Glioblastoma multiforme (GBM) is a lethal cancer that responds poorly to radiotherapy and chemotherapy. Glioma cancer-initiating cells have been shown to recapitulate the characteristic features of GBM and mediate chemotherapy and radiation resistance. However, it is unknown whether the cancer-initiating cells contribute to the profound immune suppression in GBM patients. Recent studies have found that the activated form of signal transducer and activator of transcription 3 (STAT3) is a key mediator in GBM immunosuppression. We isolated and generated CD133+ cancer-initiating single colonies from GBM patients and investigated their immune-suppressive properties. We found that the cancer-initiating cells inhibited T-cell proliferation and activation, induced regulatory T cells, and triggered T-cell apoptosis. The STAT3 pathway is constitutively active in these clones and the immunosuppressive properties were markedly diminished when the STAT3 pathway was blocked in the cancer-initiating cells. These findings indicate that cancer-initiating cells contribute to the immune evasion of GBM and that blockade of the STAT3 pathway has therapeutic potential. Mol Cancer Ther; 9(1); 67–78

Malignant gliomas express tumor-associated and tumor-specific antigens that should make these tumors detectable to the immune system (1). However, there is a distinct lack of immune-mediated tumor eradication in glioma patients, and most attempts at immunotherapy have met with little clinical success (2). Many factors work in concert to inhibit antiglioma immunity, including immunosuppressive cytokines such as interleukin (IL)-10, transforming growth factor (TGF)-β, and prostaglandin E2 (PGE2), the induction of regulatory T cells (Treg), and the downmodulation of costimulation molecules by antigen-presenting cells resulting in loss of T-cell effector function—all of which are operational in glioblastoma multiforme (GBM) patients (reviewed in ref. 3). Although central nervous system (CNS) tumors are recognized by the immune system, this is insufficient for their suppression or eradication. Primed CD8+ cytotoxic T cells gain CNS access (4); however, the lack of tumor eradication indicates that the T cells mediating adaptive immune responses are deficient in malignant glioma patients and are functionally impaired within the local tumor microenvironment (59).

Cancer-initiating cells are a heterogeneous population of undifferentiated cells with the capacity for self-renewal and a high proliferative potential. GBMs contain cancer-initiating cells that are multipotent and can recapitulate the characteristics of GBM including high motility, diversity of progeny, tendency to migrate along white matter tracts, and expression of immature antigenic phenotypes such as epidermal growth factor receptor and nestin (10). Cancer-initiating cells may express CD133 (11), although cancer-initiating cells that do not express CD133 have been identified (1214), indicating that this is not a definitive marker for the identification of cancer-initiating cells. Furthermore, the expression of CD133 is heterogeneous within neurospheres and can alter with subsequent passage and cloning (15). Characteristics of cancer-initiating cells also include high levels of the antiapoptotic genes, the ability to form neurospheres, nonadherence, possession of marker characteristics for astrocytic, neuronal, and oligodendroglial lineages (16), and tumorigencity in vivo. The cancer-initiating cells are believed to confer the resistance to chemotherapy and radiation observed in GBM (17, 18). Because significant T-cell immune suppression is induced by the GBM and because glioblastoma-associated cancer-initiating cells are a therapeutically resistant population, the question arises as to the participation of cancer-initiating cells in inhibiting T-cell responses.

The signal transducer and activator of transcription 3 (STAT3) pathway is a potent regulator of anti-inflammatory responses through its suppression of macrophage activation (19, 20), reduction of the cellular cytotoxicity of natural killer cells and neutrophils, and reduction of the expression of MHC II, CD80, CD86, and IL-12 in dendritic cells rendering them unable to stimulate T cells and generate antitumor immunity (21). The ablation of STAT3 activity in only the immune cells results in marked antitumor effects in vivo, indicating that STAT3 expression within the immune cells is what restrains antitumor eradication (21). Recent evidence shows that GBM-associated immunosuppression is linked to the upregulation of STAT3 in tumor cells (22). Many growth factors and cytokines, including IL-6 and epidermal growth factor, activate Janus kinase 2, which subsequently activates STAT3 by phosphorylation of the tyrosine residue in the transactivation domain. Phosphorylated STAT3 (p-STAT3), which is overexpressed in most cancers including gliomas (23, 24), then translocates into the nucleus and induces a variety of transcriptional factors that propagate tumorigenesis (25) and upregulate tumor-mediated immunosuppressive factors (26). These factors include IL-10 (27, 28) that adversely influences Th1-mediated cytotoxic immune responses at multiple levels and is essential for Treg function (29, 30), vascular endothelial growth factor (31) that inhibits dendritic cell maturation and activation by inhibiting costimulatory molecule expression (32), PGE2 (33) that induces the immune-suppressive Th17 cell (34), and TGF-β (35) that induces Tregs, inhibits T-cell proliferation, and downmodulates the IL-2 receptor (reviewed in ref. 36), to name just a few. These STAT3-regulated tumor-secreted factors then activate STAT3 in diverse immune cells including both innate immune cells and T cells (21). Because STAT3 target genes encode many factors that activate STAT3 in the immune cells, a feed-forward mechanism for constitutive activation of STAT3 in both the tumor cells and the immune cells within the tumor microenvironment is initiated. Cumulatively, these data indicate that the STAT3 pathway is a molecular hub of tumor-mediated immune suppression.

The inhibition of STAT3 activity in only the hematopoietic cells of tumor-bearing mice can induce potent multicomponent antitumor immune responses (21). We have previously found that inhibition of STAT3 with WP1066, which blocks the Janus kinase 2/STAT3 interaction and subsequent phosphorylation of STAT3 at tyrosine705 (p-STAT3; ref. 37), results in the upregulation of costimulatory molecules (CD80 and CD86) on human microglia, secretion of proinflammatory cytokines essential for T-effector responses, and activation and proliferation of T cells (22). Thus, STAT3 blockade is a potent approach for modulating multifactorial immunosuppression and eliciting antitumor immune responses. Cancer-initiating cells have been shown to have activated p-STAT3 that can be blocked with p-STAT3 inhibitors, resulting in diminished stem cell proliferation, neurosphere formation, and depletion of CD133-positive (CD133+) cells (38). Specifically, the STAT3 inhibitor WP1066 can induce apoptosis of human glioma cells both in vitro and in vivo (39), can reverse glioma-mediated immune suppression (22), is orally bioavailable, achieves excellent CNS penetration, exerts efficacy against established intracerebral tumors with minimal toxicity (40), and can inhibit Tregs (41). Based on the aforementioned properties (42) and the pending investigational new drug application of WP1066, we selected WP1066 and small interfering RNA (siRNA) for these studies to test the hypothesis that if cancer-initiating cells mediate T-cell immune suppression, then the p-STAT3 pathway would likely be a key mediator of this immune suppression that could be reversed by blockade of the STAT3 pathway.

Human Glioma Cell Lines

Human normal astrocytes and glioma cell lines U-251 and U-87 were purchased from the American Type Culture Collection and cultured in RPMI 1640 (astrocytes), modified Eagle's medium (U-251) or modified Eagle's medium plus 0.1 mmol/L nonessential amino acids (U-87). To all media, 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin were added.

Human Tumors

Tumor tissues from newly diagnosed GBM patients (n = 9) were obtained from surgery specimens and were graded pathologically according to the WHO classification system by a neuropathologist. 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.

Human Glioma Cancer-Initiating Cell Derivation

GBM specimens were processed within 4 h after resection. They were washed and disassociated mechanically. After a single-cell suspension was prepared, erythrocytes were lysed using 1× RBC lysis buffer (eBioscience). Trypan blue staining confirmed >80% cell viability. Glioma-associated cancer-initiating cells were cultured in DMEM F-12 containing 20 ng/mL of epidermal growth factor, basic fibroblast growth factor (Sigma), and B27 (1:50; Invitrogen) as a neural stem cell–permissive medium (neurosphere medium) and passaged every 5 to 7 d. CD133 expression on glioma-associated cancer-initiating cells was confirmed before use.

