Wnt Signaling and Colon Cancer Stem Cells Figure 4. Macrophage-derived factors do not protect from TRAIL induced apoptosis in the absence of the mutant b-catenin allele. A: B: HCT116WT and HCT116MT cells were treated with TRAIL in the absence or the presence of IL1b as indicated and the localization of b-catenin was determined by immunofluorescence in cells treated with TRAIL in the absence or the presence of IL-1b or TNF as indicated. B: The extent of caspase 8 activation and the cleavage of PARP and b-catenin were determined by immunoblotting. doi:10.1371/journal.pone.0045462.g004 We showed earlier that macrophage-derived factors failed to stabilize Snail in HCT116 cells expressing dnTCF4, consistent with the notion that active Wnt signaling is required for stabilization of Snail [13]. Here we compared the ability of macrophage-derived factors to stabilize Snail in HCT116 cells that had a deletion of either the WT or the MT b-catenin allele. As in parental HCT116 cells, macrophages, IL1b and TNF stabilized Snail in HCT116MT cells (Fig. 5C). In contrast, macrophage- derived factors failed to stabilize Snail in HCT116WT cells (Fig. 5C), consistent with the inability of macrophages to enhance Wnt signaling in these cells (Fig. 1). We silenced Snail in HCT116 cells and confirmed that, consistent with our published data [13], macrophages and IL1b failed to stimulate Wnt signaling in the absence of Snail (Fig. 5D). Tumor cells activate macrophages via a Wnt-dependent factor We demonstrated that tumor-derived factor(s) stimulate mac- rophages to secrete a variety of soluble factors, including IL1b, a cytokine that we established was required for the crosstalk between tumor cells and macrophages [10]. We showed that HCT116 cells expressing dnTCF4 failed to stimulate peripheral blood monocytes to secrete IL1b (Fig. 6A), confirming that Wnt signaling is required for the expression of the tumor derived factor that stimulates macrophages. Likewise, silencing of Snail, a Wnt target gene, in HCT116 or Hke-3 cells resulted in inability of these cells to trigger IL1b production in transformed THP1 monocytes (Fig. 6B). Together, these data confirmed that colon cancer cells activate macrophages via a Wnt/Snail-regulated factor, which is required for the production of IL1b. Discussion Although appropriately stimulated macrophages have the inherent ability to kill tumor cells, it has become clear that macrophages recruited to the tumor microenvironment and shaped by tumor-derived factors lose the ability to destroy tumor cells. Moreover, it has been established that tumor associated macrophages (TAMs) provide soluble factors that promote the growth, survival and metastatic spread of colon cancer cells [22– 24]. Several macrophage-derived factors have been identified that regulate the growth of tumor cells. We showed that macrophage- derived IL1b promotes Wnt signaling in colon cancer cells and thus regulates their growth and survival [10,13]. In turn, cancer cells secrete factors that attract and activate myeloid cells, and thus PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e45462