RESUMO
Chromothripsis is a form of genomic instability characterized by the occurrence of tens to hundreds of clustered DNA double-strand breaks in a one-off catastrophic event. Rearrangements associated with chromothripsis are detectable in numerous tumor entities and linked with poor prognosis in some of these, such as Sonic Hedgehog medulloblastoma, neuroblastoma and osteosarcoma. Hence, there is a need for therapeutic strategies eliminating tumor cells with chromothripsis. Defects in DNA double-strand break repair, and in particular homologous recombination repair, have been linked with chromothripsis. Targeting DNA repair deficiencies by synthetic lethality approaches, we performed a synergy screen using drug libraries (n = 375 compounds, 15 models) combined with either a PARP inhibitor or cisplatin. This revealed a synergistic interaction between the HDAC inhibitor romidepsin and PARP inhibition. Functional assays, transcriptome analyses and in vivo validation in patient-derived xenograft mouse models confirmed the efficacy of the combinatorial treatment.
Assuntos
Neoplasias Ósseas , Neoplasias Cerebelares , Cromotripsia , Osteossarcoma , Animais , Neoplasias Ósseas/genética , Linhagem Celular Tumoral , DNA , Reparo do DNA , Proteínas Hedgehog/genética , Humanos , Camundongos , Osteossarcoma/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêuticoRESUMO
Serum calcium isotopes (δ44/42Ca) have been suggested as a non-invasive and sensitive Ca balance marker. Quantitative δ44/42Ca changes associated with Ca flux across body compartment barriers relative to the dietary Ca and the correlation of δ44/42CaSerum with bone histology are unknown. We analyzed Ca and δ44/42Ca by mass-spectrometry in rats after two weeks of standard-Ca-diet (0.5%) and after four subsequent weeks of standard- and of low-Ca-diet (0.25%). In animals on a low-Ca-diet net Ca gain was 61 ± 3% and femur Ca content 68 ± 41% of standard-Ca-diet, bone mineralized area per section area was 68 ± 15% compared to standard-Ca-diet. δ44/42Ca was similar in the diets, and decreased in feces and urine and increased in serum in animals on low-Ca-diet. δ44/42CaBone was higher in animals on low-Ca-diet, lower in the diaphysis than the metaphysis and epiphysis, and unaffected by gender. Independent of diet, δ44/42CaBone was similar in the femora and ribs. At the time of sacrifice, δ44/42CaSerum inversely correlated with intestinal Ca uptake and histological bone mineralization markers, but not with Ca content and bone mineral density by µCT. In conclusion, δ44/42CaBone was bone site specific, but mechanical stress and gender independent. Low-Ca-diet induced marked changes in feces, serum and urine δ44/42Ca in growing rats. δ44/42CaSerum inversely correlated with markers of bone mineralization.
Assuntos
Calcificação Fisiológica , Cálcio , Animais , Densidade Óssea , Cálcio/análise , Isótopos de Cálcio , Cálcio da Dieta , Dieta , RatosRESUMO
Mutations in codon 132 of isocitrate dehydrogenase (IDH) 1 are frequent in diffuse glioma, acute myeloid leukemia, chondrosarcoma and intrahepatic cholangiocarcinoma. These mutations result in a neomorphic enzyme specificity which leads to a dramatic increase of intracellular D-2-hydroxyglutarate (2-HG) in tumor cells. Therefore, mutant IDH1 protein is a highly attractive target for inhibitory drugs. Here, we describe the development and properties of BAY 1436032, a pan-inhibitor of IDH1 protein with different codon 132 mutations. BAY 1436032 strongly reduces 2-HG levels in cells carrying IDH1-R132H, -R132C, -R132G, -R132S and -R132L mutations. Cells not carrying IDH mutations were unaffected. BAY 1436032 did not exhibit toxicity in vitro or in vivo. The pharmacokinetic properties of BAY 1436032 allow for oral administration. In two independent experiments, BAY 1436032 has been shown to significantly prolong survival of mice intracerebrally transplanted with human astrocytoma carrying the IDH1R132H mutation. In conclusion, we developed a pan-inhibitor targeting tumors with different IDH1R132 mutations.
