RESUMO
BACKGROUND AND PURPOSE: The poor delivery of drugs to infiltrating tumor cells contributes to therapeutic failure in glioblastoma. During the early phase of an anti-angiogenic treatment, a remodeling of the tumor vasculature could occur, leading to a more functional vessel network that could enhance drug delivery. However, the restructuration of blood vessels could increase the proportion of normal endothelial cells that could be a barrier for the free diffusion of drugs. The net balance, in favor or not, of a better delivery of compounds during the course of an antiangiogenic treatment remains to be established. This study explored whether cediranib and thalidomide could modulate perfusion and vessel permeability in the brain U87 tumor mouse model. METHODS: The dynamic evolution of the diffusion of agents outside the tumor core using the fluorescent dye Evans Blue in histology and Gd-DOTA using dynamic contrast-enhanced (DCE)-MRI. CD31 labelling of endothelial cells was used to measure the vascular density. RESULTS AND INTERPRETATION: Cediranib and thalidomide effectively reduced tumor size over time. The accessibility of Evans Blue outside the tumor core continuously decreased over time. The vascular density was significantly decreased after treatment while the proportion of normal vessels remained unchanged over time. In contrast to histological studies, DCE-MRI did not tackle any significant change in hemodynamic parameters, in the core or margins of the tumor, whatever the parameter used or the pharmacokinetic model used. While cediranib and thalidomide were effective in decreasing the tumor size, they were ineffective in transiently increasing the delivery of agents in the core and the margins of the tumor.
Assuntos
Inibidores da Angiogênese , Neoplasias Encefálicas , Glioblastoma , Quinazolinas , Talidomida , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/irrigação sanguínea , Talidomida/farmacologia , Talidomida/uso terapêutico , Animais , Inibidores da Angiogênese/farmacologia , Camundongos , Quinazolinas/farmacologia , Quinazolinas/farmacocinética , Quinazolinas/uso terapêutico , Humanos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Imageamento por Ressonância Magnética , Ensaios Antitumorais Modelo de Xenoenxerto , Permeabilidade Capilar/efeitos dos fármacos , Camundongos Nus , Linhagem Celular Tumoral , IndóisRESUMO
Glioblastoma is the most aggressive tumor in the central nervous system, with a survival rate of less than 15 months despite multimodal therapy. Tumor recurrence frequently occurs after removal. Tumoral angiogenesis, the formation of neovessels, has a positive impact on tumor progression and invasion, although there are controversial results in the specialized literature regarding its impact on survival. This study aims to correlate the immunoexpression of angiogenesis markers (CD34, CD105) with the proliferation index Ki67 and p53 in primary and secondary glioblastomas. This retrospective study included 54 patients diagnosed with glioblastoma at the Pathology Department of County Emergency Clinical Hospital Târgu MureÈ. Microvascular density was determined using CD34 and CD105 antibodies, and the results were correlated with the immunoexpression of p53, IDH1, ATRX and Ki67. The number of neoformed blood vessels varied among cases, characterized by different shapes and calibers, with endothelial cells showing modified morphology and moderate to marked pleomorphism. Neovessels with a glomeruloid aspect, associated with intense positivity for CD34 or CD105 in endothelial cells, were observed, characteristic of glioblastomas. Mean microvascular density values were higher for the CD34 marker in all cases, though there were no statistically significant differences compared to CD105. Mutant IDH1 and ATRX glioblastomas, wild-type p53 glioblastomas, and those with a Ki67 index above 20% showed a more abundant microvascular density, with statistical correlations not reaching significance. This study highlighted a variety of percentage intervals of microvascular density in primary and secondary glioblastomas using immunohistochemical markers CD34 and CD105, respectively, with no statistically significant correlation between evaluated microvascular density and p53 or Ki67.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Isocitrato Desidrogenase , Antígeno Ki-67 , Densidade Microvascular , Neovascularização Patológica , Proteína Supressora de Tumor p53 , Proteína Nuclear Ligada ao X , Humanos , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioblastoma/irrigação sanguínea , Glioblastoma/genética , Proteína Supressora de Tumor p53/metabolismo , Antígeno Ki-67/metabolismo , Feminino , Pessoa de Meia-Idade , Masculino , Idoso , Adulto , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/genética , Proteína Nuclear Ligada ao X/metabolismo , Proteína Nuclear Ligada ao X/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Estudos Retrospectivos , Endoglina/metabolismo , Endoglina/genética , Antígenos CD34/metabolismo , Biomarcadores Tumorais/metabolismo , Imuno-HistoquímicaRESUMO
