RESUMO
Determination of tumor oxygenation at the microvascular level will provide important insight into tumor growth, angiogenesis, necrosis and therapeutic response and will facilitate to develop protocols for studying tumor behavior. The non-ionizing near infrared spectroscopy (NIRS) technique has the potential to differentiate lesion and hemoglobin dynamics; however, it has a limited spatial resolution. On the other hand, magnetic resonance imaging (MRI) has achieved high spatial resolution with excellent tissue discrimination but is more susceptible to limited ability to monitor the hemoglobin dynamics. In the present work, the vascular status and the pathophysiological changes that occur during tumor vascularization are studied in an orthotopic brain tumor model. A noninvasive multimodal approach based on the NIRS technique, namely steady state diffuse optical spectroscopy (SSDOS) along with MRI, is applied for monitoring the concentrations of oxyhemoglobin, deoxyhemoglobin and water within tumor region. The concentrations of oxyhemoglobin, deoxyhemoglobin and water within tumor vasculature are extracted at 15 discrete wavelengths in a spectral window of 675-780 nm. We found a direct correlation between tumor size, intratumoral microvessel density and tumor oxygenation. The relative decrease in tumor oxygenation with growth indicates that though blood vessels infiltrate and proliferate the tumor region, a hypoxic trend is clearly present.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/diagnóstico , Diagnóstico por Computador/métodos , Imageamento por Ressonância Magnética/métodos , Neovascularização Patológica/diagnóstico , Espectrofotometria Infravermelho/métodos , Neoplasias Encefálicas/metabolismo , Hemoglobinas/análise , Humanos , Neovascularização Patológica/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Integração de SistemasRESUMO
PURPOSE: EMD 121974, a potent cyclic RGD peptide inhibitor of alphav-integrins, demonstrated effectiveness in suppressing brain tumor growth in both preclinical models and phases I/II clinical trials. The ability to non-invasively evaluate alphav-integrin expression provides a novel and unique way to better understand brain tumor angiogenesis in relationship to alphav-integrin expression, and allow for direct assessment of anti-integrin treatment efficacy. PROCEDURES: We developed a F-18-labeled RGD peptide [F-18]FB-RGD and performed serial microPET imaging scans to follow brain tumor growth and angiogenesis as a function of time in an orthotopic U87MG glioblastoma xenograft model in athymic nude mice. RESULTS: The tumor was barely visible on microPET at the size of Assuntos
Neoplasias Encefálicas/diagnóstico por imagem
, Neoplasias Encefálicas/metabolismo
, Radioisótopos de Flúor
, Glioblastoma/diagnóstico por imagem
, Glioblastoma/metabolismo
, Oligopeptídeos/farmacocinética
, Tomografia por Emissão de Pósitrons
, Animais
, Radioisótopos de Flúor/química
, Radioisótopos de Flúor/farmacocinética
, Humanos
, Imuno-Histoquímica
, Marcação por Isótopo/métodos
, Camundongos
, Camundongos Nus
, Conformação Molecular
, Transplante de Neoplasias
, Oligopeptídeos/química
, Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo
, Sensibilidade e Especificidade
, Transplante Heterólogo
RESUMO
UNLABELLED: The alphav-integrins, cell adhesion molecules that are highly expressed on activated endothelial cells and tumor cells but not on dormant endothelial cells or normal cells, present an attractive target for tumor imaging and therapy. We previously coupled a cyclic Arg-Gly-Asp (RGD) peptide, c(RGDyK), with 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) and labeled the RGD-DOTA conjugate with 64Cu (half-life, 12.8 h; 19% beta+) for solid tumor targeting, with high tumor-to-background contrast. The rapid tumor washout rate and persistent liver and kidney retention of this tracer prompted us to optimize the tracer for improved pharmacokinetic behavior. In this study, we introduced a polyethylene glycol (PEG; molecular weight, 3,400) moiety between DOTA and RGD and evaluated the 64Cu-DOTA-PEG-RGD tracer for microPET imaging in brain tumor models. METHODS: DOTA was activated in situ and conjugated with RGD-PEG-NH2 under slightly basic conditions. alphavbeta3-Integrin-binding affinity was evaluated with a solid-phase receptor-binding assay in the presence of 125I-echistatin. Female nude mice bearing subcutaneous U87MG glioblastoma xenografts were administered 64Cu-DOTA-PEG-RGD, and the biodistributions of the radiotracer were evaluated from 30 min to 4 h after injection. microPET (20 min of static imaging at 1 h after injection) and then quantitative autoradiography were used for tumor visualization and quantification. The same tracer was also applied