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BACKGROUND: High-grade gliomas have a poor prognosis and do not respond well to treatment. Effective cancer immune responses depend on functional immune cells, which are typically absent from the brain. This study aimed to evaluate the safety and activity of two adenoviral vectors expressing HSV1-TK (Ad-hCMV-TK) and Flt3L (Ad-hCMV-Flt3L) in patients with high-grade glioma. METHODS: In this dose-finding, first-in-human trial, treatment-naive adults aged 18-75 years with newly identified high-grade glioma that was evaluated per immunotherapy response assessment in neuro-oncology criteria, and a Karnofsky Performance Status score of 70 or more, underwent maximal safe resection followed by injections of adenoviral vectors expressing HSV1-TK and Flt3L into the tumour bed. The study was conducted at the University of Michigan Medical School, Michigan Medicine (Ann Arbor, MI, USA). The study included six escalating doses of viral particles with starting doses of 1×1010 Ad-hCMV-TK viral particles and 1×109 Ad-hCMV-Flt3L viral particles (cohort A), and then 1×1011 Ad-hCMV-TK viral particles and 1×109 Ad-hCMV-Flt3L viral particles (cohort B), 1×1010 Ad-hCMV-TK viral particles and 1×1010 Ad-hCMV-Flt3L viral particles (cohort C), 1×1011 Ad-hCMV-TK viral particles and 1×1010 Ad-hCMV-Flt3L viral particles (cohort D), 1×1010 Ad-hCMV-TK viral particles and 1×1011 Ad-hCMV-Flt3L viral particles (cohort E), and 1×1011 Ad-hCMV-TK viral particles and 1×1011 Ad-hCMV-Flt3L viral particles (cohort F) following a 3+3 design. Two 1 mL tuberculin syringes were used to deliver freehand a mix of Ad-hCMV-TK and Ad-hCMV-Flt3L vectors into the walls of the resection cavity with a total injection of 2 mL distributed as 0·1 mL per site across 20 locations. Subsequently, patients received two 14-day courses of valacyclovir (2 g orally, three times per day) at 1-3 days and 10-12 weeks after vector administration and standad upfront chemoradiotherapy. The primary endpoint was the maximum tolerated dose of Ad-hCMV-Flt3L and Ad-hCMV-TK. Overall survival was a secondary endpoint. Recruitment is complete and the trial is finished. The trial is registered with ClinicalTrials.gov, NCT01811992. FINDINGS: Between April 8, 2014, and March 13, 2019, 21 patients were assessed for eligibility and 18 patients with high-grade glioma were enrolled and included in the analysis (three patients in each of the six dose cohorts); eight patients were female and ten were male. Neuropathological examination identified 14 (78%) patients with glioblastoma, three (17%) with gliosarcoma, and one (6%) with anaplastic ependymoma. The treatment was well-tolerated, and no dose-limiting toxicity was observed. The maximum tolerated dose was not reached. The most common serious grade 3-4 adverse events across all treatment groups were wound infection (four events in two patients) and thromboembolic events (five events in four patients). One death due to an adverse event (respiratory failure) occurred but was not related to study treatment. No treatment-related deaths occurred during the study. Median overall survival was 21·3 months (95% CI 11·1-26·1). INTERPRETATION: The combination of two adenoviral vectors demonstrated safety and feasibility in patients with high-grade glioma and warrants further investigation in a phase 1b/2 clinical trial. FUNDING: Funded in part by Phase One Foundation, Los Angeles, CA, The Board of Governors at Cedars-Sinai Medical Center, Los Angeles, CA, and The Rogel Cancer Center at The University of Michigan.
