Verlene Henry

Glioma-Associated Cancer-Initiating Cells Induce Immunosuppression

Purpose: Glioblastoma multiforme is a lethal cancer that responds poorly to therapy. Glioblastoma multiforme cancer-initiating cells have been shown to mediate resistance to both chemotherapy and radiation; however, it is unknown to what extent these cells contribute to the profound immunosuppression in glioblastoma multiforme patients and if strategies that alter their differentiation state can reduce this immunosuppression. Experimental Design: We isolated a subpopulation of cells from glioblastoma multiforme that possessed the capacity for self-renewal, formed neurospheres in vitro, were capable of pluripotent differentiation, and could initiate tumors in vivo. The immune phenotype of these cells was characterized including the elaboration of immunosuppressive cytokines and chemokines by ELISA. Functional immunosuppressive properties were characterized based on the inhibition of T-cell proliferation and effector responses, triggering of T-cell apoptosis, and induction of FoxP3+ regulatory T cells. On altering their differentiation state, the immunosuppressive phenotype and functional assays were reevaluated. Results: We found that the cancer-initiating cells markedly inhibited T-cell proliferation and activation, induced regulatory T cells, and triggered T-cell apoptosis that was mediated by B7-H1 and soluble Galectin-3. These immunosuppressive properties were diminished on altering the differentiation of the cancer-initiating cells. Conclusion: Cancer-initiating cells contribute to tumor evasion of the immunosurveillance and approaches that alter the differentiation state may have immunotherapeutic potential. Clin Cancer Res; 16(2); 461–73

Glioblastoma Cancer-Initiating Cells Inhibit T-Cell Proliferation and Effector Responses by the Signal Transducers and Activators of Transcription 3 Pathway

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

Glioblastoma resistance to anti-VEGF therapy is associated with myeloid cell infiltration, stem cell accumulation, and a mesenchymal phenotype

Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis. Inhibiting the VEGF-VEGF receptor (R) signal transduction pathway in glioblastoma has recently been shown to delay progression, but the relative benefit and mechanisms of response and failure of anti-VEGF therapy and VEGFR inhibitors are not well understood. The purpose of our study was to evaluate the relative effectiveness of VEGF sequestration and/or VEGFR inhibition on orthotopic tumor growth and the mechanism(s) of treatment resistance. We evaluated, not only, the effects of anti-VEGF therapy (bevacizumab), anti-VEGFR therapy (sunitinib), and the combination on the survival of mice bearing orthotopic gliomas, but also the differential effects of the treatments on tumor vascularity, cellular proliferation, mesenchymal and stem cell markers, and myeloid cell infiltration using flow cytometry and immunohistochemistry. Bevacizumab significantly prolonged survival compared with the control or sunitinib alone. Both antiangiogenic agents initially reduced infiltration of macrophages and tumor vascularity. However, multitargeted VEGFR inhibition, but not VEGF sequestration, rapidly created a vascular gradient and more rapidly induced tumor hypoxia. Re-infiltration of macrophages was associated with the induction of hypoxia. Combination treatment with bevacizumab and sunitinib improved animal survival compared with bevacizumab therapy alone. However, at the time of tumor progression, a significant increase in CD11b(+)/Gr1(+) granulocyte infiltration was observed, and tumors developed aggressive mesenchymal features and increased stem cell marker expression. Collectively, our results demonstrate a more prolonged decrease in tumor vascularity with bevacizumab than with sunitinib, associated with a delay in the development of hypoxia and sustained reduction of infiltrated myeloid cells.

Acquired Resistance to Anti-VEGF Therapy in Glioblastoma Is Associated with a Mesenchymal Transition

Purpose: Antiangiogenic therapy reduces vascular permeability and delays progression but may ultimately promote an aggressive treatment-resistant phenotype. The aim of the present study was to identify mechanisms responsible for glioblastoma resistance to antiangiogenic therapy. Experimental Design: Glioma stem cell (GSC) NSC11 and U87 cell lines with acquired resistance to bevacizumab were developed from orthotopic xenografts in nude mice treated with bevacizumab. Genome-wide analyses were used to identify changes in tumor subtype and specific factors associated with resistance. Results: Mice with established parental NSC11 and U87 cells responded to bevacizumab, whereas glioma cell lines derived at the time of acquired resistance to anti-VEGF therapy were resistant to bevacizumab and did not have prolongation of survival compared with untreated controls. Gene expression profiling comparing anti-VEGF therapy-resistant cell lines to untreated controls showed an increase in genes associated with a mesenchymal origin, cellular migration/invasion, and inflammation. Gene-set enrichment analysis showed that bevacizumab-treated tumors showed a highly significant correlation to published mesenchymal gene signatures. Mice bearing resistant tumors showed significantly greater infiltration of myeloid cells in NSC11- and U87-resistant tumors. Invasion-related genes were also upregulated in both NSC11 and U87 resistant cells which had higher invasion rates in vitro compared with their respective parental cell lines. Conclusions: Our studies identify multiple proinflammatory factors associated with resistance and identify a proneural to mesenchymal transition in tumors resistant to antiangiogenic therapy. Clin Cancer Res; 19(16); 4392–403. ©2013 AACR.

