cancer stem cells and the role of coaction of CSCs and EPCs in the growth and metastasis of colon cancer. Materials and Methods Ethics Statement All experiments involving human participants (including collec- tion of human umbilical cord blood and colon cancer samples) have been approved by the Medical Research Ethics Committee of Sun Yat-sen University, and conducted according to the principles expressed in the Declaration of Helsinki. All participants involved in the study signed the informed consent forms and all animal experiments were conducted according to relevant national and international guidelines. And this project was approved by the Medical Research Animal Ethics Committee of Sun Yat-sen University. Cell Culture HCT116 (ATCC, CCL-247) and HT29 (ATCC, HTB-38) colon cancer cell lines were maintained in DMEM/F12 supple- mented with 10% FBS, 200 U/ml penicillin, 200 mg/ml strepto- mycin. Tumorsphere media (also called as serum free medium, SFM) was composed of DMEM/F12 media supplemented with 16B27 (Invitrogen), EGF (20 ng/ml, Peprotech), bFGF (10 ng/ ml, Peprotech), routine insulin (5 mg/ml, Invitrogen), 200 U/ml penicillin, and 200 mg/ml streptomycin. For 3D suspension culture, HCT116 and HT29 cells grown in two dimensional monolayer were digested with trypsin, resuspended, and then seeded at a density of 26106 cells in SFM in 100 mm ultra-low attachment dishes (Corning) at 37uC in a humidified 5% CO2 ? 95% air atmosphere. Detection of CD133 Expression by Flow Cytometry The cells derived from monolayer cultures and suspension spheres on day 7 after primary culture were detected for the expression of CD133. The cells were washed twice in cold PBS, and subsequently cell suspensions were incubated at 4uC with 1:10 FITC-conjugated mouse monoclonal antihuman CD133 Ab (Miltenyi biotec) for 45 minutes in the dark. After incubation, the cells were washed twice in cold PBS with 1% BSA and resuspended in 400 ml cold PBS with 1% BSA for flow cytometry analysis within 1 hour. Self-renew and Multi-differentiation Assay Spheroid cells were dissociated into single cells and plated in 6- well plates at 100 cells per well in 2 ml SFM. To evaluate the self- renew capacity of spheroid cells, cell immunofluorescence staining of Lgr5 and CK20 were performed after 7 days’ culturing. Cell differentiation was induced by culturing in DMEM/F12 supple- mented with 10% FBS in common flasks. Four weeks later, the same cell immunofluorescence staining was performed, and the expression of CD133 was determined by FCM as described before. Cell Proliferation and Colony Formation Assay Single-cell suspensions of spheroid and adherent cells were prepared and seeded at 16104 cells per well in 96-well plates, and cultured in either SFM or serum-containing media for 24 h. Cell proliferation was performed on day 1, 2, 3, 4, 5, 6, and 7 using Cell Counting Kit-8 (Dojindo, Japan). The plate colony formation assay was carried out to determine the colony formation efficacy of cell in the four groups. Single-cell suspensions were plated at 16102 cells per well in 6-well plates, and cultured in serum- containing media at 37uC in 5% CO2 for 2 weeks. Then the plates Coaction of CCSCs/EPCs Promotes Development of CC were stained with Giemsa-Wright’s stain. The number of colonies within each well was counted and photographed. The experiments were independently performed at least three times. Homogeneity and Heterogeneity Adhesion Assay The adhesion ability of cells to ECM was tested using fibronectin (FN)-coated 96-well plates (Corning). Single-cell suspensions of spheroid and adherent cells were plated (105/well), and incubated at 37uC for 2 h, then washed with PBS to remove the non-adhesive cells. Delt OD of 570 nm wavelength was determined to reflect the FN-adherent cells using Cell Counting Kit-8 (Dojindo, Japan). The adhesion ability of cells to homoge- neous cells was tested using monolayer cells-paved 96-well plates. Spheroid and adherent cells were plated at 105 per well, and incubated at 37uC for 60 min, 90 min, and 120 min. Then the non-adhesive cells were counted and homotypic adhesive activity was calculated using the formula: homotypic adhesion (%) = (total cell number - non-adhesive cells)/total cells6100%. Migration and Invasion Assays Migration and invasion assays were performed in 24-well plate transwell chambers with 8 mm-pore polycarbonate filter inserts (Corning). A total of 56104 or 105 dissociated spheroid or adherent cells were seeded on uncoated or Matrigel-coated inserts in 100 ml of serum-free medium inserts for migration or invasion assays respectively. The lower chambers were filled with 0.5 ml of 10% FBS-supplemented DMEM/F12 medium. After 24 h and/or 48 h, cells on the upper side of the filter were removed and the cells on the lower surface of the insert were fixed and stained with crystal violet. The number of stained cells was counted under a light microscope. Assays were performed in triplicates. Western Blot Analysis The total cell extracts were separated by 10% SDS-polyacryl- amide gel electrophoresis and transferred to Polyvinylidene Fluoride Membrane (Millipore) for ICAM-1, CXCR4 and VEGF detection. The membranes were washed in Tris-buffered saline with Tween (TBST, composed of 10 mM Tris-HCl, pH 8, 150 mM NaCl, and 0.05% Tween 20), blocked 1 h at room temperature with 5% nonfat milk in TBST, and then probed 1 h at room temperature with anti-ICAM-1 (Santa Cruz Biotechnol- ogies), anti-CXCR4 (Abcam), anti-VEGF (BD Pharmingen) and anti-GAPDH (Peprotech). After incubation with horseradish peroxide-conjugated secondary antibody, immunoreactive pro- teins were detected by ECL detection system (Millipore). Protein concentrations were determined by using the BCA protein assay kit (ThermoScientific Biosciences). Isolation and Assessment of EPCs Mononuclear cells were isolated by density gradient centrifu- gation from human umbilical cord blood and cultured in fibronectin-coated flask using M199 supplemented with 20% FBS and 1% extracts of mouse cerebral tissue. EPCs were purified basing on different adherence rate, and non-adherent cells within 24 h were transferred to another fibronectin-coated flask. After 14 days’ culturing, EPCs were incubated with PE-CD133, FITC- CD34, PE-VEGFR2 or isotype-matched IgG controls and identified by flow cytometry as described before. Cell Proliferation and Migration of EPCs Single-cell suspension of EPCs was seeded at 26104 cells per well in 96-well plates, and cultured in M199 containing 0, 20%, and 40% supernatant of spheroid or adherent cells. Cell PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e39069