RESUMO
Metabolic reprogramming has been described in rapidly growing tumors, which are thought to mostly contain fast-cycling cells (FCCs) that have impaired mitochondrial function and rely on aerobic glycolysis. Here, we characterize the metabolic landscape of glioblastoma (GBM) and explore metabolic specificities as targetable vulnerabilities. Our studies highlight the metabolic heterogeneity in GBM, in which FCCs harness aerobic glycolysis, and slow-cycling cells (SCCs) preferentially utilize mitochondrial oxidative phosphorylation for their functions. SCCs display enhanced invasion and chemoresistance, suggesting their important role in tumor recurrence. SCCs also demonstrate increased lipid contents that are specifically metabolized under glucose-deprived conditions. Fatty acid transport in SCCs is targetable by pharmacological inhibition or genomic deletion of FABP7, both of which sensitize SCCs to metabolic stress. Furthermore, FABP7 inhibition, whether alone or in combination with glycolysis inhibition, leads to overall increased survival. Our studies reveal the existence of GBM cell subpopulations with distinct metabolic requirements and suggest that FABP7 is central to lipid metabolism in SCCs and that targeting FABP7-related metabolic pathways is a viable therapeutic strategy.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Ácidos Graxos/metabolismo , Glioblastoma/metabolismo , Glicólise , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Animais , Linhagem Celular Tumoral , Proteína 7 de Ligação a Ácidos Graxos/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mitocôndrias/patologia , Proteínas de Neoplasias/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
OPINION STATEMENT: Despite aggressive surgery, radiation, and systemic chemotherapy, the prognosis for patients diagnosed with malignant brain tumors remains extremely poor, and standard treatments carry significant risks for long-term neurocognitive deficits. There is a clear and urgent need for the development of more effective treatments that will add minimal toxicity to standard therapies for invasive brain cancers. Cancer immunotherapy is a treatment modality that holds promise for the delivery of tumor-specific cytotoxicity, with the potential to eliminate brain tumor cells without harming the eloquent brain.
Assuntos
Neoplasias Encefálicas/terapia , Vacinas Anticâncer/uso terapêutico , Glioma/terapia , Imunoterapia Adotiva/métodos , Terapia Viral Oncolítica/métodos , Humanos , Imunoterapia Adotiva/efeitos adversosRESUMO
There are numerous mechanisms by which glioblastoma cells evade immunological detection, underscoring the need for strategic combinatorial treatments to achieve appreciable therapeutic effects. However, developing combination therapies is difficult due to dose-limiting toxicities, blood-brain-barrier, and suppressive tumor microenvironment. Glioblastoma is notoriously devoid of lymphocytes driven in part by a paucity of lymphocyte trafficking factors necessary to prompt their recruitment and activation. Herein, we develop a recombinant adeno-associated virus (AAV) gene therapy that enables focal and stable reconstitution of the tumor microenvironment with C-X-C motif ligand 9 (CXCL9), a powerful call-and-receive chemokine for lymphocytes. By manipulating local chemokine directional guidance, AAV-CXCL9 increases tumor infiltration by cytotoxic lymphocytes, sensitizing glioblastoma to anti-PD-1 immune checkpoint blockade in female preclinical tumor models. These effects are accompanied by immunologic signatures evocative of an inflamed tumor microenvironment. These findings support AAV gene therapy as an adjuvant for reconditioning glioblastoma immunogenicity given its safety profile, tropism, modularity, and off-the-shelf capability.
Assuntos
Quimiocina CXCL9 , Dependovirus , Terapia Genética , Glioblastoma , Inibidores de Checkpoint Imunológico , Receptor de Morte Celular Programada 1 , Microambiente Tumoral , Glioblastoma/terapia , Glioblastoma/imunologia , Dependovirus/genética , Microambiente Tumoral/imunologia , Animais , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Quimiocina CXCL9/genética , Quimiocina CXCL9/imunologia , Camundongos , Terapia Genética/métodos , Feminino , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Linhagem Celular Tumoral , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/terapia , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genéticaRESUMO
Pediatric central nervous system tumors are the most common solid malignancies in childhood, and aggressive therapy often leads to long-term sequelae in survivors, making these tumors challenging to treat. Immunotherapy has revolutionized prospects for many cancer types in adults, but the intrinsic complexity of treating pediatric patients and the scarcity of clinical studies of children to inform effective approaches have hampered the development of effective immunotherapies in pediatric settings. Here, we review recent advances and ongoing challenges in pediatric brain cancer immunotherapy, as well as considerations for efficient clinical translation of efficacious immunotherapies into pediatric settings.