Neutrophils promote the malignant glioma phenotype through S100A4.

Purpose: Antiangiogenic therapy is effective in blocking vascular permeability, inhibiting vascular proliferation, and slowing tumor growth, but studies in multiple cancer types have shown that tumors eventually acquire resistance to blockade of blood vessel growth. Currently, the mechanisms by which this resistance occurs are not well understood. Experimental Design: In this study, we evaluated the effects of neutrophils on glioma biology both in vitro and in vivo and determined target genes by which neutrophils promote the malignant glioma phenotype during anti-VEGF therapy. Results: We found that an increase in neutrophil infiltration into tumors is significantly correlated with glioma grade and in glioblastoma with acquired resistance to anti-VEGF therapy. Our data demonstrate that neutrophils and their condition media increased the proliferation rate of glioblastoma-initiating cells (GIC). In addition, neutrophils significantly increased GICs Transwell migration compared with controls. Consistent with this behavior, coculture with neutrophils promoted GICs to adopt morphologic and gene expression changes consistent with a mesenchymal signature. Neutrophil-promoting tumor progression could be blocked by S100A4 downregulation in vitro and in vivo. Furthermore, S100A4 depletion increased the effectiveness of anti-VEGF therapy in glioma. Conclusions: Collectively, these data suggest that increased recruitment of neutrophils during anti-VEGF therapy promotes glioma progression and may promote treatment resistance. Tumor progression with mesenchymal characteristics is partly mediated by S100A4, the expression of which is increased by neutrophil infiltration. Targeting granulocytes and S100A4 may be effective approaches to inhibit the glioma malignant phenotype and diminish antiangiogenic therapy resistance. Clin Cancer Res; 20(1); 187–98. ©2013 AACR.

Mechanisms of action of rapamycin in gliomas

Rapamycin has previously been shown to be efficacious against intracerebral glioma xenografts and to act in a cytostatic manner against gliomas. However, very little is known about the mechanism of action of rapamycin. The purpose of our study was to further investigate the in vitro and in vivo mechanisms of action of rapamycin, to elucidate molecular end points that may be applicable for investigation in a clinical trial, and to examine potential mechanisms of treatment failure. In the phosphatase and tensin homolog deleted from chromosome 10 (PTEN)-null glioma cell lines U-87 and D-54, but not the oligodendroglioma cell line HOG (PTEN null), doses of rapamycin at the IC50 resulted in accumulation of cells in G1, with a corresponding decrease in the fraction of cells traversing the S phase as early as 24 h after dosing. All glioma cell lines tested had markedly diminished production of vascular endothelial growth factor (VEGF) when cultured with rapamycin, even at doses below the IC50. After 48 h of exposure to rapamycin, the glioma cell lines (but not HOG cells) showed downregulation of the membrane type-1 matrix metalloproteinase (MMP) invasion molecule. In U-87 cells, MMP-2 was downregulated, and in D-54 cells, both MMP-2 and MMP-9 were downregulated after treatment with rapamycin. Treatment of established subcutaneous U-87 xenografts in vivo resulted in marked tumor regression (P < 0.05). Immunohistochemical studies of subcutaneous U-87 tumors demonstrated diminished production of VEGF in mice treated with rapamycin. Gelatin zymography showed marked reduction of MMP-2 in the mice with subcutaneous U-87 xenografts that were treated with rapamycin as compared with controls treated with phosphatebuffered saline. In contrast, treatment of established intracerebral U-87 xenografts did not result in increased median survival despite inhibition of the Akt pathway within the tumors. Also, in contrast with our findings for subcutaneous tumors, immunohistochemistry and quantitative Western blot analysis results for intracerebral U-87 xenografts indicated that there is not significant VEGF production, which suggests possible deferential regulation of the hypoxia-inducible factor 1alpha in the intracerebral compartment. These findings demonstrate that the complex operational mechanisms of rapamycin against gliomas include cytostasis, anti-VEGF, and anti-invasion activity, but these are dependent on the in vivo location of the tumor and have implications for the design of a clinical trial.