Intracranial Xenografts

Single-cell suspensions of glioma-associated cancer-initiating cells in serum-free medium at 2 × 102 cells per 5 μL were injected into the right frontal lobes of 58-wk-old nude mice using a stereotactic frame system (Kopf Instruments) as described previously (43). Mice were maintained in the M.D. Anderson Isolation Facility in accordance with Laboratory Animal Resources Commission standards and conducted according to an approved protocol, 08-06-11831.

Cloning of Glioma-Associated Cancer-Initiating Cells

Accutase-dissociated (Sigma) glioma-associated cancer-initiating cells were sorted using the CD133 cell isolation kit (Miltenyi Biotech), and >90% purity CD133+–sorted cells were seeded into 96-well plates at a theoretical density of one cell per well. After overnight culture, microscopic observation was used to identify wells that contained a single cell. These wells were monitored and the medium was changed every 5 to 7 d for 45 d before immune functional analysis.

Human Peripheral Blood Mononuclear Cells

Peripheral blood mononuclear cells (PBMC) were prepared from healthy donor blood (Gulf Coast Blood Center) and GBM patients' blood (the same patients whose glioma-associated cancer-initiating cells were isolated) by centrifugation on a Ficoll-Hypaque density gradient (Sigma-Aldrich). Aliquots of the isolated PBMCs were frozen and stored at −180°C until used. Before coculture experiments, frozen PBMCs were thawed at 37°C for 5 min and then washed with warm 10% FBS in RPMI 1640. CD3+ T cells were purified from PBMCs by negative selection using a Pan T Cell Isolation kit II (Miltenyi Biotech), according to the manufacturer's instructions.

Antibodies and Reagents

Tissue culture grade monoclonal antibodies to CD3 (OKT3) and CD28 (28.6) were obtained from eBioscience. Anti-human IL-6 (1936) and anti-human TGF-β1 (27235) antibodies were obtained from R&D Systems. WP1066 was synthesized and supplied by Waldemar Priebe, and was stored as a 10 mmol/L stock in DMSO and diluted with PBS when used. WP1066 was used at doses that could be achieved in vivo (1–5 μmol/L; ref. 40). The cell surface was stained with phycoerythrin (PE), FITC, or allophycocyanin (APC)-conjugated antibodies against the following proteins: CD3, CD4, CD8, MHC I, MHC II, CD40, CD80, CD86, B7-H1 (BD Pharmingen), and CD133 (Miltenyi Biotech). To detect intracellular cytokines, PE-conjugated antibodies against IL-2 and IFN-γ (R&D Systems) were used. Intracellular STAT3 was assessed using PE-conjugated p-STAT3 (pY705; BD Pharmingen). Appropriate isotype controls were used for each antibody.

Flow Cytometry

FITC-conjugated anti-CD4 (RPA-T4) and APC-conjugated anti-CD8 (RPA-T8) antibodies were used for cell surface staining. Subanalysis of the T-cell populations was based on the gated surface expression of CD4 and CD8. To detect forkhead box P3 (FoxP3) protein expression, the surface-stained cells were further subjected to intracellular staining with PE-conjugated monoclonal antibodies to human FoxP3 (clone PCH101, eBioscience) using staining buffers and conditions specified by the manufacturer. For intracellular cytokine staining, cells were stimulated for 6 h in the presence of 50 ng/mL phorbol myristate acetate, 500 ng/mL ionomycin (Sigma-Aldrich), and 2 μmol/L monensin (GolgiStop, BD Pharmingen). Then, the cells were incubated with FITC-conjugated anti-CD4 and APC-conjugated anti-CD8 (RPA-T8) antibodies for surface staining followed by intracellular staining using PE-conjugated anti-mouse IFN-γ (4S.B3) or PE-conjugated anti-mouse IL-2 (MQ1-17H12) antibodies and FIX/PERM buffers (BD Pharmingen) according to the manufacturer's instructions. Intracellular p-STAT3 intracellular staining was done as described previously (41). Flow cytometry acquisition was done with a FACSCaliber (Becton Dickinson) and data analysis was with FlowJo software (TreeStar).

Glioma-Associated Cancer-Initiating Cell Differentiation

Accutase-dissociated glioma-associated cancer-initiating cells were cultured in differentiation medium consisting of 10% FBS, 10 ng/mL retinoic acid, and 20 ng/mL platelet-derived growth factor-AA (both from Sigma-Aldrich; ref. 44). Confluent monolayer cells were detached every 5 to 7 d by trypsinization, and retinoic acid and platelet-derived growth factor-AA were replenished during the culture.

Immunohistochemistry

Differentiated glioma-associated cancer-initiating cells were cultured on eight-chamber slides (Nunc) at 5,000 per well. After 3 d, cells were fixed with 4% paraformaldehyde, permeabilized with 3% Triton X-100 in PBS, and then blocked with 5% horse serum. Primary antibodies were rabbit anti-GFAP (1:40; DAKO), mouse anti-galactosylceramidase (1:100; Chemicon), and mouse anti-microtubule associate protein 2 (MAP2; 1:50; Chemicon). After incubation for 90 min, the slides were washed with 5% horse serum. Secondary antibodies, goat anti-rabbit Alexa 546 (1:300; Invitrogen), and donkey anti-mouse Alexa 488 (1:300; Invitrogen) were added for 30 min. Slides were mounted using Vectashield Hard Set mounting medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories).

ELISA

Supernatants from the human glioma cell lines U-87 and U-251, and the glioma-associated cancer-initiating cells were measured for cytokine concentrations using ELISA kits per manufacturer's instructions (R&D Systems). These supernatants were collected after 5 d in culture and stored at −20°C. The supernatants were added in duplicate to appropriate precoated plates. The absorbance was measured at 450 nm with a microplate reader (Spectra Max 190; Molecular Devices), and chemokine concentrations were quantitated with SoftMax Pro software (Molecular Devices). The detection limits for chemokine C-C motif-2 (CCL-2) were 5 pg/mL, 16 pg/mL for TGF-β1, 5 pg/mL for IL-10, 1 pg/mL for IL-6, 10 pg/mL for PGE2, 10 pg/mL for soluble Fas, and 10 pg/mL for Galectin-3.

Cell Proliferation Assay and Treg Induction Assay

Glioma-associated cancer-initiating cells were plated into 48-well plates (3 × 104 cells/mL) containing 3 × 105 PBMCs/mL in the presence of 1 μg/mL prebound anti-CD3/anti-CD28 antibodies or 2.5 μg/mL phytohemagglutinin (Sigma-Aldrich). Alternatively, conditioned media from the glioma-associated cancer-initiating cells were also added to the stimulated PBMCs. After 72 h, 100 μL of cells from each well were transferred to new 96-well plates with 10 μL of Cell Counting kit-8 (Dojindo Laboratories). After incubation for 4 h at 37°C, absorbance was measured at 450 nm with a microplate reader (Spectra Max 190). To detect FoxP3+ Tregs, CD4 surface staining and then FoxP3 intracellular staining were done on immune cells cultured for 96 h.

FoxP3+ Treg Suppression Assay

Healthy donor PBMCs were labeled with 2 μmol/L carboxyfluorescein diacetate succinimidyl ester (CFSE) for 5 min at room temperature in PBS with 0.1% bovine serum albumin, and then the reaction was quenched with RPMI 1640 with 10% FBS for 10 min at 37°C. CFSE-labeled PBMCs (1 × 106/mL) and autologous T cells (1 × 105/mL), which were cultured with conditioned media from glioma-associated cancer-initiating cells for 4 d, were plated into 96-well plates in the presence of 2 × 106/mL allogeneic-irradiated PBMCs in RPMI 1640 with10% FBS in a total volume of 0.2 mL. After 72 h, the cells were harvested, and analysis of cell division was done by flow cytometry.

