RESUMEN
A network of communicating tumour cells that is connected by tumour microtubes mediates the progression of incurable gliomas. Moreover, neuronal activity can foster malignant behaviour of glioma cells by non-synaptic paracrine and autocrine mechanisms. Here we report a direct communication channel between neurons and glioma cells in different disease models and human tumours: functional bona fide chemical synapses between presynaptic neurons and postsynaptic glioma cells. These neurogliomal synapses show a typical synaptic ultrastructure, are located on tumour microtubes, and produce postsynaptic currents that are mediated by glutamate receptors of the AMPA subtype. Neuronal activity including epileptic conditions generates synchronised calcium transients in tumour-microtube-connected glioma networks. Glioma-cell-specific genetic perturbation of AMPA receptors reduces calcium-related invasiveness of tumour-microtube-positive tumour cells and glioma growth. Invasion and growth are also reduced by anaesthesia and the AMPA receptor antagonist perampanel, respectively. These findings reveal a biologically relevant direct synaptic communication between neurons and glioma cells with potential clinical implications.
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Neoplasias Encefálicas/fisiopatología , Progresión de la Enfermedad , Glioma/fisiopatología , Sinapsis/patología , Animales , Neoplasias Encefálicas/ultraestructura , Modelos Animales de Enfermedad , Glioma/ultraestructura , Humanos , Ratones , Microscopía Electrónica de Transmisión , Neuronas/fisiología , Receptores AMPA/genética , Receptores AMPA/metabolismoRESUMEN
Glioblastoma is the most common form of primary brain cancer in adults, and the disease has a serious prognosis. Although great progress has been made in molecular characteristics, no major breakthroughs in treatment have been achieved for many years. In this article we present a clinical review of current diagnostics and treatment, as well as the challenges and opportunities inherent in developing improved and more personalised treatment.
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Neoplasias Encefálicas , Glioblastoma , Humanos , Adulto , Glioblastoma/diagnóstico , Glioblastoma/terapia , Pronóstico , Neoplasias Encefálicas/diagnóstico por imagen , Neoplasias Encefálicas/terapiaRESUMEN
INTRODUCTION: Malignant peripheral nerve sheath tumor of the vestibulocochlear nerve (VN-MPNST) is exceedingly rare and carries a poor prognosis. Little is known about its underlying genetics and in particular the process of malignant transformation. There is an ongoing debate on whether the transformation is initiated by ionizing radiation. We present here the analysis and comparison of two post-radiation VN-MPNST and one undergoing spontaneous transformation. METHODS: Four tumors from three patients (radiation-naïve vestibular schwannoma before (VS) and after (VN-MPNST) malignant transformation in addition to two post-radiation VN-MPNST) were subjected to DNA whole-genome microarray and whole-exome sequencing and tumor-specific mutations were called. Mutational signatures were characterized using MuSiCa. RESULTS: The tumor genomes were characterized predominantly by copy-number aberrations with 36-81% of the genome affected. Even the VS genome was grossly aberrated. The spontaneous malignant transformation was characterized by a near-total whole-genome doubling, disappearance of NF2 mutation and new mutations in three cancer-related genes (GNAQ, FOXO4 and PDGFRB). All tumors had homozygous loss of the tumor suppressor CDKN2A. Neither mutational signature nor copy number profile was associated with ionizing radiation. CONCLUSION: The VN-MPNST genome in our cases is characterized by large copy-number aberrations and homozygous deletion of CDKN2A. Our study demonstrates a VS with genetic alterations similar to its malignant counterpart, suggesting the existence of premalignant VS. No consistent mutational signature was associated with ionizing radiation.
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Neoplasias de la Vaina del Nervio , Neuroma Acústico , Homocigoto , Humanos , Mutación/genética , Neuroma Acústico/genética , Neuroma Acústico/patología , Eliminación de Secuencia , Nervio VestibulococlearRESUMEN
An ageing population as well as improved diagnostics, monitoring and treatment mean that an increasing incidence of brain metastases can be expected. Patients with brain metastases were previously regarded as a homogenous group with a very poor prognosis. However, the current picture is more complex. The development of new treatment methods, better molecular understanding and personalised medicine require a focus on multidisciplinary collaboration to provide optimal treatment for individual patients. This clinical review article provides an overview of important factors related to the diagnosis and treatment of patients with brain metastases.