Assuntos
Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Astrocitoma/tratamento farmacológico , Benzimidazóis/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Isocitrato Desidrogenase/antagonistas & inibidores , Isocitrato Desidrogenase/genética , Compostos de Anilina/química , Compostos de Anilina/farmacocinética , Compostos de Anilina/toxicidade , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidade , Astrocitoma/enzimologia , Astrocitoma/genética , Benzimidazóis/química , Benzimidazóis/farmacocinética , Benzimidazóis/toxicidade , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/enzimologia , Neoplasias do Colo/genética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/toxicidade , Escherichia coli , Feminino , Glutaratos/metabolismo , Células HEK293 , Humanos , Isocitrato Desidrogenase/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sarcoma/tratamento farmacológico , Sarcoma/enzimologia , Sarcoma/genética , Células Sf9 , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Vasos Coronários , Eletrocoagulação/métodos , Infarto do Miocárdio/etiologia , Animais , Vasos Coronários/diagnóstico por imagem , Modelos Animais de Doenças , Ecocardiografia/métodos , Eletrocoagulação/instrumentação , Feminino , Masculino , Camundongos , Agulhas , Duração da Cirurgia , Ultrassonografia Doppler em Cores , Ultrassonografia de Intervenção/métodosRESUMO
Activation of the aryl hydrocarbon receptor (AHR) by environmental xenobiotic toxic chemicals, for instance 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), has been implicated in a variety of cellular processes such as embryogenesis, transformation, tumorigenesis and inflammation. But the identity of an endogenous ligand activating the AHR under physiological conditions in the absence of environmental toxic chemicals is still unknown. Here we identify the tryptophan (Trp) catabolite kynurenine (Kyn) as an endogenous ligand of the human AHR that is constitutively generated by human tumour cells via tryptophan-2,3-dioxygenase (TDO), a liver- and neuron-derived Trp-degrading enzyme not yet implicated in cancer biology. TDO-derived Kyn suppresses antitumour immune responses and promotes tumour-cell survival and motility through the AHR in an autocrine/paracrine fashion. The TDO-AHR pathway is active in human brain tumours and is associated with malignant progression and poor survival. Because Kyn is produced during cancer progression and inflammation in the local microenvironment in amounts sufficient for activating the human AHR, these results provide evidence for a previously unidentified pathophysiological function of the AHR with profound implications for cancer and immune biology.
Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioma/metabolismo , Glioma/patologia , Cinurenina/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Comunicação Autócrina , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Glioma/imunologia , Humanos , Cinurenina/imunologia , Cinurenina/farmacologia , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Transplante de Neoplasias , Comunicação Parácrina , Receptores de Hidrocarboneto Arílico/imunologia , Triptofano/metabolismo , Triptofano Oxigenase/deficiência , Triptofano Oxigenase/genética , Triptofano Oxigenase/metabolismoRESUMO
A hypoxic microenvironment induces resistance to alkylating agents by activating targets in the mammalian target of rapamycin (mTOR) pathway. The molecular mechanisms involved in this mTOR-mediated hypoxia-induced chemoresistance, however, are unclear. Here we identify the mTOR target N-myc downstream regulated gene 1 (NDRG1) as a key determinant of resistance toward alkylating chemotherapy, driven by hypoxia but also by therapeutic measures such as irradiation, corticosteroids, and chronic exposure to alkylating agents via distinct molecular routes involving hypoxia-inducible factor (HIF)-1alpha, p53, and the mTOR complex 2 (mTORC2)/serum glucocorticoid-induced protein kinase 1 (SGK1) pathway. Resistance toward alkylating chemotherapy but not radiotherapy was dependent on NDRG1 expression and activity. In posttreatment tumor tissue of patients with malignant gliomas, NDRG1 was induced and predictive of poor response to alkylating chemotherapy. On a molecular level, NDRG1 bound and stabilized methyltransferases, chiefly O(6)-methylguanine-DNA methyltransferase (MGMT), a key enzyme for resistance to alkylating agents in glioblastoma patients. In patients with glioblastoma, MGMT promoter methylation in tumor tissue was not more predictive for response to alkylating chemotherapy in patients who received concomitant corticosteroids.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Proteínas de Ciclo Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Glioblastoma/tratamento farmacológico , Glioma/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Neoplasias Encefálicas/metabolismo , Reparo do DNA , Glioblastoma/metabolismo , Glioma/metabolismo , Humanos , Hipóxia , Immunoblotting , Lentivirus/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Plasmídeos/metabolismo , Fatores de TempoRESUMO
BACKGROUND: Osteosarcoma (OS) is the most common pediatric primary malignant bone tumor. As the prognosis for patients following standard treatment did not improve for almost three decades, functional preclinical models that closely reflect important clinical cancer characteristics are urgently needed to develop and evaluate new treatment strategies. The objective of this study was to establish an orthotopic xenotransplanted mouse model using patient-derived tumor tissue. METHODS: Fresh tumor tissue from an adolescent female patient with osteosarcoma after relapse was surgically xenografted into the right tibia of 6 immunodeficient BALB/c Nu/Nu mice as well as cultured into medium. Tumor growth was serially assessed by palpation and with magnetic resonance imaging (MRI). In parallel, a primary cell line of the same tumor was established. Histology and high-resolution array-based comparative genomic hybridization (aCGH) were used to investigate both phenotypic and genotypic characteristics of different passages of human xenografts and the cell line compared to the tissue of origin. RESULTS: A primary OS cell line and a primary patient-derived orthotopic xenotranplanted mouse model were established. MRI analyses and histopathology demonstrated an identical architecture in the primary tumor and in the xenografts. Array-CGH analyses of the cell line and all xenografts showed highly comparable patterns of genomic progression. So far, three further primary patient-derived orthotopic xenotranplanted mouse models could be established. CONCLUSION: We report the first orthotopic OS mouse model generated by transplantation of tumor fragments directly harvested from the patient. This model represents the morphologic and genomic identity of the primary tumor and provides a preclinical platform to evaluate new treatment strategies in OS.