Apoptosis Assay

The T-cell apoptosis assay was done with the Annexin V/7AAD staining kit (BD Pharmingen). Healthy donor PBMCs were cultured with medium or glioma-associated cancer-initiating cell supernatants and then harvested by centrifugation at day 3 and 5. The cells were stained with APC-conjugated anti-CD3 antibodies and then washed twice with cold PBS and resuspended in 1× binding buffer (BD Pharmingen) at a concentration of 1 × 106 cells/mL. Next, PE-conjugated Annexin V and 7-AAD were added; the cells were incubated for 20 min at 25°C in the dark; and CD3+ T-cell apoptosis was analyzed by flow cytometry within 1 h.

STAT3 siRNA Transfection

To knock down STAT3 gene expression in glioma-associated cancer-initiating cells, STAT3 siRNA was transfected into two of our established glioma-associated cancer-initiating cell lines as described (Santa Cruz Biotech). Briefly, 2 × 105 glioma-associated cancer-initiating cells per well were seeded in 2 mL antibiotic-free medium in six-well plates and incubated for 8 h. The siRNA duplex solution (1 μg STAT3 siRNA or control siRNA) in 100 μL siRNA transfection medium were prepared and gently mixed with diluted siRNA transfection reagent and incubated for 45 min at room temperature. The mixture was then overlaid onto the cells washed by transfection medium. The cells were incubated for 5 h at 37°C; then, 1 mL of neurosphere medium was added and the cells were cultured for an additional 72 h before conducting the immune functional assays.

Statistical Analysis

All values were calculated as means and 95% confidence intervals from at least three independent experiments. The Student's t test was used to test for differences in the means between two groups. P values of <0.05 were considered to be statistically significant. All statistical analyses were done using the Statistical Package for the Social Sciences v.12.0.0 (SPSS). Error bars represent SD.

Isolation of Cancer-Initiating Cells from GBM Patients

From newly diagnosed GBM patients (n = 9) at the time of surgery, we isolated cancer-initiating cells and the patients' autologous T cells. The cancer-initiating cells from the patients expressed CD133, formed neurospheres (Fig. 1A) in serum-free medium containing epidermal growth factor and basic fibroblast growth factor after 5 to 10 days of culture, and these CD133+ cells express CD34 but not CD45, indicating that they are of endothelial origin (Fig. 1B; ref. 45). The established cancer-initiating cells were capable of differentiating into glial fibrillary acidic protein (GFAP+) astrocyte-like cells, neuron-like cells that were immunoreactive for mouse anti-microtubule associate protein 2, and galactosylceramidase-immunoreactive oligodendrocyte-like cells (Fig. 1C). When the cancer-initiating cells were injected into the right frontal lobes of 5- to 8-week-old nude mice, the mice developed tumors that were highly infiltrative along white matter tracts—a characteristic of human GBM (Fig. 1D). To further characterize the cancer-initiating cells, CD133+ cancer-initiating cells were sorted from the neurospheres and diluted for single colony formation. Over 80% of seeded single cells grew out, and 10 clones from each neurosphere were selected at random and expanded for further immunologic characterization.

Figure 1.

Characterization of human glioma-associated cancer-initiating cells. A, a representative image of neurospheres from one glioma-associated cancer-initiating cell is shown. B, the glioma-associated cancer-initiating cells were surface stained with antibodies to CD133, CD34, and CD45. Representative fluorescence-activated cell sorting histogram plots for CD133+ cells are shown for target staining (shaded line) with associated isotype controls (gray line). Percentages of the positive populations are shown. C, after 7 d of culture in differentiating medium, the glioma-associated cancer-initiating cells differentiated into GFAP+ astroglial lineage cells, mouse anti-microtubule associate protein 2+ neuronal lineage cells, and galactosylceramidase+ oligodendroglial lineage cells (magnification, ×40), indicating that the glioma-associated cancer-initiating cells have multipotent differentiation potential. D, a representative image of a glioma-associated cancer-initiating cell xenografted into the frontal lobe of a nude mouse. The tumor that developed from the glioma-associated cancer-initiating cell caused enlargement of the brain and were diffusely infiltrative (arrow on right), including into white matter tracts such as the corpus callosum (arrow on left).

Figure 1.

Characterization of human glioma-associated cancer-initiating cells. A, a representative image of neurospheres from one glioma-associated cancer-initiating cell is shown. B, the glioma-associated cancer-initiating cells were surface stained with antibodies to CD133, CD34, and CD45. Representative fluorescence-activated cell sorting histogram plots for CD133+ cells are shown for target staining (shaded line) with associated isotype controls (gray line). Percentages of the positive populations are shown. C, after 7 d of culture in differentiating medium, the glioma-associated cancer-initiating cells differentiated into GFAP+ astroglial lineage cells, mouse anti-microtubule associate protein 2+ neuronal lineage cells, and galactosylceramidase+ oligodendroglial lineage cells (magnification, ×40), indicating that the glioma-associated cancer-initiating cells have multipotent differentiation potential. D, a representative image of a glioma-associated cancer-initiating cell xenografted into the frontal lobe of a nude mouse. The tumor that developed from the glioma-associated cancer-initiating cell caused enlargement of the brain and were diffusely infiltrative (arrow on right), including into white matter tracts such as the corpus callosum (arrow on left).

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Immunologic Characterization of Cancer-Initiating Cells

To characterize their immunologic phenotype, the cancer-initiating cells (n = 5) were assessed for their expression of MHC I, MHC II, CD40, CD80, CD86, and B7-H1 by flow cytometry. The cancer-initiating cells expressed high levels of MHC I (mean, 99.3%; range 98.5–99.8%) and low levels of CD86 (mean, 6.7%; range, 5.9–7.9%) and CD40 (mean, 5.8%; range, 0.7–15.8%), but not MHC II (mean, 2.4%; range, 1.6–3.2%) or CD80 (mean, 0.6%; range, 0.2–0.6%; a representative example is shown in Fig. 2A), indicating that cancer-initiating cells lack the capacity for antigen presentation necessary to stimulate T-cell activation or proliferation. Furthermore, the inhibitory costimulatory molecule B7-H1 (mean, 31.2%; range 28.5–34.9%) was expressed, indicating that direct contact between T cells and cancer-initiating cells would be inhibitory on immune cells.

Figure 2.

Glioma-associated cancer-initiating cells are immunosuppressive of human T cells. A, immune surface phenotype of a representative glioma-associated cancer-initiating cell. The glioma-associated cancer-initiating cells were surface stained with antibodies to MHC I, MHC II, CD40, CD80, CD86, and B7-H1. Representative FACS histogram plots for one glioma-associated cancer-initiating cell are shown for target staining (shaded line) with associated isotype controls (black line). Percentages of the positive populations are shown. B, the supernatants from the glioma-associated cancer-initiating cells induce an increase in the number of CD4+FoxP3+ Tregs on both day 5 and 10. Representative FACS plots are shown. C, the glioma-associated cancer-initiating cell–induced FoxP3+ Tregs suppress T-cell proliferation. T cells that were treated with glioma-associated cancer-initiating cell supernatants were harvested, cocultured for 3 d with autologous PBMCs (labeled with CFSE, responder cells) at a 1:1 ratio in the presence of soluble anti-CD3, and subsequently analyzed via FACScan. The number above the line in each histogram represents proliferating responder cells. D, the glioma-associated cancer-initiating cell supernatants induce T-cell apoptosis after 3 d of exposure to the supernatants. Similar data were obtained after 5 d of exposure. After culturing with the glioma-associated cancer-initiating cell supernatants, T cells were stimulated with anti-CD3/CD28 and stained with 7-AAD and Annexin V. Compared with medium alone (control), the glioma-associated cancer-initiating cells enhanced T-cell apoptosis.