BACKGROUND: Vasculogenic mimicry (VM) is an enigmatic physiological feature that influences blood supply within glioblastoma (GBM) tumors for their sustained growth. Previous studies identify NFATC3, FOSL1 and HNRNPA2B1 as significant mediators of VEGFR2, a key player in vasculogenesis, and their molecular relationships may be crucial for VM in GBM. AIMS: The aim of this study was to understand how NFATC3, FOSL1 and HNRNPA2B1 collectively influence VM in GBM. METHODS: We have investigated the underlying gene regulatory mechanisms for VM in GBM cell lines U251 and U373 in vitro and in vivo. In vitro cell-based assays were performed to explore the role of NFATC3, FOSL1 and HNRNPA2B1 in GBM cell proliferation, VM and migration, in the context of RNA interference (RNAi)-mediated knockdown alongside corresponding controls. Western blotting and qRT-PCR assays were used to examine VEGFR2 expression levels. CO-IP was employed to detect protein-protein interactions, ChIP was used to detect DNA-protein complexes, and RIP was used to detect RNA-protein complexes. Histochemical staining was used to detect VM tube formation in vivo. RESULTS: Focusing on NFATC3, FOSL1 and HNRNPA2B1, we found each was significantly upregulated in GBM and positively correlated with VM-like cellular behaviors in U251 and U373 cell lines. Knockdown of NFATC3, FOSL1 or HNRNPA2B1 each resulted in decreased levels of VEGFR2, a key growth factor gene that drives VM, as well as the inhibition of proliferation, cell migration and extracorporeal VM activity. Chromatin immunoprecipitation (ChIP) studies and luciferase reporter gene assays revealed that NFATC3 binds to the promoter region of VEGFR2 to enhance VEGFR2 gene expression. Notably, FOSL1 interacts with NFATC3 as a co-factor to potentiate the DNA-binding capacity of NFATC3, resulting in enhanced VM-like cellular behaviors. Also, level of NFATC3 protein in cells was enhanced through HNRNPA2B1 binding of NFATC3 mRNA. Furthermore, RNAi-mediated silencing of NFATC3, FOSL1 and HNRNPA2B1 in GBM cells reduced their capacity for tumor formation and VM-like behaviors in vivo. CONCLUSION: Taken together, our findings identify NFATC3 as an important mediator of GBM tumor growth through its molecular and epistatic interactions with HNRNPA2B1 and FOSL1 to influence VEGFR2 expression and VM-like cellular behaviors.
Assuntos
Movimento Celular , Proliferação de Células , Glioblastoma , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , Fatores de Transcrição NFATC , Neovascularização Patológica , Proteínas Proto-Oncogênicas c-fos , Humanos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioblastoma/genética , Glioblastoma/irrigação sanguínea , Linhagem Celular Tumoral , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Fatores de Transcrição NFATC/metabolismo , Fatores de Transcrição NFATC/genética , Animais , Proliferação de Células/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Movimento Celular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Regulação Neoplásica da Expressão Gênica , Camundongos , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/irrigação sanguínea , Camundongos NusRESUMO
Glioblastoma (GBM) is a highly angiogenic malignancy of the central nervous system that resists standard antiangiogenic therapy, in part because of an alternative process to angiogenesis termed vasculogenic mimicry. Intricately linked to GBM, dysregulation of the Hippo signaling pathway leads to overexpression of YAP/TEAD and several downstream effectors involved in therapy resistance. Little is known about whether vasculogenic mimicry and the Hippo pathway intersect in the GBM chemoresistance phenotype. This study seeks to investigate the expression patterns of Hippo pathway regulators within clinically annotated GBM samples, examining their involvement in vitro regarding vasculogenic mimicry. In addition, it aims to assess the potential for pharmacological targeting of this pathway. In-silico analysis of the Hippo signaling members YAP1 , TEAD1 , AXL , NF2 , CTGF , and CYR61 transcript levels in low-grade GBM and GBM tumor tissues was done by Gene Expression Profiling Interactive Analysis. Gene expression was analyzed by real-time quantitative PCR from human U87, U118, U138, and U251 brain cancer cell lines and in clinically annotated brain tumor cDNA arrays. Transient gene silencing was performed with specific small interfering RNA. Vasculogenic mimicry was assessed using a Cultrex matrix, and three-dimensional capillary-like structures were analyzed with Wimasis. CYR61 and CTGF transcript levels were elevated in GBM tissues and were further induced when in-vitro vasculogenic mimicry was assessed. Silencing of CYR61 and CTGF , or treatment with a small-molecule TEAD inhibitor LM98 derived from flufenamic acid, inhibited vasculogenic mimicry. Silencing of SNAI1 and FOXC2 also altered vasculogenic mimicry and reduced CYR61 / CTGF levels. Pharmacological targeting of the Hippo pathway inhibits in-vitro vasculogenic mimicry. Unraveling the connections between the Hippo pathway and vasculogenic mimicry may pave the way for innovative therapeutic strategies.