to an orthotopic U87MG model for tumor detection. RESULTS: The radiotracer was synthesized with a high specific activity (14,800-29,600 GBq/mmol [400-800 Ci/mmol]). The c(RGDyK)-PEG-DOTA ligand showed intermediate binding affinity for alphavbeta3-integrin (50% inhibitory concentration, 67.5 +/- 7.8 nmol/L [mean +/- SD]). The pegylated RGD peptide demonstrated rapid blood clearance (0.57 +/- 0.15 percentage injected dose [%ID]/g [mean +/- SD] at 30 min after injection and 0.03 +/- 0.02 %ID/g at 4 h after injection). Activity accumulation in the tumor was rapid and high at early time points (2.74 +/- 0.45 %ID/g at 30 min after injection), and some activity washout was seen over time (1.62 +/- 0.18 %ID/g at 4 h after injection). Compared with (64)Cu-DOTA-RGD, this tracer showed improved in vivo kinetics, with significantly reduced liver uptake (0.99 +/- 0.08 %ID/g vs. 1.73 +/- 0.39 %ID/g at 30 min after injection and 0.58 +/- 0.07 %ID/g vs. 2.57 +/- 0.49 %ID/g at 4 h after injection). The pegylated RGD peptide showed higher renal accumulation at early time points (3.51 +/- 0.24 %ID/g vs. 2.18 +/- 0.23 %ID/g at 30 min after infection) but more rapid clearance (1.82 +/- 0.29 %ID/g vs. 2.01 +/- 0.25 %ID/g at 1 h after injection) than 64Cu-DOTA-RGD. The integrin receptor specificity of this radiotracer was demonstrated by blocking of tumor uptake by coinjection with nonradiolabeled c(RGDyK). The high tumor-to-organ ratios for the pegylated RGD peptide tracer (at 1 h after injection: tumor-to-blood ratio, 20; tumor-to-muscle ratio, 12; tumor-to-liver ratio, 2.7; and tumor-to-kidney ratio, 1.2) were confirmed by microPET and autoradiographic imaging in a subcutaneous U87MG tumor model. This tracer was also able to detect an orthotopic brain tumor in a model in which U87MG cells were implanted into the mouse forebrain. Although the magnitude of tumor uptake in the orthotopic xenograft was lower than that in the subcutaneous xenograft, the orthotopic tumor was still visualized with clear contrast from normal brain tissue. CONCLUSION: This study demonstrated the suitability of a PEG moiety for improving the in vivo kinetics of a 64Cu-RGD peptide tracer without compromising the tumor-targeting ability and specificity of the peptide. Systematic investigations of the effects of the size and geometry of PEG on tumor targeting and in vivo kinetics will lead to the development of radiotracers suitable for clinical applications such as visualizing and quantifying alphav-integrin expression by PET. In addition, the same ligand labeled with therapeutic radionuclides may be applicable for integrin-targeted internal radiotherapy.
Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/metabolismo , Integrina alfaVbeta3/metabolismo , Oligopeptídeos/farmacocinética , Animais , Radioisótopos de Cobre/química , Radioisótopos de Cobre/farmacocinética , Feminino , Glioblastoma/diagnóstico por imagem , Glioblastoma/metabolismo , Marcação por Isótopo/métodos , Taxa de Depuração Metabólica , Camundongos , Camundongos Nus , Oligopeptídeos/química , Especificidade de Órgãos , Peptídeos/química , Peptídeos/farmacocinética , Polietilenoglicóis/química , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Distribuição TecidualRESUMO
BACKGROUND: Hypertonic saline infusion dampens inflammatory responses and suppresses neutrophil-endothelial interaction by reducing adhesion molecule expression. This study tested the hypothesis that hypertonic saline attenuates tumor cell adhesion to the endothelium through a similar mechanism. METHODS: Human colon cancer cells (LS174T) were transfected with green fluorescent protein and exposed to lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-6 under hypertonic and isotonic conditions for 1 and 4 hours. Confluent human umbilical vein endothelial cells were similarly exposed. Cellular apoptosis and expression of adhesion molecules and laminin were measured by flow cytometry. Tumor cell adhesion to endothelium and laminin was assessed with fluorescence microscopy. Data are represented as mean +/- standard error of mean, and an ANOVA test was performed to gauge statistical significance, with P <.05 considered significant. RESULTS: Hypertonic exposure significantly reduced tumor cell adhesion despite the presence of the perioperative cell stressors (42 +/- 2.9 vs 172.5 +/- 12.4, P <.05), attenuated tumor cell beta-1 integrin (14.43 vs 23.84, P <.05), and endothelial cell laminin expression (22.78 +/- 2.2 vs 33.74 +/- 2.4, P <.05), but did not significantly alter cell viability. CONCLUSION: Hypertonic saline significantly attenuates tumor cell adhesion to endothelium by inhibiting adhesion molecule and laminin expression. This may halt the metastatic behavior of tumor cells shed at surgery.