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Neoplasias Encefálicas , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/terapia , Humanos , PronósticoRESUMEN
Long non-coding RNAs play critical roles in tumour progression. Through analysis of publicly available genomic datasets, we found that MIR22HG, the host gene of microRNAs miR-22-3p and miR-22-5p, is ranked among the most dysregulated long non-coding RNAs in glioblastoma. The main purpose of this work was to determine the impact of MIR22HG on glioblastoma growth and invasion and to elucidate its mechanistic function. The MIR22HG/miR-22 axis was highly expressed in glioblastoma as well as in glioma stem-like cells compared to normal neural stem cells. In glioblastoma, increased expression of MIR22HG is associated with poor prognosis. Through a number of functional studies, we show that MIR22HG silencing inhibits the Wnt/ß-catenin signalling pathway through loss of miR-22-3p and -5p. This leads to attenuated cell proliferation, invasion and in vivo tumour growth. We further show that two genes, SFRP2 and PCDH15, are direct targets of miR-22-3p and -5p and inhibit Wnt signalling in glioblastoma. Finally, based on the 3D structure of the pre-miR-22, we identified a specific small-molecule inhibitor, AC1L6JTK, that inhibits the enzyme Dicer to block processing of pre-miR-22 into mature miR-22. AC1L6JTK treatment caused an inhibition of tumour growth in vivo. Our findings show that MIR22HG is a critical inducer of the Wnt/ß-catenin signalling pathway, and that its targeting may represent a novel therapeutic strategy in glioblastoma patients.
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Glioblastoma/genética , MicroARNs/genética , Vía de Señalización Wnt/genética , beta Catenina/genética , Animales , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Glioma/genética , Masculino , Ratones Desnudos , ARN Largo no Codificante/genéticaRESUMEN
Astrocytic brain tumours, including glioblastomas, are incurable neoplasms characterized by diffusely infiltrative growth. Here we show that many tumour cells in astrocytomas extend ultra-long membrane protrusions, and use these distinct tumour microtubes as routes for brain invasion, proliferation, and to interconnect over long distances. The resulting network allows multicellular communication through microtube-associated gap junctions. When damage to the network occurred, tumour microtubes were used for repair. Moreover, the microtube-connected astrocytoma cells, but not those remaining unconnected throughout tumour progression, were protected from cell death inflicted by radiotherapy. The neuronal growth-associated protein 43 was important for microtube formation and function, and drove microtube-dependent tumour cell invasion, proliferation, interconnection, and radioresistance. Oligodendroglial brain tumours were deficient in this mechanism. In summary, astrocytomas can develop functional multicellular network structures. Disconnection of astrocytoma cells by targeting their tumour microtubes emerges as a new principle to reduce the treatment resistance of this disease.
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Astrocitoma/patología , Neoplasias Encefálicas/patología , Uniones Comunicantes/metabolismo , Animales , Astrocitoma/metabolismo , Astrocitoma/radioterapia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/radioterapia , Comunicación Celular/efectos de la radiación , Muerte Celular/efectos de la radiación , Proliferación Celular/efectos de la radiación , Extensiones de la Superficie Celular/metabolismo , Extensiones de la Superficie Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Conexina 43/metabolismo , Progresión de la Enfermedad , Proteína GAP-43/metabolismo , Uniones Comunicantes/efectos de la radiación , Glioma/metabolismo , Glioma/patología , Glioma/radioterapia , Humanos , Masculino , Ratones , Ratones Desnudos , Invasividad Neoplásica , Tolerancia a Radiación/efectos de los fármacosRESUMEN
Stem cells play a pivotal role in the developmental stages of an organism and in adulthood as well. Therefore, it is not surprising that stem cells constitute a focus of extensive research. Indeed, several decades of stem cell research have tremendously increased our knowledge on the mechanistic understandings of stem cell biology. Interestingly, revealing the fundamental principles of stem cell biology has also fostered its application for therapeutic purposes. Many of the attributes that the stem cells possess, some of which are unique, allow multifaceted exploitation of stem cells in the treatment of various diseases. Cancer, the leading cause of mortality worldwide, is one of the disease groups that has been benefited by the potentials of therapeutic applications of the stem cells. While the modi operandi of how stem cells contribute to cancer treatment are many-sided, two major principles can be conceived. One mode involves harnessing the regenerative power of the stem cells to promote the generation of blood-forming cells in cancer patients after cytotoxic regimens. A totally different kind of utility of stem cells has been exercised in another mode where the stem cells can potentially deliver a plethora of anti-cancer therapeutics in a tumor-specific manner. While both these approaches can improve the treatment of cancer patients, there exist several issues that warrant further research. This review summarizes the basic principles of the utility of the stem cells in cancer treatment along with the current trends and pinpoints the major obstacles to focus on in the future for further improvement.
