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Glioblastomas are incurable tumors infiltrating the brain. A subpopulation of glioblastoma cells forms a functional and therapy-resistant tumor cell network interconnected by tumor microtubes (TMs). Other subpopulations appear unconnected, and their biological role remains unclear. Here, we demonstrate that whole-brain colonization is fueled by glioblastoma cells that lack connections with other tumor cells and astrocytes yet receive synaptic input from neurons. This subpopulation corresponds to neuronal and neural-progenitor-like tumor cell states, as defined by single-cell transcriptomics, both in mouse models and in the human disease. Tumor cell invasion resembled neuronal migration mechanisms and adopted a Lévy-like movement pattern of probing the environment. Neuronal activity induced complex calcium signals in glioblastoma cells followed by the de novo formation of TMs and increased invasion speed. Collectively, superimposing molecular and functional single-cell data revealed that neuronal mechanisms govern glioblastoma cell invasion on multiple levels. This explains how glioblastoma's dissemination and cellular heterogeneity are closely interlinked.
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Neoplasias Encefálicas , Glioblastoma , Animais , Astrócitos/patologia , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Camundongos , Invasividade Neoplásica , Neurônios/fisiologiaRESUMO
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/fisiopatologia , Progressão da Doença , Glioma/fisiopatologia , Sinapses/patologia , Animais , Neoplasias Encefálicas/ultraestrutura , Modelos Animais de Doenças , Glioma/ultraestrutura , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Neurônios/fisiologia , Receptores de AMPA/genética , Receptores de AMPA/metabolismoRESUMO
Early and progressive colonization of the healthy brain is one hallmark of diffuse gliomas, including glioblastomas. We recently discovered ultralong (>10 to hundreds of microns) membrane protrusions [tumor microtubes (TMs)] extended by glioma cells. TMs have been associated with the capacity of glioma cells to effectively invade the brain and proliferate. Moreover, TMs are also used by some tumor cells to interconnect to one large, resistant multicellular network. Here, we performed a correlative gene-expression microarray and in vivo imaging analysis, and identified novel molecular candidates for TM formation and function. Interestingly, these genes were previously linked to normal CNS development. One of the genes scoring highest in tests related to the outgrowth of TMs was tweety-homolog 1 (TTYH1), which was highly expressed in a fraction of TMs in mice and patients. Ttyh1 was confirmed to be a potent regulator of normal TM morphology and of TM-mediated tumor-cell invasion and proliferation. Glioma cells with one or two TMs were mainly responsible for effective brain colonization, and Ttyh1 downregulation particularly affected this cellular subtype, resulting in reduced tumor progression and prolonged survival of mice. The remaining Ttyh1-deficient tumor cells, however, had more interconnecting TMs, which were associated with increased radioresistance in those small tumors. These findings imply a cellular and molecular heterogeneity in gliomas regarding formation and function of distinct TM subtypes, with multiple parallels to neuronal development, and suggest that Ttyh1 might be a promising target to specifically reduce TM-associated brain colonization by glioma cells in patients.SIGNIFICANCE STATEMENT In this report, we identify tweety-homolog 1 (Ttyh1), a membrane protein linked to neuronal development, as a potent driver of tumor microtube (TM)-mediated brain colonization by glioma cells. Targeting of Ttyh1 effectively inhibited the formation of invasive TMs and glioma growth, but increased network formation by intercellular TMs, suggesting a functional and molecular heterogeneity of the recently discovered TMs with potential implications for future TM-targeting strategies.
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Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioblastoma/metabolismo , Glioblastoma/patologia , Proteínas de Membrana/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Masculino , Camundongos , Camundongos Nus , Invasividade NeoplásicaRESUMO
Tumor microtubes (TMs) connect glioma cells to a network with considerable relevance for tumor progression and therapy resistance. However, the determination of TM-interconnectivity in individual tumors is challenging and the impact on patient survival unresolved. Here, we establish a connectivity signature from single-cell RNA-sequenced (scRNA-Seq) xenografted primary glioblastoma (GB) cells using a dye uptake methodology, and validate it with recording of cellular calcium epochs and clinical correlations. Astrocyte-like and mesenchymal-like GB cells have the highest connectivity signature scores in scRNA-sequenced patient-derived xenografts and patient samples. In large GB cohorts, TM-network connectivity correlates with the mesenchymal subtype and dismal patient survival. CHI3L1 gene expression serves as a robust molecular marker of connectivity and functionally influences TM networks. The connectivity signature allows insights into brain tumor biology, provides a proof-of-principle that tumor cell TM-connectivity is relevant for patients' prognosis, and serves as a robust prognostic biomarker.