Assuntos
Neoplasias Encefálicas , Neoplasias do Sistema Nervoso Central , Neoplasias Encefálicas/terapia , Neoplasias do Sistema Nervoso Central/terapia , Criança , Humanos , Fatores Imunológicos , Imunoterapia/efeitos adversos , SobreviventesRESUMO
Adoptive cellular therapy (ACT) is a potent strategy to boost the immune response against cancer. ACT is effective against blood cancers but faces challenges in treating solid tumors. A critical step for the success of ACT immunotherapy is to achieve efficient trafficking and persistence of T cells to solid tumors. Non-invasive tracking of the accumulation of adoptively transferred T cells to tumors would greatly accelerate development of more effective ACT strategies. We demonstrate the use of magnetic particle imaging (MPI) to non-invasively track ACT T cells in vivo in a mouse model of brain cancer. Magnetic labeling did not impair primary tumor-specific T cells in vitro, and MPI allowed the detection of labeled T cells in the brain after intravenous or intracerebroventricular administration. These results support the use of MPI to track adoptively transferred T cells and accelerate the development of ACT treatments for brain tumors and other cancers.
Assuntos
Transferência Adotiva , Neoplasias Encefálicas , Encéfalo , Rastreamento de Células , Nanopartículas de Magnetita/uso terapêutico , Linfócitos T , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/terapia , Camundongos , Camundongos Transgênicos , Linfócitos T/metabolismo , Linfócitos T/patologia , Linfócitos T/transplanteRESUMO
Histone deacetylase 6 (HDAC6) is an emerging therapeutic target that is overexpressed in glioblastoma when compared to other HDACs. HDAC6 catalyzes the deacetylation of alpha-tubulin and mediates the disassembly of primary cilia, a process required for cell cycle progression. HDAC6 inhibition disrupts glioma proliferation, but whether this effect is dependent on tumor cell primary cilia is unknown. We found that HDAC6 inhibitors ACY-1215 (1215) and ACY-738 (738) inhibited the proliferation of multiple patient-derived and mouse glioma cells. While both inhibitors triggered rapid increases in acetylated alpha-tubulin (aaTub) in the cytosol and led to increased frequencies of primary cilia, they unexpectedly reduced the levels of aaTub in the cilia. To test whether the antiproliferative effects of HDAC6 inhibitors are dependent on tumor cell cilia, we generated patient-derived glioma lines devoid of cilia through depletion of ciliogenesis genes ARL13B or KIF3A. At low concentrations, 1215 or 738 did not decrease the proliferation of cilia-depleted cells. Moreover, the differentiation of glioma cells that was induced by HDAC6 inhibition did not occur after the inhibition of cilia formation. These data suggest HDAC6 signaling at primary cilia promotes the proliferation of glioma cells by restricting their ability to differentiate. Surprisingly, overexpressing HDAC6 did not reduce cilia length or the frequency of ciliated glioma cells, suggesting other factors are required to control HDAC6-mediated cilia disassembly in glioma cells. Collectively, our findings suggest that HDAC6 promotes the proliferation of glioma cells through primary cilia.
RESUMO
Despite therapeutic advances for other malignancies, gliomas remain challenging solid tumors to treat. Complete surgical resection is nearly impossible due to gliomas' diffuse infiltrative nature, and treatment is hampered by restricted access to the tumors due to limited transport across the blood-brain barrier. Recent advances in genomic studies and next-generation sequencing techniques have led to a better understanding of gliomas and identification of potential aberrant signaling pathways. Targeting the specific genomic abnormalities via novel molecular therapies has opened a new avenue in the management of gliomas, with encouraging results in preclinical studies and early clinical trials. However, molecular characterization of gliomas revealed significant heterogeneity, which poses a challenge for targeted therapeutic approaches. In this context, leading neuro-oncology researchers and clinicians, industry innovators, and patient advocates convened at the inaugural annual Remission Summit held in Orlando, FL in February 2019 to discuss the latest advances in immunotherapy and precision medicine approaches for the treatment of adult and pediatric brain tumors and outline the unanswered questions, challenges, and opportunities that lay ahead for advancing the duration and quality of life for patients with brain tumors. Here, we provide historical context for precision medicine in other cancers, present emerging approaches for gliomas, discuss their limitations, and outline the steps necessary for future success. We focus on the advances in small molecule targeted therapy, as the use of immunotherapy as an emerging precision medicine modality for glioma treatment has recently been reviewed by our colleagues.
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Adoptive T cell therapy has proven effective against hematologic malignancies and demonstrated efficacy against a variety of solid tumors in preclinical studies and clinical trials. Nonetheless, antitumor responses against solid tumors remain modest, highlighting the need to enhance the effectiveness of this therapy. Genetic modification of T cells with RNA has been explored to enhance T-cell antigen specificity, effector function, and migration to tumor sites, thereby potentiating antitumor immunity. This review describes the rationale for RNA-electroporated T cell modifications and provides an overview of their applications in preclinical and clinical investigations for the treatment of hematologic malignancies and solid tumors.