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Células Madre Adultas , Antineoplásicos , Neoplasias , Adulto , Humanos , Neoplasias/terapia , Medicina Regenerativa , Células MadreRESUMEN
Glioblastoma (GBM) is a deadly disease with a need for deeper understanding and new therapeutic approaches. The microenvironment of glioblastoma has previously been shown to guide glioblastoma progression. In this study, astrocytes were investigated with regard to their effect on glioblastoma proliferation through correlative analyses of clinical samples and experimental in vitro and in vivo studies. Co-culture techniques were used to investigate the GBM growth enhancing potential of astrocytes. Cell sorting and RNA sequencing were used to generate a GBM-associated astrocyte signature and to investigate astrocyte-induced GBM genes. A NOD scid GBM mouse model was used for in vivo studies. A gene signature reflecting GBM-activated astrocytes was associated with poor prognosis in the TCGA GBM dataset. Two genes, periostin and serglycin, induced in GBM cells upon exposure to astrocytes were expressed at higher levels in cases with high "astrocyte signature score". Astrocytes were shown to enhance glioblastoma cell growth in cell lines and in a patient-derived culture, in a manner dependent on cell-cell contact and involving increased cell proliferation. Furthermore, co-injection of astrocytes with glioblastoma cells reduced survival in an orthotopic GBM model in NOD scid mice. In conclusion, this study suggests that astrocytes contribute to glioblastoma growth and implies this crosstalk as a candidate target for novel therapies.
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Astrocitos/metabolismo , Neoplasias Encefálicas/metabolismo , Movimiento Celular/fisiología , Glioblastoma/metabolismo , Animales , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Proliferación Celular/fisiología , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Glioblastoma/patología , Glioma/metabolismo , Humanos , Ratones Endogámicos NODRESUMEN
The microenvironment and architecture of peritumoral tissue have been suggested to affect permissiveness for infiltration of malignant cells. Astrocytes constitute a heterogeneous population of cells and have been linked to proliferation, migration, and drug sensitivity of glioblastoma (GBM) cells. Through double-immunohistochemical staining for platelet-derived growth factor receptor α (PDGFRα) and glial fibrillary acidic protein (GFAP), this study explored the intercase variability among 45 human GBM samples regarding density of GFAP+ peritumoral astrocytes and a subset of GFAP+ peritumoral astrocyte-like cells also expressing PDGFRα. Large intercase variability regarding the total peritumoral astrocyte density and the density of PDGFRα+/GFAP+ peritumoral astrocyte-like cells was detected. DNA fluorescence in situ hybridization analyses for commonly altered genetic tumor markers supported the interpretation that these cells represented a genetically unaffected host cell subset referred to as PDGFRα+/GFAP+ peritumoral astrocytes. The presence of PDGFRα+/GFAP+ peritumoral astrocytes was significantly positively correlated to older patient age and peritumoral astrocyte density, but not to other established prognostic factors. Notably, presence of PDGFRα+/GFAP+ peritumoral astrocytes, but not peritumoral astrocyte density, was associated with significantly shorter patient overall survival. The prognostic association of PDGFRα+/GFAP+ peritumoral astrocytes was confirmed in multivariable analyses. This exploratory study thus demonstrates previously unrecognized intercase variability and prognostic significance of peritumoral abundance of a novel PDGFRα+ subset of GFAP+ astrocytes. Findings suggest clinically relevant roles of the microenvironment of peritumoral GBM tissue and encourage further characterization of the novel astrocyte subset with regard to origin, function, and potential as biomarker and drug target.
