proliferation was performed on day 1, 2, 3, 4, 5, 6, and 7 using Cell Counting Kit-8 (Dojindo, Japan). Migration assays were performed to evaluate recruitment of EPCs by spheroid or adherent cells. Single-cell suspension of EPCs was inoculated at 56104 cells per well on inserts in 100 ml of serum-free medium. The lower chambers were filled with 0.5 ml of DMEM/F12 containing 40% supernatant of spheroid or adherent cells. After 24 h, migrated cells were counted as describe before. Assays were performed in triplicates. In vitro Capillary Tube Formation Supernatants of HT29, HT29 Sphere, HCT116 and HCT116 Sphere were added to 24-well plates (Corning Inc) coated with growth factor reduced Matrigel (BD Biosciences). To illustrate if tube formation capacity of EPCs can be influenced by blocking VEGF, bevacizumab (avastin, a VEGF monoclone antibody, Genentech Inc) was added to supernatant of spheroid cells at a concentration of 0.25 mg/ml as another two groups [30,31]. Then a total of 56104 EPCs were seeded and incubated at 37uC, 5% CO2 for 3 to 30 h. After 24 h, capillary tubes were counted in random fields from each well. Assays were performed in triplicates. ELISA To demonstrate the a paracrine effect of cancer stem cells and confirm the result of VEGF detection from Westerm Blot analysis, we investigated VEGF secretion by cancer stem cells using a quantitative method. VEGF concentration in supernatant of spheroid/adherent cells were determined by ELISA (R&D Systems) according to the protocol provided by the manufacturer. And also, the experiment was performed three times. In vivo Tumorigenesis To determine whether spheroid cells are more tumorigenic than their adherent counterparts in vivo and whether EPCs promote their tumorigenic capacity by increasing tumor angiogenesis, we carried out tumor development and liver metastasis experiments. Single cells were resuspended in PBS. And then 100 ml suspensions containing 26106 adherent cells, spheroid cells, or spheroid cells plus 26105 or 26104 EPCs (the spheroid cells and EPCs ratio of 10:1 and 100:1) were injected subcutaneously (s.c.) into the flanks of 4- to 6-week-old male BALB/C-nu mouse, obtained from the Jackson Laboratory. Subcutaneous tumor diameters were mea- sured with a digital caliper every two or three days, and tumor volume in mm3 was calculated using the formula: Volume = width26length60.52. Then 3 or 4 weeks later, the mice were killed and frozen tissue sections (6 mm) of subcutaneous tumors were made for immunofluorescent staining to observe the angiogenesis in each group. Here we use a non-specific polyclonal CD31 antibody to detect the neovascularization of the subcuta- neous tumor, and another monoclonal human-specific CD31 antibody for the EPCs contribution to angiogoiesis. In vivo Liver Metastasis Liver metastatic model was used to determine the metastatic potential of spheroid cells and whether EPCs promote this capacity by increasing tumor angiogenesis. Hepatic metastases were produced by intrasplenic injection of 46106 adherent cells, spheroid cells, or speroid cells plus 46105 or 26104 EPCs (the ratio of 10:1 or 100:1), and then splenectomy was performed 10 min after intrasplenic injection to avoid splend metastasis. The overall health condition was recorded in detail once a day. Liver metastasis foci were examined and measured. And tissue section was made for HE staining to confirm the pathological source. Coaction of CCSCs/EPCs Promotes Development of CC Clinical Evidence Obtained by Immunofluorescent Staining Frozen sections (6 mm) of colon cancer tissue and normal colon mucosa (n = 15, from the same patient) were fixed with 4% paraformaldehyde/PBS, treated with microwave for 10 min for immunofluorescent staining. The anti-CD31 Ab (Fisher Scientific), the anti-CD133 Ab (Abcam), the anti-Lgr5 Ab (Abcam), and the anti-CK20 Ab (Santa Cruz Biotechnologies) were used according to the manufacturer’s protocol. Counterstaining of nuclei with DAPI (Invitrogen) was also performed. The sections were incubated with according secondary DeLight448- or 555-conju- gated anti-mouse or rabbit Ab (Abcam) for 1 h at room temperature or overnight at 4uC. Images were captured with a Zeiss confocal microscope (LSM-710). Here, throughout this manuscript, data from at least three independent experiments have been analyzed to verify reproducibility of results. Statistical Analysis Statistical significance was determined by one-way ANOVA followed by Bonferroni post-hoc test for multiple comparisons or Student’s t-test. For all tests, P,0.05 or ,0.01 was considered significant or highly significant statistically. Results Generation and Characterization of Colonospheres Both HCT116 and HT29 colon cancer cells grew in large round, unattached floating spheroid colonies (termed colono- spheres) when they were cultured in serum free medium supplemented with 16B27, 20 ng/ml EGF, 10 ng/ml bFGF (Figure 1). The frequency of sphere-forming cells was determined by performing an extreme limiting dilution analysis (ELDA) for parental and colonospheres-derived HCT-116 cells. The frequen- cy to form spheres was found to be 5.5 fold higher for cells derived from colonospheres than those from the parental cell lines. The spheroid cells showed higher levels of CD133 and Lgr5 (known as colon CSC markers), but lower levels of differentiated epithelial cell marker CK20 than the corresponding parental cells. The proportion of the CD133 positive cells was found to be less than 1% in parental cell lines, but more than 80% observed in spheroid cells (Figure 2). When the colonospheres were subjected to immunofluorescence staining for Lgr5, obvious Lgr5 staining was observed on the the surface of most spheroid cells indicating the presence of CSCs in colonosphere (Figure 2). When the spheroid cells were cultured in 10% FBS containing medium, they attached to the plastic, gradually migrated from colon spheres and changed into adherent cells morphologically. Flow cytometry and immunofluorescence examination showed down-regulated CD133 and Lgr5 expression with up-regulated CK20 after 4 weeks’ culturing (Figure 2). The results suggest that CSCs were efficiently enriched in spheroid cells, and they have the capacity of self-renew and multi-differentiation. Spheroid Cells Display Higher Capacity of Proliferation and Clone Formation We further determined whether the spheroid cells have capacity of higher cell proliferation using CCK-8 assay. The proliferations of spheroid cells were higher than that of their parental counterpart after a period of adaptation to serum containing medium (Figure 3A and 3B). And the capacity of colony formation in spheroid cells groups was higher than that of their parental counterpart (Figure 3C and 3D). PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e39069