Assuntos
Neoplasias Encefálicas , Fator de Crescimento do Tecido Conjuntivo , Proteína Rica em Cisteína 61 , Glioblastoma , Fatores de Transcrição , Humanos , Glioblastoma/irrigação sanguínea , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/genética , Glioblastoma/metabolismo , Proteína Rica em Cisteína 61/genética , Proteína Rica em Cisteína 61/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Fatores de Transcrição/genética , Neovascularização Patológica/tratamento farmacológico , Fatores de Transcrição de Domínio TEA , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas de Sinalização YAPRESUMO
BACKGROUND: The accurate differentiation of primary central nervous system lymphoma (PCNSL) from glioblastoma multiforme (GBM) is clinically crucial due to the different treatment strategies between them. PURPOSE: To define magnetic resonance imaging (MRI) perfusion findings in PCNSL to make a safe distinction from GBM with dynamic contrast-enhanced (DCE) T1 and DSC T2 MRI perfusion findings. MATERIAL AND METHODS: This retrospective analysis included 19 patients with histopathologically diagnosed PCNSL and 21 individuals with GBM. DCE T1 vascular permeability perfusion values including K-trans, Ve, Kep, IAUGC, and DSC T2 perfusion values including cerebral blood volume (CBV) and cerebral blood flow (CBF) in axial sections from the pathological lesion and contralateral normal brain parenchyma were measured quantitatively using region of interest analysis. RESULTS: The study observed no statistically significant difference between patients with PCNSL (T/B cell) and GBM in the median values of DCE T1 perfusion ratios (P > 0.05). Nevertheless, the DSC T2 perfusion ratios showed a substantial distinction between the two groups. In contrast to patients with PCNSL (1.185 vs. 1.224, respectively), those with GBM had higher median levels of r-CBV and r-CBF (2.898 vs. 2.467, respectively; P 0.01). A cutoff value of ≤1.473 for r-CBV (Lesion/N) and ≤1.6005 for r-CBF (Lesion/N) was found to estimate the positivity of PCNSL. CONCLUSION: DSC T2 MRI perfusion values showed lower r-CBV and r-CBF values in PCNSL patients compared to GBM patients. According to the findings, r-CBV and r-CBF are the most accurate MRI perfusion parameters for distinguishing between PCSNL and GBM.
Assuntos
Neoplasias Encefálicas , Meios de Contraste , Glioblastoma , Linfoma , Imageamento por Ressonância Magnética , Humanos , Masculino , Feminino , Glioblastoma/diagnóstico por imagem , Glioblastoma/irrigação sanguínea , Pessoa de Meia-Idade , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/patologia , Estudos Retrospectivos , Linfoma/diagnóstico por imagem , Adulto , Idoso , Imageamento por Ressonância Magnética/métodos , Diagnóstico Diferencial , Encéfalo/diagnóstico por imagem , Encéfalo/irrigação sanguíneaRESUMO
Monocyte-derived tumor-associated macrophages (Mo-TAMs) intensively infiltrate diffuse gliomas with remarkable heterogeneity. Using single-cell transcriptomics, we chart a spatially resolved transcriptional landscape of Mo-TAMs across 51 patients with isocitrate dehydrogenase (IDH)-wild-type glioblastomas or IDH-mutant gliomas. We characterize a Mo-TAM subset that is localized to the peri-necrotic niche and skewed by hypoxic niche cues to acquire a hypoxia response signature. Hypoxia-TAM destabilizes endothelial adherens junctions by activating adrenomedullin paracrine signaling, thereby stimulating a hyperpermeable neovasculature that hampers drug delivery in glioblastoma xenografts. Accordingly, genetic ablation or pharmacological blockade of adrenomedullin produced by Hypoxia-TAM restores vascular integrity, improves intratumoral concentration of the anti-tumor agent dabrafenib, and achieves combinatorial therapeutic benefits. Increased proportion of Hypoxia-TAM or adrenomedullin expression is predictive of tumor vessel hyperpermeability and a worse prognosis of glioblastoma. Our findings highlight Mo-TAM diversity and spatial niche-steered Mo-TAM reprogramming in diffuse gliomas and indicate potential therapeutics targeting Hypoxia-TAM to normalize tumor vasculature.
