Hypoxia Potentiates Glioma Immunosuppression Figure 5. WP1066 inhibits pSTAT3 and HIF-1a expression, whereas WP744 only inhibits HIF-1a expression in gCSCs. A and B. WP744 or WP1066-treated, hypoxia and normoxia exposed gCSCs express lower levels of HIF-1a than gCSCs exposed to hypoxia and normoxia alone. The HIF-1a expression in the gCSCs cultured under normoxia and hypoxic conditions was measured by intracellular staining via flow cytometry in the presence or absence of WP744 or WP1066. Representative histograms were shown as A. The percentage changes of all 4 gCSCs under hypoxia and normoxia were compared to untreated gCSCs and then the corresponding percentage change of expression was calculated and plotted as bar graphs in B. C and D. WP1066 inhibited pSTAT3 expression in gCSCs under both normoxia and hypoxia, whereas WP744 enhances pSTAT3 expression. The pSTAT3 expression in the gCSCs under normoxia and hypoxic conditions was measured by intracellular pSTAT3 staining via flow cytometry in the presence or absence of WP744 or WP1066. Representative histograms were shown as C, and percentage changes of all 4 gCSCs under hypoxia and normoxia were plotted as bar graphs in D. In histograms, dashed line represented isotype staining and the black line staining for either HIF-1a or pSTAT3. The number in histograms represented positive percentage for pSTAT3 or HIF-1a relative to the isotype. *P,0.05. doi:10.1371/journal.pone.0016195.g005 significant impact on CCL-2 production (data not shown). In gCSC1, WP1066 and WP744 did not affect VEGF production; but hypoxia had the least substantial effect on VEGF production relative to normoxia in gCSCs (Fig. 1). We next addressed the potency of these agents in reversing hypoxia-exposed gCSC-mediated immunosuppression. At physio- logical levels that can be obtained in vivo, WP1066 was more potent than WP744 at restoring T cell proliferative responses (Fig. 6A). In contrast, WP744 was able to inhibit the hypoxia- induced CD4+FoxP3+ T cell to a greater degree than WP1066 (Fig. 6B). WP1066 and WP744 were equally effective in restoring monocyte phagocytosis inhibition (Fig. 6C). WP1066 increased phagocytosis to 86.0% in comparison to neurosphere medium positive control (range 75.6% to 97.3%, P = 0.01) and WP744 increasing phagocytosis to 90.0% (range 87.3% to 92.3%, P = 0.0006). Discussion In this investigation we demonstrated that tumor hypoxia potentiates glioma-mediated immunosuppression of both innate and adaptive immunity. We had previously shown that the gCSCs induced regulatory T cells [3]; however, during blocking experiments of TGF-b, we could not completely ablate the induction of regulatory T cells (unpublished data) indicating that other cytokines had a role in triggering the formation of regulatory T cells. Under conditions of hypoxia, there was a marked increase of HIF-1a by the gCSCs. A previous report has demonstrated that hypoxia can increase HIF-1a levels and subsequent FoxP3 expression in T cells along with their corresponding potency in suppressing effector T cells [10]. It has been proposed by Sitkovsky et al., [44] that maximal immunosuppression is only possible if the T cell receptor (TCR)-triggered activities of immunosuppressive regulatory T cells are combined with and/or enhanced by tissue hypoxia via the adenosine receptor [45]. Our studies support this contention because T cell proliferation and the induction of regulatory T cells were potentiated in hypoxic conditions. Macrophages have previously been demonstrated in hypoxic areas of tumors [46,47,48] likely recruited by tumor-elaborated factors [49,50]. However, in contrast to other investigators who found that hypoxia directly enhanced monocyte/macrophage phagocytosis [51,52,53], we found that hypoxic exposed gCSCs inhibited monocyte/macrophage phagocytosis emphasizing the importance of the tumor microenvironment and cell-to-cell interactions in determining immunological responses. The role of hypoxia on subverting macrophages to TAM/M2 phenotype and function likely involves both HIF-1a-dependent and independent processes. Hypoxic conditions increased the ability of gCSCs to induce pSTAT3 expression in monocytes – a defining characteristic of the M2 macrophage [54]. However, this did not result in an increase of monocyte/macrophage-elaborated immu- nosuppressive cytokines or further reduction in the expression of co- stimulatory and MHC molecules needed for antigen presentation on the monocytes/macrophages, indicating that hypoxia is not solely responsible for polarizing the monocytes toward a function- ally immunosuppressive phenotype. It’s also possible that after monocytes are freshly recruited into hypoxic areas of tumor, their further polarization towards M2 and otherwise tumor-supportive macrophage phenotypes (including the induction of immune suppressive and/or pro-angiogenic cytokines) may be directly mediated by the hypoxic environment itself [55], in addition to the direct activities of gCSCs. For example, although we did not observe induction of VEGF production in monocytes exposed to the supernatants from either normoxic or hypoxia-exposed gCSCs, VEGF production in macrophages has been shown to be induced directly by hypoxia [56]. In practice, we are exploiting the gCSCs as an immunosup- pressive cell population to evaluate the role of hypoxia given the gCSCs propensity to recapitulate many of the hallmark features of malignant gliomas, including immunosuppression; however it is unlikely that hypoxia exerts effects exclusively to this population. Hypoxia induced an increase in the production of the immuno- suppressive cytokines by the gCSCs such as galectin-3 and VEGF, regulatory T cell and macrophage chemoattractants sCSF-1 [19] and CCL-2 [21,57], and HIF-1a [10] but the gCSC is unlikely to be the sole source of these cytokines within the tumor microenvironment. It has also been suspected that hypoxia may influence the levels or activity of CTLA-4 and/or B7-H1 [58]. Although we did not find differences in the expression of CTLA-4 or B7-H1 under conditions of hypoxia, we can’t discount the possibility of alterations in the transcriptional activities of these receptors. To determine whether STAT3 and HIF-1a inhibitors can reverse the hypoxia-exposed gCSC-mediated immunosuppressive effects, we treated the gCSCs with WP744 and WP1066 under hypoxic conditions. WP1066 in comparison to WP744 had a more potent effect on reduction of immunosuppressive cytokines such as galectin-3, CCL-2 and sCSF-1 elaborated by the gCSCs under hypoxia likely secondary to WP1066 inhibiting the immune suppressive pathway pSTAT3. Both of these agents were capable of inhibiting HIF-1a, with WP744 being more potent. The immunological effects of inhibiting HIF-1a were most evident in the inhibition of induced FoxP3+ T cells in which WP744 exerted the greatest effect. However, WP1066, which also exert potent STAT3 inhibitor activity, exerted a greater and more global reversal of gCSC-mediated immunosuppression on effector responses, especially adaptive T cell proliferation and effector function, although this was not absolute. This indicates that hypoxia is a significant inducer of gCSC-mediated immunosup- pression but that this can’t be overcome by HIF-1a inhibitors alone or that the current generation of STAT3 inhibitors can completely restore immunological responsiveness to the levels seen in normal control. Further investigations will be necessary to ascertain how to optimize the STAT3 inhibitors in combination with other immune therapeutics in the setting of hypoxic tumor microenvironments. PLoS ONE | www.plosone.org 9 January 2011 | Volume 6 | Issue 1 | e16195