Coaction of CCSCs/EPCs Promotes Development of CC Figure 4. Cell adhesion and migratory/invasive capacity of adherent and spheroid cells. (A–C) Spheroid cells have lower capacities of both adhesions to FN (A) and to their homogeneous neighbors (B), as compared with their parental cells. The ICAM-1 expression in speroid cells was modestly down-regulated, while CXCR4 expression was up-regulated as compared to HCT116/HT29 monolayer cells respectively (C). (D and E) 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 (D). More spheroid cells were obviously observed to invade Matrigel as compared to their parental counterparts (p,0.001 and p,0.05, respectively, E). doi:10.1371/journal.pone.0039069.g004 hypothesis, the small subpopulation of cancer stem cells was bearing the whole characteristic of malignancy, including infinite proliferation, invasion and metastasis, relapse and resistance to therapy. Nowadays, the extensively used method to isolate or enrich colon cancer stem cells is cell sorting based on cell surface markers, such as CD133, Musasai-1, CD44, EpCAM, Lgr5, etc. Ricci- Vitiani L et al. reported the CD133+ cells which accounts for about 2.5% of the tumour cells are tumorgenic cells in colon cancer [33]. Subcutaneous injection of colon cancer CD133+ cells readily reproduced the original tumor in immunodeficient mice, whereas CD1332 cells did not develop tumors. However, there were still many different opinions on these cancer stem cell markers. For example, Shmelkov SV proposed that CD133 expression is not restricted to stem cells, and both CD133+ and CD1332 metastatic colon cancer cells initiate tumors [12]. Some researchers reported the dye-effluxing side population cells expressing ABCG2, an ATP-binding cassette half-transporter, could be isolated as cancer stem cells [34,35,36]. However, several studies showed evidence against an association between the SP population and cancer stem cells. SP cells isolated from MKN28 gastric cancer cells did not produce tumor in a xenograft model Figure 5. Supernatants of spheroid cells promote proliferation and migration of EPCs. (A-C) EPCs adhere to FN-coated flask after 72 hr and EPCs clones can be seen after one week (A), and present cobbles-like sign after two weeks (B). EPCs in later stage display a certain expression of CD34 and CD133, but high level expression of VEGFR2 (C). (D, E) Cell proliferation (D) and migration (E) of EPCs treated by supernatants of spheroid cells were higher than their counterpart. doi:10.1371/journal.pone.0039069.g005 PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e39069