Assuntos
Adrenomedulina , Neoplasias Encefálicas , Glioblastoma , Macrófagos Associados a Tumor , Humanos , Glioblastoma/patologia , Glioblastoma/tratamento farmacológico , Glioblastoma/irrigação sanguínea , Glioblastoma/genética , Glioblastoma/metabolismo , Animais , Adrenomedulina/genética , Adrenomedulina/metabolismo , Camundongos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Macrófagos Associados a Tumor/metabolismo , Neovascularização Patológica/genética , Microambiente Tumoral , Isocitrato Desidrogenase/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular Tumoral , Macrófagos/metabolismo , Hipóxia CelularRESUMO
Although bevacizumab (BVZ), a representative drug for anti-angiogenesis therapy (AAT), is used as a first-line treatment for patients with glioblastoma (GBM), its efficacy is notably limited. Whereas several mechanisms have been proposed to explain the acquisition of AAT resistance, the specific underlying mechanisms have yet to be sufficiently ascertained. Here, we established that inhibitor of differentiation 1 (ID1)high/activin Ahigh glioblastoma cell confers resistance to BVZ. The bipotent effect of activin A during its active phase was demonstrated to reduce vasculature dependence in tumorigenesis. In response to a temporary exposure to activin A, this cytokine was found to induce endothelial-to-mesenchymal transition via the Smad3/Slug axis, whereas prolonged exposure led to endothelial apoptosis. ID1 tumors showing resistance to BVZ were established to be characterized by a hypovascular structure, hyperpermeability, and scattered hypoxic regions. Using a GBM mouse model, we demonstrated that AAT resistance can be overcome by administering therapy based on a combination of BVZ and SB431542, a Smad2/3 inhibitor, which contributed to enhancing survival. These findings offer valuable insights that could contribute to the development of new strategies for treating AAT-resistant GBM.
Assuntos
Ativinas , Inibidores da Angiogênese , Bevacizumab , Resistencia a Medicamentos Antineoplásicos , Glioblastoma , Proteína 1 Inibidora de Diferenciação , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/metabolismo , Glioblastoma/irrigação sanguínea , Humanos , Animais , Proteína 1 Inibidora de Diferenciação/metabolismo , Proteína 1 Inibidora de Diferenciação/genética , Camundongos , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Ativinas/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linhagem Celular Tumoral , Bevacizumab/farmacologia , Bevacizumab/uso terapêutico , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Camundongos Nus , Apoptose/efeitos dos fármacosRESUMO
Purpose: Primary central nervous system vasculitis (PCNSV) is a rare inflammatory disease affecting the central nervous system. In some cases, it presents with large, solitary lesion with extensive mass effect that mimic intracranial neoplasms. This condition results in a diagnostic confusion for neuroradiologists because the differentiation is almost impossible on conventional MRI sequences. The aim of this study is to reveal the significance of dynamic susceptibility contrast (DSC) perfusion-weighted imaging in differentiating of tumefactive PCNSV (t-PCNSV) lesions from intracranial neoplasms such as glio-blastomas and metastasis. Methods: In this retrospective study, DSC of 8 patients with biopsy-proven t-PCNSV has been compared with DSC obtained in 10 patients with glioblastoma, 10 patients with metastasis, who underwent surgery and histopathological confirmation. The ratio of relative cerebral blood volume (rrCBV) was calculated by rCBV (lesion) / rCBV (controlateral normal-appearing white matter) in the gadolinium-enhancing solid areas. Results: The mean rrCBV was 0.86±0.7 (range: 0.76-0.98) in the patients with t-PCNSV, 5,16±0.79 in patients with glioblastoma (range: 3.9-6.3), and 4.27±0.73 (range: 2.8-5.3) in patients with metastases. Conclusion: DSC-PWI seems to be useful in the diagnostic work-up of t-PCSNVs. A low rrCBV, i.e. a rCBV similar or lower to that of the contralateral normal white matter, seems to be consistent with the possibility of t-PCSNV.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Vasculite do Sistema Nervoso Central , Humanos , Glioblastoma/irrigação sanguínea , Glioblastoma/patologia , Estudos Retrospectivos , Imageamento por Ressonância Magnética/métodos , Neoplasias Encefálicas/diagnóstico por imagem , Vasculite do Sistema Nervoso Central/diagnóstico por imagem , PerfusãoRESUMO
PURPOSE: The reproducibility of relative cerebral blood volume (rCBV) measurements among readers with different levels of experience is a concern. This study aimed to investigate the inter-reader reproducibility of rCBV measurement of glioblastomas using the hotspot method in dynamic susceptibility contrast perfusion magnetic resonance imaging (DSC-MRI) with various strategies. METHODS: In this institutional review board-approved single-center study, 30 patients with glioblastoma were retrospectively evaluated with DSC-MRI at a 3.0 Tesla scanner. Three groups of reviewers, including neuroradiologists, general radiologists, and radiology residents, calculated the rCBV based on the number of regions of interest (ROIs) and reference areas. For statistical analysis of feature reproducibility, the intraclass correlation coefficient (ICC) and Bland-Altman plots were used. Analyses were made among individuals, reader groups, reader-group pooling, and a population that contained all of them. RESULTS: For individuals, the highest inter-reader reproducibility was observed between neuroradiologists [ICC: 0.527; 95% confidence interval (CI): 0.21-0.74] and between residents (ICC: 0.513; 95% CI: 0.20-0.73). There was poor reproducibility in the analyses of individuals with different levels of experience (ICC range: 0.296-0.335) and in reader-wise and group-wise pooling (ICC range: 0.296-0.335 and 0.397-0.427, respectively). However, an increase in ICC values was observed when five ROIs were used. In an analysis of all strategies, the ICC for the centrum semiovale was significantly higher than that for contralateral white matter (P < 0.001). CONCLUSION: The inter-reader reproducibility of rCBV measurement was poor to moderate regardless of whether it was calculated by neuroradiologists, general radiologists, or residents, which may indicate the need for automated methods. Choosing five ROIs and using the centrum semiovale as a reference area may increase reliability for all users.