RESUMO
PURPOSE: Patient-derived cancer cell lines can be very useful to investigate genetic as well as epigenetic mechanisms of transformation and to test new drugs. In this multi-centric study, we performed genomic and transcriptomic characterization of a large set of patient-derived glioblastoma (GBM) stem-like cells (GSCs). METHODS: 94 (80 I surgery/14 II surgery) and 53 (42 I surgery/11 II surgery) GSCs lines underwent whole exome and trascriptome analysis, respectively. RESULTS: Exome sequencing revealed TP53 as the main mutated gene (41/94 samples, 44%), followed by PTEN (33/94, 35%), RB1 (16/94, 17%) and NF1 (15/94, 16%), among other genes associated to brain tumors. One GSC sample bearing a BRAF p.V600E mutation showed sensitivity in vitro to a BRAF inhibitor. Gene Ontology and Reactome analysis uncovered several biological processes mostly associated to gliogenesis and glial cell differentiation, S - adenosylmethionine metabolic process, mismatch repair and methylation. Comparison of I and II surgery samples disclosed a similar distribution of mutated genes, with an overrepresentation of mutations in mismatch repair, cell cycle, p53 and methylation pathways in I surgery samples, and of mutations in receptor tyrosine kinase and MAPK signaling pathways in II surgery samples. Unsupervised hierarchical clustering of RNA-seq data produced 3 clusters characterized by distinctive sets of up-regulated genes and signaling pathways. CONCLUSION: The availability of a large set of fully molecularly characterized GCSs represents a valuable public resource to support the advancement of precision oncology for the treatment of GBM.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/patologia , Transcriptoma , Proteínas Proto-Oncogênicas B-raf/genética , Células-Tronco Neoplásicas/patologia , Medicina de Precisão , Neoplasias Encefálicas/patologiaRESUMO
Glioblastomas (GBM) can be classified into three major transcriptional subgroups (proneural, mesenchymal, classical), underlying different molecular alterations, prognosis, and response to therapy. However, transcriptional analysis is not routinely feasible and assessment of a simplified method for glioblastoma subclassification is required. We propose an integrated molecular and immunohistochemical approach aimed at identifying GBM subtypes in routine paraffin-embedded material. RNA-sequencing analysis was performed on representative samples (n = 51) by means of a "glioblastoma transcriptional subtypes (GliTS) redux" custom gene signature including a restricted number (n = 90) of upregulated genes validated on the TCGA dataset. With this dataset, immunohistochemical profiles, based on expression of a restricted panel of gene classifiers, were integrated by a machine-learning approach to generate a GliTS based on protein quantification that allowed an efficient GliTS assignment when applied to an extended cohort (n = 197). GliTS redux maintained high levels of correspondence with the original GliTS classification using the TCGA dataset. The machine-learning approach designed an immunohistochemical (IHC)-based classification, whose concordance was 79.5% with the transcriptional- based classification, and reached 90% for the mesenchymal subgroup. Distribution and survival of GliTS were in line with reported data, with the mesenchymal subgroup given the worst prognosis. Notably, the algorithm allowed the identification of cases with comparable probability to be assigned to different GliTS, thus falling within overlapping regions and reflecting an extreme heterogeneous phenotype that mirrors the underlying genetic and biological tumor heterogeneity. Indeed, while mesenchymal and classical subgroups were well segregated, the proneural types frequently showed a mixed proneural/classical phenotype, predicted as proneural by the algorithm, but with comparable probability of being assigned to the classical subtype. These cases, characterized by concomitant high expression of EGFR and proneural biomarkers, showed lower survival. Collectively, these data indicate that a restricted panel of highly sensitive immunohistochemical markers can efficiently predict GliTS with high accuracy and significant association with different clinical outcomes.