Assuntos
Neoplasias , Linfócitos T , Humanos , Imunoterapia , Imunoterapia Adotiva , Neoplasias/terapia , RNARESUMO
BACKGROUND: The mechanisms by which primary cilia affect glioma pathogenesis are unclear. Depending on the glioma cell line, primary cilia can promote or inhibit tumor development. Here, we used piggyBac-mediated transgenesis to generate patient-derived glioblastoma (GBM) cell lines that stably express Arl13b:GFP in their cilia. This allowed us to visualize and analyze the behavior of cilia and ciliated cells during live GBM cell proliferation. RESULTS: Time-lapse imaging of Arl13b:GFP+ cilia revealed their dynamic behaviors, including distal tip excision into the extracellular milieu. Recent studies of non-cancerous cells indicate that this process occurs during the G0 phase, prior to cilia resorption and cell cycle re-entry, and requires ciliary recruitment of F-actin and actin regulators. Similarly, we observed ciliary buds associated with Ki67- cells as well as scattered F-actin+ cilia, suggesting that quiescent GBM cells may also utilize an actin network-based mechanism for ciliary tip excision. Notably, we found that the proliferation of ciliated GBM cells was promoted by exposing them to conditioned media obtained from ciliated cell cultures when compared to conditioned media collected from cilia-defective cell cultures (depleted in either KIF3A or IFT88 using CRISPR/Cas9). These results suggest that GBM cilia may release mitogenic vesicles carrying factors that promote tumor cell proliferation. Although Arl13b is implicated in tumor growth, our data suggest that Arl13b released from GBM cilia does not mediate tumor cell proliferation. CONCLUSION: Collectively, our results indicate that ciliary vesicles may represent a novel mode of intercellular communication within tumors that contributes to GBM pathogenesis. The mitogenic capacity of GBM ciliary vesicles and the molecular mediators of this phenomenon requires further investigation.
RESUMO
Neonatal or early-life seizures (ELS) are often associated with life-long neurophysiological, cognitive and behavioral deficits, but the underlying mechanisms contributing to these deficits remain poorly understood. Newborn, post-migratory cortical neurons sprout ciliary buds (procilia) that mature into primary cilia. Disruption of the growth or signaling capabilities of these cilia has been linked to atypical neurite outgrowth from neurons and abnormalities in neuronal circuitry. Here, we tested the hypothesis that generalized seizures induced by pentylenetetrazol (PTZ) or kainic acid (KA) during early postnatal development impair neuronal and/or glial ciliogenesis. Mice received PTZ (50 or 100mg/kg), KA (2mg/kg), or saline either once at birth (P0), or once daily from P0 to P4. Using immunohistochemistry and electron microscopy, the cilia of neurons and glia were examined at P7, P14, and P42. A total of 83 regions were analyzed, representing 13 unique neocortical and hippocampal regions. Neuronal cilia were identified by co-expression of NeuN and type 3 adenylyl cyclase (ACIII) or somatostatin receptor 3 (SSTR3), while glial cilia were identified by co-expression of GFAP, Arl13b, and gamma-tubulin. We found that PTZ exposure at either P0 or from P0 to P4 induced convulsive behavior, followed by acute and lasting effects on neuronal cilia lengths that varied depending on the cortical region, PTZ dose, injection frequency, and time post-PTZ. Both increases and decreases in neuronal cilia length were observed. No changes in the length of glial cilia were observed under any of the test conditions. Lastly, we found that a single KA seizure at P0 led to similar abnormalities in neuronal cilia lengths. Our results suggest that seizure(s) occurring during early stages of cortical development induce persistent and widespread changes in neuronal cilia length. Given the impact neuronal cilia have on neuronal differentiation, ELS-induced changes in ciliogenesis may contribute to long-term pathology and abnormal cortical function.