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Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Glioblastoma/genética , Glioma/genética , Neoplasias Encefálicas/genética , Proteína 1 Semelhante à Quitinase-3RESUMO
OBJECTIVE: To explore the effects of all-trans retinoic acid (ATRA) on glioma stem cell phenotype. METHODS: The glioma stem cell (GSC) from surgically resected human glioma specimens were isolated and enriched by neurosphere assay and then its differentiation was induced with all-trans retinoic acid (ATRA, 1 µmol/L) for 1 week. Markers were determined by flow cytometry, Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Side population cells were analyzed by flow cytometry. Growth characteristics were detected by neurosphere formation assay and cell cycle analysis. GSC and the differentiated cells (1×10(5)) were implanted stereotactically and intracranially into the Balb/c nude mice to compare the survival time. All data were analyzed with the SPSS software version 17.0. RESULTS: ATRA potently induced the differentiation of GSC and reduced glioma stem cell phenotype. And there were an elevated expression of glial fibrillary acidic protein (GFAP) and a reduced expression of such stem cell makers as CD133 and Nestin. The side population rate decreased. ATRA inhibited the neurosphere formation of GSC and induced the arrest of cell growth. ATRA could prolong the survival time. CONCLUSION: GSC may be differentiated efficiently by ATRA. The phenotype of GSC decreases obviously after the differentiation of ATRA and the survival time is prolonged. Thus ATRA may be applied for targeted therapies of glioma stem cell.
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Diferenciação Celular/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Tretinoína/farmacologia , Animais , Feminino , Glioma/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/citologia , Células Tumorais CultivadasRESUMO
Subsequently to the publication of the above article and a Corrigendum that addressed the issue of a misspelling of one of the authors' names (DOI: 10.3892/ijo.2019.4769; published online on April 2, 2019), the authors have subsequently discovered that Fig. 7 on p. 1079 contained a duplication in two of the panels that might cause the readers some confusion. The authors were able to re-examine the original data, repeat the experiment, and have decided to revise Fig. 7. The corrected version of Fig. 7, showing replacement data for the p-Akt and Cyclin D1 experiments, is shown on the next page. The authors confirm that these data continue to support the main conclusions presented in their paper, and are grateful to the Editor of International Journal of Oncology for allowing them this opportunity to publish a Corrigendum. They also apologize to the readership for any inconvenience caused. [the original article was published in International Journal of Oncology 10.3892/ijo.2018.4482].
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Both the perivascular niche (PVN) and the integration into multicellular networks by tumor microtubes (TMs) have been associated with progression and resistance to therapies in glioblastoma, but their specific contribution remained unknown. By long-term tracking of tumor cell fate and dynamics in the live mouse brain, differential therapeutic responses in both niches are determined. Both the PVN, a preferential location of long-term quiescent glioma cells, and network integration facilitate resistance against cytotoxic effects of radiotherapy and chemotherapy-independently of each other, but with additive effects. Perivascular glioblastoma cells are particularly able to actively repair damage to tumor regions. Population of the PVN and resistance in it depend on proficient NOTCH1 expression. In turn, NOTCH1 downregulation induces resistant multicellular networks by TM extension. Our findings identify NOTCH1 as a central switch between the PVN and network niche in glioma, and demonstrate robust cross-compensation when only one niche is targeted.