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Antineoplásicos , Glioblastoma , Glioma , Adulto , Feminino , Humanos , Masculino , Quimiorradioterapia , Terapia Genética , Glioblastoma/genética , Glioblastoma/terapia , Glioma/genética , Glioma/terapia , Adolescente , Pessoa de Meia-Idade , IdosoRESUMO
Glioblastoma is the most deadly and common primary tumor of the central nervous system. Heterogeneity in the disease causes complications from diagnosis to treatment. It has long been suggested that a stem cell and/or progenitor population may be the origin of this disease and provide the underlying heterogeneity. However, which population precisely is the cell of origin, or whether there is only one cell of origin, has remained elusive. Previous studies have shown that, with proper combinations of oncogene expression and tumor suppressor loss, three cell types have the potential to transform into glioma-neural stem cells (NSC), oligodendrocyte precursor cells (OPC), and astrocytes. In a newly published article in Cancer Research, Verma and colleagues make a convincing argument through elegant animal work that an intermediate progenitor cell population, primitive OPCs, has higher tumorigenic potential than the NSCs or OPCs. This study helps rectify the controversy around which cell populations are the most sensitive to transformation in gliomagenesis. See related article by Verma et al., p. 890.
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Células-Tronco Neurais , Células Precursoras de Oligodendrócitos , Animais , Células Precursoras de Oligodendrócitos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Diferenciação Celular/fisiologia , Oligodendroglia , Células-Tronco Neurais/metabolismoRESUMO
Immune checkpoint blockade (ICB) has revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by immune-related adverse events (irAEs). Limited understanding of irAE mechanisms hampers development of approaches to mitigate their damage. To address this, we examined whether mice gained sensitivity to anti-CTLA-4 (αCTLA-4)-mediated toxicity upon disruption of gut homeostatic immunity. We found αCTLA-4 drove increased inflammation and colonic tissue damage in mice with genetic predisposition to intestinal inflammation, acute gastrointestinal infection, transplantation with a dysbiotic fecal microbiome, or dextran sodium sulfate administration. We identified an immune signature of αCTLA-4-mediated irAEs, including colonic neutrophil accumulation and systemic interleukin-6 (IL-6) release. IL-6 blockade combined with antibiotic treatment reduced intestinal damage and improved αCTLA-4 therapeutic efficacy in inflammation-prone mice. Intestinal immune signatures were validated in biopsies from patients with ICB colitis. Our work provides new preclinical models of αCTLA-4 intestinal irAEs, mechanistic insights into irAE development, and potential approaches to enhance ICB efficacy while mitigating irAEs.
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Colite , Interleucina-6 , Camundongos , Animais , Qualidade de Vida , Colite/patologia , Imunoterapia , InflamaçãoRESUMO
Glioblastoma (GBM) is a high-grade, aggressive brain tumor with dismal median survival time of 15 months. Chromosome 6q (Ch6q) is a hotspot of genomic alterations, which is commonly deleted or hyper-methylated in GBM. Two neighboring genes in this region, QKI and PRKN have been appointed as tumor suppressors in GBM. While a genetically modified mouse model (GEMM) of GBM has been successfully generated with Qk deletion in the central nervous system (CNS), in vivo genetic evidence supporting the tumor suppressor function of Prkn has not been established. In the present study, we generated a mouse model with Prkn-null allele and conditional Trp53 and Pten deletions in the neural stem cells (NSCs) and compared the tumorigenicity of this model to our previous GBM model with Qk deletion within the same system. We find that Qk but not Prkn is the potent tumor suppressor in the frequently altered Ch6q region in GBM.
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Novel therapeutic strategies targeting glioblastoma (GBM) often fail in the clinic, partly because preclinical models in which hypotheses are being tested do not recapitulate human disease. To address this challenge, we took advantage of our previously developed spontaneous Qk/Trp53/Pten (QPP) triple-knockout model of human GBM, comparing the immune microenvironment of QPP mice with that of patient-derived tumors to determine whether this model provides opportunity for gaining insights into tumor physiopathology and preclinical evaluation of therapeutic agents. Immune profiling analyses and single-cell sequencing of implanted and spontaneous tumors from QPP mice and from patients with glioma revealed intratumoral immune components that were predominantly myeloid cells (e.g., monocytes, macrophages, and microglia), with minor populations of T, B, and NK cells. When comparing spontaneous and implanted mouse samples, we found more neutrophils and T and NK cells in the implanted model. Neutrophils and T and NK cells were increased in abundance in samples derived from human high-grade glioma compared with those derived from low-grade glioma. Overall, our data demonstrate that our implanted and spontaneous QPP models recapitulate the immunosuppressive myeloid-dominant nature of the tumor microenvironment of human gliomas. Our model provides a suitable tool for investigating the complex immune compartment of gliomas.