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Astrocitos/metabolismo , Neoplasias Encefálicas/mortalidad , Proteína Ácida Fibrilar de la Glía/metabolismo , Glioblastoma/mortalidad , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Microambiente Tumoral/fisiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Niño , Femenino , Proteína Ácida Fibrilar de la Glía/genética , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Pronóstico , Receptores del Factor de Crecimiento Derivado de Plaquetas/genética , Tasa de Supervivencia , Adulto JovenRESUMEN
Patient-based cancer models are essential tools for studying tumor biology and for the assessment of drug responses in a translational context. We report the establishment a large cohort of unique organoids and patient-derived orthotopic xenografts (PDOX) of various glioma subtypes, including gliomas with mutations in IDH1, and paired longitudinal PDOX from primary and recurrent tumors of the same patient. We show that glioma PDOXs enable long-term propagation of patient tumors and represent clinically relevant patient avatars that retain histopathological, genetic, epigenetic, and transcriptomic features of parental tumors. We find no evidence of mouse-specific clonal evolution in glioma PDOXs. Our cohort captures individual molecular genotypes for precision medicine including mutations in IDH1, ATRX, TP53, MDM2/4, amplification of EGFR, PDGFRA, MET, CDK4/6, MDM2/4, and deletion of CDKN2A/B, PTCH, and PTEN. Matched longitudinal PDOX recapitulate the limited genetic evolution of gliomas observed in patients following treatment. At the histological level, we observe increased vascularization in the rat host as compared to mice. PDOX-derived standardized glioma organoids are amenable to high-throughput drug screens that can be validated in mice. We show clinically relevant responses to temozolomide (TMZ) and to targeted treatments, such as EGFR and CDK4/6 inhibitors in (epi)genetically defined subgroups, according to MGMT promoter and EGFR/CDK status, respectively. Dianhydrogalactitol (VAL-083), a promising bifunctional alkylating agent in the current clinical trial, displayed high therapeutic efficacy, and was able to overcome TMZ resistance in glioblastoma. Our work underscores the clinical relevance of glioma organoids and PDOX models for translational research and personalized treatment studies and represents a unique publicly available resource for precision oncology.
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Neoplasias Encefálicas/tratamiento farmacológico , Glioma/tratamiento farmacológico , Xenoinjertos/inmunología , Organoides/patología , Temozolomida/uso terapéutico , Animales , Neoplasias Encefálicas/genética , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioma/genética , Xenoinjertos/efectos de los fármacos , Humanos , Ratones , Recurrencia Local de Neoplasia/genética , Organoides/inmunología , Medicina de Precisión/métodos , RatasRESUMEN
BACKGROUND: A previous study based on Norwegian Cancer Registry data suggested regional differences in overall survival (OS) after treatment for medulloblastoma (MB) and supratentorial primitive neuroectodermal tumor (CNS-PNET) in Norway. The purpose of the present study was to confirm in an extended cohort whether there were regional differences in outcome or not, and if so try to identify possible explanations. MATERIAL AND METHODS: Data from patients aged 0-20 years diagnosed with and treated for MB/CNS-PNET at all four university hospitals in Norway from 1974 to 2013 were collected and compared. RESULTS: Of 266 identified patients, 251 fulfilled inclusion criteria. MB was diagnosed in 200 and CNS-PNET in 51 patients. Five-year OS and event-free survival (EFS) were 59% and 52%, respectively. There was a significant difference in five-year OS and EFS between MB and CNS-PNET patients; 62% versus 47% (P = 0.007) and 57% versus 35% (P < 0.001). In multivariable analysis, two factors were found to significantly contribute to improved five-year OS and EFS, whereas one factor contributed to improved five-year OS only. Gross total resection (GTR) versus non-GTR (hazard ratio [HR] 0.53, P = 0.003; HR 0.46, P < 0.001) and cerebrospinal irradiation (CSI) versus non-CSI (HR 0.24, P < 0.001; HR 0.28, P < 0.001) for both, and treatment outside Oslo University Hospital for OS only (HR 0.64, P = 0.048). CONCLUSION: Survival was comparable with data from other population-based studies, and the importance of GTR and CSI was confirmed. The cause for regional survival differences could not be identified.
