colony stimulating factor (sCSF)-1 [18,19], C-C chemokine ligand (CCL)-2 [20,21] and galectin-3 [22,23] have also been shown to be associated with or induced by HIF-1a and hypoxia [24,25,26,27,28]. The STAT3 pathway, which is induced in the various immune populations, has been shown to be a potent regulator of anti- inflammatory responses in both innate [29] and adaptive immunity [30], including immunosuppression potentiated by the gCSC [2,4]. CNS macrophages (microglia), which are the dominant infiltrating immune cells in GBM and the principle mediators of innate immunity within the tumor [31,32], become tumor-associated macrophages (TAMs) when exposed to the hypoxic tumor microenvironment (TAMs) [33,34]. TAMs become polarized toward immunosuppressive and tumor supportive phenotypes (M2) via the STAT3 pathway and then contribute to angiogenesis and tumor invasion [26]. We have demonstrated that these TAMs, which arise from circulating monocytes that are recruited into the tumor microenvironment [35], can be modulated by the gCSCs by inducing an immunosuppressive phenotype via the STAT3 pathway[4]. Since HIF-1a can promote the expansion of CD133-positive gCSCs [36,37], it is possible that hypoxia induces a feed-forward mechanism of tumor-mediated immunosuppression. We suspected that hypoxia potentiates the ability of the gCSC to evade immune surveillance that could account for, in part, the discrepancy between investigators that demonstrate immunolog- ical clearance of this population [5,6,7] and the grim reality of recurrence and failure of gCSC eradication even in the setting immune therapeutic approaches. We therefore hypothesized that hypoxia would further potentiate the innate and adaptive immunosuppressive properties of gliomas which could be reversed by inhibiting hypoxia induced pathways such as pSTAT3. To determine whether we could reverse the gCSC-mediated immunosuppressive effects of hypoxia, we tested the pSTAT3 and HIF-1a inhibitor WP1066 [38] and WP744, an anthracycline compound [39] with suspected HIF-1a inhibitory effects [40]. Materials and Methods Ethical Treatment of Research Subjects and Patient Consent Each patient provided written informed consent for tumor tissues, and this study was conducted under protocol #LAB03- 0687, which was approved by the institutional review board of The University of Texas M. D. Anderson Cancer Center. Derivation of human gCSCs GBM specimens were processed within 4 hours after resection. The four separate gCSCs used in this study were isolated from four different individual GBM patients. They were washed with DMEM-F-12 medium, disassociated as previously described [41], and have been previously characterized [2,3,4]. The gCSCs were grown in neurosphere medium (DMEM-F-12 medium containing 20 ng/ml of epidermal growth factor, basic fibroblast growth factor (Sigma, St. Louis, MO), and B27 (1:50; Invitrogen, Carlsbad, CA). To minimize possible phenotypic changes in gCSCs due to prolonged maintenance in vitro and multiple passages, early passage gCSCs were frozen in neurosphere medium containing 5% DMSO and stored at 270uC. After thawing, gCSCs were used in experiments after 1 or 2 passages. Hypoxia Potentiates Glioma Immunosuppression conditions for 48 h to allow the cells to re-enter log-growth phase. The cells were then moved to a hypoxia chamber (Sanyo Manufacturing) at 1% O2 for another 24 h. Alternatively, cells were treated with 100 mM of hypoxia-mimic chemical desferriox- amine mesylate (DFX; Sigma). Antibodies and reagents Tissue-culture grade monoclonal antibodies to CD3 (OKT3) and CD28 (28.6) were obtained from eBioscience. The cell surface was stained with phycoerythrin (PE), fluorescein isothiocyanate (FITC), or allo-phycocyanin-conjugated (APC) antibodies against the following proteins: CD3, CD4, CD8, major histocompatibility complex (MHC) I, MHC II, CD40, CD80, CD86, and B7-H1 (BD Pharmingen, San Diego, CA) and CD133 (Miltenyi Biotech, Auburn, CA). To detect intracellular cytokines, PE-conjugated antibodies against interleukin (IL)-2 and interferon (IFN)-c (R&D Systems) were used. To detect pSTAT3, PE-conjugated antibodies against pSTAT3 (BD Pharmingen, San Diego, CA) were used. Appropriate isotype controls were used for each antibody. The inhibitor agents WP1066 and WP744 were synthesized and supplied by Waldemar Priebe. WP1066 was stored as a 10 mM stock solutions in dimethyl sulfoxide and diluted with PBS and WP744 was stored as a 10 mM stock solutions in N,N-dimethyla- cetamide. WP1066 is a specific and potent inhibitor of STAT3 [38] and of down-stream STAT3-regulated products (such as HIF-1a) that achieves in vivo physiological doses of 2 mM, including within the central nervous system [42]. WP744 is an anthracycline compound [39] that is suspected to inhibit HIF-1a [40] and achieves in vivo physiological doses of 0.1 mM (unpublished data). Only WP1066 possesses significant pSTAT3 inhibitory activity. The cell viability of the gCSCs based on MTT (dimethyl thiazolyl diphenyl tetrazolium) assays is not affected by doses of 2 and 0.1 mM of WP1066 or WP744, respectively. ELISA Supernatant medium from the gCSC cell cultures were assayed for cytokine concentrations using ELISA kits as described by the manufacturer (R&D Systems). These supernatants were collected from 36106 cells after 5 d in culture and stored at 220uC. The supernatants were added in duplicate to appropriate pre-coated plates. After the plates were washed, horseradish peroxidase- conjugated detection antibody was added. The substrate used for color development was tetramethylbenzidine. The optical density was measured at 450 nm with a microplate reader (Spectra Max 190; Molecular Devices, Sunnyvale, CA), and chemokine concen- trations were quantified using SoftMax Pro software (Molecular Devices). The detection limits for CC chemokine ligand 2 (CCL-2) were 5 pg/ml; TGF-b1, 16 pg/ml; IL-10, 5 pg/ml; soluble colony- stimulating factor (sCSF)-1, 8 pg/mL; VEGF, 5 pg/ml; Galectin-3, 10 pg/ml; and macrophage inhibitory cytokine (MIC)-1, 8 pg/ml. Human peripheral blood mononuclear cells (PBMCs) PBMCs were prepared from healthy donor blood (Gulf Coast Blood Center, Houston, TX). CD14+ monocytes were purified from PBMCs by positive selection using CD14 microbeads (Miltenyi Biotech, Auburn, CA), and CD3+ T cells were purified from PBMCs by negative selection using a Pan T Cell Isolation Kit II (Miltenyi Biotech, Auburn, CA), according to the manufacturer’s instructions. Hypoxia induction Dissociated gCSCs (1.56106 cells) were plated in T-25 flasks in 4 ml of neurosphere medium and cultured under normoxic Human monocyte assays 56105 CD14+ monocytes were incubated with 500 mL (16106 cells/mL) of supernatant medium from gCSCs cultured in both PLoS ONE | www.plosone.org 2 January 2011 | Volume 6 | Issue 1 | e16195