Wnt Signaling and Colon Cancer Stem Cells Figure 6. Wnt signaling in HCT116 cells is required for stimulation of monocytes. A: HCT116 transfected with an empty vector or dnTCF4 were co-cultured with primary peripheral blood monocytes (Mo) for 24 hours and the amount of IL1b was determined by ELISA. B: HCT116 or Hke-3 cells transfected with NSP (nonspecific) or Snail specific RNAi were cultured with THP1 monocytes and the amount of IL1b was determined by ELISA. doi:10.1371/journal.pone.0045462.g006 [15,16] which, in turn, interacts with b-catenin, and thus co- regulates the expression of Wnt target genes [17]. In contrast to the parental HCT116 cells, macrophage-derived factors failed to stabilize Snail in HCT116 cells with deleted MT b-catenin allele (Fig. 5C). We demonstrated that the ability of macrophages and IL1b to inhibit TRAIL-induced apoptosis and to promote clonogenic growth of tumor cells was also inhibited in tumor cells with silenced Snail expression [13], demonstrating that Snail regulates several steps in colon cancer progression and pointing to a crucial role of Snail in the crosstalk between tumor cells and macrophages. Here we show that active Wnt signaling in colon cancer cells, and Wnt-dependent stabilization of Snail specifically, are required for tumor cells to stimulate macrophages to produce IL1b (Fig. 6). Similarly, recent findings demonstrated that Snail upregulates the expression of proinflammatory mediators in oral keratinocytes, including IL6, IL8, CXCL1 and IL1b [31]. This is consistent with our finding that macrophage-derived factors protect colon cancer cells from TRAIL-induced apoptosis through stabilization of Snail [13]. Therefore, the lack of Snail stabilization in HCT116 cells with inactivated MT b-catenin allele underlies the inability of macrophages to protect these cells from TRAIL- induced apoptosis. These data confirmed that activation of Wnt signaling and subsequent stabilization of Snail in colon cancer cells significantly impacts their interaction with macrophages, and suggests a pivotal role of Snail in the crosstalk between colon cancer cells and macrophages. Together, our data strongly suggest that tumor cells stimulate macrophages via a Wnt-dependent factor. This is consistent with our finding that HCT116 cells expressing dnTCF4 failed to stimulate IL1b in macrophages (Fig. 6) and thus exhibit perturbed crosstalk with macrophages [13]. Indeed, we showed that HCT116 cells expressing dnTCF4 have increased response to TRAIL, and that macrophages do not protect these cells from TRAIL-induced apoptosis [13]. Experiments are underway to identify the tumor-derived factors which stimulate macrophages, and to determine whether the expression of Toll-Like Receptors (TLRs) on macrophages is required for the crosstalk between colon cancer cells and macrophages (Figure 7). Human colon tumors display heterogeneous levels of Wnt activity [32] and it has been shown that only cells with high levels of Wnt signaling display colon cancer stem cell (CSC) properties [33,34]. Indeed, silencing of b-catenin in HCT116 cells has been shown to decrease the number of colonospheres, which are highly enriched in CSCs [35]. Consistent with this, HCT116WT cells failed to form spheroids when plated in low serum conditions [9]. We demonstrated that macrophages and IL1b enhanced Wnt signaling and stabilized Snail in tumor cells [13], which has been shown to endow cells with stem cell-like properties [18], suggesting PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e45462