Assuntos
Neoplasias Ósseas/patologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Modelos Animais de Doenças , Osteossarcoma/patologia , Adolescente , Animais , Hibridização Genômica Comparativa , Feminino , Genótipo , Humanos , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Fenótipo , Prognóstico , Recidiva , Microtomografia por Raio-XRESUMO
The vasculature of solid tumours is morphologically aberrant and characterized by dilated and fragile vessels, intensive vessel sprouting and loss of hierarchical architecture. Constant vessel remodelling leads to spontaneous haemorrhages and increased interstitial fluid pressure in the tumour environment. Tumour-related angiogenesis supports tumour growth and is also a major obstacle for successful immune therapy as it prevents migration of immune effector cells into established tumour parenchyma. The molecular mechanisms for these angiogenic alterations are largely unknown. Here we identify regulator of G-protein signalling 5 (Rgs5) as a master gene responsible for the abnormal tumour vascular morphology in mice. Loss of Rgs5 results in pericyte maturation, vascular normalization and consequent marked reductions in tumour hypoxia and vessel leakiness. These vascular and intratumoral changes enhance influx of immune effector cells into tumour parenchyma and markedly prolong survival of tumour-bearing mice. This is the first demonstration, to our knowledge, of reduced tumour angiogenesis and improved immune therapeutic outcome on loss of a vascular gene function and establishes a previously unrecognized role of G-protein signalling in tumour angiogenesis.
Assuntos
Neovascularização Patológica/prevenção & controle , Neoplasias Pancreáticas/irrigação sanguínea , Neoplasias Pancreáticas/imunologia , Proteínas RGS/deficiência , Proteínas RGS/metabolismo , Animais , Permeabilidade Capilar , Hipóxia Celular/fisiologia , Feminino , Masculino , Camundongos , Oxigênio/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas RGS/genéticaRESUMO
OBJECTIVE: Fibrillin-1 hypomorphic mice (mgR/mgR) are accepted as a model of Marfan syndrome. Phenotypic investigations of this mouse have not previously included quantification of phenotypic features and detailed examinations of the histopathology other than in the ascending aorta. METHODS: We developed a quantitative polymerase chain reaction assay to genotype the mice. Necropsy was performed on 50 male mice after natural death. We then sacrificed 10 mgR/mgR and 10 wild-type mice at 14-19 weeks to perform in vivo computed tomographic scans (n = 3) and microscopic examinations (n = 7). Four aortic segments (ascending, descending, pararenal, and infrarenal aorta) were excised. Each segment was divided into four subsegments and analyzed with Van Gieson staining. The number of elastin breaks and internal aortic diameter were determined twice in randomized, blinded fashion. RESULTS: Computed tomographic scans of mgR/mgR mice revealed aneurysm formation in the ascending aorta and kyphoscoliosis. Elastolysis was present in all four aortic segments of mgR/mgR but was rarely observed in wild-type mice (P < .001). The diameter of the ascending aorta was larger in mgR/mgR than in wild-type mice (P = .01), but para- and infrarenal aortic diameter were even smaller in mgR/mgR mice (P < .001 and P = .01, respectively). Exploratory gene expression analysis showed a number of differentially expressed genes with overrepresentation of immune-related functions. Quantitative polymerase chain reaction analysis confirmed upregulation of selected genes in both the ascending aorta and the abdominal aorta. CONCLUSIONS: Our findings suggest that mgR/mgR mice could be a useful model to study aortic abnormalities in segments other than the ascending aorta in order to understand the molecular mechanisms of aortic disease in Marfan syndrome.