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/diagnóstico por imagem , Glioblastoma/irrigação sanguínea , Glioblastoma/patologia , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/patologia , Volume Sanguíneo Cerebral , Reprodutibilidade dos Testes , Estudos Retrospectivos , Meios de Contraste , Angiografia por Ressonância Magnética/métodos , Perfusão , Imageamento por Ressonância Magnética/métodosRESUMO
Vasculogenic mimicry (VM) is an endothelium-independent tumor microcirculation that provides adequate blood supply for tumor growth. The presence of VM greatly hinders the treatment of glioblastoma (GBM) with anti-angiogenic drugs. Therefore, targeting VM formation may be a feasible therapeutic strategy for GBM. The research aimed to evaluate the roles of BUD13, CDK12, MBNL1 in regulating VM formation of GBM. BUD13 and CDK12 were upregulated and MBNL1 was downregulated in GBM tissues and cells. Knockdown of BUD13, CDK12, or overexpression of MBNL1 inhibited GBM VM formation. METTL3 enhanced the stability of BUD13 mRNA and upregulated its expression through m6A methylation. BUD13 enhanced the stability of CDK12 mRNA and upregulated its expression. CDK12 phosphorylated MBNL1, thereby regulating VM formation of GBM. The simultaneous knockdown of BUD13, CDK12, and overexpression of MBNL1 reduced the volume of subcutaneously transplanted tumors in nude mice and prolonged the survival period. Thus, the BUD13/CDK12/MBNL1 axis plays a crucial role in regulating VM formation of GBM and provides a potential target for GBM therapy.
Assuntos
Quinases Ciclina-Dependentes , Proteínas de Ligação a DNA , Glioblastoma , Metiltransferases , Proteínas de Ligação a RNA , Animais , Camundongos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Glioblastoma/irrigação sanguínea , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Metilação , Camundongos Nus , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Microcirculação/genética , Microcirculação/fisiologia , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismoRESUMO
Glioblastoma multiforme (GBM) is a highly vascularized malignant brain tumor. Our previous study showed that prostate-specific membrane antigen (PSMA) promotes angiogenesis of GBM. However, the specific mechanism underlying GBM-induced PSMA upregulation remains unclear. In this study, we demonstrate that the GBM-secreted cytokine phosphoprotein 1 (SPP1) can regulate the expression of PSMA in human umbilical vein endothelial cells (HUVECs). Our mechanistic study further reveals that SPP1 regulates the expression of PSMA through the transcription factor HIF1α. Moreover, SPP1 promotes HUVEC migration and tube formation. In addition, HIF1α knockdown reduces the expression of PSMA in HUVECs and blocks the ability of SPP1 to promote HUVEC migration and tube formation. We further confirm that SPP1 is abundantly expressed in GBM, is associated with poor prognosis, and has high clinical diagnostic value with considerable sensitivity and specificity. Collectively, our findings identify that the GBM-secreted cytokine SPP1 upregulates PSMA expression in endothelial cells via the transcription factor HIF1α, providing insight into the angiogenic process and promising candidates for targeted GBM therapy.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Humanos , Masculino , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Glioblastoma/genética , Glioblastoma/irrigação sanguínea , Glioblastoma/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Patológica/metabolismo , Osteopontina/metabolismo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Growing evidence indicates fractal analysis (FA) has potential as a computational tool to assess tumor microvasculature in glioblastoma (GBM). As fractal parameters of microvasculature have shown to be reliable quantitative biomarkers in brain tumors, there has been similar success in measuring the architecture of tumor tissue using FA in other tumor types. However, evaluating fractal parameters of tissue structure in relation to the microvasculature has not yet been implemented in GBM. We aimed to assess the utility of this methodology in quantifying structural characteristics of GBM cytoarchitecture and vascularity by correlating fractal parameters with gene expression. METHODS: Formalin-fixed paraffin-embedded specimens were retrospectively collected from 43 patients following resection of a newly diagnosed GBM; 4 normal brain specimens were obtained from epilepsy surgeries as controls. Tumor samples were processed using FA employing a software-based box-counting method algorithm and custom messenger RNA expression assays. Fractal parameters were then correlated with clinical features, outcomes, and a panel of 92 genes associated with vascularity and angiogenesis. RESULTS: Statistical analysis demonstrated that fractal-based indices were not adequate parameters for distinction of GBM cytoarchitecture compared with normal brain specimens. Correlation analysis of our gene expression findings suggested that hematoxylin and eosin-based FA may have adequate sensitivity to detect associations with vascular gene expression. CONCLUSIONS: The combination of neuropathological assessment and histology does not provide optimized data for FA in GBM. However, an association between FA and gene expression in GBM of genes pertaining to cytoarchitecture and angiogenesis warrants further investigation.