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Glioblastoma , Glioma , Animais , Modelos Animais de Doenças , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Macrófagos , Camundongos , Microambiente TumoralRESUMO
Intra-tumoral heterogeneity is a hallmark of glioblastoma that challenges treatment efficacy. However, the mechanisms that set up tumor heterogeneity and tumor cell migration remain poorly understood. Herein, we present a comprehensive spatiotemporal study that aligns distinctive intra-tumoral histopathological structures, oncostreams, with dynamic properties and a specific, actionable, spatial transcriptomic signature. Oncostreams are dynamic multicellular fascicles of spindle-like and aligned cells with mesenchymal properties, detected using ex vivo explants and in vivo intravital imaging. Their density correlates with tumor aggressiveness in genetically engineered mouse glioma models, and high grade human gliomas. Oncostreams facilitate the intra-tumoral distribution of tumoral and non-tumoral cells, and potentially the collective invasion of the normal brain. These fascicles are defined by a specific molecular signature that regulates their organization and function. Oncostreams structure and function depend on overexpression of COL1A1. Col1a1 is a central gene in the dynamic organization of glioma mesenchymal transformation, and a powerful regulator of glioma malignant behavior. Inhibition of Col1a1 eliminates oncostreams, reprograms the malignant histopathological phenotype, reduces expression of the mesenchymal associated genes, induces changes in the tumor microenvironment and prolongs animal survival. Oncostreams represent a pathological marker of potential value for diagnosis, prognosis, and treatment.
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Neoplasias Encefálicas , Glioblastoma , Glioma , Animais , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Glioma/patologia , Camundongos , Análise Espaço-Temporal , Microambiente Tumoral/genéticaRESUMO
Although pharmacological stimulation of TLRs has anti-tumor effects, it has not been determined whether endogenous stimulation of TLRs can lead to tumor rejection. Herein, we demonstrate the existence of an innate anti-glioma NK-mediated circuit initiated by glioma-released miR-1983 within exosomes, and which is under the regulation of galectin-1 (Gal-1). We demonstrate that miR-1983 is an endogenous TLR7 ligand that activates TLR7 in pDCs and cDCs through a 5'-UGUUU-3' motif at its 3' end. TLR7 activation and downstream signaling through MyD88-IRF5/IRF7 stimulates secretion of IFN-ß. IFN-ß then stimulates NK cells resulting in the eradication of gliomas. We propose that successful immunotherapy for glioma could exploit this endogenous innate immune circuit to activate TLR7 signaling and stimulate powerful anti-glioma NK activity, at least 10-14 days before the activation of anti-tumor adaptive immunity.
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Galectina 1 , Glioma , Receptor 7 Toll-Like , Galectina 1/genética , Glioma/genética , Humanos , Fatores Reguladores de Interferon , Interferon beta , Células Matadoras Naturais/metabolismo , Licenciamento , MicroRNAs , Receptor 7 Toll-Like/genéticaRESUMO
Glioblastoma multiforme (GBM), the most aggressive brain cancer, recurs because glioblastoma stem cells (GSCs) are resistant to all standard therapies. We showed that GSCs, but not normal astrocytes, are sensitive to lysis by healthy allogeneic natural killer (NK) cells in vitro. Mass cytometry and single-cell RNA sequencing of primary tumor samples revealed that GBM tumor-infiltrating NK cells acquired an altered phenotype associated with impaired lytic function relative to matched peripheral blood NK cells from patients with GBM or healthy donors. We attributed this immune evasion tactic to direct cell-to-cell contact between GSCs and NK cells via αv integrin-mediated TGF-ß activation. Treatment of GSC-engrafted mice with allogeneic NK cells in combination with inhibitors of integrin or TGF-ß signaling or with TGFBR2 gene-edited allogeneic NK cells prevented GSC-induced NK cell dysfunction and tumor growth. These findings reveal an important mechanism of NK cell immune evasion by GSCs and suggest the αv integrin/TGF-ß axis as a potentially useful therapeutic target in GBM.