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Plasticidade Celular/fisiologia , Glioma/metabolismo , Microambiente Tumoral/fisiologia , Animais , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioma/patologia , Humanos , Camundongos , Células-Tronco Neoplásicas , Pericitos/metabolismo , Receptor Notch1/genéticaRESUMO
BACKGROUND: Malignant gliomas including glioblastomas are characterized by a striking cellular heterogeneity, which includes a subpopulation of glioma cells that becomes highly resistant by integration into tumor microtube (TM)-connected multicellular networks. METHODS: A novel functional approach to detect, isolate, and characterize glioma cell subpopulations with respect to in vivo network integration is established, combining a dye staining method with intravital two-photon microscopy, Fluorescence-Activated Cell Sorting (FACS), molecular profiling, and gene reporter studies. RESULTS: Glioblastoma cells that are part of the TM-connected tumor network show activated neurodevelopmental and glioma progression gene expression pathways. Importantly, many of them revealed profiles indicative of increased cellular stemness, including high expression of nestin. TM-connected glioblastoma cells also had a higher potential for reinitiation of brain tumor growth. Long-term tracking of tumor cell nestin expression in vivo revealed a stronger TM network integration and higher radioresistance of the nestin-high subpopulation. Glioblastoma cells that were both nestin-high and network-integrated were particularly able to adapt to radiotherapy with increased TM formation. CONCLUSION: Multiple stem-like features are strongly enriched in a fraction of network-integrated glioma cells, explaining their particular resilience.
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Neoplasias Encefálicas , Glioblastoma , Glioma , Encéfalo , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Glioblastoma/genética , Glioma/genética , Humanos , Células-Tronco Neoplásicas , Nestina/genéticaRESUMO
The effects of RNAi-mediated gene silencing of LRIG3 expression on cell cycle and survival of human glioma cell line GL15 and the possible mechanisms were explored. The plasmids pGenesil2-LRIG3-shRNA1 and pGenesil2-LRIG3-shRNA2 were transfected into GL15 glioma cells respectively by using Metafectine, and the transfected cells that stably suppressed LRIG3 expression were selected by G418. The control cells were transfected with negative control shRNA. The changes in LRIG3 mRNA and protein levels were measured by RT-PCR and Western blot. The apoptosis rate and cell cycle were analyzed by flow cytometry. As compared with the negative shRNA-transfected GL15 cells, LRIG3 mRNA expression in GL15 cells transfected with pGenesil2-LRIG3-shRNA1 and pGenesil2-LRIG3-shRNA2 was silenced by 52.4%, 63.8%, and LRIG3 protein expression was reduced by 50.9% and 67.4% respectively. The LRIG3-specific siRNA transfected cells had higher proliferation rate than control cells. Cell cycle analysis showed that silencing LRIG3 increased the percentage of G(2)/M phase cells and the proliferation index significantly (P<0.01). Silencing LRIG3 could inhibit the apoptosis of GL15 cells (P<0.05). These findings suggest that the siRNA targeting LRIG3 gene shows a dramatic inhibitory effect on RNA transcription and protein expression, then promoting the proliferation of GL15 cells, arresting GL15 cells in G(2)/M phase, and suppressing apoptosis of GL15 cells.
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Glioma/genética , Proteínas de Membrana/metabolismo , RNA Interferente Pequeno/genética , Apoptose/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Glioma/patologia , Humanos , Proteínas de Membrana/genética , Interferência de RNARESUMO
Following the publication of this article, the authors have realized that the name of the second author was misspelt: "Minghai Dong" should have appeared as "Minhai Dong". The correct information for the authors on this paper is presented above. The authors regret that this error made it into print, andapologize to the readership for any inconvenience caused. [the original article was published in International Journal of Oncology 53: 10691082, 2018; DOI: 10.3892/ijo.2018.4482].
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[This corrects the article DOI: 10.1155/2015/183821.].