Assuntos
Glioblastoma , Biomarcadores , Amarelo de Eosina-(YS) , Formaldeído , Fractais , Glioblastoma/irrigação sanguínea , Glioblastoma/genética , Glioblastoma/cirurgia , Hematoxilina , Humanos , Neovascularização Patológica/patologia , RNA Mensageiro , Estudos RetrospectivosRESUMO
The bloodbrain barrier represents a significant challenge for the treatment of high-grade gliomas, and our understanding of drug transport across this critical biointerface remains limited. To advance preclinical therapeutic development for gliomas, there is an urgent need for predictive in vitro models with realistic bloodbrain-barrier vasculature. Here, we report a vascularized human glioblastoma multiforme (GBM) model in a microfluidic device that accurately recapitulates brain tumor vasculature with self-assembled endothelial cells, astrocytes, and pericytes to investigate the transport of targeted nanotherapeutics across the bloodbrain barrier and into GBM cells. Using modular layer-by-layer assembly, we functionalized the surface of nanoparticles with GBM-targeting motifs to improve trafficking to tumors. We directly compared nanoparticle transport in our in vitro platform with transport across mouse brain capillaries using intravital imaging, validating the ability of the platform to model in vivo bloodbrain-barrier transport. We investigated the therapeutic potential of functionalized nanoparticles by encapsulating cisplatin and showed improved efficacy of these GBM-targeted nanoparticles both in vitro and in an in vivo orthotopic xenograft model. Our vascularized GBM model represents a significant biomaterials advance, enabling in-depth investigation of brain tumor vasculature and accelerating the development of targeted nanotherapeutics.
Assuntos
Barreira Hematoencefálica , Neoplasias Encefálicas , Permeabilidade Capilar , Glioblastoma , Nanopartículas , Animais , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/metabolismo , Células Endoteliais/metabolismo , Glioblastoma/irrigação sanguínea , Glioblastoma/metabolismo , Humanos , Camundongos , Microfluídica , Nanopartículas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Angiogenesis is a key physiological process that plays a key role in glioblastoma (GBM) progression and displays therapeutic resistance to antiangiogenic therapies. In this study, we aimed to identify whether vascular endothelial growth factor receptor 2(VEGFR2)monoclonal antibodies(mab)could inhibit tumorigenicity and the formation of vascular mimicry (VM) in GBM. The bioinformatic analysis from TCGA, CGGA, and TCPA databases and Immunohistochemistry (IHC) revealed that VEGFR2 is highly expressed in glioma tissues and results in a poor prognosis and is positively associated with VM markers (CD34 and PAS). The anti-VEGFR2 monoclonal antibodies(MSB0254)could inhibit the invasion, migration, and VM formation of U251 and primary glioma cells in vitro. In vivo, MSB0254 (m) could not only inhibit the growth of transplanted tumors of U251 and GL261 cells, but also significantly inhibit the expression of CD34, VEGFR2, Ki67, MMP2, MMP9 and reduce the expression of CD34/PAS and inhibit VM formation. The VEGFR2 monoclonal antibody could inhibit the angiogenesis and tumor growth of GBM by blocking the signaling pathway mediated by VEGFR2. It may become a new supplementary treatment for GBM.