Assuntos
Aorta/anormalidades , Aorta/metabolismo , Modelos Animais de Doenças , Síndrome de Marfan , Proteínas dos Microfilamentos/biossíntese , Animais , Aorta/patologia , Fibrilina-1 , Fibrilinas , Masculino , Camundongos , Proteínas dos Microfilamentos/genéticaRESUMO
In a strategy to identify novel genes involved in glioma pathogenesis by molecular characterization of chromosomal translocation breakpoints, we identified the KIAA1797 gene, encoding a protein with an as yet undefined function, to be disrupted by a 7;9 translocation in a primary glioblastoma culture. Array-based comparative genomic hybridization detected deletions involving KIAA1797 in around half of glioblastoma cell lines and glioblastomas investigated. Quantification of messenger RNA levels in human tissues demonstrated highest KIAA1797 expression in brain, reduced levels in all glioblastoma cell lines and most glioblastomas and similar levels in glial and neuronal cells by analysis of different hippocampal regions from murine brain. Antibodies against KIAA1797 were generated and showed similar protein levels in cortex and subcortical white matter of human brain, while levels were significantly reduced in glioblastomas with KIAA1797 deletion. By immunofluorescence of astrocytoma cells, KIAA1797 co-localized with vinculin in focal adhesions. Physical interaction between KIAA1797 and vinculin was demonstrated via co-immunoprecipitation. Functional in vitro assays demonstrated a significant decrease in colony formation, migration and invasion capacity of LN18 and U87MG glioma cells carrying a homozygous KIAA1797 deletion ectopically expressing KIAA1797 compared with mock-transduced cells. In an in vivo orthotopic xenograft mouse model, U87MG tumour lesions expressing KIAA1797 had a significantly reduced volume compared to tumours not expressing KIAA1797. In summary, the frequently deleted KIAA1797 gene encodes a novel focal adhesion complex protein with tumour suppressor function in gliomas, which we name 'focadhesin'. Since KIAA1797 genetic variation has been implicated in Alzheimer's disease, our data are also relevant for neurodegeneration.
Assuntos
Neoplasias Encefálicas/genética , Adesões Focais/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Genes Supressores de Tumor/fisiologia , Glioblastoma/genética , Animais , Animais Recém-Nascidos , Encéfalo/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Hibridização Genômica Comparativa , Inibidor de Quinase Dependente de Ciclina p15/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Modelos Animais de Doenças , Feminino , Adesões Focais/imunologia , Adesões Focais/metabolismo , Gadolínio , Regulação Neoplásica da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imunoprecipitação , Técnicas In Vitro , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Neuroglia/metabolismo , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Transfecção , Ensaio Tumoral de Célula-Tronco/métodos , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Vinculina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The contribution of microRNAs to the initiation, progression, and metastasis of medulloblastoma (MB) remains poorly understood. Metastatic dissemination at diagnosis is present in about 30% of MB patients, and is associated with a dismal prognosis. Using microRNA expression profiling, we demonstrate that the retinal miR-183-96-182 cluster on chromosome 7q32 is highly overexpressed in non-sonic hedgehog MBs (non-SHH-MBs). Expression of miR-182 and miR-183 is associated with cerebellar midline localization, and miR-182 is significantly overexpressed in metastatic MB as compared to non-metastatic tumors. Overexpression of miR-182 in non-SHH-MB increases and knockdown of miR-182 decreases cell migration in vitro. Xenografts overexpressing miR-182 invaded adjacent normal tissue and spread to the leptomeninges, phenotypically reminiscent of clinically highly aggressive large cell anaplastic MB. Hence, our study provides strong in vitro and in vivo evidence that miR-182 contributes to leptomeningeal metastatic dissemination in non-SHH-MB. We therefore reason that targeted inhibition of miR-182 may prevent leptomeningeal spread in patients with non-SHH-MB.