Figure 2.

Glioma-associated cancer-initiating cells are immunosuppressive of human T cells. A, immune surface phenotype of a representative glioma-associated cancer-initiating cell. The glioma-associated cancer-initiating cells were surface stained with antibodies to MHC I, MHC II, CD40, CD80, CD86, and B7-H1. Representative FACS histogram plots for one glioma-associated cancer-initiating cell are shown for target staining (shaded line) with associated isotype controls (black line). Percentages of the positive populations are shown. B, the supernatants from the glioma-associated cancer-initiating cells induce an increase in the number of CD4+FoxP3+ Tregs on both day 5 and 10. Representative FACS plots are shown. C, the glioma-associated cancer-initiating cell–induced FoxP3+ Tregs suppress T-cell proliferation. T cells that were treated with glioma-associated cancer-initiating cell supernatants were harvested, cocultured for 3 d with autologous PBMCs (labeled with CFSE, responder cells) at a 1:1 ratio in the presence of soluble anti-CD3, and subsequently analyzed via FACScan. The number above the line in each histogram represents proliferating responder cells. D, the glioma-associated cancer-initiating cell supernatants induce T-cell apoptosis after 3 d of exposure to the supernatants. Similar data were obtained after 5 d of exposure. After culturing with the glioma-associated cancer-initiating cell supernatants, T cells were stimulated with anti-CD3/CD28 and stained with 7-AAD and Annexin V. Compared with medium alone (control), the glioma-associated cancer-initiating cells enhanced T-cell apoptosis.

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To evaluate what immunosuppressive cytokines the cancer-initiating cells were elaborating, the cancer-initiating cells (n = 4) and established glioma cell lines were assayed for immunosuppressive cytokines such as TGF-β1, IL-6, IL-10, PGE2, soluble Fas, and vascular endothelial growth factor by ELISA. The cancer-initiating cells did not produce any appreciable IL-6, IL-10, or soluble Fas, but did produce TGF-β1 (38.5–118 pg/mL) and the Treg chemokine attractant CCL-2 (12.8–1134 pg/mL; Table 1).

Table 1.

Immunosuppressive cytokines and chemokines are elaborated by glioma cell lines and glioma-associated cancer-initiating cells

SourceCytokine (pg/mL)
TGF-β1VEGFIL-6PGE2CCL-2FasIL-10Galectin-3
U-87 2,473 3,920 43,568 231 4,401 467 
U-251 1,597 1,708 70 80 10,586 175 
Cancer-initiating cells (n = 4) 76 (38.5–118) 49 (22–98) 75 (54–95) 457 (12.8–1,134) 2,440 (1,645–3,183) 
SourceCytokine (pg/mL)
TGF-β1VEGFIL-6PGE2CCL-2FasIL-10Galectin-3
U-87 2,473 3,920 43,568 231 4,401 467 
U-251 1,597 1,708 70 80 10,586 175 
Cancer-initiating cells (n = 4) 76 (38.5–118) 49 (22–98) 75 (54–95) 457 (12.8–1,134) 2,440 (1,645–3,183) 

NOTE: For U-87 and U-251, the values are actual values from one experiment, whereas cancer-initiating cell values are means with ranges.

Abbreviations: VEGF, vascular endothelial growth factor; CCL-2, chemokine C-C motif-2.

Cancer-Initiating Cells Inhibit T-Cell Activation and Proliferation

To determine if the cancer-initiating cells could inhibit the proliferation and elaboration of proinflammatory responses of immune cells, PBMCs from healthy donors were activated with anti-CD3/CD28 or phytohemagglutinin in the presence of conditioned medium obtained from 3-day cultures of cancer-initiating cells, and T-cell proliferation was assessed by flow cytometry. The media from all of the CD133+ clones (n = 20, generated from two GBM specimens, and 10 clones from each) potently inhibited T-cell proliferation by 91 ± 12% (P = 0.0005). No inhibition of T-cell proliferation was detected when the conditioned medium was obtained from either normal human astrocytes or the U-87 cell line. To ascertain if the supernatants could also inhibit the elaboration of the proinflammatory cytokines IL-2 and IFN-γ from the CD4+ T-cell helper and the CD8+ T-cell effector subset, the cancer-initiating cell supernatants were coincubated with the healthy donors' PBMCs in the presence of antiCD3/CD28 stimulation, and the percentages of CD4+ and CD8+ T cells producing IL-2 and IFN-γ were determined by intracellular staining via flow cytometry. The production of both IFN-γ and IL-2 by the CD4+ and CD8+ T-cell subsets was suppressed by the cancer-initiating cell supernatants (Table 2). These data show that cancer-initiating cells suppress T-cell proliferative and proinflammatory responses.

Table 2.

Effects of cancer-initiating supernatants on cytokine production by CD4 or CD8 T-cell subsets

Experimental conditionCancer-initiating cell supernatantT-cell subsetIL-2–producing cells (%)Change compared with medium (%)γ-IFN–producing cells (%)Change compared with medium (%)
Medium None CD4 60.5 — 28.3 — 
Medium None CD8 64.2 — 53.9 — 
Supernatant 9–29 CD4 21.4 ↓ 65 17.5 ↓ 38 
Supernatant 9–29 CD8 6.1 ↓ 90 49.4 ↓ 8 
Supernatant 6–15 CD4 36.5 ↓ 40 19.9 ↓ 30 
Supernatant 6–15 CD8 14.5 ↓ 76 43.5 ↓ 19 
Supernatant 11–28 CD4 31.8 ↓ 47 22.3 ↓ 21 
Supernatant 11–28 CD8 9.2 ↓ 85 50.3 ↓ 7 
Experimental conditionCancer-initiating cell supernatantT-cell subsetIL-2–producing cells (%)Change compared with medium (%)γ-IFN–producing cells (%)Change compared with medium (%)
Medium None CD4 60.5 — 28.3 — 
Medium None CD8 64.2 — 53.9 — 
Supernatant 9–29 CD4 21.4 ↓ 65 17.5 ↓ 38 
Supernatant 9–29 CD8 6.1 ↓ 90 49.4 ↓ 8 
Supernatant 6–15 CD4 36.5 ↓ 40 19.9 ↓ 30 
Supernatant 6–15 CD8 14.5 ↓ 76 43.5 ↓ 19 
Supernatant 11–28 CD4 31.8 ↓ 47 22.3 ↓ 21 
Supernatant 11–28 CD8 9.2 ↓ 85 50.3 ↓ 7 

Cancer-Initiating Cells Induce Tregs and T-Cell Apoptosis

Because the cancer-initiating cells were producing TGF-β, we next determined if these cells could induce Tregs. Incubation with supernatants from the cancer-initiating cells (18 of 20) markedly expanded the number of CD4+FoxP3+ Tregs in healthy donor PBMCs by 128 ± 51% (P = 0.0007; representative example in Fig. 2B). These FoxP3+ Tregs were functionally suppressive in autologous T-cell proliferation assays (Fig. 2C). All of the cancer-initiating supernatants (n = 20) were able to increase immune cell apoptosis by 144 ± 29% (P = 0.0001) in healthy donor PBMCs. Furthermore, when the GBM patients' PBMCs were coincubated with the respective patients' cancer-initiating cells, as predicted from the phenotypic expression of B7-H1, both preapoptosis and apoptosis were induced in the immune cells (representative example in Fig. 2D). Conditioned media from normal human astrocytes and the U-87 glioma cell line did not induce T-cell apoptosis. Activated immune cells also underwent apoptosis when cocultured with conditioned medium from cancer-initiating cells, indicating that activation did not protect immune cells from the apoptosis induced by conditioned medium from cancer-initiating cells. This indicates that cancer-initiating cells can mediate immunosuppression by apoptotic elimination of immune cells, regardless of their activation state, likely by both secretion of product(s) and direct cell-to-cell contact.