Assuntos
Adesão Celular/efeitos dos fármacos , Comunicação Celular/efeitos dos fármacos , Neoplasias do Colo/fisiopatologia , Células Endoteliais/efeitos dos fármacos , Solução Salina Hipertônica/farmacologia , Moléculas de Adesão Celular/biossíntese , Linhagem Celular Tumoral , Humanos , Laminina/biossínteseRESUMO
The primary growth factor receptors involved in angiogenesis and lymphomagenesis can be grouped into the vascular endothelial growth factor (VEGF) receptors and related families. Inhibition of VEGF and other growth factors, including c-Abl, c-Kit, platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and insulin-like growth factor (IGF), or their receptors containing tyrosine kinase domains by antiangiogenesis drugs disrupts cell survival signal transduction pathways and may contribute to the proapoptotic pathways in malignant cells. However, clinical trials suggest that signal transduction inhibitors have considerable antitumor activity when used as single agents only for a short time, most likely due to the development of drug resistance by the host or by the tumor cells. In order to prevent this problem and to augment their antitumor efficacy, these agents could be administered in combination with cytotoxic antineoplastic drugs. We hypothesized that the combination of the antiangiogenesis tyrosine kinase inhibitors with cytotoxic drugs would produce synergistic drug regimens. Two human T-lymphoblastic leukemia cell lines that express VEGF-R1, CEM/0 (wild-type, WT) and the drug-resistant clone CEM/ara-C/I/ASNase-0.5-2, were utilized in the drug combination studies. NSC 680410, a tyrosine kinase inhibitor given at 0.1 to 1 microM for 72 h, inhibited VEGF secretion and leukemic cell growth at 90% of vehicle-treated control cultures with an IC50 value of less than 1 microM. The cytotoxic drugs idarubicin (IDA), fludarabine (Fludara), and cytosine arabinoside (ara-C) were used for the various drug combinations. One-, two-, three-, and four-drug treatments were tested. Cell viability was documented by the MTT assay and photomicrographic estimation of apoptotic cells. Both the combination index (CI) and isobologram evaluations demonstrated strong synergism between these drugs and the tyrosine kinase inhibitor. NSC 680410 was highly synergistic with IDA, IDA + ara-C, and IDA + Fludara + ara-C, over the respective cytotoxic drug regimens at concentrations easily achieved in patient plasma. NSC 680410 potentiated the activity of IDA in both leukemia cell lines by 17.8- and 221.4-fold in the WT and drug-resistant line, respectively. The activity of NSC 680410 + IDA + ara-C was also potentiated by 58.8-fold in the WT line, and the activity of NSC 680410 + IDA + Fludara + ara-C by 2.4- and 6.47x10(6)-fold in the WT and drug-resistant lines, respectively. The results suggest that IDA was not needed for optimal synergistic activity in the CEM/0 cells, but IDA was a necessary component to obtain drug synergism in the drug-resistant clone. Similarly, STI571 (imatinib mesylate, Gleevec), the p210(bcr/abl) tyrosine kinase inhibitor, demonstrated synergism with Fludara + ara-C or IDA + ara-C. Most importantly STI571 showed synergism with NSC 680410, suggesting that these drugs inhibit different tyrosine kinase domains in human leukemia cells. Lastly, pretreatment of leukemic cells with NSC 680410 showed additivity with gamma radiation in comparison to either treatment modality alone. The data, taken together, suggest that by inhibiting the pro-survival signal transduction pathway (VEGF-R1) and DNA replication by cytotoxic drugs, leukemic cells undergo apoptosis in a synergistic manner. In conclusion, the combinations of antiangiogenesis and DNA-damaging cytotoxic drugs are highly synergistic regimens in both WT and drug-resistant leukemic cell lines and they should be examined further.