Assuntos
Inibidores da Angiogênese , Anticorpos Monoclonais , Glioblastoma , Neovascularização Patológica , Receptor 2 de Fatores de Crescimento do Endotélio Vascular , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células , Glioblastoma/irrigação sanguínea , Glioblastoma/terapia , Humanos , Neovascularização Patológica/terapia , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Glioblastoma is a common central nervous system tumor and despite considerable advancements in treatment patient prognosis remains poor. Angiogenesis is a significant prognostic factor in glioblastoma, antiangiogenic treatments represent a promising therapeutic approach. Vascular endothelial growth factor A (VEGFA) is a predominant regulator of angiogenesis and mounting evidence suggests that the Wnt signaling pathway serves a significant role in tumor angiogenesis. As a positive regulator of the Wnt/ßcatenin signaling pathway, frequently rearranged in advanced Tcell lymphomas1 (FRAT1) is highly expressed in human glioblastoma and is significantly associated with glioblastoma growth, invasion and migration, as well as poor patient prognosis. Bioinformatics analysis demonstrated that both VEGFA and FRAT1 were highly expressed in most tumor tissues and associated with prognosis. However, whether and how FRAT1 is involved in angiogenesis remains to be elucidated. In the present study, the relationship between FRAT1 and VEGFA in angiogenesis was investigated using the human glioblastoma U251 cell line. Small interfering RNAs (siRNAs) were used to silence FRAT1 expression in U251 cells, and the mRNA and protein expression levels of VEGFA, as well as the concentration of VEGFA in U251 cell supernatants, were determined using reverse transcriptionquantitative PCR, western blotting and ELISA. A tube formation assay was conducted to assess angiogenesis. The results demonstrated that siRNA knockdown significantly decreased the protein expression levels of FRAT1 in U251 cells and markedly decreased the mRNA and protein expression levels of VEGFA. Furthermore, the concentration of VEGFA in the cell supernatant was significantly reduced and angiogenesis was suppressed. These results suggested that FRAT1 may promote VEGFA secretion and angiogenesis in human glioblastoma cells via the Wnt/ßcatenin signaling pathway, supporting the potential use of FRAT1 as a promising therapeutic target in human glioblastoma.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Encefálicas/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Neovascularização Patológica/genética , Proteínas Proto-Oncogênicas/genética , Fator A de Crescimento do Endotélio Vascular/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Western Blotting , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Glioblastoma/irrigação sanguínea , Glioblastoma/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
BACKGROUND AND PURPOSE: Differentiation between glioblastoma multiforme (GBM) and solitary brain metastasis (SBM) remains a challenge in neuroradiology with up to 40% of the cases to be incorrectly classified using only conventional MRI. The inclusion of perfusion MRI parameters provides characteristic features that could support the distinction of these pathological entities. On these grounds, we aim to use a perfusion gradient in the peritumoral edema. METHODS: Twenty-four patients with GBM or an SBM underwent conventional and perfusion MR imaging sequences before tumors' surgical resection. After postprocessing of the images, quantification of dynamic susceptibility contrast (DSC) perfusion parameters was made. Three concentric areas around the tumor were defined in each case. The monocompartimental and pharmacokinetics parameters of perfusion MRI were analyzed in both series. RESULTS: DSC perfusion MRI models can provide useful information for the differentiation between GBM and SBM. It can be observed that most of the perfusion MR parameters (relative cerebral blood volume, relative cerebral blood flow, relative Ktrans, and relative volume fraction of the interstitial space) clearly show higher gradient for GBM than SBM. GBM also demonstrates higher heterogeneity in the peritumoral edema and most of the perfusion parameters demonstrate higher gradients in the area closest to the enhancing tumor. CONCLUSION: Our results show that there is a difference in the perfusion parameters of the edema between GBM and SBM demonstrating a vascularization gradient. This could help not only for the diagnosis, but also for planning surgical or radiotherapy treatments delineating the real extension of the tumor.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/patologia , Meios de Contraste , Diagnóstico Diferencial , Edema/diagnóstico , Glioblastoma/irrigação sanguínea , Glioblastoma/diagnóstico por imagem , Humanos , Imageamento por Ressonância Magnética/métodos , PerfusãoRESUMO
Glioblastoma (GBM) is the most aggressive type of brain tumor. Microvascular proliferation and abnormal vasculature are the hallmarks of the GBM, aggravating disease progression and increasing patient morbidity. Here, we uncovered a key role of ETS1 on vascular abnormality in glioblastoma. ETS1 was upregulated in endothelial cells from human tumors compared to endothelial cells from paired control brain tissue. Knockdown of Ets1 in mouse brain endothelial cells inhibited cell migration and proliferation, and suppressed expression of genes associated with vascular abnormality in GBM. ETS1 upregulation in tumor ECs was dependent on TGFß signaling, and targeting TGFß signaling by inhibitor decreased tumor angiogenesis and vascular abnormality in CT-2A glioma model. Our results identified ETS1 as a key factor regulating tumor angiogenesis, and suggested that TGFß inhibition may suppress the vascular abnormality driven by ETS1.