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Neoplasias Cerebelosas/mortalidad , Meduloblastoma/mortalidad , Tumores Neuroectodérmicos Primitivos/mortalidad , Neoplasias Supratentoriales/mortalidad , Adolescente , Neoplasias Cerebelosas/terapia , Niño , Preescolar , Terapia Combinada/métodos , Supervivencia sin Enfermedad , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Meduloblastoma/terapia , Tumores Neuroectodérmicos Primitivos/terapia , Noruega/epidemiología , Estudios Retrospectivos , Neoplasias Supratentoriales/terapia , Análisis de Supervivencia , Resultado del Tratamiento , Adulto JovenRESUMEN
Mitochondrial complex I deficiency occurs in the substantia nigra of individuals with Parkinson's disease. It is generally believed that this phenomenon is caused by accumulating mitochondrial DNA damage in neurons and that it contributes to the process of neurodegeneration. We hypothesized that if these theories are correct, complex I deficiency should extend beyond the substantia nigra to other affected brain regions in Parkinson's disease and correlate tightly with neuronal mitochondrial DNA damage. To test our hypothesis, we employed a combination of semiquantitative immunohistochemical analyses, Western blot and activity measurements, to assess complex I quantity and function in multiple brain regions from an extensively characterized population-based cohort of idiopathic Parkinson's disease (n = 18) and gender and age matched healthy controls (n = 11). Mitochondrial DNA was assessed in single neurons from the same areas by real-time PCR. Immunohistochemistry showed that neuronal complex I deficiency occurs throughout the Parkinson's disease brain, including areas spared by the neurodegenerative process such as the cerebellum. Activity measurements in brain homogenate confirmed a moderate decrease of complex I function, whereas Western blot was less sensitive, detecting only a mild reduction, which did not reach statistical significance at the group level. With the exception of the substantia nigra, neuronal complex I loss showed no correlation with the load of somatic mitochondrial DNA damage. Interestingly, α-synuclein aggregation was less common in complex I deficient neurons in the substantia nigra. We show that neuronal complex I deficiency is a widespread phenomenon in the Parkinson's disease brain which, contrary to mainstream theory, does not follow the anatomical distribution of neurodegeneration and is not associated with the neuronal load of mitochondrial DNA mutation. Our findings suggest that complex I deficiency in Parkinson's disease can occur independently of mitochondrial DNA damage and may not have a pathogenic role in the neurodegenerative process.
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Encéfalo/metabolismo , Complejo I de Transporte de Electrón/deficiencia , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , Neuronas/metabolismo , Enfermedad de Parkinson/metabolismo , Anciano , Anciano de 80 o más Años , Encéfalo/patología , Daño del ADN , ADN Mitocondrial/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mitocondrias/patología , Enfermedades Mitocondriales/patología , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Neuronas/patología , Enfermedad de Parkinson/patología , Estudios Prospectivos , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , alfa-Sinucleína/metabolismoRESUMEN
Low-grade, pilocytic astrocytomas are treated by resection, but additional therapy is necessary for those tumors with anaplastic features. Arsenic trioxide (As2O3) is emerging as an effective chemotherapeutic agent for treatment of malignant glioblastoma multiforme, where Cathepsin L silencing enables lower, less harmful As2O3 concentrations to achieve the desired cytotoxic effect. Here, we evaluated the effects of As2O3 combined with stable Cathepsin L shRNA silencing on cell viability/metabolic activity, and apoptosis in primary cultures of recurrent malignantly transformed pilocytic astrocytoma (MPA). These cells expressed high Cathepsin L levels, and when grown as monolayers and spheroids, they were more resistant to As2O3 than the U87MG glioblastoma cell line. Caspases 3/7 activity in MPA58 spheroids was not significantly affected by As2O3, possibly due to higher chemoresistance of primary biopsy tissue of less malignant astrocytoma versus the malignant U87MG cell line. However, As2O3 treatment was cytotoxic to MPA spheroids after silencing of Cathepsin L expression. While Cathepsin L silencing only slightly decreased the live/dead cell ratio in As2O3-treated MPA-si spheroids under our experimental conditions, there was an increase in As2O3-mediated apoptosis in MPA-si spheroids, as indicated by elevated caspases 3/7 activity. Therefore, Cathepsin L silencing by gene manipulation can be applied when a more aggressive approach is needed in treatment of pilocytic astrocytomas with anaplastic features.