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The leucinerich repeats and immunoglobulinlike domains (LRIG) gene family, comprising LRIG1, 2 and 3, encodes integral membrane proteins. It has been well established that LRIG1 negatively regulates multiple growth factor signaling pathways and is considered to be a tumor suppressor; however, the biological functions of LRIG2 remain largely unexplored. It was previously demonstrated that LRIG2 positively regulates epidermal growth factor receptor (EGFR) signaling, the most common aberrant receptor tyrosine kinase (RTK) signaling in glioblastoma multiforme (GBM), which promotes GBM growth. In the present study, the effect of LRIG2 on the proliferation of GBM cells was further addressed, as well as the possible mechanisms underlying the regulatory effect of LRIG2 on plateletderived growth factor receptor ß (PDGFRß) signaling, another common oncogenic RTK signaling pathway in GBM. First, the expression levels of endogenous LRIG2 and PDGFRß were found to vary notably in human GBM, and the LRIG2 expression level was positively correlated with the expression level of PDGFRß. Furthermore, to the best of our knowledge, this is the first study to demonstrate that LRIG2 promoted the PDGFBBinduced proliferation of GBM cells in vitro and in vivo through regulating the PDGFRß signalingmediated cell cycle progression. Mechanistically, LRIG2 has the ability to physically interact with PDGFRß, promoting the total expression and the activation of PDGFRß, and enhancing its downstream signaling pathways of Akt and signal transducer and activator of transcription 3 and the effectors of key regulators of cell cycle progression, resulting in increased GBM cell proliferation. Collectively, these data indicated that LRIG2 may serve as a tumor promoter gene in gliomagenesis by positively regulating PDGFRß signaling, another important oncogenic RTK signaling pathway, in addition to the previously reported EGFR signaling in GBM modulated by LRIG2, and validated LRIG2 as a promising therapeutic target for the treatment of GBM characterized by multiple aberrant RTK signaling.
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Neoplasias Encefálicas/patologia , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glicoproteínas de Membrana/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Adulto , Idoso , Animais , Neoplasias Encefálicas/cirurgia , Carcinogênese/patologia , Pontos de Checagem do Ciclo Celular/genética , Divisão Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Receptores ErbB/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Glioblastoma/patologia , Glioblastoma/cirurgia , Humanos , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Active angiogenesis is the basic pathological feature of glioma. Tumor angiogenesis is involved in vascular endothelial cell migration to the tumor tissue and in the formation of tube-like structures. The present study aimed to investigate the role of leucine-rich repeats and immunoglobulin-like domains 2 (LRIG2) in glioma angiogenesis. Glioma (n=50) and normal brain (n=20) tissue samples were collected from patients to detect the expression of LRIG2, epidermal growth factor receptor (EGFR), vascular endothelial growth factor A (VEGF-A), and cluster of differentiation 31 (CD31) using immunohistochemistry. In addition, the association between the expression of LRIG2 in glioma tissue and the microvessel density (MVD) was analyzed. In vitro, the expression of LRIG2 in human glioma U87 and U251 cell lines was knocked down. Subsequently, cell migration and tube formation assays of human umbilical vein endothelial cells (HUVECs) were performed using a coculture system. The protein expression levels of LRIG2, EGFR, phosphorylated-EGFR and VEGF-A were determined using western blotting. The results demonstrated that the expression levels of LRIG2, EGFR, VEGF-A and CD31 were highly upregulated in glioma tissue samples. Furthermore, LRIG2 expression in glioma tissue samples was significantly correlated with the MVD. In vitro, the downregulation of LRIG2 inhibited HUVEC migration and tube formation induced by coculture with glioma cells. The downregulation of LRIG2 resulted in decreased expression of EGFR and VEGF-A. The effects of the LRIG2 knockdown were reversed following EGF treatment. These findings suggest that LRIG2 is a potential target for the inhibition of glioma angiogenesis, which is possibly mediated via the EGFR/VEGF-A signaling pathway.
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Triptolide is a principal diterpene triepoxide from the Chinese medical plant Tripterygium wilfordii Hook. f., whose extracts have been utilized in dealing with diverse diseases in traditional Chinese medicine for centuries. Recently, the antitumor effect of triptolide has been found in several pre-clinical neoplasm models, but its effect on pituitary corticotroph adenomas has not been investigated so far. In this study, we are aiming to figure out the antitumor effect of triptolide and address the underlying molecular mechanism in AtT20 murine corticotroph cell line. Our results demonstrated that triptolide inhibited cell viability and colony number of AtT20 cells in a dose- and time-dependent pattern. Triptolide also suppressed proopiomelanocortin (Pomc) mRNA expression and extracellular adrenocorticotropic hormone (ACTH) secretion in AtT20 cells. Flow cytometry prompted that triptolide leaded to G2/M phase arrest, apoptosis program and mitochondrial membrane depolarization in AtT20 cells. Moreover, dose-dependent activation of caspase-3 and decreased Bcl2/Bax proportion were observed after triptolide treatment. By western blot analysis we found that triptolide impeded phosphorylation of NF-κB p65 subunit and extracellular signal-regulated kinase (ERK), along with reduction of cyclin D1, without any impact on other NF-κB related protein expression like total p65, p50, IκB-α, p-IκB-α. Furthermore, the mouse xenograft model revealed the inhibition of tumor growth and hormone secretion after triptolide administration. Altogether this compound might be a potential pharmaceutical choice in managing Cushing's disease.