Assuntos
Glioblastoma/genética , Neovascularização Patológica/genética , Proteína Proto-Oncogênica c-ets-1/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Células Endoteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/irrigação sanguínea , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Camundongos , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Proteína Proto-Oncogênica c-ets-1/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Glioblastoma (GBM) is the most malignant tumor in the brain. In addition to the vascular pattern with thin-walled vessels and findings of sprouting angiogenesis, GBM presents a bizarre microvasculature (BM) formed by vascular clusters, vascular garlands, and glomeruloid bodies. The mechanisms in BM morphogenesis are not well known. Our objective was to assess the role of pericyte/endothelial proliferation and intussusceptive angiogenic mechanisms in the formation of the BM. For this purpose, we studied specimens of 66 GBM cases using immunochemistry and confocal microscopy. In the BM, the results showed (a) transitional forms between the BM patterns, mostly with prominent pericytes covering all the abluminal endothelial cell (EC) surface of the vessels, (b) a proliferation index high in the prominent pericytes and low in ECs (47.85 times higher in pericytes than in ECs), (c) intravascular pillars (hallmark of intussusceptive angiogenesis) formed by transcapillary interendothelial bridges, endothelial contacts of opposite vessel walls, and vessel loops, and (d) the persistence of these findings in complex glomeruloid bodies. In conclusion, disproportion in pericyte/EC proliferation and mechanisms of intussusceptive angiogenesis participate in BM formation. The contributions have morphogenic and clinical interest since pericytes and intussusceptive angiogenesis can condition antiangiogenic therapy in GBM.
Assuntos
Células Endoteliais/patologia , Glioblastoma/irrigação sanguínea , Glioblastoma/patologia , Neovascularização Patológica/patologia , Pericitos/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proliferação de Células , Feminino , Humanos , Masculino , Microvasos/patologia , Pessoa de Meia-Idade , Neuroglia/patologia , Adulto JovemRESUMO
Malignant glioma is one of the most lethal cancers with rapid progression, high recurrence, and poor prognosis in the central nervous system. Fatty acid-binding protein 6 (FABP6) is a bile acid carrier protein that is overexpressed in colorectal cancer. This study aimed to assess the involvement of FABP6 expression in the progression of malignant glioma. Immunohistochemical analysis revealed that FABP6 expression was higher in glioma than in normal brain tissue. After the knockdown of FABP6, a decrease in the migration and invasion abilities of glioma cells was observed. The phosphorylation of the myosin light chain was inhibited, which may be associated with migration ability. Moreover, expression levels of invasion-related proteins, matrix metalloproteinase-2 (MMP-2) and cathepsin B, were reduced. Furthermore, tube formation was inhibited in the human umbilical vein endothelial cells with a decreased concentration of vascular endothelial growth factor (VEGF) in the conditioned medium after the knockdown of FABP6. The phosphorylation of the extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p65 were also decreased after FABP6 reduction. Finally, the bioluminescent images and immunostaining of MMP-2, cluster of differentiation 31 (CD31), and the VEGF receptor 1 (VEGFR1) revealed attenuated tumor progression in the combination of the FABP6-knocked-down and temozolomide (TMZ)-treated group in an orthotopic xenograft mouse tumor model. This is the first study that revealed the impact of FABP6 on the invasion, angiogenesis, and progression of glioma. The results of this study show that FABP6 may be a potential therapeutic target combined with TMZ for malignant gliomas.
Assuntos
Proteínas de Ligação a Ácido Graxo/antagonistas & inibidores , Hormônios Gastrointestinais/antagonistas & inibidores , Glioblastoma/irrigação sanguínea , Glioblastoma/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Neovascularização Patológica/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Células Clonais , Progressão da Doença , Matriz Extracelular/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Hormônios Gastrointestinais/genética , Hormônios Gastrointestinais/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Humanos , Camundongos Nus , Invasividade Neoplásica , Fosforilação , RNA Interferente Pequeno/metabolismo , Temozolomida/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Vasculogenic mimicry (VM) is a functional microcirculation pattern formed by aggressive tumor cells. Thus far, no effective drugs have been developed to target VM. Glioblastoma (GBM) is the most malignant form of brain cancer and is a highly vascularized tumor. Vasculogenic mimicry represents a means whereby GBM can escape anti-angiogenic therapies. METHODS: Here, using an in vitro tube formation assay on Matrigel, we evaluated the ability of N6-isopentenyladenosine (iPA) to interfere with vasculogenic mimicry (VM). RhoA activity was assessed using a pull-down assay, while the modulation of the adherens junctions proteins was analyzed by Western blot analysis. RESULTS: We found that iPA at sublethal doses inhibited the formation of capillary-like structures suppressing cell migration and invasion of U87MG, U343MG, and U251MG cells, of patient-derived human GBM cells and GBM stem cells. iPA reduces the vascular endothelial cadherin (VE-cadherin) expression levels in a dose-dependent manner, impairs the vasculogenic mimicry network by modulation of the Src/p120-catenin pathway and inhibition of RhoA-GTPase activity. CONCLUSIONS: Taken together, our results revealed iPA as a promising novel anti-VM drug in GBM clinical therapeutics.