Ling Yuan Kong

Glioma cancer stem cells induce immunosuppressive macrophages/microglia

Macrophages (MΦs)/microglia that constitute the dominant tumor-infiltrating immune cells in glioblastoma are recruited by tumor-secreted factors and are induced to become immunosuppressive and tumor supportive (M2). Glioma cancer stem cells (gCSCs) have been shown to suppress adaptive immunity, but their role in innate immunity with respect to the recruitment and polarization of MΦs/microglia is unknown. The innate immunosuppressive properties of the gCSCs were characterized based on elaborated MΦ inhibitory cytokine-1 (MIC-1), transforming growth factor (TGF-β1), soluble colony-stimulating factor (sCSF), recruitment of monocytes, inhibition of MΦ/microglia phagocytosis, induction of MΦ/microglia cytokine secretion, and the inhibition of T-cell proliferation. The role of the signal transducer and activator of transcription 3 (STAT3) in mediating innate immune suppression was evaluated in the context of the functional assays. The gCSCs produced sCSF-1, TGF-β1, and MIC-1, cytokines known to recruit and polarize the MΦs/microglia to become immunosuppressive. The gCSC-conditioned medium polarized the MΦ/microglia to an M2 phenotype, inhibited MΦ/microglia phagocytosis, induced the secretion of the immunosuppressive cytokines interleukin-10 (IL-10) and TGF-β1 by the MΦs/microglia, and enhanced the capacity of MΦs/microglia to inhibit T-cell proliferation. The inhibition of phagocytosis and the secretion of IL-10 were reversed when the STAT3 pathway was blocked in the gCSCs. The gCSCs modulate innate immunity in glioblastoma by inducing immunosuppressive MΦs/microglia, and this capacity can be reversed by inhibiting phosphorylated STAT3.

A Novel Small Molecule Inhibitor of Signal Transducers and Activators of Transcription 3 Reverses Immune Tolerance in Malignant Glioma Patients

Overcoming the profound immunosuppression in patients with solid cancers has impeded efficacious immunotherapy. Signal transducers and activators of transcription 3 (STAT3) has recently emerged as a potential target for effective immunotherapy, and in this study, we describe a novel small molecule inhibitor of STAT3 that can penetrate the central nervous system (CNS) in mice and in physiologically relevant doses in vitro and reverse tolerance in immune cells isolated from glioblastoma multiforme (GBM) patients. Specifically, it induces the expression of costimulatory molecules on peripheral macrophages and tumor-infiltrating microglia, stimulates the production of the immune-stimulatory cytokines interleukin 2 (IL-2), IL-4, IL-12, and IL-15, and induces proliferation of effector T cells from GBM patients that are refractory to CD3 stimulation. We show that the functional enhancement of immune responses after STAT3 inhibition is accompanied by up-regulation of several key intracellular signaling molecules that critically regulate T-cell and monocyte activation. Specifically, the phosphorylation of Syk (Tyr352) in monocytes and ZAP-70 (Tyr319) in T cells are enhanced by the STAT-3 inhibitor in marked contrast to toll-like receptor and T-cell receptor agonists, respectively. This novel small molecule STAT3 inhibitor has tremendous potential for clinical applications with its penetration into the CNS, easy parental administration, direct tumor cytotoxicity, and potent immune adjuvant responses in immunosuppressed cancer patients.

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

Association Between Imatinib-Resistant BCR-ABL Mutation-Negative Leukemia and Persistent Activation of LYN Kinase

BACKGROUND Imatinib is a tyrosine kinase inhibitor that is used to treat chronic myelogenous leukemia (CML). BCR-ABL mutations are associated with failure of imatinib treatment in many CML patients. LYN kinase regulates survival and responsiveness of CML cells to inhibition of BCR-ABL kinase, and differences in LYN regulation have been found between imatinib-sensitive and -resistant CML cell lines. METHODS We evaluated cells from 12 imatinib-resistant CML patients with mutation-negative BCR-ABL and from six imatinib-sensitive patients who discontinued therapy because of imatinib intolerance. Phosphorylation of BCR-ABL and LYN was assessed in patient cells and cell lines by immunoblotting with activation state-specific antibodies, co-immunoprecipitation studies, and mass spectroscopy analysis of phosphopeptides. Cell viability, caspase activation, and apoptosis were also measured. Mutations were analyzed by sequencing. The effect of silencing LYN with short interfering RNAs (siRNAs) or reducing activation by treatment with tyrosine kinase inhibitors was evaluated in cell lines and patient cells. RESULTS Imatinib treatment suppressed LYN phosphorylation in cells from imatinib-sensitive CML patients and imatinib-sensitive cell lines. Imatinib treatment blocked BCR-ABL signaling but did not suppress LYN phosphorylation in cells from imatinib-resistant patients, and persistent activation of LYN kinase was not associated with mutations in LYN kinase or its carboxyl-terminal regulatory domains. Unique LYN phosphorylation sites (tyrosine-193 and tyrosine-459) and associated proteins (c-Cbl and p80) were identified in cells from imatinib-resistant patients. Reducing LYN expression (siRNA) or activation (dasatinib) was associated with loss of cell survival and cytogenetic or complete hematologic responses in imatinib-resistant disease. CONCLUSIONS LYN activation was independent of BCR-ABL in cells from imatinib-resistant patients. Thus, LYN kinase may be involved in imatinib resistance in CML patients with mutation-negative BCR-ABL and its direct inhibition is consistent with clinical responses in these patients.