Assuntos
Neoplasias Encefálicas/classificação , Neoplasias Encefálicas/metabolismo , Perfilação da Expressão Gênica , Glioblastoma/classificação , Glioblastoma/metabolismo , Idoso , Algoritmos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/genética , Análise por Conglomerados , Estudos de Coortes , Feminino , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/estatística & dados numéricos , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Humanos , Imuno-Histoquímica , Aprendizado de Máquina , Masculino , Pessoa de Meia-Idade , RNA-SeqRESUMO
Glioblastoma (GBM) is the most malignant brain tumor of adults and is characterized by extensive cell dissemination within the brain parenchyma and enhanced angiogenesis. Effective preclinical modeling of these key features suffers from several shortcomings. Aim of this study was to determine whether modulating the expression of extracellular matrix (ECM) modifiers in proneural (PN) and mesenchymal (MES) cancer stem cells (CSCs) and in conventional glioma cell lines (GCLs) might improve tumor invasion and vascularization. To this end, we selected secreted, acidic and rich in cysteine-like 1 (SPARCL1) as a potential mediator of ECM remodeling in GBM. SPARCL1 transcript and protein expression was assessed in PN and MES CSCs as well as GCLs, in their xenografts and in patient-derived specimens by qPCR, WB and IHC. SPARCL1 expression was then enforced in both CSCs and GCLs by lentiviral-based transduction. The effect of SPARCL1 gain-of-function on microvascular proliferation, microglia activation and advanced imaging features was tested in intracranial xenografts by IHC and MRI and validated by chorioallantoic membrane (CAM) assays. SPARCL1 expression significantly enhanced the infiltrative and neoangiogenic features of PN and MES CSC/GCL-induced tumors, with the concomitant activation of inflammatory responses associated with the tumor microenvironment, thus resulting in experimental GBMs that reproduced both the parenchymal infiltration and the increased microvascular density, typical of GBM. Overall, these results indicate that SPARCL1 overexpression might be instrumental for the generation of CSC-derived preclinical models of GBM in which the main pathognomonic hallmarks of GBMs are retrievable, making them suitable for effective preclinical testing of therapeutics.
Assuntos
Neoplasias Encefálicas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glioblastoma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neovascularização Patológica/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Feminino , Xenoenxertos , Humanos , Camundongos , Microglia/metabolismoRESUMO
microRNAs (miRNAs) are short noncoding RNAs, which regulate gene expression post-transcriptionally and play crucial roles in relevant biological and pathological processes. Here, we investigated the putative role of miRNAs in modulating the tumor-initiating potential of mouse medulloblastoma (MB)-derived cancer stem cells (CSCs). We first subjected bona fide highly tumorigenic (HT) CSCs as well as lowly tumorigenic MB CSCs and normal neural stem cells to miRNA profiling, which identified a HT CSC-specific miRNA signature. Next, by cross-checking CSC mRNA/miRNA profiles, we pinpointed miR-135a as a potential tumor suppressor gene, which was strongly downregulated in HT CSCs as well as in the highly malignant experimental tumors derived from them. Remarkably, enforced expression of miR-135a in HT CSCs strongly inhibited tumorigenesis by repressing the miR-135a direct target gene Arhgef6. Considering the upregulation of Arhgef6 in human MBs and its involvement in mediating experimental medulloblastomagenesis, its efficient suppression by miR-135a might make available an effective therapeutic strategy to selectively impair the tumorigenic potential of MB CSCs. Stem Cells 2015;33:1377-1389.
Assuntos
Carcinogênese/patologia , Meduloblastoma/patologia , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Animais , Carcinogênese/genética , Agregação Celular , Transformação Celular Neoplásica/genética , Regulação para Baixo , Perfilação da Expressão Gênica , Meduloblastoma/genética , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Células-Tronco Neoplásicas/patologia , Células-Tronco Neurais/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
The dogma that the genesis of new cells is a negligible event in the adult mammalian brain has long influenced our perception and understanding of the origin and development of CNS tumours. The discovery that new neurons and glia are produced throughout life from neural stem cells provides new possibilities for the candidate cells of origin of CNS neoplasias. The emerging hypothesis is that alterations in the cellular and genetic mechanisms that control adult neurogenesis might contribute to brain tumorigenesis, thereby allowing the identification of new therapeutic strategies.