Assuntos
Adamantano/análogos & derivados , Adamantano/farmacologia , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Hidroquinonas/farmacologia , Leucemia-Linfoma de Células T do Adulto/tratamento farmacológico , Piperazinas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Fosfato de Vidarabina/análogos & derivados , Antimetabólitos Antineoplásicos/farmacologia , Benzamidas , Linhagem Celular Tumoral , Citarabina/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Ensaio de Imunoadsorção Enzimática , Humanos , Mesilato de Imatinib , Leucemia-Linfoma de Células T do Adulto/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fosfato de Vidarabina/farmacologiaRESUMO
PURPOSE: NSC 680410, the novel adamantyl ester of AG957, an inhibitor of the p210bcr/abl tyrosine kinase (CML, Ph(+)) and possibly other kinases, was tested for antitumor activity in ten human leukemia and human glioblastoma cell lines. METHODS: CEM/0, seven ara-C- and/or ASNase-resistant clones, Jurkat/0, the myelomonocytic line U937 and U87 MG glioblastoma cell lines were used for these studies. The drug-resistant leukemic clones lack p53, express bcl-2 and VEGF-R1, and thus are refractory to apoptosis. Since tyrosine kinases drive many proliferative pathways and these activities are increased in many leukemic cells, we hypothesized that NSC 680410 may induce cytotoxicity in drug-resistant leukemia clones, independently of p210bcr/abl expression. RESULTS: NSC 680410 exhibited significant antileukemic activity in CEM/0, Jurkat E6-1, and in the drug-resistant leukemic cell lines. The IC(50) values in nine leukemia lines ranged from 17 to 216 n M. Western blot analyses after NSC 680410 treatment demonstrated caspase-3 cleavage and ELISAs showed a fivefold upregulation of its activity in cellular extracts. In addition, U87 MG human glioblastoma cells, which express VEGF-R1, were treated with the Flt-1/Fc chimera, a specific inhibitor of VEGF, and showed 30-43% cell kill in the MTT assay. Furthermore, the combination of NSC 680410 plus Flt-1/Fc chimera demonstrated an eightfold synergism against U87 MG cells in vitro. To verify this observation in vivo, athymic mice were inoculated orthotopically into the caudate putamen with 10(6) U87 MG cells. On day 3, five mice per group were treated i.p. with either 8.3 mg/kg NSC 680410 daily for three doses per week for 4 weeks alone or in combination with one dose of Flt-1/Fc chimera 100 mg/kg subcutaneously. Treatment with NSC 680410 alone produced no weight changes and increased the median survival to 133%, whereas treatment with NSC680410 plus Flt-1/Fc chimera increased survival to 142% over control. Control animals had large intra- and extracranial tumors while the NSC 680410-treated mice had small, only intracranial tumors with necrotic centers. The combination treatment resulted in small residual tumors around the needle track, indicating significant inhibition of tumor growth. CONCLUSIONS: These studies demonstrate that the tyrosine kinase inhibitor NSC 680410 has significant antileukemic activity in p53-null, drug-resistant human leukemia cell lines, as well as significant antitumor activity in combination with Flt-1/Fc chimera against U87 MG glioblastoma brain tumors implanted in situ in athymic mice.
Assuntos
Adamantano/análogos & derivados , Adamantano/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Glioblastoma/tratamento farmacológico , Hidroquinonas/uso terapêutico , Leucemia de Células T/tratamento farmacológico , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Caspase 3 , Caspases/metabolismo , DNA de Neoplasias/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fatores de Crescimento Endotelial/metabolismo , Ensaio de Imunoadsorção Enzimática , Proteínas da Matriz Extracelular/farmacologia , Feminino , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Leucemia de Células T/genética , Leucemia de Células T/metabolismo , Linfocinas/metabolismo , Camundongos , Camundongos Nus , Cadeias Pesadas de Miosina , Transplante de Neoplasias , Miosina não Muscular Tipo IIB , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fatores de Crescimento do Endotélio VascularRESUMO
Brain tumors are highly angiogenesis dependent. The cell adhesion receptor integrin alpha(v)beta(3) is overexpressed in glioma and activated endothelial cells and plays an important role in brain tumor growth, spread and angiogenesis. Suitably labeled alpha(v)beta(3)-integrin antagonists may therefore be useful for imaging brain tumor associated angiogenesis. Cyclic RGD peptide c(RGDyK) was labeled with (18)F via N-succinimidyl-4-[(18)F]fluorobenzoate through the side-chain epsilon-amino group of the lysine residue. The radiotracer was evaluated in vivo for its tumor targeting efficacy and pharmacokinetics in subcutaneously implanted U87MG and orthotopically implanted U251T glioblastoma nude mouse models by means of microPET, quantitative autoradiography and direct tissue sampling. The N-4-[(18)F]fluorobenzoyl-RGD ([(18)F]FB-RGD) was produced in less than 2 h with 20-25% decay-corrected yields and specific activity of 230 GBq/micromol at end of synthesis. The tracer showed very rapid blood clearance and both hepatobiliary and renal excretion. Tumor-to-muscle uptake ratio at 30 min was approximately 5 in the subcutaneous U87MG tumor model. MicroPET imaging with the orthotopic U251T brain tumor model revealed very high tumor-to-brain ratio, with virtually no uptake in the normal brain. Successful blocking of tumor uptake of [(18)F]FB-RGD in the presence of excess amount of c(RGDyK) revealed receptor specific activity accumulation. Hence, N-4-[(18)F]fluorobenzoyl labeled cyclic RGD peptide [(18)F]FB-RGD is a potential tracer for imaging alpha(v)beta(3)-integrin positive tumors in brain and other anatomic locations.