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Arsenicales/farmacología , Astrocitoma/tratamiento farmacológico , Neoplasias Encefálicas/tratamiento farmacológico , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Catepsina L/genética , Óxidos/farmacología , Neoplasias de la Médula Espinal/tratamiento farmacológico , Animales , Apoptosis/genética , Trióxido de Arsénico , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Resistencia a Antineoplásicos , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/genética , Activación Enzimática/inmunología , Glioblastoma/tratamiento farmacológico , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Óxidos/toxicidad , Interferencia de ARN , ARN Interferente Pequeño/genética , Esferoides Celulares , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Little is known about the role of glial host cells in brain tumours. However, supporting stromal cells have been shown to foster tumour growth in other cancers. METHODS: We isolated stromal cells from patient-derived glioblastoma (GBM) xenografts established in GFP-NOD/scid mice. With simultaneous removal of CD11b+ immune and CD31+ endothelial cells by fluorescence activated cell sorting (FACS), we obtained a population of tumour-associated glial cells, TAGs, expressing markers of terminally differentiaed glial cell types or glial progenitors. This cell population was subsequently characterised using gene expression analyses and immunocytochemistry. Furthermore, sphere formation was assessed in vitro and their glioma growth-promoting ability was examined in vivo. Finally, the expression of TAG related markers was validated in human GBMs. RESULTS: TAGs were highly enriched for the expression of glial cell proteins including GFAP and myelin basic protein (MBP), and immature markers such as Nestin and O4. A fraction of TAGs displayed sphere formation in stem cell medium. Moreover, TAGs promoted brain tumour growth in vivo when co-implanted with glioma cells, compared to implanting only glioma cells, or glioma cells and unconditioned glial cells from mice without tumours. Genome-wide microarray analysis of TAGs showed an expression profile distinct from glial cells from healthy mice brains. Notably, TAGs upregulated genes associated with immature cell types and self-renewal, including Pou3f2 and Sox2. In addition, TAGs from highly angiogenic tumours showed upregulation of angiogenic factors, including Vegf and Angiopoietin 2. Immunohistochemistry of three GBMs, two patient biopsies and one GBM xenograft, confirmed that the expression of these genes was mainly confined to TAGs in the tumour bed. Furthermore, their expression profiles displayed a significant overlap with gene clusters defining prognostic subclasses of human GBMs. CONCLUSIONS: Our data demonstrate that glial host cells in brain tumours are functionally distinct from glial cells of healthy mice brains. Furthermore, TAGs display a gene expression profile with enrichment for genes related to stem cells, immature cell types and developmental processes. Future studies are needed to delineate the biological mechanisms regulating the brain tumour-host interplay.
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Neoplasias Encefálicas/metabolismo , Encéfalo/metabolismo , Glioblastoma/metabolismo , Transcriptoma , Animales , Biomarcadores de Tumor , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos NOD , Ratones SCID , Análisis por Micromatrices , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Gene therapeutic strategies with suicide genes are currently investigated in clinical trials for brain tumors. Previously, we have shown that lentiviral vectors delivering the suicide gene HSV-Tk to experimental brain tumors promote a highly significant treatment effect and thus are promising vectors for clinical translation. METHODS: In the present study, we tested lentiviral vectors delivering the suicide gene HSV-Tk.007, a highly active mutant of HSV-Tk, to rat brains as a preclinical toxicity study. We injected 10(6) vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped functional lentiviral particles harboring the suicide gene HSV-Tk.007 into the brain of healthy, immunocompetent rats. During prodrug treatment with ganciclovir (GCV), we measured weight and assessed the behavior of the rats in an open field test. After 14 days of GCV treatment, we analyzed HSV-Tk.007 expression in different brain cell populations, as well as inflammatory responses and apoptosis. RESULTS: During prodrug treatment with GCV, behavior experiments did not reveal differences between the treated rats and the control groups. Analysis of HSV-Tk expression in different brain cell populations showed that transduced normal brain cells survived GCV treatment. There were no statistically significant differences in the number of transduced cells between treatment and control groups. Furthermore, inflammatory responses and apoptosis of brain cells were not observed. CONCLUSIONS: We show that HSV-Tk.007-mediated suicide gene therapy is not toxic to normal brain cells. This observation is of high relevance for the translation of lentivirus-mediated suicide gene therapies into the clinic for the treatment of brain tumor patients. Copyright © 2016 John Wiley & Sons, Ltd.
