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1.
Int J Cancer ; 151(4): 590-606, 2022 08 15.
Article in English | MEDLINE | ID: mdl-35411591

ABSTRACT

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.


Subject(s)
Bone Neoplasms , Cerebellar Neoplasms , Chromothripsis , Osteosarcoma , Animals , Bone Neoplasms/genetics , Cell Line, Tumor , DNA , DNA Repair , Hedgehog Proteins/genetics , Humans , Mice , Osteosarcoma/genetics , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use
2.
Int J Mol Sci ; 23(14)2022 Jul 14.
Article in English | MEDLINE | ID: mdl-35887143

ABSTRACT

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.


Subject(s)
Calcification, Physiologic , Calcium , Animals , Bone Density , Calcium/analysis , Calcium Isotopes , Calcium, Dietary , Diet , Rats
3.
Acta Neuropathol ; 133(4): 629-644, 2017 04.
Article in English | MEDLINE | ID: mdl-28124097

ABSTRACT

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.


Subject(s)
Aniline Compounds/pharmacology , Antineoplastic Agents/pharmacology , Astrocytoma/drug therapy , Benzimidazoles/pharmacology , Brain Neoplasms/drug therapy , Isocitrate Dehydrogenase/antagonists & inhibitors , Isocitrate Dehydrogenase/genetics , Aniline Compounds/chemistry , Aniline Compounds/pharmacokinetics , Aniline Compounds/toxicity , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/toxicity , Astrocytoma/enzymology , Astrocytoma/genetics , Benzimidazoles/chemistry , Benzimidazoles/pharmacokinetics , Benzimidazoles/toxicity , Brain Neoplasms/enzymology , Brain Neoplasms/genetics , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/physiology , Colonic Neoplasms/drug therapy , Colonic Neoplasms/enzymology , Colonic Neoplasms/genetics , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/toxicity , Escherichia coli , Female , Glutarates/metabolism , HEK293 Cells , Humans , Isocitrate Dehydrogenase/metabolism , Mice, Inbred BALB C , Mice, Nude , Mutation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sarcoma/drug therapy , Sarcoma/enzymology , Sarcoma/genetics , Sf9 Cells , Xenograft Model Antitumor Assays
5.
Nature ; 478(7368): 197-203, 2011 Oct 05.
Article in English | MEDLINE | ID: mdl-21976023

ABSTRACT

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.


Subject(s)
Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Glioma/metabolism , Glioma/pathology , Kynurenine/metabolism , Receptors, Aryl Hydrocarbon/metabolism , Animals , Autocrine Communication , Brain Neoplasms/genetics , Brain Neoplasms/immunology , Cell Line, Tumor , Cell Survival , Disease Progression , Gene Expression Regulation, Neoplastic , Glioma/genetics , Glioma/immunology , Humans , Kynurenine/immunology , Kynurenine/pharmacology , Ligands , Mice , Mice, Inbred C57BL , Mice, Nude , Neoplasm Transplantation , Paracrine Communication , Receptors, Aryl Hydrocarbon/immunology , Tryptophan/metabolism , Tryptophan Oxygenase/deficiency , Tryptophan Oxygenase/genetics , Tryptophan Oxygenase/metabolism
6.
Proc Natl Acad Sci U S A ; 111(1): 409-14, 2014 01 07.
Article in English | MEDLINE | ID: mdl-24367102

ABSTRACT

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.


Subject(s)
Antineoplastic Agents, Alkylating/pharmacology , Brain Neoplasms/drug therapy , Cell Cycle Proteins/metabolism , Drug Resistance, Neoplasm , Gene Expression Regulation, Neoplastic , Glioblastoma/drug therapy , Glioma/drug therapy , Intracellular Signaling Peptides and Proteins/metabolism , O(6)-Methylguanine-DNA Methyltransferase/pharmacology , TOR Serine-Threonine Kinases/metabolism , Animals , Brain Neoplasms/metabolism , DNA Repair , Glioblastoma/metabolism , Glioma/metabolism , Humans , Hypoxia , Immunoblotting , Lentivirus/metabolism , Mice , Mice, Nude , Neoplasm Transplantation , Plasmids/metabolism , Time Factors
7.
J Transl Med ; 13: 136, 2015 Apr 30.
Article in English | MEDLINE | ID: mdl-25926029

ABSTRACT

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.