Novel MET/TIE2/VEGFR2 inhibitor altiratinib inhibits tumor growth and invasiveness in bevacizumab-resistant glioblastoma mouse models

BACKGROUND Glioblastoma highly expresses the proto-oncogene MET in the setting of resistance to bevacizumab. MET engagement by hepatocyte growth factor (HGF) results in receptor dimerization and autophosphorylation mediating tumor growth, invasion, and metastasis. Evasive revascularization and the recruitment of TIE2-expressing macrophages (TEMs) are also triggered by anti-VEGF therapy. METHODS We investigated the activity of altiratinib (a novel balanced inhibitor of MET/TIE2/VEGFR2) against human glioblastoma stem cell lines in vitro and in vivo using xenograft mouse models. The biological activity of altiratinib was assessed in vitro by testing the expression of HGF-stimulated MET phosphorylation as well as cell viability after altiratinib treatment. Tumor volume, stem cell and mesenchymal marker levels, microvessel density, and TIE2-expressing monocyte infiltration were evaluated in vivo following treatment with a control, bevacizumab alone, bevacizumab combined with altiratinib, or altiratinib alone. RESULTS In vitro, HGF-stimulated MET phosphorylation was completely suppressed by altiratinib in GSC17 and GSC267, and altiratinib markedly inhibited cell viability in several glioblastoma stem cell lines. More importantly, in multiple xenograft mouse models, altiratinib combined with bevacizumab dramatically reduced tumor volume, invasiveness, mesenchymal marker expression, microvessel density, and TIE2-expressing monocyte infiltration compared with bevacizumab alone. Furthermore, in the GSC17 xenograft model, altiratinib combined with bevacizumab significantly prolonged survival compared with bevacizumab alone. CONCLUSIONS Together, these data suggest that altiratinib may suppress tumor growth, invasiveness, angiogenesis, and myeloid cell infiltration in glioblastoma. Thus, altiratinib administered alone or in combination with bevacizumab may overcome resistance to bevacizumab and prolong survival in patients with glioblastoma.

Suppression of RAF/MEK or PI3K synergizes cytotoxicity of receptor tyrosine kinase inhibitors in glioma tumor-initiating cells

BackgroundThe majority of glioblastomas have aberrant receptor tyrosine kinase (RTK)/RAS/phosphoinositide 3 kinase (PI3K) signaling pathways and malignant glioma cells are thought to be addicted to these signaling pathways for their survival and proliferation. However, recent studies suggest that monotherapies or inappropriate combination therapies using the molecular targeted drugs have limited efficacy possibly because of tumor heterogeneities, signaling redundancy and crosstalk in intracellular signaling network, indicating necessity of rationale and methods for efficient personalized combination treatments. Here, we evaluated the growth of colonies obtained from glioma tumor-initiating cells (GICs) derived from glioma sphere culture (GSC) in agarose and examined the effects of combination treatments on GICs using targeted drugs that affect the signaling pathways to which most glioma cells are addicted.MethodsHuman GICs were cultured in agarose and treated with inhibitors of RTKs, non-receptor kinases or transcription factors. The colony number and volume were analyzed using a colony counter, and Chou-Talalay combination indices were evaluated. Autophagy and apoptosis were also analyzed. Phosphorylation of proteins was evaluated by reverse phase protein array and immunoblotting.ResultsIncreases of colony number and volume in agarose correlated with the Gompertz function. GICs showed diverse drug sensitivity, but inhibitions of RTK and RAF/MEK or PI3K by combinations such as EGFR inhibitor and MEK inhibitor, sorafenib and U0126, erlotinib and BKM120, and EGFR inhibitor and sorafenib showed synergy in different subtypes of GICs. Combination of erlotinib and sorafenib, synergistic in GSC11, induced apoptosis and autophagic cell death associated with suppressed Akt and ERK signaling pathways and decreased nuclear PKM2 and β-catenin in vitro, and tended to improve survival of nude mice bearing GSC11 brain tumor. Reverse phase protein array analysis of the synergistic treatment indicated involvement of not only MEK and PI3K signaling pathways but also others associated with glucose metabolism, fatty acid metabolism, gene transcription, histone methylation, iron transport, stress response, cell cycle, and apoptosis.ConclusionInhibiting RTK and RAF/MEK or PI3K could induce synergistic cytotoxicity but personalization is necessary. Examining colonies in agarose initiated by GICs from each patient may be useful for drug sensitivity testing in personalized cancer therapy.