Assuntos
Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/diagnóstico por imagem , Glioblastoma/diagnóstico por imagem , Glioblastoma/metabolismo , Neovascularização Patológica , Oligopeptídeos/farmacocinética , Animais , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Estudos de Viabilidade , Feminino , Radioisótopos de Flúor/farmacocinética , Humanos , Integrina alfaVbeta3/metabolismo , Taxa de Depuração Metabólica , Camundongos , Camundongos Nus , Especificidade de Órgãos , Projetos Piloto , Cintilografia , Compostos Radiofarmacêuticos/farmacocinética , Distribuição TecidualRESUMO
OBJECTIVE: To improve currently used techniques to implant tumor cells into the parenchyma of the mouse brain. MATERIALS AND METHODS: The stereotactic injection of 0.5 to 5 microl of indigo carmine over 5 to 40 minutes into the caudate/putamen nuclei of the mouse was done followed by sacrifice and examination of the brain injection site. 1 microl containing 10(5) U87MG glioma cells were stereotactically implanted into the caudate/ putamen nuclei over 20 minutes. The animals were sacrificed from one hour to 63 days after implantation and the brain examined and tumor size measured. RESULTS: An injection of 1 microl of indigo carmine over 20 minutes produced a spherical deposit of dye within the caudate/putamen nuclei. Larger volumes of indigo carmine or shorter injection times resulted in dye spreading along the injection tract or into the ventricles or subarachnoid space. Using the results of the dye studies, the same parameters were used to successfully inject and confine the glioma cells to the caudate/putamen nuclei in 30 of 32 mice. No tumor was found in 2 animals and appears to be explained by obstruction of the injection cannula. The tumor cells appeared viable an hour after injection. However by day three, considerable necrosis of tumor cells were noted, the effects of which resolved by day five. On day six, the injection site was comparable to that at one hour. In the early phase, until the fifth week, tumor volume doubling time was ten days while afterward it was only five days. CONCLUSION: The technique described allows the highly accurate and reproducible introduction of a given number of cells into a specific area of the mouse brain. This should reduce the intragroup variability, be it control or therapeutic, allowing better assessment of outcome with fewer number of mice.
Assuntos
Neoplasias Encefálicas/patologia , Núcleo Caudado/patologia , Glioma/patologia , Transplante de Neoplasias/métodos , Putamen/patologia , Animais , Linhagem Celular Tumoral , Estudos de Viabilidade , Feminino , Camundongos , Camundongos Nus , Reprodutibilidade dos Testes , Técnicas Estereotáxicas , Fatores de Tempo , Carga TumoralRESUMO
Blood vessels are critical to normal mammalian development, tissue repair, and growth and treatment of cancer. Mouse research models enable mechanistic studies of blood vessels. We detail how to perfuse mice with fluorescent tomato lectin or the lipophilic fluorophore DiI. We provide details on how to image fluorescently labeled blood vessels.
Assuntos
Vasos Sanguíneos/ultraestrutura , Microscopia Confocal/métodos , Neoplasias/patologia , Neovascularização Patológica/patologia , Animais , Vasos Sanguíneos/patologia , Modelos Animais de Doenças , Corantes Fluorescentes , Proteínas de Fluorescência Verde , Humanos , CamundongosRESUMO
Plasminogen activator inhibitor-1 (PAI-1) paradoxically enhances tumor progression and angiogenesis; however, the mechanism supporting this role is not known. Here we provide evidence that PAI-1 is essential to protect endothelial cells (ECs) from FasL-mediated apoptosis. In the absence of host-derived PAI-1, human neuroblastoma cells implanted in PAI-1-deficient mice form smaller and poorly vascularized tumors containing an increased number of apoptotic ECs. We observed that knockdown of PAI-1 in ECs enhances cell-associated plasmin activity and increases spontaneous apoptosis in vitro. We further demonstrate that plasmin cleaves FasL at Arg144-Lys145, releasing a soluble proapoptotic FasL fragment from the surface of ECs. The data provide a mechanism explaining the proangiogenic activity of PAI-1.