Assuntos
Neoplasias Encefálicas/patologia , Neoplasias do Sistema Nervoso Central/patologia , Células-Tronco Neoplásicas/citologia , Animais , Encéfalo/anatomia & histologia , Encéfalo/patologia , Humanos , Modelos Biológicos , Neurônios/metabolismoRESUMO
The mammalian target of rapamycin (mTOR) pathway is a central controller of growth and homeostasis, and, as such, is implicated in disease states where growth is deregulated, namely cancer, metabolic diseases, and hamartoma syndromes like tuberous sclerosis complex (TSC). Accordingly, mTOR is also a pivotal regulator of the homeostasis of several distinct stem cell pools in which it finely tunes the balance between stem cell self-renewal and differentiation. mTOR hyperactivation in neural stem cells (NSCs) has been etiologically linked to the development of TSC-associated neurological lesions, such as brain hamartomas and benign tumors. Animal models generated by deletion of mTOR upstream regulators in different types of NSCs reproduce faithfully some of the TSC neurological alterations. Thus, mTOR dysregulation in NSCs seems to be responsible for the derangement of their homeostasis, thus leading to TSC development. Here we review recent advances in the molecular dissection of the mTOR cascade, its involvement in the maintenance of stem cell compartments, and in particular the implications of mTOR hyperactivation in NSCs in vivo and in vitro.
Assuntos
Células-Tronco Neurais/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Síndrome do Hamartoma Múltiplo/metabolismo , Síndrome do Hamartoma Múltiplo/patologia , Humanos , Doenças Metabólicas/metabolismo , Doenças Metabólicas/patologia , Neoplasias/metabolismo , Neoplasias/patologia , Células-Tronco Neurais/patologia , Transdução de SinaisRESUMO
Bone marrow-derived cells contribute to tumor angiogenesis. Here, we demonstrate that monocytes expressing the Tie2 receptor (Tie2-expressing monocytes [TEMs]) (1) are a distinct hematopoietic lineage of proangiogenic cells, (2) are selectively recruited to spontaneous and orthotopic tumors, (3) promote angiogenesis in a paracrine manner, and (4) account for most of the proangiogenic activity of myeloid cells in tumors. Remarkably, TEM knockout completely prevented human glioma neovascularization in the mouse brain and induced substantial tumor regression. Besides TEMs and endothelial cells (ECs), Tie2 expression distinguished a rare population of tumor stroma-derived mesenchymal progenitors representing a primary source of tumor pericytes. Therefore, Tie2 expression characterizes three distinct cell types required for tumor neovascularization: ECs, proangiogenic cells of hematopoietic origin, and pericyte precursors of mesenchymal origin.
Assuntos
Glioblastoma/patologia , Monócitos/fisiologia , Pericitos/patologia , Receptor TIE-2/fisiologia , Animais , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Humanos , Mesoderma/citologia , Mesoderma/patologia , Camundongos , Camundongos Nus , Camundongos SCID , Camundongos Transgênicos , Neovascularização Patológica/fisiopatologia , Neoplasias Pancreáticas/patologia , Células-Tronco/patologia , Transplante HeterólogoRESUMO
Glioblastoma (GBM) is a common and deadly form of brain tumor in adults. Dysregulated metabolism in GBM offers an opportunity to deploy metabolic interventions as precise therapeutic strategies. To identify the molecular drivers and the modalities by which different molecular subgroups of GBM exploit metabolic rewiring to sustain tumor progression, we interrogated the transcriptome, the metabolome, and the glycoproteome of human subgroup-specific GBM sphere-forming cells (GSC). L-fucose abundance and core fucosylation activation were elevated in mesenchymal (MES) compared with proneural GSCs; this pattern was retained in subgroup-specific xenografts and in subgroup-affiliated human patient samples. Genetic and pharmacological inhibition of core fucosylation significantly reduced tumor growth in MES GBM preclinical models. Liquid chromatography-mass spectrometry (LC-MS)-based