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Encéfalo/metabolismo , Genes Transgénicos Suicidas/genética , Terapia Genética/métodos , Simplexvirus/enzimología , Timidina Quinasa/metabolismo , Animales , Antivirales/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Encéfalo/citología , Encéfalo/efectos de los fármacos , Línea Celular Tumoral , Ganciclovir/farmacología , Humanos , Lentivirus/genética , Microscopía Confocal , Actividad Motora/efectos de los fármacos , Actividad Motora/genética , Mutación , Ratas , Simplexvirus/genética , Timidina Quinasa/genéticaRESUMEN
OBJECTIVE: Polymerase gamma (POLG) mutations are a common cause of mitochondrial disease and have also been linked to neurodegeneration and aging. We studied the molecular mechanisms underlying POLG-related neurodegeneration using postmortem tissue from a large number of patients. METHODS: Clinical information was available from all subjects. Formalin-fixed and frozen brain tissue from 15 patients and 23 controls was studied employing a combination of histopathology, immunohistochemistry, and molecular studies of microdissected neurons. RESULTS: The primary consequence of POLG mutation in neurons is mitochondrial DNA depletion. This was already present in infants with little evidence of neuronal loss or mitochondrial dysfunction. With longer disease duration, we found an additional, progressive accumulation of mitochondrial DNA deletions and point mutations accompanied by increasing numbers of complex I-deficient neurons. Progressive neurodegeneration primarily affected the cerebellar systems and dopaminergic cells of the substantia nigra. Superimposed on this chronic process were acute, focal cortical lesions that correlated with epileptogenic foci and that showed massive neuronal loss. INTERPRETATION: POLG mutations appear to compromise neuronal respiration via a combination of early and stable depletion and a progressive somatic mutagenesis of the mitochondrial genome. This leads to 2 distinct but overlapping biological processes: a chronic neurodegeneration reflected clinically by progressive ataxia and cognitive impairment, and an acute focal neuronal necrosis that appears to be related to the presence of epileptic seizures. Our findings offer an explanation of the acute-on-chronic clinical course of this common mitochondrial encephalopathy.
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ADN Polimerasa Dirigida por ADN/efectos adversos , ADN Polimerasa Dirigida por ADN/genética , Degeneración Nerviosa/enzimología , Degeneración Nerviosa/patología , Adolescente , Adulto , Cerebelo/enzimología , Cerebelo/patología , Corteza Cerebral/enzimología , Corteza Cerebral/patología , Niño , ADN Polimerasa gamma , ADN Mitocondrial/genética , Progresión de la Enfermedad , Humanos , Lactante , Persona de Mediana Edad , Encefalomiopatías Mitocondriales/enzimología , Encefalomiopatías Mitocondriales/genética , Encefalomiopatías Mitocondriales/patología , Mutación/genética , Sustancia Negra/enzimología , Sustancia Negra/patología , Adulto JovenRESUMEN
Anti-angiogenic therapy in glioblastoma (GBM) has unfortunately not led to the anticipated improvement in patient prognosis. We here describe how human GBM adapts to bevacizumab treatment at the metabolic level. By performing (13)C6-glucose metabolic flux analysis, we show for the first time that the tumors undergo metabolic re-programming toward anaerobic metabolism, thereby uncoupling glycolysis from oxidative phosphorylation. Following treatment, an increased influx of (13)C6-glucose was observed into the tumors, concomitant to increased lactate levels and a reduction of metabolites associated with the tricarboxylic acid cycle. This was confirmed by increased expression of glycolytic enzymes including pyruvate dehydrogenase kinase in the treated tumors. Interestingly, L-glutamine levels were also reduced. These results were further confirmed by the assessment of in vivo metabolic data obtained by magnetic resonance spectroscopy and positron emission tomography. Moreover, bevacizumab led to a depletion in glutathione levels indicating that the treatment caused oxidative stress in the tumors. Confirming the metabolic flux results, immunohistochemical analysis showed an up-regulation of lactate dehydrogenase in the bevacizumab-treated tumor core as well as in single tumor cells infiltrating the brain, which may explain the increased invasion observed after bevacizumab treatment. These observations were further validated in a panel of eight human GBM patients in which paired biopsy samples were obtained before and after bevacizumab treatment. Importantly, we show that the GBM adaptation to bevacizumab therapy is not mediated by clonal selection mechanisms, but represents an adaptive response to therapy.