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Glioblastoma/imunologia , Integrinas/imunologia , Células Matadoras Naturais/imunologia , Proteínas de Neoplasias/imunologia , Células-Tronco Neoplásicas/imunologia , Fator de Crescimento Transformador beta/imunologia , Animais , Feminino , Glioblastoma/genética , Glioblastoma/patologia , Glioblastoma/terapia , Xenoenxertos , Humanos , Integrinas/genética , Células Matadoras Naturais/patologia , Masculino , Camundongos , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Células-Tronco Neoplásicas/patologia , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/imunologia , Fator de Crescimento Transformador beta/genéticaRESUMO
PURPOSE: Patients with central nervous system (CNS) tumors are typically treated with radiotherapy, but this is not curative and results in the upregulation of phosphorylated STAT3 (p-STAT3), which drives invasion, angiogenesis, and immune suppression. Therefore, we investigated the combined effect of an inhibitor of STAT3 and whole-brain radiotherapy (WBRT) in a murine model of glioma. EXPERIMENTAL DESIGN: C57BL/6 mice underwent intracerebral implantation of GL261 glioma cells, WBRT, and treatment with WP1066, a blood-brain barrier-penetrant inhibitor of the STAT3 pathway, or the two in combination. The role of the immune system was evaluated using tumor rechallenge strategies, immune-incompetent backgrounds, immunofluorescence, immune phenotyping of tumor-infiltrating immune cells (via flow cytometry), and NanoString gene expression analysis of 770 immune-related genes from immune cells, including those directly isolated from the tumor microenvironment. RESULTS: The combination of WP1066 and WBRT resulted in long-term survivors and enhanced median survival time relative to monotherapy in the GL261 glioma model (combination vs. control P < 0.0001). Immunologic memory appeared to be induced, because mice were protected during subsequent tumor rechallenge. The therapeutic effect of the combination was completely lost in immune-incompetent animals. NanoString analysis and immunofluorescence revealed immunologic reprograming in the CNS tumor microenvironment specifically affecting dendritic cell antigen presentation and T-cell effector functions. CONCLUSIONS: This study indicates that the combination of STAT3 inhibition and WBRT enhances the therapeutic effect against gliomas in the CNS by inducing dendritic cell and T-cell interactions in the CNS tumor.
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Neoplasias Encefálicas/terapia , Comunicação Celular/imunologia , Quimiorradioterapia/métodos , Glioma/terapia , Fator de Transcrição STAT3/antagonistas & inibidores , Animais , Apresentação de Antígeno/efeitos dos fármacos , Apresentação de Antígeno/efeitos da radiação , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/efeitos da radiação , Linhagem Celular Tumoral/ultraestrutura , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/efeitos da radiação , Modelos Animais de Doenças , Glioma/imunologia , Glioma/patologia , Humanos , Memória Imunológica/efeitos dos fármacos , Camundongos , Piridinas/administração & dosagem , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/efeitos da radiação , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Microambiente Tumoral/efeitos da radiação , Tirfostinas/administração & dosagemRESUMO
PURPOSE: Anti-programmed cell death protein 1 (PD-1) therapy has demonstrated inconsistent therapeutic results in patients with glioblastoma (GBM) including those with profound impairments in CD8 T-cell effector responses. EXPERIMENTAL DESIGN: We ablated the CD8α gene in BL6 mice and intercrossed them with Ntv-a mice to determine how CD8 T cells affect malignant progression in forming endogenous gliomas. Tumor-bearing mice were treated with PD-1 to determine the efficacy of this treatment in the absence of T cells. The tumor microenvironment of treated and control mice was analyzed by IHC and FACS. RESULTS: We observed a survival benefit in immunocompetent mice with endogenously arising intracranial glioblastomas after intravenous administration of anti-PD-1. The therapeutic effect of PD-1 administration persisted in mice even after genetic ablation of the CD8 gene (CD8-/-). CD11b+ and Iba1+ monocytes and macrophages were enriched in the glioma microenvironment of the CD8-/- mice. The macrophages and microglia assumed a proinflammatory M1 response signature in the setting of anti-PD-1 blockade through the elimination of PD-1-expressing macrophages and microglia in the tumor microenvironment. Anti-PD-1 can inhibit the proliferation of and induce apoptosis of microglia through antibody-dependent cellular cytotoxicity, as fluorescently labeled anti-PD-1 was shown to gain direct access to the glioma microenvironment. CONCLUSIONS: Our results show that the therapeutic effect of anti-PD-1 blockade in GBM may be mediated by the innate immune system, rather than by CD8 T cells. Anti-PD-1 immunologically modulates innate immunity in the glioma microenvironment-likely a key mode of activity.