Assuntos
Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Meduloblastoma/genética , Meduloblastoma/patologia , Neoplasias Meníngeas/secundário , MicroRNAs/genética , Adolescente , Animais , Ensaios de Migração Celular , Proliferação de Células , Criança , Pré-Escolar , Estudos de Coortes , Biologia Computacional , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas Hedgehog/genética , Humanos , Masculino , Neoplasias Meníngeas/genética , Camundongos , Camundongos SCID , Análise em Microsséries , Transplante Heterólogo/métodos , Células Tumorais Cultivadas , Proteínas Wnt/genética , Adulto JovemRESUMO
Glioblastoma multiforme (GBM) is one of the most common and malignant brain tumors in adulthood with a median survival of only 15 months. This poor prognosis is related to GBM's ability to extensively infiltrate the surrounding brain parenchyma resulting in diffuse spread of neoplastic cells in the brain, responsible for high rate of recurrence. CD44 (Cluster of Differentiation 44) is a transmembrane protein, overexpressed in multiple cancer types, including gliomas, and implicated in cell motility, proliferation and angiogenesis. Multiple studies have investigated the role of CD44 in GBM cells and have highlighted a link between tumor malignancy and CD44 expression. However up to date, little is known of the role of CD44 on cells from the tumor microenvironment (TME). Here, we have investigated a potential role of CD44 in the TME in regards to GBM invasiveness. Using an ex-vivo organotypic brain slice invasion assay, we show that absence of CD44 from the TME impairs the ability of glioma cells to invade the surrounding brain parenchyma. By deleting CD44 in the astrocytic, endothelial and myeloid compartments, we show that it is specifically CD44 expression in myeloid cells that is responsible for the observed phenotype. Combining in vivo studies in cell-specific knock-out mice and in vitro analyses on primary microglia we demonstrate that myeloid CD44 is implicated in Toll Like Receptor 2 signaling and is a major regulator of Matrix metalloproteinase 9 expression.
RESUMO
Ras-associated protein 1 (Rap1), a small GTPase, attracted attention because of its involvement in several aspects of cell adhesion, including integrin- and cadherin-mediated adhesion. Yet, the role of Rap1 genes and of Rap1 effectors for angiogenesis has not been investigated. Human umbilical vein endothelial cells (HUVECs) express Rap1a and Rap1b mRNA. To determine the contribution of Rap1 activity for angiogenesis, we overexpressed Rap1GAP1, a GTPase-activating protein that inhibits Rap1 activity. Overexpression of Rap1GAP1 significantly blocked angiogenic sprouting and tube-forming activity of HUVECs as well as migration and integrin-dependent adhesion. Silencing of Rap1a, Rap1b, or both significantly blocked HUVECs sprouting under basal and basic fibroblast growth factor-stimulated conditions and reduced HUVEC migration and integrin-dependent adhesion. We found that Rap1a and Rap1b are essential for the conformational activation of beta(1)-integrins in endothelial cells. Furthermore, silencing of Rap1a and Rap1b prevented phosphorylation of tyrosine 397 in focal adhesion kinase (FAK) and vascular endothelial growth factor-induced Akt1-activation. Rap1a(-/-)-deficient and Rap1a(+/-) heterozygote mice displayed reduced neovascularization after hind limb ischemia compared with wild-type mice. Silencing of RAPL significantly blocked the Rap1-induced sprouting of HUVECs, suggesting that the angiogenic activity of Rap1 is partly mediated by RAPL. Our data demonstrate a critical role of Rap1 in the regulation of beta(1)-integrin affinity, adhesion, and migration in endothelial cells and in postnatal neovascularization.
Assuntos
Movimento Celular/fisiologia , Células Endoteliais/enzimologia , Integrina beta1/metabolismo , Neovascularização Fisiológica/fisiologia , Veias Umbilicais/enzimologia , Proteínas rap de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Reguladoras de Apoptose , Adesão Celular/fisiologia , Células Endoteliais/citologia , Fatores de Crescimento de Fibroblastos/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Inativação Gênica , Membro Posterior/irrigação sanguínea , Membro Posterior/enzimologia , Humanos , Isquemia/enzimologia , Camundongos , Camundongos Knockout , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-akt , Veias Umbilicais/citologiaRESUMO
Incompletely resectable ependymomas are associated with poor prognosis despite intensive radio- and chemotherapy. Novel treatments have been difficult to develop due to the lack of appropriate models. Here, we report on the generation of a high-risk cytogenetic group 3 and molecular group C ependymoma model (DKFZ-EP1NS) which is based on primary ependymoma cells obtained from a patient with metastatic disease. This model displays stem cell features such as self-renewal capacity, differentiation capacity, and specific marker expression. In vivo transplantation showed high tumorigenic potential of these cells, and xenografts phenotypically recapitulated the original tumor in a niche-dependent manner. DKFZ-EP1NS cells harbor transcriptome plasticity, enabling a shift from a neural stem cell-like program towards a profile of primary ependymoma tumor upon in vivo transplantation. Serial transplantation of DKFZ-EP1NS cells from orthotopic xenografts yielded secondary tumors in half the time compared with the initial transplantation. The cells were resistant to temozolomide, vincristine, and cisplatin, but responded to histone deacetylase inhibitor (HDACi) treatment at therapeutically achievable concentrations. In vitro treatment of DKFZ-EP1NS cells with the HDACi Vorinostat induced neuronal differentiation associated with loss of stem cell-specific properties. In summary, this is the first ependymoma model of a cytogenetic group 3 and molecular subgroup C ependymoma based on a human cell line with stem cell-like properties, which we used to demonstrate the differentiation-inducing therapeutic potential of HDACi.