Assuntos
Córtex Cerebral , Convulsivantes/toxicidade , Ácido Caínico/toxicidade , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Pentilenotetrazol/toxicidade , Convulsões/induzido quimicamente , Fatores Etários , Animais , Animais Recém-Nascidos , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Camundongos , Microscopia Imunoeletrônica , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/fisiologia , Neuroglia/ultraestrutura , Neurônios/metabolismo , Neurônios/ultraestrutura , Convulsões/patologiaRESUMO
A better understanding of the molecules implicated in the growth and survival of glioblastoma (GBM) cells and their response to temozolomide (TMZ), the standard-of-care chemotherapeutic agent, is necessary for the development of new therapies that would improve the outcome of current GBM treatments. In this study, we characterize the role of pericentriolar material 1 (PCM1), a component of centriolar satellites surrounding centrosomes, in GBM cell proliferation and sensitivity to genotoxic agents such as TMZ. We show that PCM1 is expressed around centrioles and ciliary basal bodies in patient GBM biopsies and derived cell lines and that its localization is dynamic throughout the cell cycle. To test whether PCM1 mediates GBM cell proliferation and/or response to TMZ, we used CRISPR/Cas9 genome editing to generate primary GBM cell lines depleted of PCM1. These PCM1-depleted cells displayed reduced AZI1 satellite protein localization and significantly decreased proliferation, which was attributable to increased apoptotic cell death. Furthermore, PCM1-depleted lines were more sensitive to TMZ toxicity than control lines. The increase in TMZ sensitivity may be partly due to the reduced ability of PCM1-depleted cells to form primary cilia, as depletion of KIF3A also ablated GBM cells' ciliogenesis and increased their sensitivity to TMZ while preserving PCM1 localization. In addition, the co-depletion of KIF3A and PCM1 did not have any additive effect on TMZ sensitivity. Together, our data suggest that PCM1 plays multiple roles in GBM pathogenesis and that associated pathways could be targeted to augment current or future anti-GBM therapies.
RESUMO
KIF3A, a component of the kinesin-2 motor, is necessary for the progression of diverse tumor types. This is partly due to its role in regulating ciliogenesis and cell responsiveness to sonic hedgehog (SHH). Notably, primary cilia have been detected in human glioblastoma multiforme (GBM) tumor biopsies and derived cell lines. Here, we asked whether disrupting KIF3A in GBM cells affected ciliogenesis, in vitro growth and responsiveness to SHH, or tumorigenic behavior in vivo. We used a lentiviral vector to create three patient-derived GBM cell lines expressing a dominant negative, motorless form of Kif3a (dnKif3a). In all unmodified lines, we found that most GBM cells were capable of producing ciliated progeny and that dnKif3a expression in these cells ablated ciliogenesis. Interestingly, unmodified and dnKif3a-expressing cell lines displayed differential sensitivities and pathway activation to SHH and variable tumor-associated survival following mouse xenografts. In one cell line, SHH-induced cell proliferation was prevented in vitro by either expressing dnKif3a or inhibiting SMO signaling using cyclopamine, and the survival times of mice implanted with dnKif3a-expressing cells were increased. In a second line, expression of dnKif3a increased the cells' baseline proliferation while, surprisingly, sensitizing them to SHH-induced cell death. The survival times of mice implanted with these dnKif3a-expressing cells were decreased. Finally, expression of dnKif3a in a third cell line had no effect on cell proliferation, SHH sensitivity, or mouse survival times. These findings indicate that KIF3A is essential for GBM cell ciliogenesis, but its role in modulating GBM cell behavior is highly variable.
Assuntos
Carcinogênese/patologia , Cílios/fisiologia , Genes Dominantes/genética , Glioblastoma/patologia , Proteínas Hedgehog/metabolismo , Cinesinas/antagonistas & inibidores , Adulto , Idoso , Animais , Apoptose , Western Blotting , Carcinogênese/genética , Carcinogênese/metabolismo , Proliferação de Células , Glioblastoma/genética , Glioblastoma/metabolismo , Proteínas Hedgehog/genética , Humanos , Técnicas Imunoenzimáticas , Cinesinas/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The harvest of intact cerebral microvessel yields could permit the in vitro characterization of mechanisms that underlie numerous vascular-linked central nervous system (CNS) phenomena. Here, we test (1) the effect of mild enzyme digestion on microvessel purity and yield; and then (2) the effect of variable centrifugation and filtration methods on microvessel yields. The brains of female Sprague-Dawley rats (4 weeks-old; n=38) were removed rapidly and homogenized. In Experiments 1 and 2, brain homogenates were incubated in DMEM or a solution of papain (2.5 U/ml), DNAse I (250 U/ml) and dispase II (1 U/ml) in DMEM for 15 min at 37 °C before microvessels were purified using differential (20% Ficoll) and then discontinuous (15/20% Dextran) centrifugation (@3500 × g) and collected with glass bead column filtration. Enzymatic digestion decreased microvessel yields (27 vs. 12 k/g tissue; p=0.053) but increased microvessel purity by decreasing adherent cells (p=0.002), which included NF-L(+) neurons (p<0.05) and GFAP+ astrocytes (p<0.001) and astrocyte endfeet (p<0.01). After one week in culture, >85% of harvested cells morphologically resembled microvessels and expressed the vascular proteins lectin and/or RECA-1. Finally, microvessels yields decreased when discontinuous centrifugation was omitted or nylon mesh filtration was employed. In summary, we found that digesting brain homogenates enzymatically could improve the purity of harvested microvessels that could be cultured for at least a week.