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Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Inibidores de Checkpoint Imunológico/farmacologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Macrófagos Associados a Tumor/efeitos dos fármacos , Animais , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Glioblastoma/imunologia , Glioblastoma/patologia , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunidade Inata/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Macrófagos Associados a Tumor/imunologiaRESUMO
BACKGROUND: High-grade gliomas are aggressive and immunosuppressive brain tumors. Molecular mechanisms that regulate the inhibitory immune tumor microenvironment (TME) and glioma progression remain poorly understood. Fyn tyrosine kinase is a downstream target of the oncogenic receptor tyrosine kinase pathway and is overexpressed in human gliomas. Fyn's role in vivo in glioma growth remains unknown. We investigated whether Fyn regulates glioma initiation, growth and invasion. METHODS: We evaluated the role of Fyn using genetically engineered mouse glioma models (GEMMs). We also generated Fyn knockdown stem cells to induce gliomas in immune-competent and immune-deficient mice (nonobese diabetic severe combined immunodeficient gamma mice [NSG], CD8-/-, CD4-/-). We analyzed molecular mechanism by RNA sequencing and bioinformatics analysis. Flow cytometry was used to characterize immune cellular infiltrates in the Fyn knockdown glioma TME. RESULTS: We demonstrate that Fyn knockdown in diverse immune-competent GEMMs of glioma reduced tumor progression and significantly increased survival. Gene ontology (GO) analysis of differentially expressed genes in wild-type versus Fyn knockdown gliomas showed enrichment of GOs related to immune reactivity. However, in NSG and CD8-/- and CD4-/- immune-deficient mice, Fyn knockdown gliomas failed to show differences in survival. These data suggest that the expression of Fyn in glioma cells reduces antiglioma immune activation. Examination of glioma immune infiltrates by flow cytometry displayed reduction in the amount and activity of immune suppressive myeloid derived cells in the Fyn glioma TME. CONCLUSIONS: Gliomas employ Fyn mediated mechanisms to enhance immune suppression and promote tumor progression. We propose that Fyn inhibition within glioma cells could improve the efficacy of antiglioma immunotherapies.
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Neoplasias Encefálicas , Glioma , Animais , Neoplasias Encefálicas/genética , Glioma/genética , Imunidade , Camundongos , Proteínas Proto-Oncogênicas c-fyn/genética , Receptores Proteína Tirosina Quinases , Microambiente TumoralRESUMO
CNS immune defenses are marshaled and dominated by brain resident macrophages and microglia, which are the innate immune sentinels and frontline host immune barriers against various pathogenic insults. These myeloid lineage cells are the predominant immune population in gliomas and can constitute up to 30-50% of the total cellular composition. Parenchymal microglial cells and recruited monocyte-derived macrophages from the periphery exhibit disease-specific phenotypic characteristics with spatial and temporal distinctions and are heterogeneous subpopulations based on their molecular signatures. A preponderance of myeloid over lymphoid lineage cells during CNS inflammation, including gliomas, is a contrasting feature of brain immunity relative to peripheral immunity. Herein we discuss glioma-associated macrophage and microglia immune biology in the context of their identity, molecular drivers of recruitment, nomenclature and functional paradoxes, therapeutic reprogramming and polarization strategies, relevant challenges, and our perspectives on therapeutic modulation.