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Ependimoma/patologia , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Modelos Biológicos , Neoplasias Supratentoriais/patologia , Animais , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Inibidores de Histona Desacetilases/administração & dosagem , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Técnicas In Vitro , Injeções Intraventriculares , Camundongos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/patologia , Fenótipo , Transplante Heterólogo , VorinostatRESUMO
BACKGROUND: Radiation-induced myelopathy is a severe and irreversible complication that occurs after a long symptom-free latency time if the spinal cord was exposed to a significant irradiation dose during tumor treatment. As carbon ions are increasingly investigated for tumor treatment in clinical trials, their effect on normal tissue needs further investigation to assure safety of patient treatments. Magnetic resonance imaging (MRI)-visible morphological alterations could serve as predictive markers for medicinal interventions to avoid severe side effects. Thus, MRI-visible morphological alterations in the rat spinal cord after high dose photon and carbon ion irradiation and their latency times were investigated. METHODS: Rats whose spinal cords were irradiated with iso-effective high photon (n = 8) or carbon ion (n = 8) doses as well as sham-treated control animals (n = 6) underwent frequent MRI measurements until they developed radiation-induced myelopathy (paresis II). MR images were analyzed for morphological alterations and animals were regularly tested for neurological deficits. In addition, histological analysis was performed of animals suffering from paresis II compared to controls. RESULTS: For both beam modalities, first morphological alterations occurred outside the spinal cord (bone marrow conversion, contrast agent accumulation in the musculature ventral and dorsal to the spinal cord) followed by morphological alterations inside the spinal cord (edema, syrinx, contrast agent accumulation) and eventually neurological alterations (paresis I and II). Latency times were significantly shorter after carbon ions as compared to photon irradiation. CONCLUSIONS: Irradiation of the rat spinal cord with photon or carbon ion doses that lead to 100% myelopathy induced a comparable fixed sequence of MRI-visible morphological alterations and neurological distortions. However, at least in the animal model used in this study, the observed MRI-visible morphological alterations in the spinal cord are not suited as predictive markers to identify animals that will develop myelopathy as the time between MRI-visible alterations and the occurrence of myelopathy is too short to intervene with protective or mitigative drugs.
Assuntos
Radioterapia com Íons Pesados/efeitos adversos , Imageamento por Ressonância Magnética/métodos , Fótons/efeitos adversos , Lesões por Radiação/etiologia , Doenças da Medula Espinal/etiologia , Medula Espinal/efeitos da radiação , Animais , Feminino , Fótons/uso terapêutico , Lesões por Radiação/diagnóstico por imagem , Ratos , Ratos Sprague-Dawley , Tempo de Reação , Pele/efeitos da radiação , Medula Espinal/patologia , Doenças da Medula Espinal/diagnóstico por imagemRESUMO
BACKGROUND: Medulloblastomas with chromothripsis developing in children with Li-Fraumeni Syndrome (germline TP53 mutations) are highly aggressive brain tumors with dismal prognosis. Conventional photon radiotherapy and DNA-damaging chemotherapy are not successful for these patients and raise the risk of secondary malignancies. We hypothesized that the pronounced homologous recombination deficiency in these tumors might offer vulnerabilities that can be therapeutically utilized in combination with high linear energy transfer carbon ion radiotherapy. METHODS: We tested high-precision particle therapy with carbon ions and protons as well as topotecan with or without PARP inhibitor in orthotopic primary and matched relapsed patient-derived xenograft models. Tumor and normal tissue underwent longitudinal morphological MRI, cellular (markers of neurogenesis and DNA damage-repair), and molecular characterization (whole-genome sequencing). RESULTS: In the primary medulloblastoma model, carbon ions led to complete response in 79% of animals irrespective of PARP inhibitor within a follow-up period of 300 days postirradiation, as detected by MRI and histology. No sign of neurologic symptoms, impairment of neurogenesis or in-field carcinogenesis was detected in repair-deficient host mice. PARP inhibitors further enhanced the effect of proton irradiation. In the postradiotherapy relapsed tumor model, median survival was significantly increased after carbon ions (96 days) versus control (43 days, P < .0001). No major change in the clonal composition was detected in the relapsed model. CONCLUSION: The high efficacy and favorable toxicity profile of carbon ions warrants further investigation in primary medulloblastomas with chromothripsis. Postradiotherapy relapsed medulloblastomas exhibit relative resistance compared to treatment-naïve tumors, calling for exploration of multimodal strategies.