Assuntos
Apoptose , Células Endoteliais/metabolismo , Proteína Ligante Fas/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Serpinas/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Células Cultivadas , Células Endoteliais/imunologia , Células Endoteliais/patologia , Fibrinolisina/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/metabolismo , Neuroblastoma/irrigação sanguínea , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fragmentos de Peptídeos/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Interferência de RNA , RNA Interferente Pequeno , Receptores de Superfície Celular/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Serpina E2 , Serpinas/deficiência , Serpinas/genética , Fatores de Tempo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Receptor fas/metabolismoRESUMO
In vivo bioluminescent imaging using cells expressing Renilla luciferase is becoming increasingly common. Hindrances to the more widespread use of Renilla luciferase are the high autoluminescence of its natural substrate, coelenterazine, in plasma, the relatively high absorbance by tissue of the light emitted by the enzyme-substrate reaction; rapid clearance of the substrate; and significant cost. These factors, save for the cost, which has its own limiting effect on use, can combine to reduce the sensitivity of in vivo assays utilizing this reporter system, and methods of increasing light output or decreasing autoluminescence could be of great benefit. A number of analogs of coelenterazine are being investigated may accomplish one or both of these goals. In this study that we report on the testing of two new substrate analogs, EnduRen and ViViren, manufactured by Promega Corporation, in an orthotopic murine model of human glioblastoma expressing Renilla luciferase. We have tested these analogs in this cell line both in vitro and in vivo, and find that the substrate viviren results in significantly greater light output than the natural substrate or the other analog EnduRen. This new substrate could be valuable for studies where greater sensitivity is important.
Assuntos
Glioblastoma/diagnóstico , Imidazóis , Luciferases de Renilla , Substâncias Luminescentes , Pirazinas , Animais , Linhagem Celular Tumoral , Feminino , Glioblastoma/metabolismo , Humanos , Imidazóis/administração & dosagem , Injeções Intraperitoneais , Injeções Intravenosas , Luciferases de Renilla/biossíntese , Substâncias Luminescentes/administração & dosagem , Camundongos , Camundongos Nus , Pirazinas/administração & dosagem , Especificidade por SubstratoRESUMO
OBJECTIVE: To determine the effect of the angiogenesis inhibitor Cilengitide (EMD 121974) on glioblastoma growth and associated angiogenesis in the brains of nude mice. METHODS: Human glioblastoma cells (10 U87MG cells) in 1 mul of medium were stereotactically injected during a 20-minute period into the caudate/putamen of nude mice. The mice were intraperitoneally treated daily with Cilengitide or solvent (control) beginning 5 days after tumor injection. The mice were sacrificed from 1 hour to 63 days after tumor implantation and examined for tumor size, vascularity, apoptosis, and cell replication. RESULTS: This injection technique resulted in a highly reproducible, localized, spherical tumor cell placement in the parenchyma without reflux into the subarachnoid space or penetration into the ventricle. Serial brain sections showed the tumor size remained unchanged at 1 to 2 mm for approximately 30 to 40 days. Thereafter, the control tumors showed exponential growth to a volume of 120 mm, with death of the mice at approximately 8 to 9 weeks. Serial staining for Ki-67, a marker for cell replication, and CD31, an indicator for angiogenesis, demonstrated an increase in proportion to the growth of the tumor. In contrast, the tumor volume in Cilengitide-treated mice stayed unchanged at 1 to 2 mm during the entire length of the experiment, with staining for Ki-67 and CD31 remaining low. CONCLUSION: This standardized brain tumor model is highly reproducible and useful for testing new treatment regimens. Cilengitide is highly effective in suppressing blood vessel growth, thereby controlling orthotopic growth of this glioblastoma cell line.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Glioblastoma/irrigação sanguínea , Glioblastoma/tratamento farmacológico , Neovascularização Patológica/patologia , Neovascularização Patológica/prevenção & controle , Venenos de Serpentes/administração & dosagem , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Resultado do TratamentoRESUMO
Matrix ligation of integrins alphavbeta3/alphavbeta5 is critical for endothelial survival and angiogenesis. We have previously shown that ceramide, a proapoptotic lipid second messenger, increases during endothelial anoikis (detachment-induced apoptosis). We now show that RGDfV, an integrin alphavbeta3/alphavbeta5 cyclic function-blocking peptide, increased ceramide and decreased sphingomyelin in human brain microvascular endothelial cells (HBMECs) plated on vitronectin, suggesting that sphingomyelin hydrolysis contributes to RGDfV-induced ceramide increase. Desipramine and imipramine, inhibitors of acid sphingomyelinase (ASMase), suppressed RGDfV-induced ceramide increase. Importantly, desipramine, imipramine, and a third ASMase inhibitor, SR33557, but not inhibitors of neutral sphingomyelinase, suppressed RGDfV-induced apoptosis, suggesting that ASMase was required for integrin-mediated apoptosis. Myriocin, an inhibitor of de novo ceramide synthesis, had no effect on RGDfV-induced HBMEC apoptosis. Interestingly, ASMase inhibitors also suppressed the RGDfV-induced loss of spreading on vitronectin. RGDfV induced a similar increase in ceramide and apoptosis in HBMECs on poly-l-lysine or vitronectin, although cells detached only from vitronectin, indicating that cell detachment was not required for RGDfV-induced apoptosis. Our results suggest involvement of ASMase and ceramide in endothelial apoptosis induced by inhibition of integrins alphavbeta3/alphavbeta5, and propose a novel molecular mechanism for the antiangiogenic effect of RGDfV.