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Neoplasias Encefálicas/imunologia , Glioma/imunologia , Macrófagos/imunologia , Microglia/imunologia , Animais , HumanosRESUMO
Craniopharyngioma is a rare tumor in adults. Although histologically benign, it can be locally aggressive and may require additional therapeutic modalities to surgical resection. Analyses including next generation sequencing, chromogenic and in situ hybridization, immunohistochemistry, and gene amplification were used to profile craniopharyngiomas (n=6) for frequently altered therapeutic targets. Four of six patients had the BRAF V600E missense mutation, frequent in the papillary craniopharyngioma subtype. One patient had a missense mutation in the WNT pathway, specifically CTNNB1, often associated with the adamantinomatous subtype. Craniopharyngiomas lacked microsatellite instability, had low tumor mutational burden, but did express PD-L1 protein, indicating potential therapeutic value for immune checkpoint inhibition. We identified mutations not previously described, including an E318K missense mutation in the MITF gene, an R1407 frameshift in the SETD2 gene of the PIK3CA pathway, R462H in the NF2 gene, and a I463V mutation in TSC2. Two patients testing positive for EGFR expression were negative for the EGFRvIII variant. Herein, we identified several alterations such as those in BRAF V600E and PD-L1, which may be considered as targets for combination therapy of residual craniopharygiomas.
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Background: Glioblastoma (GBM) is an aggressive and highly vascular tumor with median survival below 2 years. Despite advances in surgery, radiotherapy, and chemotherapy, survival has improved modestly. To combat glioma vascular proliferation, anti-angiogenic agents targeting vascular endothelial growth factor (VEGF) were introduced. Preclinically these agents were effective, yet they did not improve overall survival in phase III trials. We tested the hypothesis that ganciclovir (GCV)-mediated killing of proliferating endothelial cells expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK) would have direct antitumor effects, and whether vessel ablation would affect the antitumor activity of anti-VEGF antibodies and radiotherapy. Methods: Proliferating endothelial cells were eliminated using GCV-mediated killing of proliferating endothelial cells expressing HSV1-TK (in Tie2-TK-IRES-GFP mice). Syngeneic NRAS/p53 (NP) gliomas were implanted into the brains of Tie2-TK-IRES-GFP mice. Endothelial proliferation activates the Tie2 promoter and HSV1-TK expression. Administration of GCV kills proliferating tumor endothelial cells and slows tumor growth. The effects of endothelial cell ablation on anti-angiogenic therapy were examined using anti-VEGF antibodies or irradiation. Results: GCV administration reduced tumor growth and vascular density, increased tumor apoptosis, and prolonged survival. Anti-VEGF antibodies or irradiation also prolonged survival. Surprisingly, combining GCV with irradiation, or with anti-VEGF antibodies, reduced their individual therapeutic effects. Conclusion: GCV-mediated killing of proliferating endothelial cells expressing HSV1-TK, anti-VEGF antibodies, or irradiation all reduced growth of a murine glioma. However, elimination of microvascular proliferation decreased the efficacy of anti-VEGF or irradiation therapy. We conclude that, in our model, the integrity of proliferating vessels is necessary for the antiglioma effects of anti-VEGF and radiation therapy.