Cancer-Initiating Cells Induce the Expression of p-STAT3 in Immune Cells

The expression of p-STAT3 by tumor cells triggers a cascade of transcriptional factors that have been shown to activate p-STAT3 in immune cells (26). In addition, cancer-initiating cells have been shown to express p-STAT3 (38). The p-STAT3 expression of cancer-initiating cells can be inhibited with the STAT3 siRNA (Fig. 3A), and this treatment did not affect cell viability based on trypan blue exclusion. The supernatants from cancer-initiating cells (n = 3) were capable of increasing p-STAT3 expression in immune cells by 64.7 ± 12.5% (P = 0.006; representative in Fig. 3B). When the cancer-initiating cells were treated with the STAT3 siRNA and then healthy donor PBMCs (n = 3) were subsequently exposed to conditioned media from these glioma-associated cancer-initiating cells, the induction of p-STAT3 was significantly diminished in the immune cells (Fig. 3B). Similar data were obtained with the small molecular inhibitor of the p-STAT3 pathway WP1066.

Figure 3.

Glioma-associated cancer-initiating cells express p-STAT3 and induce p-STAT3 expression in human immune cells. A, STAT3 siRNA–treated glioma-associated cancer-initiating cells express lower p-STAT3 compared with control siRNA. B, glioma-associated cancer-initiating cell supernatants enhance the expression of p-STAT3 in PBMCs, and the supernatants from STAT3 siRNA–treated cancer-initiating cells reduce p-STAT3 level in PBMCs. p-STAT3 expression in normal donor PBMCs was measured by intracellular p-STAT3 (pY705) staining via flow cytometry after 3 d of coculture with cancer-initiating cell supernatants in the presence of anti-CD3/CD28 stimulation. Representative histograms are shown from three independent experiments. Black line, p-STAT3; gray shade, isotype control.

Figure 3.

Glioma-associated cancer-initiating cells express p-STAT3 and induce p-STAT3 expression in human immune cells. A, STAT3 siRNA–treated glioma-associated cancer-initiating cells express lower p-STAT3 compared with control siRNA. B, glioma-associated cancer-initiating cell supernatants enhance the expression of p-STAT3 in PBMCs, and the supernatants from STAT3 siRNA–treated cancer-initiating cells reduce p-STAT3 level in PBMCs. p-STAT3 expression in normal donor PBMCs was measured by intracellular p-STAT3 (pY705) staining via flow cytometry after 3 d of coculture with cancer-initiating cell supernatants in the presence of anti-CD3/CD28 stimulation. Representative histograms are shown from three independent experiments. Black line, p-STAT3; gray shade, isotype control.

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Blockade of p-STAT3 Can Restore T-Cell Function

To determine if p-STAT3 blockade can change the immunosuppressive functions of the cancer-initiating cells, the cancer-initiating cells were treated with the STAT3 siRNA and phenotypically and functionally characterized. Treatment did not alter the expression of MHC II, CD40, CD80, CD86, or B7-H1 (data not shown). In contrast to the marked inhibition of T-cell proliferation with the supernatant from the cancer-initiating cells, the supernatants from siRNA-treated cancer-initiating cells did not inhibit T-cell proliferation (Fig. 4A); there was restoration of the number of T cells secreting IL-2 and IFN-γ; T-cell apoptosis was inhibited (Fig. 4B); and there was a reduction in glioma-associated cancer-initiating cells–induced FoxP3+ Tregs (Fig. 4C). Identical data were obtained with the small molecular inhibitor of the p-STAT3 pathway WP1066 (Fig. 4D–F). The treatment of cancer-initiating cells (n = 3) with the STAT3 siRNA decreased the production of Galectin-3 from 1,967 ± 1,151 to 920 ± 423 pg/mL (P < 0.05).

Figure 4.

Immunosuppression mediated by glioma-associated cancer-initiating cells is reversed with p-STAT3 inhibition. A, two glioma-associated cancer-initiating cell lines were transfected with STAT3 siRNA or control siRNA. After 3 d, the glioma-associated cancer-initiating cells were harvested for p-STAT3 staining, and conditioned media were collected for conducting T-cell immune function assays. Inhibition of T-cell proliferation mediated by glioma-associated cancer-initiating cells is reversed with STAT3 siRNA inhibition. Proliferation of T cells from normal donor was measured with cell division of CFSE-labeled T cells via flow cytometry after 5 d of culture. B, siRNA-treated glioma-associated cancer-initiating cells reduces T-cell apoptosis. Cultured T cells on day 5 from (A) were analyzed for apoptosis. C, siRNA-treated glioma-associated cancer-initiating cells reduce the generation of FoxP3+ Tregs. Cultured T cells on day 5 from (A) were stained for CD4 and FoxP3, and FACS data were converted into bar graphs showing fold change in the percentage of FoxP3+ Tregs versus medium alone (control; set at baseline of 1). In A to C, media only served as negative controls (black columns). Columns, mean from three independent experiments; bars, SD. P < 0.05 for all control and STAT3 siRNA comparisons. D, similar to the STAT-3 siRNA, the inhibition of T-cell proliferation mediated by glioma-associated cancer-initiating cells is reversed by WP1066. Proliferation of the T cells was measured by CCK-8 staining after culturing the T cells with the supernatants from the cancer-initiating cells or the cancer-initiating cells treated with WP1066 for 4 d. The results are averages from three independent experiments. *, P < 0.05. E, treatment of glioma-associated cancer-initiating cells with WP1066 reduces the generation of FoxP3+ Tregs. F, inhibition of the proinflammatory cytokine IL-2 and IFN-y by the T cells in the presence of supernatant from glioma-associated cancer-initiating cells is partially reversed by WP1066. Intracellular cytokine staining of CD3+ T cells from D for IL-2 and IFN-y was done on day 4. In E and F, FACS plots from one representative glioma-associated cancer-initiating cell experiment were shown but similar results were obtained with glioma-associated cancer-initiating cells from three other patients.

Figure 4.

Immunosuppression mediated by glioma-associated cancer-initiating cells is reversed with p-STAT3 inhibition. A, two glioma-associated cancer-initiating cell lines were transfected with STAT3 siRNA or control siRNA. After 3 d, the glioma-associated cancer-initiating cells were harvested for p-STAT3 staining, and conditioned media were collected for conducting T-cell immune function assays. Inhibition of T-cell proliferation mediated by glioma-associated cancer-initiating cells is reversed with STAT3 siRNA inhibition. Proliferation of T cells from normal donor was measured with cell division of CFSE-labeled T cells via flow cytometry after 5 d of culture. B, siRNA-treated glioma-associated cancer-initiating cells reduces T-cell apoptosis. Cultured T cells on day 5 from (A) were analyzed for apoptosis. C, siRNA-treated glioma-associated cancer-initiating cells reduce the generation of FoxP3+ Tregs. Cultured T cells on day 5 from (A) were stained for CD4 and FoxP3, and FACS data were converted into bar graphs showing fold change in the percentage of FoxP3+ Tregs versus medium alone (control; set at baseline of 1). In A to C, media only served as negative controls (black columns). Columns, mean from three independent experiments; bars, SD. P < 0.05 for all control and STAT3 siRNA comparisons. D, similar to the STAT-3 siRNA, the inhibition of T-cell proliferation mediated by glioma-associated cancer-initiating cells is reversed by WP1066. Proliferation of the T cells was measured by CCK-8 staining after culturing the T cells with the supernatants from the cancer-initiating cells or the cancer-initiating cells treated with WP1066 for 4 d. The results are averages from three independent experiments. *, P < 0.05. E, treatment of glioma-associated cancer-initiating cells with WP1066 reduces the generation of FoxP3+ Tregs. F, inhibition of the proinflammatory cytokine IL-2 and IFN-y by the T cells in the presence of supernatant from glioma-associated cancer-initiating cells is partially reversed by WP1066. Intracellular cytokine staining of CD3+ T cells from D for IL-2 and IFN-y was done on day 4. In E and F, FACS plots from one representative glioma-associated cancer-initiating cell experiment were shown but similar results were obtained with glioma-associated cancer-initiating cells from three other patients.

