constitutively active in many tumor cells and was found to have an important role in oncogenesis [7]. In addition STAT3 was found to have a profound role in regulating the immune responses in the tumor micro-environment. In tumor cells, STAT3 activation has been linked to both inhibition of pro-inflammatory cytokine secretion and induction of anti-inflammatory cytokine secretion, such as IL-10 and VEGF [8]. These latter anti-inflammatory cytokines can, in turn, induce STAT3 activation within neighbor- ing DCs, thereby influencing their functional maturation [8]. Collectively, tumor cells were shown to secrete soluble factors that activate STAT3 and suppress DCs function. An effective anti-tumor immune response requires the partic- ipation of the host APCs, mostly DCs and macrophages [7]. Macrophages were shown to be recruited to the tumor site by cytokines, chemokines and other tumor derived factors, and once within the tumor environment they are skewed toward an anti- inflammatory phenotype and thereby promote tumor growth [9]. DCs in the tumor microenvironment are usually found in an immature state, characterized by an insufficient level of expression of MHC class II complexes, co-stimulatory molecules such as CD80 and CD86 and IL-12. These cells are not only poor antigen-presenting cells, but could further induce immune tolerance [10,11]. Tolerogenic DCs are generated in the absence of inflammatory mediators, and recent studies have shown that DCs can be further blocked from becoming effective antigen presenting cells by tumor secreted factors that inhibit their maturation [10,11]. We have previously described that human Mesenchymal Stem Cells (hMSC) inhibit T cells indirectly by contact-dependent induction of regulatory APCs (both monocytes and DCs) with T cell suppressive properties [12]. Accordingly, when APCs came into productive contact with hMSC, the secretion of pro- inflammatory cytokines such as TNF-a and IL-12 were inhibited while the anti-inflammatory cytokine IL-10 was induced. Under these circumstances, IFN-c secretion and T- cell proliferation were inhibited [12]. This contact-dependent induction of regulatory APCs was dependent on the activation of STAT3 in these cells and could be blocked with specific STAT3 inhibitors [13]. In the present study, we demonstrated that tumor cells modulate APCs function by using two parallel mechanisms for the activation of STAT3; the first involving secretion of soluble cytokines as previously described [8,14], and second involving a novel pathway that is based on cell-cell interaction. While these two distinct pathways activate STAT3 within APCs, they result in disparate phenotypic outcomes. A contact-dependent mechanism may provide a molecular explanation for how the surrounding microenvironment influences APCs maturation in a focused and unique way. Results Cancer cells inhibit TNF-a and IFN-c secretion while inducing IL-10 production Recent studies have suggested that tumor cells secrete tumor- derived factors that induce STAT3 activity within neighboring APCs, effecting their maturation and function [8]. Based upon our previous insight into a contact-dependent effect of hMSC on APC phenotype and function [12], along with the linking of this effect to STAT3 activation [13], we now tested whether a similar contact- dependent STAT3 activation is also induced in tumor cell-APC interaction. We started by co-culturing two different tumor cell lines (MCF-7 breast carcinoma; PC3 prostate carcinoma) with a mixture of monocytes, T cells, and the superantigen SEB as activator. Both Contact Induced Regulatory DC tumor cell lines successfully inhibited IFN-c and TNF-a secretion (Supporting data Fig. S1A) and promoted IL-10 secretion, with the latter, primarily produced by the monocytes, as determined using immunofluorescence and flow cytometry (Supporting data Fig. S1B). Increased STAT3 activity in monocytes:tumor cells co- cultures is dependent on both cell:cell contact and tumor cells secreted factor(s) We next assessed STAT3 activity in these co-cultures using STAT3 transcription factor assay. As shown in figure 1, we observed an increase in STAT3, but not STAT1 (not shown), activity in tumor cells-monocyte co-cultures, as compared to each cell type cultured individually. We further assessed the contribu- tion of soluble and contact-dependent factors to the increase in STAT3 activation in tumor cell-APC co-cultures. There is soluble factor-dependence of STAT3 induced activity in the tumor cells- monocyte co-cultures based on the increase in STAT3 activity in monocytes when both PC3 and MCF-7 were replaced with their conditioned media (Fig. 1 and 2A, lower panels), or when monocytes and the tumor cells were on opposite sides of a transwell membrane (not shown). However, the effect of conditioned media was lower than the combined effect achieved when the two cell types were co-cultured (Fig. 1 and 2A). Therefore, to eliminate interference from tumor cytokines in the co-cultures, tumor cell conditioned media was washed away just prior to adding the monocytes. Under these conditions, we observed significant increase in STAT3 activity after co-culturing the cells compared to its activity in each cell type cultured Figure 1. Increased STAT3 activity in co-cultures is dependent on both cell:cell contact and secreted factor(s). Confluent cultures of MCF-7 (2.56104/well) in a 24 well plate were incubated for 24 hours, then the cells were either left untreated or their conditioned media was collected and replaced with fresh media (without conditioned medium –c.m). Monocytes (2.56104 cells/well) were either added to tumor cell’s cultures (both untreated and cells that received fresh media-c.m.) or were incubated alone or with tumor cell’s conditioned medium (c.m). After 2 hours, nuclear extracts were prepared and tested for STAT3 activity using Non-Radioactive STAT3 Transcription Factor Assay. Comparable results were obtained in two independent experiments. doi:10.1371/journal.pone.0006846.g001 PLoS ONE | www.plosone.org 2 August 2009 | Volume 4 | Issue 8 | e6846