glycoproteomic screening indicated that most MES-restricted core-fucosylated proteins are involved in therapeutically relevant GBM pathological processes, such as extracellular matrix interaction, cell adhesion, and integrin-mediated signaling. Selective L-fucose accumulation in MES GBMs was observed using preclinical minimally invasive PET, implicating this metabolite as a potential subgroup-restricted biomarker.Overall, these findings indicate that L-fucose pathway activation in MES GBM is a subgroup-specific dependency that could provide diagnostic markers and actionable therapeutic targets. SIGNIFICANCE: Metabolic characterization of subgroup-specific glioblastoma (GBM) sphere-forming cells identifies the L-fucose pathway as a vulnerability restricted to mesenchymal GBM, disclosing a potential precision medicine strategy for targeting cancer metabolism.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/patologia , Fucose/metabolismo , Transdução de Sinais , Neoplasias Encefálicas/patologia , Células-Tronco Neoplásicas/patologia , Linhagem Celular TumoralRESUMO
The synthetic purine reversine has been shown to possess a dual activity as it promotes the de-differentiation of adult cells, including fibroblasts, into stem-cell-like progenitors, but it also induces cell growth arrest and ultimately cell death of cancer cells, suggesting its possible application as an anti-cancer agent. Aim of this study was to investigate the mechanism underneath reversine selectivity in inducing cell death of cancer cells by a comparative analysis of its effects on several tumor cells and normal dermal fibroblasts. We found that reversine is lethal for all cancer cells studied as it induces cell endoreplication, a process that malignant cells cannot effectively oppose due to aberrations in cell cycle checkpoints. On the other hand, normal cells, like dermal fibroblasts, can control reversine activity by blocking the cell cycle, entering a reversible quiescent state. However, they can be induced to become sensitive to the molecule when key cell cycle proteins, e.g., p53, are silenced.
Assuntos
Antineoplásicos/farmacologia , Morfolinas/farmacologia , Purinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Benzotiazóis/farmacologia , Western Blotting , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular , Morte Celular , Desdiferenciação Celular , Proliferação de Células , Forma Celular/efeitos dos fármacos , Sobrevivência Celular , Endorreduplicação , Ativação Enzimática , Fibroblastos/efeitos dos fármacos , Fibrossarcoma/genética , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Citometria de Fluxo , Inativação Gênica , Células HeLa , Humanos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genéticaRESUMO
We investigated the potential of antibody-vectorialized superparamagnetic iron oxide (SPIO) particles as cellular specific magnetic resonance contrast agents to image lymphocyte populations within the central nervous system (CNS), with the final goal of obtaining a reliable tool for noninvasively detecting and tracking specific cellular populations in vivo. We used superparamagnetic particles bound to a monoclonal antibody. The particle is the contrast agent, by means of its T2* relaxation properties; the antibody is the targeting vector, responsible for homing the particle to target a surface antigen. To investigate the efficiency of particle vectorialization by these antibodies, we compared two types of antibody-vectorialized CD3-specific particles in vivo. We successfully employed vectorialized SPIO particles to image B220⺠cells in a murine model of B-cell lymphoma. Likewise, we were able to identify CD3⺠infiltrates in a murine model of multiple sclerosis. The specificity of the technique was confirmed by immunohistochemistry and electron microscopy of corresponding sections. Our findings suggest that indirect binding of the antibody to a streptavidinated particle allows for enhanced particle vectorialization compared to covalent binding of the antibody to the particle.