Assuntos
Neoplasias Cerebelares , Cromotripsia , Radioterapia com Íons Pesados , Síndrome de Li-Fraumeni , Meduloblastoma , Animais , Carbono , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/radioterapia , Humanos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/radioterapia , CamundongosRESUMO
The role of vascular endothelial growth factor (VEGF) in renal fibrosis, tubular cyst formation, and glomerular diseases is incompletely understood. We studied a new conditional transgenic mouse system [Pax8-rtTA/(tetO)(7)VEGF], which allows increased tubular VEGF production in adult mice. The following pathology was observed. The interstitial changes consisted of a ubiquitous proliferation of peritubular capillaries and fibroblasts, followed by deposition of matrix leading to a unique kind of fibrosis, ie, healthy tubules amid a capillary-rich dense fibrotic tissue. In tubular segments with high expression of VEGF, cysts developed that were surrounded by a dense network of peritubular capillaries. The glomerular effects consisted of a proliferative enlargement of glomerular capillaries, followed by mesangial proliferation. This resulted in enlarged glomeruli with loss of the characteristic lobular structure. Capillaries became randomly embedded into mesangial nodules, losing their filtration surface. Serum VEGF levels were increased, whereas endogenous VEGF production by podocytes was down-regulated. Taken together, this study shows that systemic VEGF interferes with the intraglomerular cross-talk between podocytes and the endocapillary compartment. It suppresses VEGF secretion by podocytes but cannot compensate for the deficit. VEGF from podocytes induces a directional effect, attracting the capillaries to the lobular surface, a relevant mechanism to optimize filtration surface. Systemic VEGF lacks this effect, leading to severe deterioration in glomerular architecture, similar to that seen in diabetic nephropathy.
Assuntos
Cistos , Glomerulonefrite , Nefropatias , Glomérulos Renais , Túbulos Renais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Capilares/citologia , Capilares/metabolismo , Capilares/patologia , Cistos/metabolismo , Cistos/patologia , Fibrose/metabolismo , Fibrose/patologia , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Humanos , Hibridização In Situ , Nefropatias/metabolismo , Nefropatias/patologia , Glomérulos Renais/citologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Camundongos , Camundongos Transgênicos , Podócitos/citologia , Podócitos/metabolismo , Podócitos/patologiaRESUMO
Sustained growth of solid tumours can rely on both the formation of new and the co-option of existing blood vessels. Current models suggest that binding of angiopoietin-2 (Ang-2) to its endothelial Tie2 receptor prevents receptor phosphorylation, destabilizes blood vessels, and promotes vascular permeability. In contrast, binding of angiopoietin-1 (Ang-1) induces Tie2 receptor activation and supports the formation of mature blood vessels covered by pericytes. Despite the intense research to decipher the role of angiopoietins during physiological neovascularization and tumour angiogenesis, a mechanistic understanding of angiopoietin function on vascular integrity and remodelling is still incomplete. We therefore assessed the vascular morphology of two mouse mammary carcinoma xenotransplants (M6378 and M6363) which differ in their natural angiopoietin expression. M6378 displayed Ang-1 in tumour cells but no Ang-2 in tumour endothelial cells in vivo. In contrast, M6363 tumours expressed Ang-2 in the tumour vasculature, whereas no Ang-1 expression was present in tumour cells. We stably transfected M6378 mouse mammary carcinoma cells with human Ang-1 or Ang-2 and investigated the consequences on the host vasculature, including ultrastructural morphology. Interestingly, M6378/Ang-2 and M6363 tumours displayed a similar vascular morphology, with intratumoural haemorrhage and non-functional and abnormal blood vessels. Pericyte loss was prominent in these tumours and was accompanied by increased endothelial cell apoptosis. Thus, overexpression of Ang-2 converted the vascular phenotype of M6378 tumours into a phenotype similar to M6363 tumours. Our results support the hypothesis that Ang-1/Tie2 signalling is essential for vessel stabilization and endothelial cell/pericyte interaction, and suggest that Ang-2 is able to induce a switch of vascular phenotypes within tumours.