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Bevacizumab/farmacologia , Células Endoteliais/metabolismo , Ganciclovir/farmacologia , Glioma/prevenção & controle , Radiação Ionizante , Simplexvirus/genética , Timidina Quinase/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Antivirais/farmacologia , Terapia Combinada , Glioma/irrigação sanguínea , Glioma/patologia , Humanos , Camundongos , Camundongos Transgênicos , Taxa de Sobrevida , Timidina Quinase/genética , Células Tumorais CultivadasRESUMO
Purpose: One likely cause of treatment failure in glioblastoma is the persistence of glioma stem-like cells (GSLCs) which are highly resistant to therapies currently employed. We found that CXCL12 has highest expression in glioma cells derived from neural progenitor cells (NPC). The development and molecular signature of NPC-derived glioblastomas were analyzed and the therapeutic effect of blocking CXCL12 was tested.Experimental Design: Tumors were induced by injecting DNA into the lateral ventricle of neonatal mice, using the Sleeping Beauty transposase method. Histology and expression of GSLC markers were analyzed during disease progression. Survival upon treatment with pharmacologic (plerixafor) or genetic inhibition of CXCR4 was analyzed. Primary neurospheres were generated and analyzed for proliferation, apoptosis, and expression of proteins regulating survival and cell-cycle progression.Results: Tumors induced from NPCs display histologic features of human glioblastoma and express markers of GSLC. In vivo, inhibiting the CXCL12/CXCR4 signaling axis results in increased survival of tumor-bearing animals. In vitro, CXCR4 blockade induces apoptosis and inhibits cell-cycle progression, downregulates molecules regulating survival and proliferation, and also blocks the hypoxic induction of HIF-1α and CXCL12. Exogenous administration of CXCL12 rescues the drug-induced decrease in proliferation.Conclusions: This study demonstrates that the CXCL12/CXCR4 axis operates in glioblastoma cells under hypoxic stress via an autocrine-positive feedback mechanism, which promotes survival and cell-cycle progression. Our study brings new mechanistic insight and encourages further exploration of the use of drugs blocking CXCL12 as adjuvant agents to target hypoxia-induced glioblastoma progression, prevent resistance to treatment, and recurrence of the disease. Clin Cancer Res; 23(5); 1250-62. ©2016 AACR.
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Quimiocina CXCL12/genética , Glioblastoma/genética , Recidiva Local de Neoplasia/genética , Receptores CXCR4/genética , Animais , Apoptose/genética , Hipóxia Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Camundongos , Terapia de Alvo Molecular , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/patologia , Células-Tronco Neurais/patologia , Transdução de Sinais , Transposases/genéticaRESUMO
Improved molecular understanding is needed for rational treatment of diffuse intrinsic pontine gliomas (DIPG). Here, using multi-focal paired tumor and germline exome DNA and RNA sequencing, we uncovered phosphatase and tensin homolog (PTEN) loss as a clonal mutation in the case of a 6-year-old boy with a diffuse intrinsic pontine glioma, and incorporated copy number alteration analyses to provide a more detailed understanding of clonal evolution in diffuse intrinsic pontine gliomas. As well, using the PedcBioPortal, we found alterations in PTEN in 16 of 326 (4.9%) cases of pediatric high-grade glioma (3 of 154 (1.9%) brainstem) for which full sequencing data was available. Our data strengthens the association with PTEN loss in diffuse intrinsic pontine gliomas and provides further argument for the inclusion of PTEN in future targeted sequencing panels for pediatric diffuse intrinsic pontine gliomas and for the development and optimization of mTOR/PI3K inhibitors with optimal central nervous system penetration.
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Glioblastoma (GBM) is a highly invasive brain tumor. Perivascular invasion, autovascularization and vascular co-option occur throughout the disease and lead to tumor invasion and progression. The molecular basis for perivascular invasion, i.e., the interaction of glioma tumor cells with endothelial cells is not well characterized. Recent studies indicate that glioma cells have increased expression of CXCR4. We investigated the in-vivo role of CXCR4 in perivascular invasion of glioma cells using shRNA-mediated knock down of CXCR4. We show that primary cultures of human glioma stem cells HF2303 and mouse glioma GL26-Cit cells exhibit significant migration towards human (HBMVE) and mouse (MBVE) brain microvascular endothelial cells. Blocking CXCR4 on tumor cells with AMD3100 in-vitro, inhibits migration of GL26-Cit and HF2303 toward MBVE and HBMVE cells. Additionally, genetic down regulation of CXCR4 in mouse glioma GL26-Cit cells inhibits their in-vitro migration towards MBVE cells; in an in-vivo intracranial mouse model, these cells display reduced tumor growth and perivascular invasion, leading to increased survival. Quantitative analysis of brain sections showed that CXCR4 knockdown tumors are less invasive. Lastly, we tested the effects of radiation on CXCR4 knock down GL26-Cit cells in an orthotopic brain tumor model. Radiation treatment increased apoptosis of CXCR4 downregulated tumor cells and prolonged median survival. In summary, our data suggest that CXCR4 signaling is critical for perivascular invasion of GBM cells and targeting this receptor makes tumors less invasive and more sensitive to radiation therapy. Combination of CXCR4 knock down and radiation treatment might improve the efficacy of GBM therapy.