Assuntos
Anticorpos Monoclonais , Encéfalo/citologia , Dextranos , Imageamento por Ressonância Magnética/métodos , Nanopartículas de Magnetita , Linfócitos T/citologia , Animais , Dextranos/administração & dosagem , Encefalomielite Autoimune Experimental/diagnóstico , Feminino , Humanos , Injeções , Linfoma de Células B/diagnóstico , Nanopartículas de Magnetita/administração & dosagem , Camundongos , Sensibilidade e EspecificidadeRESUMO
T cell receptor (TCR)-based therapy has the potential to induce durable clinical responses in patients with cancer by targeting intracellular tumor antigens with high sensitivity and by promoting T cell survival. However, the need for TCRs specific for shared oncogenic antigens and the need for manufacturing protocols able to redirect T cell specificity while preserving T cell fitness remain limiting factors. By longitudinal monitoring of T cell functionality and dynamics in 15 healthy donors, we isolated 19 TCRs specific for Wilms' tumor antigen 1 (WT1), which is overexpressed by several tumor types. TCRs recognized several peptides restricted by common human leukocyte antigen (HLA) alleles and displayed a wide range of functional avidities. We selected five high-avidity HLA-A*02:01-restricted TCRs, three that were specific to the less explored immunodominant WT137-45 and two that were specific to the noncanonical WT1-78-64 epitopes, both naturally processed by primary acute myeloid leukemia (AML) blasts. With CRISPR-Cas9 genome editing tools, we combined TCR-targeted integration into the TCR α constant (TRAC) locus with TCR ß constant (TRBC) knockout, thus avoiding TCRαß mispairing and maximizing TCR expression and function. The engineered lymphocytes were enriched in memory stem T cells. A unique WT137-45-specific TCR showed antigen-specific responses and efficiently killed AML blasts, acute lymphoblastic leukemia blasts, and glioblastoma cells in vitro and in vivo in the absence of off-tumor toxicity. T cells engineered to express this receptor are being advanced into clinical development for AML immunotherapy and represent a candidate therapy for other WT1-expressing tumors.
Assuntos
Leucemia Mieloide Aguda , Proteínas WT1 , Antígenos de Neoplasias , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T , Proteínas WT1/genética , Proteínas WT1/metabolismoRESUMO
Glioblastoma (GBM) represents the most common and aggressive tumor of the brain. Despite the fact that several studies have recently addressed the molecular mechanisms underlying the disease, its etiology and pathogenesis are still poorly understood. GBM displays poor prognosis and its resistance to common therapeutic approaches makes it a highly recurrent tumor. Several studies have identified a subpopulation of tumor cells, known as GBM cancer stem cells (CSCs) characterized by the ability of self-renewal, tumor initiation and propagation. GBM CSCs have been shown to survive GBM chemotherapy and radiotherapy. Thus, targeting CSCs represents a promising approach to treat GBM. Recent evidence has shown that GBM is characterized by a dysregulated expression of microRNA (miRNAs). In this study we have investigated the difference between human GBM CSCs and their paired autologous differentiated tumor cells. Array-based profiling and quantitative Real-Time PCR (qRT-PCR) were performed to identify miRNAs differentially expressed in CSCs. The Cancer Genome Atlas (TCGA) data were also interrogated, and functional interpretation analysis was performed. We have identified 14 miRNAs significantly differentially expressed in GBM CSCs (p < 0.005). MiR-21 and miR-95 were among the most significantly deregulated miRNAs, and their expression was also associated to patient survival. We believe that the data provided here carry important implications for future studies aiming at elucidating the molecular mechanisms underlying GBM.
RESUMO
The poor transport of molecular and nanoscale agents through the blood-brain barrier together with tumour heterogeneity contribute to the dismal prognosis in patients with glioblastoma multiforme. Here, a biodegradable implant (µMESH) is engineered in the form of a micrometre-sized poly(lactic-co-glycolic acid) mesh laid over a water-soluble poly(vinyl alcohol) layer. Upon poly(vinyl alcohol) dissolution, the flexible poly(lactic-co-glycolic acid) mesh conforms to the resected tumour cavity as docetaxel-loaded nanomedicines and diclofenac molecules are continuously and directly released into the adjacent tumour bed. In orthotopic brain cancer models, generated with a conventional, reference cell line and patient-derived cells, a single µMESH application, carrying 0.75 mg kg-1 of docetaxel and diclofenac, abrogates disease recurrence up to eight months after tumour resection, with no appreciable adverse effects. Without tumour resection, the µMESH increases the median overall survival (â¼30 d) as compared with the one-time intracranial deposition of docetaxel-loaded nanomedicines (15 d) or 10 cycles of systemically administered temozolomide (12 d). The µMESH modular structure, for the independent coloading of different molecules and nanomedicines, together with its mechanical flexibility, can be exploited to treat a variety of cancers, realizing patient-specific dosing and interventions.