Assuntos
Angiopoietina-1/metabolismo , Angiopoietina-2/farmacologia , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neovascularização Patológica/patologia , Angiopoietina-1/análise , Angiopoietina-2/metabolismo , Animais , Linhagem Celular Tumoral , Células Endoteliais/patologia , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neovascularização Patológica/metabolismo , Pericitos/patologia , Fenótipo , Receptor TIE-2/metabolismo , Transplante HeterólogoRESUMO
PURPOSE: To evaluate and compare the material characteristics of a novel type of radiopaque doxorubicin-loaded microsphere (V-100) with radiopaque and non-radiopaque doxorubicin-loaded microspheres. MATERIALS AND METHODS: The prototype V-100 featuring inherent radiopacity and three available commercial controls (DC-Bead-LUMI™-70-150, Embozene-Tandem™-100 and DC-Bead™-M1) were analyzed before and after doxorubicin loading (37.5 mg doxorubicin/1 ml microspheres) in suspension with aqua and/or aqua/iodixanol-320. Study goals included inherent radiopacity [e.g., using conventional computed tomography (CT)], doxorubicin loading efficacy, morphology using light and fluorescence microscopy, size distribution using laser diffraction/light scattering, time-in-suspension, rheological properties using rheometer analysis, and microsphere stability observed over a period of 5 days after doxorubicin loading. RESULTS: V-100 showed good inherent radiopacity without adverse imaging artifacts. Under conventional CT, the quantitative radiopacity was as follows: 480.4 ± 2.9HU for V-100, 2432.7 ± 3.2HU for DC-Bead-LUMI™-70-150, 118.1 ± 3.0HU for Embozene-Tandem™-100, and 19.8 ± 1.5HU for DC-Bead™-M1. All of the types of microspheres showed a similar loading efficiency (> 98%) after 24 h; however, there were slower doxorubicin loading velocities for the radiopaque microspheres. The doxorubicin-loaded V-100 and Embozene-Tandem™-100 showed typical narrow-sized distributions. In aqua/iodixanol-320 suspension, doxorubicin-loaded V-100 showed the best suspension features and ideal deformability and elasticity characteristics. Similar to other microspheres, doxorubicin-loaded V-100 was very stable and storable for at least 5 days. CONCLUSION: V-100 is a promising novel type of radiopaque doxorubicin-loaded microsphere. Compared with the controls, V-100 shows good inherent radiopacity without adverse imaging artifacts and with comparable doxorubicin loading efficacy. Further advantages of V-100 include narrow-sized distribution and excellent suspension, rheology, and stability features.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Portadores de Fármacos/administração & dosagem , Sistemas de Liberação de Medicamentos/instrumentação , Microesferas , Técnicas In VitroRESUMO
Angiogenesis is essential for the development of malignant tumors and provides important targets for tumor diagnosis and therapy. To noninvasively assess the angiogenic profile of tumors, novel alpha(v)beta(3) integrin-targeted ultrasmall superparamagnetic iron oxide particles (USPIOs) were designed and their specific uptake by endothelial cells was evaluated in vitro and in vivo. USPIOs were coated with 3-aminopropyltrimethoxysilane (APTMS) and conjugated with Arg-Gly-Asp (RGD) peptides. Accumulation in human umbilical vein endothelial cells (HUVECs) was evaluated using Prussian blue staining, transmission electron microscopy, magnetic resonance (MR) imaging, and inductively coupled plasma mass spectrometry. Uptake of RGD-USPIO by HUVECs was significantly increased when compared with unlabeled USPIO and could be competitively inhibited by addition of unbound RGD. The ability of the RGD-USPIO to noninvasively distinguish tumors with high (HaCaT-ras-A-5RT3) and lower (A431) area fractions of alpha(v)beta(3) integrin-positive vessels was evaluated using a 1.5-T MR scanner. Indeed, after RGD-USPIO injection, there was a more pronounced decrease in T(2) relaxation times in HaCaT-ras-A-5RT3 tumors than in A431 tumors. Furthermore, T(2)*-weighted images clearly identified the heterogeneous arrangement of vessels with alpha(v)beta(3) integrins in HaCaT-ras-A-5RT3 tumors by an irregular signal intensity decrease. In contrast, in A431 tumors with predominantly small and uniformly distributed vessels, the signal intensity decreased more homogeneously. In summary, RGD-coupled, APTMS-coated USPIOs efficiently label alpha(v)beta(3) integrins expressed on endothelial cells. Furthermore, these molecular MR imaging probes are capable of distinguishing tumors differing in the degree of alpha(v)beta(3) integrin expression and in their angiogenesis profile even when using a clinical 1.5-T MR scanner.