MiR-124 inhibits STAT3 signaling to enhance T cell-mediated immune clearance of glioma

miRNAs (miR) have been shown to modulate critical gene transcripts involved in tumorigenesis, but their role in tumor-mediated immunosuppression is largely unknown. On the basis of miRNA gene expression in gliomas using tissue microarrays, in situ hybridization, and molecular modeling, miR-124 was identified as a lead candidate for modulating STAT3 signaling, a key pathway mediating immunosuppression in the tumor microenvironment. miR-124 is absent in all grades and pathologic types of gliomas. Upon upregulating miR-124 in glioma cancer stem cells (gCSC), the STAT3 pathway was inhibited, and miR-124 reversed gCSC-mediated immunosuppression of T-cell proliferation and induction of forkhead box P3 (Foxp3)(+) regulatory T cells (Treg). Treatment of T cells from immunosuppressed glioblastoma patients with miR-124 induced marked effector response including upregulation of interleukin (IL)-2, IFN-γ, and TNF-α. Both systemic administration of miR-124 or adoptive miR-124-transfected T-cell transfers exerted potent anti-glioma therapeutic effects in clonotypic and genetically engineered murine models of glioblastoma and enhanced effector responses in the local tumor microenvironment. These therapeutic effects were ablated in both CD4(+)- and CD8(+)-depleted mice and nude mouse systems, indicating that the therapeutic effect of miR-124 depends on the presence of a T-cell-mediated antitumor immune response. Our findings highlight the potential application of miR-124 as a novel immunotherapeutic agent for neoplasms and serve as a model for identifying miRNAs that can be exploited as immunotherapeutics.

Glioma-Associated Cytomegalovirus Mediates Subversion of the Monocyte Lineage to a Tumor Propagating Phenotype

Purpose: Cytomegalovirus (CMV) has been ubiquitously detected within high-grade gliomas, but its role in gliomagenesis has not been fully elicited. Experimental Design: Glioblastoma multiforme (GBM) tumors were analyzed by flow cytometry to determine CMV antigen expression within various glioma-associated immune populations. The glioma cancer stem cell (gCSC) CMV interleukin (IL)-10 production was determined by ELISA. Human monocytes were stimulated with recombinant CMV IL-10 and levels of expression of p-STAT3, VEGF (vascular endothelial growth factor), TGF-β, viral IE1, and pp65 were determined by flow cytometry. The influence of CMV IL-10–treated monocytes on gCSC biology was ascertained by functional assays. Results: CMV showed a tropism for macrophages (MΦ)/microglia and CD133+ gCSCs within GBMs. The gCSCs produce CMV IL-10, which induces human monocytes (the precursor to the central nervous system MΦs/microglia) to assume an M2 immunosuppressive phenotype (as manifested by downmodulation of the major histocompatibility complex and costimulatory molecules) while upregulating immunoinhibitory B7-H1. CMV IL-10 also induces expression of viral IE1, a modulator of viral replication and transcription in the monocytes. Finally, the CMV IL-10–treated monocytes produced angiogenic VEGF, immunosuppressive TGF-β, and enhanced migration of gCSCs. Conclusions: CMV triggers a feedforward mechanism of gliomagenesis by inducing tumor-supportive monocytes. Clin Cancer Res; 17(14); 4642–9. ©2011 AACR.

