Wnt signaling in HCT116 cells [10]. We showed that macro- phage-derived factors stimulated Wnt signaling in an NF-kB- dependent manner [11]. Consistent with this, constitutive activa- tion of IKK2 in intestinal epithelial cells, which is sufficient to induce intestinal tumors in mice, triggers mobilization of macrophages and the expression of a variety of proinflammatory cytokines, including TNF and IL1b [12], and cells with constitutive activation of IKK2 displayed activated Wnt signaling. We showed that macrophage-derived factors stabilize Snail in colon cancer cells [13], a transcription factor that promotes the invasive and metastatic phenotype through induction of an epithelial mesenchymal transition (EMT) [14]. Snail is a Wnt target gene [15,16], but it has also been shown to interact with b- catenin and to co-stimulate Wnt target genes [15–17], indicating an intricate interplay between Wnt signaling and Snail. In addition, Snail regulates growth and survival of tumor cells, and has been linked to the formation of cancer stem cells [18]. Increased Wnt signaling and stabilization of Snail therefore strongly suggest that macrophage-derived factors increase the stem cell properties of cancer cells, which contribute to metastasis and display resistance to therapy. Consistent with this, we showed that macrophage-derived factors protected tumor cells from TRAIL-induced apoptosis [13]. Wnt Signaling and Colon Cancer Stem Cells Here we show that the ability of HCT116 cells to stimulate macrophages is completely dependent on the presence of the endogenous mutant b-catenin and subsequent stabilization of Snail in epithelial cells, corroborating our hypothesis that acquisition of oncogenic mutations in intestinal epithelial cells alters their interaction with their microenvironment. We showed that colon cancer cells stimulate macrophages through the product of a Wnt target gene. Our data suggest that Snail regulates the expression of this gene and thus plays a crucial role in the crosstalk between colon cancer cells and macrophages. Results Mutations in b-catenin, but not kras, alter colon cancer cell interactions with macrophages We reported that colon cancer cells, via a soluble factor, activate macrophages to secrete IL1b, which in turn promotes b-catenin/ TCF4 transcriptional activity in the cancer cells and stimulates their growth [10]. To establish whether acquisition of the oncogenic kRas mutation in tumor cells alters their interaction with macrophages, we performed experiments in HCT116 and Hke-3 cells, isogenic cell lines that differ only by the presence of the mutant kRas allele [19]. We showed that both HCT116 and Figure 1. The presence of the mutant kRas in tumor cells does not alter their interaction with macrophages. A: HCT116 and Hke-3 cells were compared for their ability to induce IL1b in peripheral blood monocytes (Mo). The ability of THP1 macrophages and IL1b to induce Wnt signaling in HCT116 and HKe-3 cells (B), to promote clonogenic growth (C) and to protect from TRAIL-induced apoptosis (D) was measured. doi:10.1371/journal.pone.0045462.g001 PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e45462