Assuntos
Implantes Absorvíveis , Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias Encefálicas/tratamento farmacológico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular , Diclofenaco/farmacocinética , Diclofenaco/farmacologia , Docetaxel/farmacocinética , Docetaxel/farmacologia , Implantes de Medicamento/farmacocinética , Implantes de Medicamento/farmacologia , Feminino , Humanos , Camundongos , Camundongos Nus , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacocinética , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Glioblastoma (GBM) is a highly aggressive tumor of the brain. Despite the efforts, response to current therapies is poor and 2-years survival rate ranging from 6-12%. Here, we evaluated the preclinical efficacy of Metformin (MET) as add-on therapy to Temozolomide (TMZ) and the ability of [18F]FLT (activity of thymidine kinase 1 related to cell proliferation) and [18F]VC701 (translocator protein, TSPO) Positron Emission Tomography (PET) radiotracers to predict tumor response to therapy. Indeed, TSPO is expressed on the outer mitochondrial membrane of activated microglia/macrophages, tumor cells, astrocytes and endothelial cells. TMZ-sensitive (Gli36ΔEGFR-1 and L0627) or -resistant (Gli36ΔEGFR-2) GBM cell lines representative of classical molecular subtype were tested in vitro and in vivo in orthotopic mouse models. Our results indicate that in vitro, MET increased the efficacy of TMZ on TMZ-sensitive and on TMZ-resistant cells by deregulating the balance between pro-survival (bcl2) and pro-apoptotic (bax/bad) Bcl-family members and promoting early apoptosis in both Gli36ΔEGFR-1 and Gli36ΔEGFR-2 cells. In vivo, MET add-on significantly extended the median survival of tumor-bearing mice compared to TMZ-treated ones and reduced the rate of recurrence in the TMZ-sensitive models. PET studies with the cell proliferation radiopharmaceutical [18F]FLT performed at early time during treatment were able to distinguish responder from non-responder to TMZ but not to predict the duration of the effect. On the contrary, [18F]VC701 uptake was reduced only in mice treated with MET plus TMZ and levels of uptake negatively correlated with animals' survival. Overall, our data showed that MET addition improved TMZ efficacy in GBM preclinical models representative of classical molecular subtype increasing survival time and reducing tumor relapsing rate. Finally, results from PET imaging suggest that the reduction of cell proliferation represents a common mechanism of TMZ and combined treatment, whereas only the last was able to reduce TSPO. This reduction was associated with the duration of treatment response. TSPO-ligand may be used as a complementary molecular imaging marker to predict tumor microenvironment related treatment effects.
RESUMO
Medulloblastoma (MB), one of the most malignant brain tumors of childhood, comprises distinct molecular subgroups, with p53 mutant sonic hedgehog-activated (SHH-activated) MB patients having a very severe outcome that is associated with unfavorable histological large cell/anaplastic (LC/A) features. To identify the molecular underpinnings of this phenotype, we analyzed a large cohort of MB developing in p53-deficient Ptch+/- SHH mice that, unexpectedly, showed LC/A traits that correlated with mTORC1 hyperactivation. Mechanistically, mTORC1 hyperactivation was mediated by a decrease in the p53-dependent expression of mTORC1 negative regulator Tsc2. Ectopic mTORC1 activation in mouse MB cancer stem cells (CSCs) promoted the in vivo acquisition of LC/A features and increased malignancy; accordingly, mTORC1 inhibition in p53-mutant Ptch+/- SHH MB and CSC-derived MB resulted in reduced tumor burden and aggressiveness. Most remarkably, mTORC1 hyperactivation was detected only in p53-mutant SHH MB patient samples, and treatment with rapamycin of a human preclinical model phenocopying this subgroup decreased tumor growth and malignancy. Thus, mTORC1 may act as a specific druggable target for this subset of SHH MB, resulting in the implementation of a stringent risk stratification and in the potentially rapid translation of this precision medicine approach into the clinical setting.