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The immunologic properties of human cancer-initiating cells have not been defined previously, and to our knowledge, this is the first study to show that these cells mediate many of the key features of immunosuppression and suggests a possible mechanism for resistance to immunotherapy. To investigate the immune properties of glioma-associated cancer-initiating cells, we used two different approaches. In the first, the supernatants from glioma-associated cancer-initiating cells were used in immunologic assays with T cells from healthy donors to determine the effects of glioma-associated cancer-initiating cells in the absence of pre-existing T-cell immunosuppression while avoiding allogeneic responses that could confound the interpretation of the data. In the second approach, using GBM patients' T cells and the respective patients' glioma-associated cancer-initiating cells, allogeneic interactions would not confound the data, allowing for analysis of direct cell-to-cell contact; however, pre-existing immune suppression in the patient T cells might dampen the extent of immunosuppression exerted by the glioma-associated cancer-initiating cells. Regardless of the experimental approaches, the data showed that the glioma-associated cancer-initiating cells inhibit T cell–mediated responses.

More specifically, although the glioma-associated cancer-initiating cells expressed MHC I, they lacked MHC II, CD40, and CD80 expression, which would be anticipated to induce T-cell anergy (46), and this was confirmed in our functional assays of T-cell proliferation. The glioma-associated cancer-initiating cells expressed the costimulatory inhibitory molecule B7-H1, which has previously been shown to be a key factor mediating immune resistance in gliomas (47) and can induce T-cell apoptosis (48). Thus, it was not unexpected to find in the direct cell-to-cell contact experiments that the glioma-associated cancer-initiating cells induced T-cell apoptosis in the scenario of B7-H1 expression. Additionally, it was not entirely surprising to see that cancer-initiating cells could induce Tregs that were functionally active and have been shown to contribute to the immune suppression in malignant glioma patients (4951) because the cancer-initiating cells elaborated TGF-β (10–100 pg/mL) at levels sufficient to induce Tregs (52). Interestingly, the cancer-initiating cells also produced PGE2, which has been shown to regulate Th17 cell function (34). The Th17 immune cell population has recently been shown to promote tumor growth through the IL-6-STAT3 pathway (53). Likely, additional STAT3-mediated cancer-initiating mechanisms of immune suppression will be identified possibly with cytokine microarrays. Although the glioma cell lines U-87 and U-251 also produce TGF-β1, they failed to show significant inhibition of T-cell proliferation that is likely secondary to the elaborated IL-6 being produced, which is a potent driver for T-cell proliferation (54), indicating a complex interplay among the various secreted components. Cumulatively, these data indicate that in addition to the previously identified key role of glioma-associated cancer-initiating cells in mediating radiation resistance (17) and chemotherapy resistance (11, 55, 56), glioma-associated cancer-initiating cells also contribute to T-cell immune suppression by multiple mechanisms.

In this report, we also show that the supernatants from the glioma-associated cancer-initiating cells could increase the number of immune cells expressing p-STAT3, likely by one or more of the previously characterized STAT3-transcriptionally controlled tumor-secreted factors. The treatment of the STAT3 expressing glioma-associated cancer-initiating cells with STAT3 siRNA or WP1066 diminished the percentage of immune cells expressing p-STAT3 that were exposed to the supernatant from the glioma-associated cancer-initiating cells. Although treatment of glioma-associated cancer-initiating cells with STAT3 siRNA or WP1066 did not alter the immunologic phenotype of the glioma-associated cancer-initiating cells, as reflected by the levels of expression of MHC or costimulatory molecules, the treatment of the glioma-associated cancer-initiating cells with either STAT3 siRNA or WP1066 did partially restore T-cell proliferation and effector function secondary to decreased T-cell apoptosis and Tregs. In the case of the PBMCs exposed to the supernatants from the cancer-initiating cells treated with the STAT3 inhibitors, the decrease in Tregs could possibly be secondary to residual STAT3 siRNA or WP1066 in the medium that blocks FoxP3 expression in human CD4+CD25+ Tregs (57). Or alternatively, the blockade of the STAT3 pathway in the cancer-initiating cells resulted in the downmodulation of secreted factors that induce Tregs. In the case of the PBMCs exposed to the supernatants from the cancer-initiating cells treated with the STAT3 inhibitors, the decrease in T-cell apoptosis is likely secondary to a decrease in Galectin-3. Soluble Galectin-3 has been shown to induce T-cell apoptosis (58), is expressed in glioma cell lines but not normal astrocytes or oligodendrocytes (59), and has been shown to enhance glioma proliferation and migration (60). Upon treatment with the STAT3 inhibitors, we found a decrease in the amount of Galectin-3 production that was responsible for the T-cell apoptosis. However, because we observed only partial blockade of T-cell apoptosis with the STAT-3 inhibitors, thus accounting for the incomplete restoration of T-cell proliferation and effector function, a STAT-3–independent pathway such as B7-H1 expression is likely also operational in the cancer-initiating, cell-mediated T-cell apoptosis.

Although we have shown that blockade of the STAT3 pathway with siRNA and the small-molecule inhibitor WP1066 could reverse cancer-initiating, cell-mediated immune suppression, other molecules such as JSI-124 (61) and NSC 74859 (62), among many others, targeting the STAT3 pathway could also potentially exert the same effects. A variety of molecular inhibitors of STAT3 have been devised. These molecular inhibitors interfere with the signaling of STAT3 by blocking the ligand-receptor interaction and activation sites of STAT3, dimerization, nuclear translocation, DNA binding, and gene transcription. Conceivably, the inhibitors of the STAT3 pathway may be able to overcome cancer-initiating, cell-mediated chemoradiation and radiation therapeutic resistance in addition to immune suppression, but this awaits more detailed study. Further investigations will be necessary to ascertain how to optimize the STAT3 inhibitors in combination with other immune therapeutics. Finally, because our data indicate that the STAT3 pathway is a significant molecular pathway mediating cancer-initiating cell-mediated immune suppression, newer murine model systems are under development. These murine model systems are immune competent and have constitutive overactivation of the STAT3 pathway under the GFAP promoter. These models may ultimately be more applicable to glioma tumorigenesis and may more accurately recapitulate heterogeneous human gliomas as opposed to clonotypic xenografts. These murine models may also provide an enriched population of murine glioma cancer-initiating cells in which agents that block the STAT3 pathway could be tested.

No potential conflicts of interest were disclosed.

We thank Lamonne Crutcher for the assistance in obtaining tissue specimens and Melissa Burkett and Adelina “Keats” Fuentes for the editorial assistance.