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

Purpose: Activation of signal transducers and activators of transcription 3 (STAT3) has been identified as a central mediator of melanoma growth and metastasis. We hypothesized that WP1066, a novel STAT3 blockade agent, has marked antitumor activity, even against the melanoma metastasis to brain, a site typically refractory to therapies. Experimental Design: The antitumor activities and related mechanisms of WP1066 were investigated both in vitro on melanoma cell lines and in vivo on mice with subcutaneously syngeneic melanoma or with intracerebral melanoma tumors. Results: WP1066 achieved an IC50 of 1.6, 2.3, and 1.5 μmol/L against melanoma cell line A375, B16, and B16EGFRvIII, respectively. WP1066 suppressed the phosphorylation of Janus-activated kinase 2 and STAT3 (Tyr705) in these cells. Tumor growth in mice with subcutaneously established syngeneic melanoma was markedly inhibited by WP1066 compared with that in controls. Long-term survival (>78 days) was observed in 80% of mice with established intracerebral syngeneic melanoma treated with 40 mg/kg of WP1066 in contrast to control mice who survived for a median of 15 days. Although WP1066 did not induce immunologic memory or enhance humoral responses to EGFRvIII, this compound reduced the production of immunosuppressive cytokines and chemokines (transforming growth factor-β, RANTES, MCP-1, vascular endothelial growth factor), markedly inhibited natural and inducible Treg proliferation, and significantly increased cytotoxic immune responses of T cells. Conclusions: The antitumor cytotoxic effects of WP1066 and its ability to induce antitumor immune responses suggest that this compound has potential for the effective treatment of melanoma metastatic to brain.

effect of mir-142-3p on the M2 Macrophage and therapeutic efficacy Against Murine Glioblastoma

BACKGROUND The immune therapeutic potential of microRNAs (miRNAs) in the context of tumor-mediated immune suppression has not been previously described for monocyte-derived glioma-associated macrophages, which are the largest infiltrating immune cell population in glioblastomas and facilitate gliomagenesis. METHODS An miRNA microarray was used to compare expression profiles between human glioblastoma-infiltrating macrophages and matched peripheral monocytes. The effects of miR-142-3p on phenotype and function of proinflammatory M1 and immunosuppressive M2 macrophages were determined. The therapeutic effect of miR-142-3p was ascertained in immune-competent C57BL/6J mice harboring intracerebral GL261 gliomas and in genetically engineered Ntv-a mice bearing high-grade gliomas. Student t test was used to evaluate the differences between ex vivo datasets. Survival was analyzed with the log-rank test and tumor sizes with linear mixed models and F test. All statistical tests were two-sided. RESULTS miR-142-3p was the most downregulated miRNA (approximately 4.95-fold) in glioblastoma-infiltrating macrophages. M2 macrophages had lower miR-142-3p expression relative to M1 macrophages (P = .03). Overexpression of miR-142-3p in M2 macrophages induced selective modulation of transforming growth factor beta receptor 1, which led to subsequent preferential apoptosis in the M2 subset (P = .01). In vivo miR-142-3p administration resulted in glioma growth inhibition (P = .03, n = 5) and extended median survival (miR-142-3p-treated C57BL/6J mice vs scramble control: 31 days vs 23.5 days, P = .03, n = 10; miR-142-3p treated Ntv-a mice vs scramble control: 32 days vs 24 days, P = .03, n = 9), with an associated decrease in infiltrating macrophages (R (2) = .303). CONCLUSIONS These data indicate a unique role of miR-142-3p in glioma immunity by modulating M2 macrophages through the transforming growth factor beta signaling pathway.

Inhibition of p-STAT3 Enhances IFN-α Efficacy against Metastatic Melanoma in a Murine Model

Purpose: Melanoma is a common and deadly tumor that upon metastasis to the central nervous system has a median survival duration of <6 months. Activation of the signal transducer and activator of transcription 3 (STAT3) has been identified as a key mediator that drives the fundamental components of melanoma malignancy, including immune suppression in melanoma patients. We hypothesized that WP1193, a novel inhibitor of STAT3 signaling, would enhance the antitumor activity of IFN-α against metastatic melanoma. Experimental Design: Combinational therapy of STAT3 blockade agents with IFN-α was investigated in a metastatic and an established syngeneic intracerebral murine tumor model of melanoma. The immunologic in vivo mechanisms of efficacy were investigated by T-cell and natural killer (NK) cell cytotoxic assays. Results: IFN-α immunotherapy was synergistic with WP1193 showing marked in vivo efficacy against metastatic and established intracerebral melanoma. At autopsy, it was noted that there was a decreased trend in mice with melanoma developing leptomeningeal disease treated with combinational therapy. The combinational approach enhanced both NK-mediated and T-cell–mediated antitumor cytotoxicity. Conclusions: The immune modulatory effects of STAT3 blockade can enhance the therapeutic efficacy of IFN-α immunotherapy by enhancing both innate and adaptive cytotoxic T-cell activities. This combination therapy has the potential in the treatment of metastatic melanoma that is typically refractory to this type of immune therapeutic approach. Clin Cancer Res; 16(9); 2550–61. ©2010 AACR.