Subject(s)
Bone Neoplasms/pathology , Cell Culture Techniques , Cell Line, Tumor , Disease Models, Animal , Osteosarcoma/pathology , Adolescent , Animals , Comparative Genomic Hybridization , Female , Genotype , Humans , Magnetic Resonance Imaging , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Transplantation , Phenotype , Prognosis , Recurrence , X-Ray Microtomography
8.
Nature ; 453(7193): 410-4, 2008 May 15.
Article in English | MEDLINE | ID: mdl-18418378

ABSTRACT

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.


Subject(s)
Neovascularization, Pathologic/prevention & control , Pancreatic Neoplasms/blood supply , Pancreatic Neoplasms/immunology , RGS Proteins/deficiency , RGS Proteins/metabolism , Animals , Capillary Permeability , Cell Hypoxia/physiology , Female , Male , Mice , Oxygen/metabolism , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , RGS Proteins/genetics
9.
J Vasc Surg ; 57(6): 1628-36, 1636.e1-3, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23294503

ABSTRACT

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.


Subject(s)
Aorta/abnormalities , Aorta/metabolism , Disease Models, Animal , Marfan Syndrome , Microfilament Proteins/biosynthesis , Animals , Aorta/pathology , Fibrillin-1 , Fibrillins , Male , Mice , Microfilament Proteins/genetics
10.
Brain ; 135(Pt 4): 1027-41, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22427331

ABSTRACT

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.


Subject(s)
Brain Neoplasms/genetics , Focal Adhesions/genetics , Gene Expression Regulation, Neoplastic/physiology , Genes, Tumor Suppressor/physiology , Glioblastoma/genetics , Animals , Animals, Newborn , Brain/metabolism , Cell Line, Tumor , Cell Movement/genetics , Comparative Genomic Hybridization , Cyclin-Dependent Kinase Inhibitor p15/metabolism , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Disease Models, Animal , Female , Focal Adhesions/immunology , Focal Adhesions/metabolism , Gadolinium , Gene Expression Regulation, Neoplastic/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Immunoprecipitation , In Vitro Techniques , Magnetic Resonance Imaging , Male , Mice , Mice, Inbred C57BL , Mice, Nude , Neuroglia/metabolism , Neurons/metabolism , RNA, Messenger/metabolism , Transfection , Tumor Stem Cell Assay/methods , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Vinculin/metabolism , Xenograft Model Antitumor Assays
11.
Acta Neuropathol ; 123(4): 529-38, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22134538

ABSTRACT

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.


Subject(s)
Cerebellar Neoplasms/genetics , Cerebellar Neoplasms/pathology , Medulloblastoma/genetics , Medulloblastoma/pathology , Meningeal Neoplasms/secondary , MicroRNAs/genetics , Adolescent , Animals , Cell Migration Assays , Cell Proliferation , Child , Child, Preschool , Cohort Studies , Computational Biology , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Hedgehog Proteins/genetics , Humans , Male , Meningeal Neoplasms/genetics , Mice , Mice, SCID , Microarray Analysis , Transplantation, Heterologous/methods , Tumor Cells, Cultured , Wnt Proteins/genetics , Young Adult
12.
Front Oncol ; 12: 969787, 2022.
Article in English | MEDLINE | ID: mdl-35992852

ABSTRACT

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.

13.
Blood ; 113(2): 488-97, 2009 Jan 08.
Article in English | MEDLINE | ID: mdl-18805968

ABSTRACT

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.


Subject(s)
Cell Movement/physiology , Endothelial Cells/enzymology , Integrin beta1/metabolism , Neovascularization, Physiologic/physiology , Umbilical Veins/enzymology , rap GTP-Binding Proteins/metabolism , rap1 GTP-Binding Proteins/metabolism , Adaptor Proteins, Signal Transducing , Animals , Apoptosis Regulatory Proteins , Cell Adhesion/physiology , Endothelial Cells/cytology , Fibroblast Growth Factors/metabolism , Focal Adhesion Kinase 1/metabolism , GTPase-Activating Proteins/metabolism , Gene Silencing , Hindlimb/blood supply , Hindlimb/enzymology , Humans , Ischemia/enzymology , Mice , Mice, Knockout , Monomeric GTP-Binding Proteins/metabolism , Phosphorylation/physiology , Proto-Oncogene Proteins c-akt , Umbilical Veins/cytology
14.
Acta Neuropathol ; 122(5): 637-50, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21863243

ABSTRACT

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.