Grant Support: The Anthony Bullock III Foundation (A.B. Heimberger), the Dr. Marnie Rose Foundation (A.B. Heimberger), the University of Texas M.D. Anderson Cancer Center (A.B. Heimberger), and the NIH grant CA120813-01 (A.B. Heimberger).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1
Kurpad
SN
,
Zhao
XG
,
Wikstrand
CJ
,
Batra
SK
,
McLendon
RE
,
Bigner
DD
. 
Tumor antigens in astrocytic gliomas [Review]
.
Glia
1995
;
15
:
244
56
.
2
Rosenberg
SA
,
Yang
JC
,
Restifo
NP
. 
Cancer immunotherapy: moving beyond current vaccines
.
Nat Med
2004
;
10
:
909
15
.
3
Dey
M
,
Hussain
SF
,
Heimberger
AB
. 
The role of glioma microenvironment in immune modulation: potential targets for intervention
.
Lett Drug Des Discov
2006
;
3
:
443
51
.
4
Hickey
WF
,
Hsu
BL
,
Kimura
H
. 
T-lymphocyte entry into the central nervous system
.
J Neurosci Res
1991
;
28
:
254
60
.
5
Morford
LA
,
Elliott
LH
,
Carlson
SL
,
Brooks
WH
,
Roszman
TL
. 
T cell receptor-mediated signaling is defective in T cells obtained from patients with primary intracranial tumors
.
J Immunol
1997
;
159
:
4415
25
.
6
Fontana
A
,
Hengartner
H
,
de Tribolet
N
,
Weber
E
. 
Glioblastoma cells release interleukin 1 and factors inhibiting interleukin 2-mediated effects
.
J Immunol
1984
;
132
:
1837
44
.
7
Elliott
LH
,
Brooks
WH
,
Roszman
TL
. 
Cytokinetic basis for the impaired activation of lymphocytes from patients with primary intracranial tumors
.
J Immunol
1984
;
132
:
1208
15
.
8
Miescher
S
,
Whiteside
TL
,
de Tribolet
N
,
von Fliedner
V
. 
In situ characterization, clonogenic potential, and antitumor cytolytic activity of T lymphocytes infiltrating human brain cancers
.
J Neurosurg
1988
;
68
:
438
48
.
9
Ashkenazi
E
,
Deutsch
M
,
Tirosh
R
,
Weinreb
A
,
Tsukerman
A
,
Brodie
C
. 
A selective impairment of the IL-2 system in lymphocytes of patients with glioblastomas: increased level of soluble IL-2R and reduced protein tyrosine phosphorylation
.
Neuroimmunomodulation
1997
;
4
:
49
56
.
10
Sanai
N
,
Alvarez-Buylla
A
,
Berger
MS
. 
Neural stem cells and the origin of gliomas
.
N Engl J Med
2005
;
353
:
811
22
.
11
Liu
G
,
Yuan
X
,
Zeng
Z
, et al
. 
Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma
.
Mol Cancer
2006
;
5
:
67
79
.
12
Sun
Y
,
Kong
W
,
Falk
A
, et al
. 
CD133 (Prominin) negative human neural stem cells are clonogenic and tripotent
.
PLoS One
2009
;
4
:
e5498
.
13
Shmelkov
SV
,
Butler
JM
,
Hooper
AT
, et al
. 
CD133 expression is not restricted to stem cells, and both CD133+ and CD133− metastatic colon cancer cells initiate tumors
.
J Clin Invest
2008
;
118
:
2111
20
.
14
Bleau
AM
,
Hambardzumyan
D
,
Ozawa
T
, et al
. 
PTEN/PI3K/Akt pathway regulates the side population phenotype and ABCG2 activity in glioma tumor stem-like cells
.
Cell Stem Cell
2009
;
4
:
226
35
.
15
Gunther
HS
,
Schmidt
NO
,
Phillips
HS
, et al
. 
Glioblastoma-derived stem cell-enriched cultures form distinct subgroups according to molecular and phenotypic criteria
.
Oncogene
2008
;
27
:
2897
909
.
16
Galli
R
,
Binda
E
,
Orfanelli
U
, et al
. 
Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma
.
Cancer Res
2004
;
64
:
7011
21
.
17
Bao
S
,
Wu
Q
,
McLendon
RE
, et al
. 
Glioma stem cells promote radioresistance by preferential activation of the DNA damage response
.
Nature
2006
;
444
:
756
60
.
18
Murat
A
,
Migliavacca
E
,
Gorlia
T
, et al
. 
Stem cell-related “self-renewal” signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma
.
J Clin Oncol
2008
;
26
:
3015
24
.
19
Lang
R
,
Patel
D
,
Morris
JJ
,
Rutschman
RL
,
Murray
PJ
. 
Shaping gene expression in activated and resting primary macrophages by IL-10
.
J Immunol
2002
;
169
:
2253
63
.
20
O'Farrell
AM
,
Liu
Y
,
Moore
KW
,
Mui
AL
. 
IL-10 inhibits macrophage activation and proliferation by distinct signaling mechanisms: evidence for Stat3-dependent and -independent pathways
.
EMBO J
1998
;
17
:
1006
18
.
21
Kortylewski
M
,
Kujawski
M
,
Wang
T
, et al
. 
Inhibiting Stat3 signaling in the hematopoietic system elicits multicomponent antitumor immunity
.
Nat Med
2005
;
11
:
1314
21
.
22
Hussain
SF
,
Kong
L-Y
,
Jordan
J
, et al
. 
A novel small molecule inhibitor of signal transducers and activators of transcription 3 reverses immune tolerance in malignant glioma patients
.
Cancer Res
2007
;
67
:
9630
6
.
23
Brantley
EC
,
Nabors
LB
,
Y
GG
, et al
. 
Loss of protein inhibitors of activated STAT-3 expression in glioblastoma multiforme tumors: implications for STAT-3 activation and gene expression
.
Clin Cancer Res
2008
;
14
:
4694
704
.
24
Abou-Ghazal
M
,
Yang
DS
,
Qiao
W
, et al
. 
The incidence, correlation with tumor-infiltrating inflammation, and prognosis of phosphorylated STAT3 expression in human gliomas
.
Clin Cancer Res
2008
;
14
:
8228
35
.
25
Yu
H
,
Jove
R
. 
The STATs of cancer-new molecular targets come of age
.
Nat Rev Cancer
2004
;
4
:
97
105
.
26
Yu
H
,
Kortylewski
M
,
Pardoll
D
. 
Crosstalk between cancer and immune cells: role of STAT3 in the tumour microenvironment
.
Nat Rev Immunol
2007
;
7
:
41
51
.
27
Steinbrink
K
,
Wolfl
M
,
Jonuleit
H
,
Knop
J
,
Enk
AH
. 
Induction of tolerance by IL-10-treated dendritic cells
.
J Immunol
1997
;
159
:
4772
80
.
28
Williams
L
,
Bradley
L
,
Smith
A
,
Foxwell
B
. 
Signal transducer and activator of transcription 3 is the dominant mediator of the anti-inflammatory effects of IL-10 in human macrophages
.
J Immunol
2004
;
172
:
567
76
.
29
DiMeo
D
,
Tian
J
,
Zhang
J
,
Narushima
S
,
Berg
DJ
. 
Increased interleukin-10 production and Th2 skewing in the absence of 5-lipoxygenase
.
Immunology
2008
;
123
:
250
62
.
30
Asseman
C
,
Mauze
S
,
Leach
MW
,
Coffman
RL
,
Powrie
F
. 
An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation
.
J Exp Med
1999
;
190
:
995
1004
.
31
Niu
G
,
Bowman
T
,
Huang
M
, et al
. 
Roles of activated Src and Stat3 signaling in melanoma tumor cell growth
.
Oncogene