Assuntos
Proteínas Hedgehog/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Meduloblastoma/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Meduloblastoma/patologia , CamundongosRESUMO
In this work we optimized a novel approach for combining in vivo MRI and ex vivo high-resolution fluorescence microscopy that involves: (i) a method for slicing rat brain tissue into sections with the same thickness and spatial orientation as in in vivo MRI, to better correlate in vivo MRI analyses with ex-vivo imaging via scanning confocal microscope and (ii) an improved clearing protocol compatible with lipophilic dyes that highlight the neurovascular network, to obtain high tissue transparency while preserving tissue staining and morphology with no significant tissue shrinkage or expansion. We applied this methodology in two rat models of glioblastoma (GBM; U87 human glioma cells and patient-derived human glioblastoma cancer stem cells) to demonstrate how vital the information retrieved from the correlation between MRI and confocal images is and to highlight how the increased invasiveness of xenografts derived from cancer stem cells may not be clearly detected by standard in vivo MRI approaches. The protocol studied in this work could be implemented in pre-clinical GBM research to further the development and validation of more predictive and translatable MR imaging protocols that can be used as critical diagnostic and prognostic tools. The development of this protocol is part of the quest for more efficacious treatment approaches for this devastating and still uncurable disease. In particular, this approach could be instrumental in validating novel MRI-based techniques to assess cellular infiltration beyond the macroscopic tumor margins and to quantify neo-angiogenesis.
Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Meios de Contraste , Corantes Fluorescentes , Glioblastoma/diagnóstico por imagem , Aumento da Imagem/métodos , Imageamento por Ressonância Magnética/métodos , Neuroimagem/métodos , Imagem Óptica/métodos , Animais , Neoplasias Encefálicas/irrigação sanguínea , Linhagem Celular Tumoral , Modelos Animais de Doenças , Glioblastoma/irrigação sanguínea , Humanos , Interações Hidrofóbicas e Hidrofílicas , Neovascularização Patológica , RatosRESUMO
Galectin-3 (Gal-3) is an extracellular matrix glycan-binding protein with several immunosuppressive and pro-tumor functions. The role of Galectin-3 in cancer stem-like cells (CSCs) is poorly investigated. Here, we show that prostate CSCs also colonizing prostate-draining lymph nodes of transgenic adenocarcinoma of the mouse prostate (TRAMP) mice overexpress Gal-3. Gal-3 contributes to prostate CSC-mediated immune suppression because either Gal-3 silencing in CSCs, or co-culture of CSCs and T cells in the presence of the Gal-3 inhibitor N-Acetyl-D-lactosamine rescued T cell proliferation. N-Acetyl-D-lactosamine also rescued the proliferation of T cells in prostate-draining lymph nodes of TRAMP mice affected by prostate intraepithelial neoplasia. Additionally, Gal-3 impacted prostate CSC tumorigenic and metastatic potential in vivo, as Gal-3 silencing in prostate CSCs reduced both primary tumor growth and secondary invasion. Gal-3 was also found expressed in more differentiated prostate cancer cells, but with different intracellular distribution as compared to CSCs, which suggests different functions of Gal-3 in the two cell populations. In fact, the prevalent nuclear and cytoplasmic distribution of Gal-3 in prostate CSCs made them less susceptible to apoptosis, when compared to more differentiated prostate cancer cells, in which Gal-3 was predominantly intra-cytoplasmic. Finally, we found Gal-3 expressed in human and mouse prostate intraepithelial neoplasia lesions and in metastatic lymph nodes. All together, these findings identify Gal-3 as a key molecule and a potential therapeutic target already in the early phases of prostate cancer progression and metastasis.