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Corticotrofos/metabolismo , Corticotrofos/patologia , Diterpenos/farmacologia , NF-kappa B/metabolismo , Fenantrenos/farmacologia , Neoplasias Hipofisárias/metabolismo , Neoplasias Hipofisárias/patologia , Transdução de Sinais , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Clonais , Corticotrofos/efeitos dos fármacos , Compostos de Epóxi/farmacologia , Feminino , Hormônios/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Nus , Mitocôndrias/metabolismo , Pró-Opiomelanocortina/genética , Pró-Opiomelanocortina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
This study investigates the biokinetics of LGT proteome, a potential biomarker of severe TBI, in serum of severe TBI patients. The LGT proteome presents in the serum of severe TBI patients. The abundance diversity of LGT proteome is closely associated with pathologic condition of TBI patients. Serum LGT proteome may be used as a promising marker for evaluating severity of severe TBI.
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Proteínas Sanguíneas/metabolismo , Lesões Encefálicas/sangue , Proteoma/metabolismo , Índices de Gravidade do Trauma , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Hedyotis diffusa Willd (Rubiaceae) (HDW) has been widely applied for the treatment of tumors, inflammation and toxication in traditional Chinese medicine. The antitumor effect of HDW on glioblastoma has been rarely reported. We aim to evaluate the activity of this extract and explore the underlying mechanism in U87 human glioblastoma cell line. MATERIALS AND METHODS: Cytotoxicity of HDW extract on U87 cells was measured by MTT assay. Apoptosis, cell cycle arrest and mitochondrial membrane potential (MMP) collapse induced by HDW extract were determined by flow cytometry. Caspase activity was analyzed based on colorimetric assay with a microplate spectrophotometer. Protein expression was examined by Western blot. RESULTS: HDW extract suppressed U87 cells growth in a dose- and time-dependent manner. Flow cytometry showed that HDW extract induced significant apoptosis, S/G2-M phase arrest and MMP collapse in U87 cells. Furthermore, dose-dependent activation of caspase-3, Bcl-2, Bax and ERK was observed with HDW extract treatment. Decreased Bcl-2/Bax ratio and Akt suppression were readily found as well. CONCLUSIONS: Induction of mitochondria-mediated apoptosis played an essential role in antitumor activity of HDW extract in U87 cells, in which ERKs and Akt signaling proteins were also involved. These findings contributed to the feasibility of using HDW extract in glioblastoma treatment and the understanding of the molecular mechanism.
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Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glioblastoma/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Rubiaceae/química , Neoplasias Encefálicas/enzimologia , Linhagem Celular Tumoral , Citometria de Fluxo , Glioblastoma/enzimologia , Humanos , Extratos VegetaisRESUMO
Glioma stem cells (GSCs), characterized by self-renewal, multi-potentiality and tumorigenicity, are responsible for the tumor propagation, recurrence and resistance to traditional treatments, representing a critical therapeutic target. Neural stem cells (NSCs) possess inherent tropism to brain tumor cells and inhibit their growth. However, there is a limited understanding of the mechanism underlying NSC tropism and the effect of NSC migration on GSC stemness phenotypes. In the present study, we showed that GSCs exhibited enhanced chemotaxis for NSC tropism compared with their differentiated cells. Chemokines secreted by GSCs contributed to the targeted migration of NSCs. Hypoxia enhanced NSC tropism via the upregulated chemokine expression of GSCs, such as VEGF, EGF and bFGF. In vitro migration of NSCs induced GSC differentiation and reduced stem-like phenotypes. Moreover, in vivo data provided direct evidence that transplanted NSCs could migrate to GSCs from either the homolateral or contralateral brain injection site, which prolonged the survival of grafted mice. Taken together, these findings show that NSCs preferentially migrate to GSCs and reduce their stemness phenotypes, raising the intriguing possibility that the targeted migration of NSCs can be applied as a novel therapeutic strategy to target these intractable brain tumors.
