developed liver metastasis in spheroid cells group, but only one mouse (1/5) did in adherent group. Metastatic tumors were also confirmed as colon cancers with hematoxylin-eosin staining (figure not shown). The result of liver metastatic foci counting showed the similar results (Figure 7M). The overall conditions of the mice in spheroid cells group are worse than those in adherent cells group, and some of them developed ascites and severe emaciation. And there were no significant difference between spheroid cells plus EPCs (with ratio of 10:1 and 100:1) groups and spheroid cells groups (data not shown). Clinical Evidence for Cancer Stem Cells Promote Angiogenesis in Human Colon Cancers To support our experimental findings, we examined whether colon cancer stem cells promote angiogenesis within human colon cancers, and the normal mucosa from the same patient as control. The immunofluorescent staining of CD133/Lgr5 (colon cancer stem cell marker) and blood vessel EC marker CD31 were performed. As we all know, there are more microvessels in cancer tissue than normal tissue. In addition to this, we found that higher microvessel density in the area enriching cancer stem cells in the fresh specimen of human colon cancer (Figure 8). Discussion In the 70s of the last century, Fidler IJ et al. [32] proposed the concept of tumor heterogeneity, which presume there were different phenotypes in the tumor tissue. The cancer stem cell theory was shaped based on this concept. Cancer stem cell is generally defined as a small sub-population of tumor cells bearing self-renewal and multi-directional differentiation potential [11]. Up to now, the existence of cancer stem cells has been confirmed in several solid tumors, including colon cancer. According to this Figure 3. Proliferation and clone formation of adherent and spheroid cells of HCT-116 and HT29. (A–B) Proliferation of HCT116 and HT29 spheroid cells were higher than that of their parental counterparts after a period of adaptation to serum containing medium. (C–D) The capacity of colony formation in spheroid cells groups was higher than that of their parental counterpart. doi:10.1371/journal.pone.0039069.g003 PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e39069