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Apoptose , Neoplasias Encefálicas/metabolismo , Movimento Celular , Técnicas de Silenciamento de Genes , Glioblastoma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Interferência de RNA , Receptores CXCR4/metabolismo , Animais , Apoptose/efeitos da radiação , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/radioterapia , Linhagem Celular Tumoral , Movimento Celular/efeitos da radiação , Proliferação de Células , Técnicas de Cocultura , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/patologia , Glioblastoma/radioterapia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Invasividade Neoplásica , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/efeitos da radiação , Tolerância a Radiação , Receptores CXCR4/genética , Transdução de Sinais , Fatores de Tempo , Transfecção , Carga Tumoral , Células Tumorais CultivadasRESUMO
Pediatric high-grade glioma (HGG, WHO Grade III and IV) is a devastating brain tumor with a median survival of less than two years. PDGFRA is frequently mutated/ amplified in pediatric HGG, but the significance of this finding has not been fully characterized. We hypothesize that alterations of PDGFRA will promote distinct prognostic and treatment implications in pediatric HGG. In order to characterize the impact of PDGFR pathway alterations, we integrated genomic data from pediatric HGG patients (n=290) from multiple pediatric datasets and sequencing platforms. Integration of multiple human datasets showed that PDGFRA mutation, but not amplification, was associated with older age in pediatric HGG (P= <0.0001). In multivariate analysis, PDGFRA mutation was correlated with worse prognosis (P = 0.026), while PDGFRA amplification was not (P = 0.11). By Kaplan-Meier analysis, non-brainstem HGG with PDGFRA amplification carried a worse prognosis than non-brainstem HGG without PDGFRA amplification (P = 0.021). There were no pediatric patients with PDGFRA-amplified HGG that survived longer than two years. Additionally, we performed paired molecular profiling (germline / tumor / primary cell culture) and targeting of an infant thalamic HGG with amplification and outlier increased expression of PDGFRA. Dasatinib inhibited proliferation most effectively. In summary, integration of the largest genomic dataset of pediatric HGG to date, allowed us to highlight that PDGFRA mutation is found in older pediatric patients and that PDGFRA amplification is prognostic in non-brainstem HGG. Future precision-medicine based clinical trials for pediatric patients with PDGFRA-altered HGG should consider the optimized delivery of dasatinib.
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Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Amplificação de Genes , Glioma/genética , Mutação , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Adolescente , Adulto , Fatores Etários , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Proliferação de Células/efeitos dos fármacos , Criança , Pré-Escolar , Feminino , Seguimentos , Glioma/tratamento farmacológico , Glioma/patologia , Humanos , Lactente , Masculino , Gradação de Tumores , Taxa de Sobrevida , Células Tumorais Cultivadas , Adulto JovemRESUMO
Glioma cells grow in two phenotypic forms, as adherent monolayers and as free floating "neurospheres/tumorspheres", using specific media supplements. Whether each phenotype is irreversible remains unknown. Herein we show that both states are reversible using patient derived glioblastoma cell cultures (i.e., HF2303, IN859, MGG8, IN2045). Both phenotypic states differ in proliferation rate, invasion, migration, chemotaxis and chemosensitivity. We used microarrays to characterize gene expression across the patient derived glioblastoma cell cultures, to find specific inhibitors of the sphere population. Traditional chemotherapeutics (i.e., doxorubicin or paclitaxel) inhibit rapidly dividing adherent cells; it has been more challenging to inhibit the growth of the sphere phenotype. PRKG1, known to induce apoptosis when activated, is increased in all patient derived glioblastoma spheres. Stimulation of PRKG1 activity preferentially reduced cell viability in the sphere phenotype. Computational network and gene ontology analysis identified novel potential target genes linked to the PRKG1 expression node.