2002
;
21
:
7001
10
.
32
Gabrilovich
DI
,
Chen
HL
,
Girgis
KR
, et al
. 
Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells [erratum appears in Nat Med 1996 Nov;2:1267]
.
Nat Med
1996
;
2
:
1096
103
.
33
Rummel
C
,
Sachot
C
,
Poole
S
,
Luheshi
GN
. 
Circulating interleukin-6 induces fever through a STAT3-linked activation of COX-2 in the brain
.
Am J Physiol Regul Integr Comp Physiol
2006
;
291
:
R1316
26
.
34
Boniface
K
,
Bak-Jensen
KS
,
Li
Y
, et al
. 
Prostaglandin E2 regulates Th17 cell differentiation and function through cyclic AMP and EP2/EP4 receptor signaling
.
J Exp Med
2009
;
206
:
535
48
.
35
Kinjyo
I
,
Inoue
H
,
Hamano
S
, et al
. 
Loss of SOCS3 in T helper cells resulted in reduced immune responses and hyperproduction of interleukin 10 and transforming growth factor-β1
.
J Exp Med
2006
;
203
:
1021
31
.
36
Heimberger
AB
,
Bigner
DD
,
Sampson
JJ
. 
Biological principles of brain tumor immunotherapy
. In:
Liau
LM
,
Becker
DP
,
Cloughesy
TF
,
Bigner
DD
, editors.
Brain Tumor Immunotherapy
.
Totowa (NJ)
:
Humana Press Inc.
; 
2000
, p.
101
30
.
37
Ferrajoli
A
,
Faderl
S
,
Van
Q
, et al
. 
WP1066 disrupts Janus kinase-2 and induces caspase-dependent apoptosis in acute myelogenous leukemia cells
.
Cancer Res
2007
;
67
:
11291
9
.
38
Sherry
MM
,
Reeves
A
,
Wu
JK
,
Cochran
BH
. 
STAT3 is required for proliferation and maintenance of multipotency in glioblastoma stem cells
.
Stem Cells
2009
;
27
:
2383
92
.
39
Iwamaru
A
,
Szymanski
S
,
Iwado
E
, et al
. 
A novel inhibitor of the STAT3 pathway induces apoptosis in malignant glioma cells both in vitro and in vivo
.
Oncogene
2007
;
26
:
2435
44
.
40
Kong
LY
,
Abou-Ghazal
MK
,
Wei
J
, et al
. 
A novel inhibitor of signal transducers and activators of transcription 3 activation is efficacious against established central nervous system melanoma and inhibits regulatory T cells
.
Clin Cancer Res
2008
;
14
:
5759
68
.
41
Kong
L-K
,
Wei
J
,
Sharma
AK
, et al
. 
A novel phosphorylated STAT3 inhibitor enhances T cell cytotoxicity against melanoma through inhibition of regulatory T cells
.
Cancer Immunol Immunother
2008
;
58
:
1023
32
.
42
Heimberger
AB
,
Priebe
W
. 
Small molecular inhibitors of p-STAT3: novel agents for treatment of primary and metastatic CNS cancers
.
Recent Patents CNS Drug Discov
2008
;
3
:
179
88
.
43
Heimberger
AB
,
Crotty
LE
,
Archer
GE
, et al
. 
Epidermal growth factor receptor VIII peptide vaccination is efficacious against established intracerebral tumors
.
Clin Cancer Res
2003
;
9
:
4247
54
.
44
Lee
A
,
Kessler
JD
,
Read
TA
, et al
. 
Isolation of neural stem cells from the postnatal cerebellum
.
Nat Neurosci
2005
;
8
:
723
9
.
45
Parant
O
,
Dubernard
G
,
Challier
JC
, et al
. 
CD34+ cells in maternal placental blood are mainly fetal in origin and express endothelial markers
.
Lab Invest
2009
;
89
:
915
23
.
46
Gomez
GG
,
Kruse
CA
. 
Mechanisms of malignant glioma immune resistance and sources of immunosuppression
.
Gene Ther Mol Biol
2006
;
10a
:
133
46
.
47
Parsa
AT
,
Waldron
JS
,
Panner
A
, et al
. 
Loss of tumor suppressor PTEN function increases B7-1 expression and immunoresistance in glioma
.
Nat Med
2007
;
1
:
84
8
.
48
Dong
H
,
Strome
SE
,
Salomao
DR
, et al
. 
Tumor-associated B7-1 promotes T-cell apoptosis: a potential mechanism of immune evasion
.
Nat Med
2002
;
8
:
793
800
.
49
Fecci
PE
,
Mitchell
DA
,
Whitesides
JF
, et al
. 
Increased regulatory T-cell fraction amidst a diminished CD4 compartment explains cellular immune defects in patients with malignant glioma
.
Cancer Res
2006
;
66
:
3294
302
.
50
Heimberger
AB
,
Reina-Ortiz
C
,
Yang
DS
, et al
. 
Incidence and prognostic impact of FoxP3+ regulatory T cells in human gliomas
.
Clin Cancer Res
2008
;
14
:
5166
72
.
51
Jacobs
JF
,
Idema
AJ
,
Bol
KF
, et al
. 
Regulatory T cells and the PD-L1/PD-1 pathway mediate immune suppression in malignant human brain tumors
.
Neuro Oncol
2009
;
11
:
394
402
.
52
Wei
J
,
Duramad
O
,
Perng
OA
,
Reiner
SL
,
Liu
YJ
,
Qin
FX
. 
Antagonistic nature of T helper 1/2 developmental programs in opposing peripheral induction of Foxp3+ regulatory T cells
.
Proc Natl Acad Sci U S A
2007
;
104
:
18169
74
.
53
Wang
L
,
Yi
T
,
Kortylewski
M
,
Pardoll
DM
,
Zeng
D
,
Yu
H
. 
IL-17 can promote tumor growth through an IL-6-Stat3 signaling pathway
.
J Exp Med
2009
;
206
:
1457
64
.
54
Sawa
S
,
Kamimura
D
,
Jin
GH
, et al
. 
Autoimmune arthritis associated with mutated interleukin (IL)-6 receptor gp130 is driven by STAT3/IL-7-dependent homeostatic proliferation of CD4+ T cells
.
J Exp Med
2006
;
203
:
1459
70
.
55
Ghods
AJ
,
Irvin
D
,
Liu
G
, et al
. 
Spheres isolated from 9L gliosarcoma rat cell line possess chemoresistant and aggressive cancer stem-like cells
.
Stem Cells
2007
;
25
:
1645
53
.
56
Kang
SK
,
Park
JB
,
Cha
SH
. 
Multipotent, dedifferentiated cancer stem-like cells from brain gliomas
.
Stem Cells Dev
2006
;
15
:
423
35
.
57
Zorn
E
,
Nelson
EA
,
Mohseni
M
, et al
. 
IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo
.
Blood
2006
;
108
:
1571
9
.
58
Peng
W
,
Wang
HY
,
Miyahara
Y
,
Peng
G
,
Wang
RF
. 
Tumor-associated galectin-3 modulates the function of tumor-reactive T cells
.
Cancer Res
2008
;
68
:
7228
36
.
59
Kuklinski
S
,
Pesheva
P
,
Heimann
C
, et al
. 
Expression pattern of galectin-3 in neural tumor cell lines
.
J Neurosci Res
2000
;
60
:
45
57
.
60
Strik
HM
,
Schmidt
K
,
Lingor
P
, et al
. 
Galectin-1 expression in human glioma cells: modulation by ionizing radiation and effects on tumor cell proliferation and migration
.
Oncol Rep
2007
;
18
:
483
8
.
61
Turkson
J
,
Jove
R
. 
STAT proteins: novel molecular targets for cancer drug discovery
.
Oncogene
2000
;
19
:
6613
26
.
62
Lin
L
,
Amin
R
,
Gallicano
GI
, et al
. 
The STAT3 inhibitor NSC 74859 is effective in hepatocellular cancers with disrupted TGF-β signaling
.
Oncogene
2009
;
28
:
961
72
.