Wnt Signaling and Colon Cancer Stem Cells Figure 7. A proposed model for the interaction of tumor cells with macrophages: Colon cancer cells produce a soluble factor, a product of a Wnt regulated gene, which through binding to an unknown receptor stimulates macrophages to release IL1b. IL1b in turn binds IL1R on tumor cells and stimulates Wnt signaling. We showed that macrophage-derived factor stabilize Snail in tumor cells, which further promotes Wnt signaling and is required for the ability of tumor cells to activate macrophages to produce IL1b. doi:10.1371/journal.pone.0045462.g007 that macrophage- derived factors contribute to heterogeneity of colon tumors by expanding the fraction of tumor cells that have cancer stem cell properties. Likewise, myofibroblast-derived factors have been shown to impose the CSC phenotype by promoting Wnt signaling in tumor cells [34] and periostin, a component of the extracellular matrix, promotes the stemness of cancer cells and increases their ability to form metastasis by upregulating Wnt signaling [36]. Because an active Wnt signaling is a signature of colon cancer stem cells, it is possible that cancer stem cells have an exclusive ability to interact with the cells in the tumor microenvironment. Consistent with this, cancer associated fibroblasts promote the invasiveness of CD133+ colon cancer stem cells more efficiently than that of CD1332 cells [37]. This is likely to contribute to the unique ability of cancer stem cells to propagate tumor growth and to confer resistance to therapeutic approaches. Materials and Methods Cell lines and co-culture experiments The HCT116 and Hke-3 colorectal carcinoma cell lines, which differ only by the presence of the mutant k-Ras allele [19], were cultured in MEM, supplemented with 10% FCS. HCT116 with disrupted WT or MT b-catenin allele were provided by Kenneth Kinzler [8]. The human monocytic cell line, THP1, was cultured in RPMI. Normal human monocytes, .90% CD14 and CD11c positive and less than 1% anti T cell receptor positive, were purchased from Astarte Biologics (Redmond, WA). Tumor cells and monocytes/macrophages were co-cultured separated by transwell inserts of a polycarbonate membrane with 0.4 mM pore size, which preclude direct cell-cell contact, but permit the exchange of soluble factors (Corning Incorporated, Lowell, MA). For clonogenic assays, HCT116 and Hke-3 cells were seeded at a density of 200 cells per well of a six well plate alone or together with peripheral blood monocytes for 7 days as described previously [10,11]. Colonies were fixed and stained with 6% glutaraldehyde and 0.5% crystal violet and counted using Total Lab 1.1 software (Nonlinear Dynamics, Durham, NC, USA). Apoptosis assay Cells were treated with recombinant TRAIL (50 ng/ml, the optimal concentration for the induction of apoptosis in HCT116 cells) alone or in the presence of macrophages, IL1b (5 ng/ml) or TNFa (10 ng/ml) for 7 hours. Cells were resuspended in hypotonic buffer (0.1% Triton X-100, 0.1% sodium citrate) and stained with propidium iodide (50 mg/ml) for 4 hours at 4uC as described [38]. Samples were analyzed by flow cytometry and cell cycle distribution and the extent of apoptosis (cells with a subG1 DNA content) were analyzed by the Modfit software. Cells were treated with TRAIL for ,7 hours and were collected for evaluation based on morphological criteria. We confirmed the apoptotic nature of cells by biochemical analysis of cell lysates. Statistical analysis was performed using unpaired Student’s t test, with values ,0.05 considered statistically significant. PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e45462