Assuntos
Apoptose , Ceramidas/metabolismo , Endotélio Vascular/citologia , Integrina alfaVbeta3/antagonistas & inibidores , Integrinas/antagonistas & inibidores , Receptores de Vitronectina/antagonistas & inibidores , Inibidores da Angiogênese/farmacologia , Encéfalo/irrigação sanguínea , Encéfalo/citologia , Humanos , Integrina alfaVbeta3/fisiologia , Integrinas/fisiologia , Oligopeptídeos/farmacologia , Receptores de Vitronectina/fisiologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/fisiologia , Esfingomielinas/metabolismoRESUMO
Species-specific urokinase receptor (uPAR) ligands with improved pharmacokinetics were generated by site-specific mutagenesis and amino-terminal pegylation. These molecules were used to probe the role of uPAR in brain tumor progression and angiogenesis. The ligands blocked endothelial cell tube formation in Matrigel in a species-specific manner and reduced both baseline and uPA amino-terminal fragment-stimulated cell migration on vitronectin gradients. Treatment of U87MG gliomas implanted orthotopically in mice with single species-specific or combination uPAR ligands resulted in significant decreases in tumor size, which translated to increases in survival time, and which were most significant when the murine-specific ligand was included. Further analysis of tumors showed that the reduced sizes were correlated with a decrease in tumor cell proliferation and mean vessel density and an increase in tumor cell apoptosis. In addition, a large increase in collagen deposition was observed in the treated groups. Statistical analysis showed that the combination therapy demonstrated a clear synergy as compared to the individual agent treatments. These results suggest that the major role of the uPAR system in brain tumor progression is in the stromal compartment and particularly in neovascularization, a hallmark of invasive brain tumors.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Glioma/tratamento farmacológico , Receptores de Superfície Celular/metabolismo , Animais , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Glioma/irrigação sanguínea , Glioma/metabolismo , Glioma/patologia , Humanos , Ligantes , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neovascularização Patológica , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Polietilenoglicóis/farmacologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Especificidade da Espécie , Transplante Heterólogo , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/farmacologiaRESUMO
We have previously labeled cyclic RGD peptide c(RGDyK) with fluorine-18 through conjugation labeling via a prosthetic 4-[18F]fluorobenzoyl moiety and applied this [18F]FB-RGD radiotracer for alphav-integrin expression imaging in different preclinical tumor models with good tumor-to-background contrast. However, the unfavorable hepatobiliary excretion and rapid tumor washout rate of this tracer limit its potential clinical applications. The aims of this study were to modify the [18F]FB-RGD tracer by inserting a heterobifunctional poly(ethylene glycol) (PEG, M.W. =3,400) between the 18F radiolabel and the RGD moiety and to test this [18F]FB-PEG-RGD tracer for brain tumor targeting and in vivo kinetics. [18F]FB-PEG-RGD was prepared by coupling the RGD-PEG conjugate with N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) under slightly basic conditions (pH=8.5). The radiochemical yield was about 20-30% based on the active ester [18F]SFB, and specific activity was over 100 GBq/micromol. This tracer had fast blood clearance, rapid and high tumor uptake in the subcutaneous U87MG glioblastoma model (5.2+/-0.5%ID/g at 30 min p.i.). Moderately rapid tumor washout was observed, with the activity accumulation decreased to 2.2+/-0.4%ID/g at 4 h p.i. MicroPET and autoradiography imaging showed a very high tumor-to-background ratio and limited activity accumulation in the liver, kidneys and intestinal tracts. U87MG tumor implanted into the mouse forebrain was well visualized with [18F]FB-PEG-RGD. Although uptake in the orthotopic tumor was significantly lower (P<0.01) than in the subcutaneous tumor, the maximum tumor-to-brain ratio still reached 5.0+/-0.6 due to low normal brain background. The results of H&E staining post mortem agreed with the anatomical information obtained from non-invasive microPET imaging. In conclusion, PEGylation suitably modifies the physiological behavior of the RGD peptide. [18F]FB-PEG-RGD gave improved tumor retention and in vivo kinetics compared with [18F]FB-RGD.
Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/metabolismo , Glioblastoma/diagnóstico por imagem , Glioblastoma/metabolismo , Neovascularização Patológica/diagnóstico por imagem , Neovascularização Patológica/metabolismo , Peptídeos Cíclicos/farmacocinética , Polietilenoglicóis/farmacocinética , Tomografia por Emissão de Pósitrons/métodos , Animais , Encéfalo/irrigação sanguínea , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Feminino , Taxa de Depuração Metabólica , Camundongos , Camundongos Nus , Especificidade de Órgãos , Compostos Radiofarmacêuticos/farmacocinética , Distribuição Tecidual , Contagem Corporal TotalRESUMO
Orthotopic brain tumor growth is inhibited in athymic mice by the daily systemic administration of the alpha v-integrin antagonist EMD 121974. This compound, a cyclic RGD-penta-peptide, is a potent inhibitor of angiogenesis, which induces apoptosis of growing endothelial cells through inhibition of their alpha v-integrin interaction with the matrix proteins vitronectin and tenascin. Here we show that EMD 121974 also induces apoptosis in the alpha v-integrin-expressing tumor cell lines U87 MG and DAOY by detaching them from vitronectin and tenascin, matrix proteins known to be essential for brain tumor growth and invasion. These matrix proteins are shown to be produced by the brain tumor cells in vitro and in vivo. Furthermore, only tumor cells expressing alpha v-integrins responded to the treatment with EMD 121974, after xenotransplantation into the forebrain of nude mice, supporting the importance of tumor cell-matrix interactions in tumor cell survival in the brain. Thus, the alpha v-antagonist EMD 121974 suppresses brain tumor growth through induction of apoptosis in both brain capillary and brain tumor cells by preventing their interaction with the matrix proteins vitronectin and tenascin. The dual action of this peptide explains its potent growth suppression of orthotopically transplanted brain tumors.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Integrinas/antagonistas & inibidores , Meduloblastoma/patologia , Peptídeos Cíclicos/farmacologia , Receptores de Vitronectina/antagonistas & inibidores , Animais , Neoplasias Encefálicas/metabolismo , Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Colágeno/metabolismo , Citometria de Fluxo , Imunofluorescência , Glioblastoma/metabolismo , Humanos , Técnicas Imunoenzimáticas , Marcação In Situ das Extremidades Cortadas , Integrinas/metabolismo , Meduloblastoma/metabolismo , Camundongos , Camundongos Nus , Receptores de Vitronectina/metabolismo , Venenos de Serpentes , Tenascina/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologia , Vitronectina/metabolismoRESUMO
In vivo imaging methodologies allow for serial measurement of tumor size, circumventing the need for sacrificing mice at given time points. In orthotopically transplanted murine models of brain tumors, cross-section micro-MRI allows for visualization and measurement of the physically inaccessible tumors. To allow for long resident times of a contrast agent in the tumor, intraperitoneal administration was used as a route of injection for contrast-enhanced micro-MRI, and a simple method for relative tumor volume measurements was examined. A strategy for visualizing the variability of the delayed tumor enhancement was developed. These strategies were applied to monitor the growth of brain tumors xenotransplanted into nude mice and either treated with the antiangiogenic peptide EMD 121974 or an inactive control peptide. Each mouse was used as its own control. Serial imaging was done weekly, beginning at Day 7 after tumor cell implantation and continued for 7 weeks. Images obtained were reconstructed on the MRI instrument. The image files were transferred off line to be postprocessed to assess tumor growth (volume) and variability in enhancement (three-dimensional [3-D] intensity models). In a small study, tumor growth and response to treatment were followed using this methodology and the high-resolution images displayed in 3-D allowed for straightforward qualitative assessment of variable enhancement related to vascular factors and tumor age.