Interferon-regulatory factor-1 (IRF1) regulates bevacizumab induced autophagy

Purpose Antiangiogenic therapy is commonly being used for the treatment of glioblastoma. However, the benefits of angiogenesis inhibitors are typically transient and resistance often develops. Determining the mechanism of treatment failure of the VEGF monoclonal antibody bevacizumab for malignant glioma would provide insight into approaches to overcome therapeutic resistance. Experimental Design In this study, we evaluated the effects of bevacizumab on the autophagy of glioma cells and determined target genes involving in the regulation of bevacizumab-induced autophagy. Results We demonstrated that bevacizumab treatment increased expression of autophagy markers and autophagosome formation in cell culture experiments as well as in in vivo studies. Gene expression profile analysis performed on murine xenograft models of glioblastoma showed increased transcriptional levels of STAT1/IRF1 signaling in bevacizumab resistant tumors compared to control tumors. In vitro experiments showed that bevacizumab treatment increased IRF1 expression in a dose and time dependent manner, which was coincident with bevacizumab-mediated autophagy. Down regulation of IRF1 by shRNA blocked autophagy and increased AIF-dependent apoptosis in bevacizumab-treated glioma cells. Consistently, IRF1 depletion increased the efficacy of anti-VEGF therapy in a glioma xenograft model, which was due to less bevacizumab-promoted autophagy and increased apoptosis in tumors with down-regulated IRF1. Conclusions These data suggest that IRF1 may regulate bevacizumab-induced autophagy, and may be one important mediator of glioblastoma resistant to bevacizumab.

Targeting intercellular adhesion molecule-1 prolongs survival in mice bearing bevacizumab-resistant glioblastoma

Intercellular cell adhesion molecule 1 (ICAM-1; also known as CD54) is overexpressed in bevacizumab-resistant glioblastoma. In the present study, we tested our hypothesis that highly expressed ICAM-1 mediates glioblastoma’s resistance to antiangiogenic therapy. We validated ICAM-1 overexpression in tumors resistant to antiangiogenic therapy using real-time polymerase chain reaction, immunohistochemistry, and Western blotting. We also detected ICAM1 expression in most glioma stem cells (GSCs). We investigated the mechanism of ICAM-1 overexpression after bevacizumab treatment and found that ICAM-1 protein expression was markedly increased in a time-dependent manner in GSC11 and GSC17 cells under hypoxic conditions in vitro. We also found that hypoxia induced ICAM-1 overexpression through the up-regulation of phosphorylated signal transducer and activator of transcription (p-STAT3). Hypoxia-induced p-STAT3 increased the mRNA transcription of ICAM-1, which we could inhibit with the STAT3 inhibitor AZD1480. Next, we used GFP-tagged ICAM-1 shRNA lentivirus to knock down ICAM-1 in GSC11 and GSC17 glioma cell lines. Then, we injected shICAM-1 GSC11 and scramble glioma stem cells into the brains of nude mice. Mice bearing tumors from shICAM-1 GSC11 cells survived significantly longer than mice injected with control cells did. The tumor sizes was significantly decreased in mice bearing tumors from shICAM-1 cells than that in mice bearing tumors from GFP-tagged GSC11 control cells. Knocking down ICAM-1 suppressed tumor invasion in vitro and in vivo and inhibited macrophage infiltration to the tumor site in bevacizumab-treated mice. Our findings suggest that ICAM-1 is a potentially important mediator of tumor migration and invasion in bevacizumab-resistant glioblastoma. Targeting ICAM-1 may provide a new strategy for enhancing the efficacy of antiangiogenic therapy against glioblastoma and preventing the invasive phenotype of the disease.Intercellular cell adhesion molecule 1 (ICAM-1; also known as CD54) is overexpressed in bevacizumab-resistant glioblastoma. In the present study, we tested our hypothesis that highly expressed ICAM-1 mediates glioblastomas resistance to antiangiogenic therapy. We validated ICAM-1 overexpression in tumors resistant to antiangiogenic therapy using real-time polymerase chain reaction, immunohistochemistry, and Western blotting. We also detected ICAM1 expression in most glioma stem cells (GSCs). We investigated the mechanism of ICAM-1 overexpression after bevacizumab treatment and found that ICAM-1 protein expression was markedly increased in a time-dependent manner in GSC11 and GSC17 cells under hypoxic conditions in vitro. We also found that hypoxia induced ICAM-1 overexpression through the up-regulation of phosphorylated signal transducer and activator of transcription (p-STAT3). Hypoxia-induced p-STAT3 increased the mRNA transcription of ICAM-1, which we could inhibit with the STAT3 inhibitor AZD1480. Next, we used GFP-tagged ICAM-1 shRNA lentivirus to knock down ICAM-1 in GSC11 and GSC17 glioma cell lines. Then, we injected shICAM-1 GSC11 and scramble glioma stem cells into the brains of nude mice. Mice bearing tumors from shICAM-1 GSC11 cells survived significantly longer than mice injected with control cells did. The tumor sizes was significantly decreased in mice bearing tumors from shICAM-1 cells than that in mice bearing tumors from GFP-tagged GSC11 control cells. Knocking down ICAM-1 suppressed tumor invasion in vitro and in vivo and inhibited macrophage infiltration to the tumor site in bevacizumab-treated mice. Our findings suggest that ICAM-1 is a potentially important mediator of tumor migration and invasion in bevacizumab-resistant glioblastoma. Targeting ICAM-1 may provide a new strategy for enhancing the efficacy of antiangiogenic therapy against glioblastoma and preventing the invasive phenotype of the disease.