Wnt Signaling and Colon Cancer Stem Cells Figure 2. Interaction of HCT116 cells with macrophages is altered by mutations in b-catenin. A: Isogenic HCT116 cell lines with the deleted WT (HCT116MT) or MT b-catenin allele (HCT116WT) were transfected with the TOP-LUC reporter gene and were treated with increasing concentrations of IL1b or were cultured with THP1 macrophages as indicated. B: HCT116 cells were transfected with the NF-kB-reporter gene and were treated with TNF and IL1b as indicated. doi:10.1371/journal.pone.0045462.g002 Hke-3 cells induced IL1b in peripheral blood monocytes, precursors of the tumor associated macrophages (Fig. 1A). Accordingly, macrophages and IL1b stimulated Wnt signaling (Fig. 1B), promoted growth (Fig. 1C) and protected HCT116 and Hke-3 cells from TRAIL-induced apoptosis (Fig. 1D). These studies demonstrated that the presence of oncogenic kRas in tumor cells does not have a significant impact on their interaction with tumor associated macrophages. HCT116 cells are also heterozygous for b-catenin, containing one wild type (WT) allele and one mutant (MT) allele with a 3-bp deletion that eliminates the serine residue at codon 45 [7], resulting in b-catenin stabilization and activation of Wnt signaling. To determine whether the presence of the activated, MT b- catenin in tumor cells modulates their interaction with macro- phages we performed experiments in HCT116 cells with a deletion of either the WT (HCT116MT) or MT b-catenin allele (HCT116WT) [8]. The isogenic cell lines were treated with IL1b or were cultured with THP1 macrophages. As in parental HCT116 cells, macrophages and IL1b induced Wnt signaling in HCT116MT cells (Fig. 2A). In contrast, HCT116WT cells, which showed lower Wnt signaling, failed to respond to IL1b or to macrophages with increased Wnt signaling (Fig. 2A). The ability of IL1b and TNF to activate NF-kB, the major signaling pathway activated by these cytokines, was not affected by the status of b- catenin (Fig. 2B), demonstrating pathway specificity, and is consistent with our previous finding that NFkB signaling is upstream of Wnt signaling [11]. These results demonstrated that the presence of the MT b-catenin allele in tumor cells, which results in constitutive b-catenin/TCF4 activity, alters their interaction with macrophages. Macrophages fail to protect HCT116 cells that lack the MT b-catenin allele from TRAIL induced apoptosis We demonstrated that macrophage-derived factors protect colon cancer cells from TRAIL-induced apoptosis through a mechanism dependent on Wnt signaling [13], suggesting that HCT116WT cells (lacking the mutant b-catenin allele), which did not respond to macrophages with enhanced Wnt signaling (Fig. 1), may display an altered response to TRAIL. We treated the parental HCT116 cells, HCT116WT and HCT116MT cells with TRAIL in the absence or the presence of THP1 macrophages or IL1b. As shown in Fig. 3, HCT116 cells that lack the mutant b- catenin allele (HCT116WT) displayed increased sensitivity to PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e45462