Assuntos
Adenocarcinoma/metabolismo , Galectina 3/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasia Prostática Intraepitelial/metabolismo , Neoplasias da Próstata/metabolismo , Evasão Tumoral , Adenocarcinoma/genética , Adenocarcinoma/imunologia , Adenocarcinoma/secundário , Animais , Proteínas Sanguíneas , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Cocultura , Galectina 3/genética , Galectinas , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática , Ativação Linfocitária , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Neoplásicas/imunologia , Neoplasia Prostática Intraepitelial/genética , Neoplasia Prostática Intraepitelial/imunologia , Neoplasia Prostática Intraepitelial/secundário , Neoplasias da Próstata/genética , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Transdução de Sinais , Microambiente TumoralRESUMO
In this work we showed that genotype-related patterns of hexosaminidase activity, isoenzyme composition, gene expression and ganglioside metabolism observed during embryonic and postnatal brain development are recapitulated during the progressive stages of neural precursor cell (NPC) differentiation to mature glia and neurons in vitro. Further, by comparing NPCs and their differentiated progeny established from Tay-Sachs (TS) and Sandhoff (SD) animal models with the wild-type counterparts, we studied the events linking the accumulation of undegraded substrates to hexosaminidase activity. We showed that similarly to what observed in brain tissues in TS NPCs and progeny, the stored GM2 was partially converted by sialidase to GA2, which can be then degraded in the lysosomes to its components. The latter can be used in a salvage pathway for the formation of GM3. Interestingly, results obtained from ganglioside feeding assays and from measurement of lysosomal sialidase activity suggest that a similar pathway might work also in the SD model.
Assuntos
Encéfalo/metabolismo , Modelos Animais de Doenças , Gangliosidoses GM2/metabolismo , Neurônios/metabolismo , Células-Tronco/metabolismo , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Encéfalo/patologia , Diferenciação Celular/fisiologia , Células Cultivadas , Gangliosidoses GM2/patologia , Camundongos , Camundongos Mutantes Neurológicos , Neurogênese/fisiologia , Neurônios/patologia , Células-Tronco/patologiaRESUMO
Glioblastoma multiforme (GBM) is the most invasive and undifferentiated type of brain tumour, and so surgical interventions are ineffective. We found that GluR2 is absent in fast-growing GBM-derived tumour stem cells and high-grade glioma specimens, but is expressed in slow-growing stem cells and low-grade glioma specimens. More remarkably, GluR2 overexpression in U-87MG cells inhibits proliferation by inactivating extracellular signal-regulated kinase (ERK)1/2-Src phosphorylation and induces apoptosis. Mechanistically, we observed that the scaffold protein GRIP is essential for the effect of GluR2 on ERK-Src inactivation. These findings indicate that the absence of the GluR2 subunit favours malignancy.
Assuntos
Apoptose/fisiologia , Caspase 3/metabolismo , Caspase 6/metabolismo , Glioma/fisiopatologia , Sistema de Sinalização das MAP Quinases/fisiologia , Receptores de AMPA/metabolismo , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ratos , Quinases da Família src/metabolismoRESUMO
Recent observations have suggested that extensive culturing of adult neural stem cells (ANSCs) by exploiting the NeuroSphere assay might select for aggressive cell clones, endowed with neoplastic potential, that overgrow the rest of the native stem cells. However, a detailed study of the propensity of ANSCs to transform has never been thoroughly undertaken. Here, we report the first demonstration that ANSCs can be propagated in vitro for over a year, maintaining a strikingly stable profile with regard to self-renewal, differentiation, growth factor dependence, karyotype, and molecular profiling. Most importantly, the long-term culturing of ANSCs did not result in the formation of tumors in vivo, even when ANSCs were transduced with Myc and Ras oncogenes. The cancer resistance could depend on specific mechanisms aimed at protecting ANSCs and preserved by optimal nonstressful culture conditions. In conclusion, besides a plentiful and safe source of cells for therapeutic applications, ANSCs provide an ideal model to study aging and cancer in the context of stemness.