Hypoxia Potentiates Glioma-Mediated Immunosuppression Jun Wei1., Adam Wu1., Ling-Yuan Kong 1 , Yongtao Wang 1 , Gregory Fuller2, Isabella Fokt3, Giovanni Melillo4, Waldemar Priebe3, Amy B. Heimberger1* 1 Department of Neurosurgery, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America, 2 Department of Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America, 3 Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America, 4 National Cancer Institute, Frederick, Maryland, United States of America Abstract Glioblastoma multiforme (GBM) is a lethal cancer that exerts potent immune suppression. Hypoxia is a predominant feature of GBM, but it is unclear to the degree in which tumor hypoxia contributes to this tumor-mediated immunosuppression. Utilizing GBM associated cancer stem cells (gCSCs) as a treatment resistant population that has been shown to inhibit both innate and adaptive immune responses, we compared immunosuppressive properties under both normoxic and hypoxic conditions. Functional immunosuppression was characterized based on production of immunosuppressive cytokines and chemokines, the inhibition of T cell proliferation and effector responses, induction of FoxP3+ regulatory T cells, effect on macrophage phagocytosis, and skewing to the immunosuppressive M2 phenotype. We found that hypoxia potentiated the gCSC-mediated inhibition of T cell proliferation and activation and especially the induction of FoxP3+T cells, and further inhibited macrophage phagocytosis compared to normoxia condition. These immunosuppressive hypoxic effects were mediated by signal transducer and activator of transcription 3 (STAT3) and its transcriptionally regulated products such as hypoxia inducible factor (HIF)-1a and vascular endothelial growth factor (VEGF). Inhibitors of STAT3 and HIF-1a down modulated the gCSCs’ hypoxia-induced immunosuppressive effects. Thus, hypoxia further enhances GBM-mediated immunosuppression, which can be reversed with therapeutic inhibition of STAT3 and HIF-1a and also helps to reconcile the disparate findings that immune therapeutic approaches can be used successfully in model systems but have yet to achieve generalized successful responses in the vast majority of GBM patients by demonstrating the importance of the tumor hypoxic environment. Citation: Wei J, Wu A, Kong L-Y, Wang Y, Fuller G, et al. (2011) Hypoxia Potentiates Glioma-Mediated Immunosuppression. PLoS ONE 6(1): e16195. doi:10.1371/ journal.pone.0016195 Editor: Holger K. Eltzschig, University of Colorado Denver, United States of America Received August 2, 2010; Accepted December 14, 2010; Published January 20, 2011 Copyright: 2011 Wei et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Anthony Bullock III Foundation (ABH), the Mitchell Foundation (ABH), the Dr. Marnie Rose Foundation (ABH), and the U. S. National Institutes of Health grants CA120813-03, A177225-01, P50 CA093459 (ABH), and P50 CA093459 (WP). The funding organizations had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: W. Priebe holds patents on WP744, and W. Priebe and A. B. Heimberger hold patents on WP1066. * E-mail: aheimber@mdanderson.org . These authors contributed equally to this work. Introduction We and other investigators have recently demonstrated that the GBM-associated cancer stem cells (gCSCs) participate in tumor- mediated immunosuppression by both secreted and membrane- associated mechanisms and inhibit both innate and adaptive immunity [1,2,3,4]. Specifically, gCSCs markedly inhibited adaptive T cell proliferation and activation and induced regulatory T cells by both direct cell-to-cell contact and secreted soluble factors in both donor and autologous T cell assays with similar results [2,3]. Furthermore, the gCSCs also induce macrophage/ microglia to assume an immunosuppressive M2 phenotype mediated by the phosphorylated signal transducer and activator of transcription (pSTAT3) pathway with impaired innate functions that could be blocked by WP1066 [4]. These findings conflict with those that suggest that the gCSC may be targeted using a variety of immunotherapeutic approaches [5,6,7]. The reconciliation of this disparity may reside in environmental factors that are minimized or negated in cell lines that are perpetuated in vitro. Because gCSCs are a heterogeneous cell population, and immunosuppressive properties provide no survival benefit in a cell-culture environ- ment, selective pressures may favor the loss of immunosuppressive phenotypes over time. This important consideration may falsely lead an investigator to implement an immune therapeutic strategy based on preclinical data that has artificially minimized the influence of tumor mediated immunosuppression. Hypoxic microenvironments are a frequent characteristic of GBM that are the consequences of morphologically and functionally inappropriate neovascularization, irregular blood flow, anemia, and the high oxygen consumption of rapidly proliferating malignant cells [8]. These hypoxic microenviron- ments are a powerful stimulus for the expression of genes involved in tumor cell proliferation and angiogenesis [9]. Tumor hypoxia can activate the pSTAT3 immunosuppressive pathway thereby triggering the downstream synthesis of hypoxia inducible factor (HIF)-1a that can then induce regulatory T cells [10] and vascular endothelial growth factor (VEGF)[11,12], which in addition to promoting angiogenesis is also immunosuppressive [13,14]. In addition, other glioma-elaborated immune suppressive cytokines such as transforming growth factor (TGF)-b [15,16,17], soluble PLoS ONE | www.plosone.org 1 January 2011 | Volume 6 | Issue 1 | e16195