Subject(s)
Cell Differentiation/drug effects , Ependymoma/pathology , Histone Deacetylase Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Models, Biological , Supratentorial Neoplasms/pathology , Animals , Cell Line, Tumor , Gene Expression Profiling , Histone Deacetylase Inhibitors/administration & dosage , Humans , Hydroxamic Acids/administration & dosage , In Vitro Techniques , Injections, Intraventricular , Mice , Neural Stem Cells/drug effects , Neural Stem Cells/pathology , Phenotype , Transplantation, Heterologous , Vorinostat
15.
Radiat Oncol ; 16(1): 63, 2021 Mar 31.
Article in English | MEDLINE | ID: mdl-33789720

ABSTRACT

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.


Subject(s)
Heavy Ion Radiotherapy/adverse effects , Magnetic Resonance Imaging/methods , Photons/adverse effects , Radiation Injuries/etiology , Spinal Cord Diseases/etiology , Spinal Cord/radiation effects , Animals , Female , Photons/therapeutic use , Radiation Injuries/diagnostic imaging , Rats , Rats, Sprague-Dawley , Reaction Time , Skin/radiation effects , Spinal Cord/pathology , Spinal Cord Diseases/diagnostic imaging
16.
Neuro Oncol ; 23(12): 2028-2041, 2021 12 01.
Article in English | MEDLINE | ID: mdl-34049392

ABSTRACT

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.


Subject(s)
Cerebellar Neoplasms , Chromothripsis , Heavy Ion Radiotherapy , Li-Fraumeni Syndrome , Medulloblastoma , Animals , Carbon , Cerebellar Neoplasms/drug therapy , Cerebellar Neoplasms/genetics , Cerebellar Neoplasms/radiotherapy , Humans , Medulloblastoma/drug therapy , Medulloblastoma/radiotherapy , Mice
17.
Am J Pathol ; 175(5): 1883-95, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19834063

ABSTRACT

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.


Subject(s)
Cysts , Glomerulonephritis , Kidney Diseases , Kidney Glomerulus , Kidney Tubules , Vascular Endothelial Growth Factor A/metabolism , Animals , Capillaries/cytology , Capillaries/metabolism , Capillaries/pathology , Cysts/metabolism , Cysts/pathology , Fibrosis/metabolism , Fibrosis/pathology , Glomerulonephritis/metabolism , Glomerulonephritis/pathology , Humans , In Situ Hybridization , Kidney Diseases/metabolism , Kidney Diseases/pathology , Kidney Glomerulus/cytology , Kidney Glomerulus/metabolism , Kidney Glomerulus/pathology , Kidney Tubules/cytology , Kidney Tubules/metabolism , Kidney Tubules/pathology , Mice , Mice, Transgenic , Podocytes/cytology , Podocytes/metabolism , Podocytes/pathology
18.
J Pathol ; 217(4): 571-80, 2009 03.
Article in English | MEDLINE | ID: mdl-19116989

ABSTRACT

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.


Subject(s)
Angiopoietin-1/metabolism , Angiopoietin-2/pharmacology , Mammary Neoplasms, Experimental/blood supply , Neovascularization, Pathologic/pathology , Angiopoietin-1/analysis , Angiopoietin-2/metabolism , Animals , Cell Line, Tumor , Endothelial Cells/pathology , Mammary Neoplasms, Experimental/metabolism , Mice , Mice, Nude , Neoplasm Transplantation , Neovascularization, Pathologic/metabolism , Pericytes/pathology , Phenotype , Receptor, TIE-2/metabolism , Transplantation, Heterologous
19.
Cardiovasc Intervent Radiol ; 43(4): 636-647, 2020 Apr.
Article in English | MEDLINE | ID: mdl-31965224

ABSTRACT

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.


Subject(s)
Antibiotics, Antineoplastic/administration & dosage , Doxorubicin/administration & dosage , Drug Carriers/administration & dosage , Drug Delivery Systems/instrumentation , Microspheres , In Vitro Techniques
20.
Cancer Res ; 67(4): 1555-62, 2007 Feb 15.
Article in English | MEDLINE | ID: mdl-17308094

ABSTRACT

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.


Subject(s)
Carcinoma, Squamous Cell/blood supply , Endothelial Cells/metabolism , Ferric Compounds/pharmacokinetics , Integrin alphaVbeta3/metabolism , Magnetic Resonance Imaging/methods , Neoplasms, Experimental/blood supply , Oligopeptides/pharmacokinetics , Animals , Carcinoma, Squamous Cell/metabolism , Cells, Cultured , Humans , Integrin alphaVbeta3/biosynthesis , Mice , Mice, Nude , Neoplasms, Experimental/metabolism , Neovascularization, Pathologic/diagnosis , Neovascularization, Pathologic/metabolism , Particle Size , Propylamines/pharmacokinetics , Silanes/pharmacokinetics
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