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Neoplasias Encefálicas/genética , Movimento Celular/genética , Glioma/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neurais/metabolismo , Animais , Neoplasias Encefálicas/patologia , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Glioma/patologia , Humanos , Camundongos , Células-Tronco Neoplásicas/patologia , Células-Tronco Neurais/patologiaRESUMO
The human leucine-rich repeats and immunoglobulin-like domains (LRIG) gene family contains LRIG1, 2 and 3, encoding integral membrane proteins with an ectodomain, a transmembrane domain and a cytoplasmic tail. LRIG1 negatively regulates multiple receptor tyrosine kinases signaling including the epidermal growth factor receptor (EGFR) and is a proposed tumor suppressor. The soluble LRIG1 ectodomain is demonstrated to be shed naturally and inhibit the progression of glioma. However, little is known regarding the functions of LRIG2. In oligodendroglioma, LRIG2 expression is associated with poor survival, suggesting that LRIG2 might have different functions compared with LRIG1. Since soluble LRIG1 ectodomain has a similar function to the full-length LRIG1, we hypothesize that the different roles exerted by LRIG2 and LRIG1 result from the difference of their ectodomains. Here, we addressed the functions of LRIG2 and LRIG2 ectodomain in the proliferation and apoptosis of glioma and the possible underlying mechanisms. Firstly, we found that LRIG2 expression levels positively correlated with the grade of glioma. Further, we demonstrated for the first time that soluble LRIG2 ectodomain was capable of being released from glioblastoma cells and exerted a pro-proliferative effect. Overexpression of LRIG2 ectodomain promoted the proliferation and inhibited the apoptosis of glioblastoma cells in vitro and in vivo in a similar manner to the full-length LRIG2. Both full-length LRIG2 and LRIG2 ectodomain were found to physically interact with EGFR, enhance the activation of EGFR and its downstream PI3 K/Akt pathway. To our knowledge, this is the first report demonstrating that soluble LRIG2 ectodomain is capable of being released from glioblastoma cells and exerts a similar role to the full-length LRIG2 in the regulation of EGFR signaling in the progression of glioblastoma. LRIG2 ectodomain, with potent pro-tumor effects, holds promise for providing a new therapeutic target for the treatment of glioblastoma.
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Apoptose , Proliferação de Células , Glioblastoma/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Feminino , Glioblastoma/patologia , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , ProteóliseRESUMO
Glioblastoma, the most common and lethal type of intracranial tumor, is characterized by extensive heterogeneity at the cellular and molecular levels. The discovery of glioma stem cells (GSCs) lends support to a new paradigm in tumor biology. In the present study, we aimed to clarify the validity of using U251 glioma cells as a source of GSC culture and critically evaluate the heterogeneous stem-like phenotypes of these cells when grown under various culture conditions. The findings suggested that U251 cells (U251-Adh, U251-SC-Sph and U251-SC-Adh) showed distinctive growth patterns and self-renewal capacity. The U251 glioma cell line is endowed with certain GSC phenotypes that may be moderately enriched in vitro when transferred into stem cell culture conditions, although this is not sustainable and reproducible in vivo. Notably, glioma cells are plastic in response to their environment. The reversible adaptive plasticity contributes to the GSC heterogeneity, which may lead to the heterogeneity of glioblastoma and the differing responses to current therapies. Therefore, an improved understanding of GSC heterogeneity is urgently required for designing more effective therapies against this highly malignant brain tumor.
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The LRIG1 [leucine-rich repeats and immunoglobulin-like domains (LRIG)] gene is not universally downregulated in human cancers, and its role in tumorigenesis and the development of glioma has not been well addressed. In this study, we used short hairpin RNA (shRNA)-triggered RNA interference (RNAi) to block LRIG1 gene expression in the GL15 human glioma cell line. Specific downregulation of LRIG1 by shRNA resulted in significantly enhanced capabilities of proliferation, inhibition of apoptosis and invasion in the GL15 cells. LRIG1 repression induced marked activation of epidermal growth factor receptor (EGFR), protein kinase B (Akt) and c-Myc signaling molecules. Our results demonstrated that RNAi against LRIG1 may effectively downregulate LRIG1 gene expression. LRIG1 functions as a tumor suppressor in the pathogenesis of glioma via EGFR/Akt/c-Myc activation.