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Inhibidores de la Angiogénesis/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Adulto , Anciano , Animales , Bevacizumab , Encéfalo/diagnóstico por imagen , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Neoplasias Encefálicas/diagnóstico por imagen , Femenino , Glioblastoma/diagnóstico por imagen , Glutamina/metabolismo , Glutatión/metabolismo , Glucólisis/efectos de los fármacos , Humanos , L-Lactato Deshidrogenasa/metabolismo , Ácido Láctico/metabolismo , Masculino , Ratones SCID , Ratones Transgénicos , Persona de Mediana Edad , Trasplante de Neoplasias , Estrés Oxidativo/efectos de los fármacos , Cintigrafía , Ratas DesnudasRESUMEN
The role of mitochondria in the pathogenesis of neurodegeneration is an area of intense study. It is known that defects in proteins involved in mitochondrial quality control can cause Parkinson's disease, and there is increasing evidence linking mitochondrial dysfunction, and particularly mitochondrial DNA abnormalities, to neuronal loss in the substantia nigra. Mutations in the catalytic subunit of polymerase gamma are among the most common causes of mitochondrial disease and owing to its role in mitochondrial DNA homeostasis, polymerase gamma defects are often considered a paradigm for mitochondrial diseases generally. Yet, despite this, parkinsonism is uncommon with polymerase gamma defects. In this study, we investigated structural and functional changes in the substantia nigra of 11 patients with polymerase gamma encephalopathy. We characterized the mitochondrial DNA abnormalities and examined the respiratory chain in neurons of the substantia nigra. We also investigated nigrostriatal integrity and function using a combination of post-mortem and in vivo functional studies with dopamine transporter imaging and positron emission tomography. At the cellular level, dopaminergic nigral neurons of patients with polymerase gamma encephalopathy contained a significantly lower copy number of mitochondrial DNA (depletion) and higher levels of deletions than normal control subjects. A selective and progressive complex I deficiency was seen and this was associated with a severe and progressive loss of the dopaminergic neurons of the pars compacta. Dopamine transporter imaging and positron emission tomography showed that the degree of nigral neuronal loss and nigrostriatal depletion were severe and appeared greater even than that seen in idiopathic Parkinson's disease. Despite this, however, none of our patients showed any signs of parkinsonism. The additional presence of both thalamic and cerebellar dysfunction in our patients suggested that these may play a role in counteracting the effects of basal ganglia dysfunction and prevent the development of clinical parkinsonism.
Asunto(s)
Cuerpo Estriado/patología , ADN Polimerasa Dirigida por ADN/genética , Enfermedades Mitocondriales/genética , Degeneración Nerviosa/genética , Trastornos Parkinsonianos/genética , Sustancia Negra/patología , Adolescente , Adulto , Cuerpo Estriado/metabolismo , ADN Polimerasa gamma , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/patología , Mutación , Degeneración Nerviosa/patología , Neuronas/metabolismo , Neuronas/patología , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/patología , Sustancia Negra/metabolismoRESUMEN
Bevacizumab, an antibody against vascular endothelial growth factor (VEGF), is a promising, yet controversial, drug in human glioblastoma treatment (GBM). Its effects on tumor burden, recurrence, and vascular physiology are unclear. We therefore determined the tumor response to bevacizumab at the phenotypic, physiological, and molecular level in a clinically relevant intracranial GBM xenograft model derived from patient tumor spheroids. Using anatomical and physiological magnetic resonance imaging (MRI), we show that bevacizumab causes a strong decrease in contrast enhancement while having only a marginal effect on tumor growth. Interestingly, dynamic contrast-enhanced MRI revealed a significant reduction of the vascular supply, as evidenced by a decrease in intratumoral blood flow and volume and, at the morphological level, by a strong reduction of large- and medium-sized blood vessels. Electron microscopy revealed fewer mitochondria in the treated tumor cells. Importantly, this was accompanied by a 68% increase in infiltrating tumor cells in the brain parenchyma. At the molecular level we observed an increase in lactate and alanine metabolites, together with an induction of hypoxia-inducible factor 1α and an activation of the phosphatidyl-inositol-3-kinase pathway. These data strongly suggest that vascular remodeling induced by anti-VEGF treatment leads to a more hypoxic tumor microenvironment. This favors a metabolic change in the tumor cells toward glycolysis, which leads to enhanced tumor cell invasion into the normal brain. The present work underlines the need to combine anti-angiogenic treatment in GBMs with drugs targeting specific signaling or metabolic pathways linked to the glycolytic phenotype.