RESUMO
Tumor invasion requires intense interactions with stromal cells and a profound extracellular matrix remodelling by matrix metalloproteinases (MMPs). Here, we assessed the specific contribution of fibroblasts to tumor invasion, MMPs, tissue inhibitors of MMPs and angiogenesis-related cytokine expression in organotypic cultures of highly malignant HaCaT-ras A-5RT3 cells, with and without MMP inhibition. Collagen degradation, the hallmark of tumor invasion, was dependent on fibroblasts and active MMP-2. Additionally, MMP blockade down-regulated VEGF-A and up-regulated PDGF-BB. These results were paralleled in xenotransplants in vivo, demonstrating strong inhibitory effects of MMP blockade on tumor invasion and vascularization, as shown by the almost complete absence of VEGF-A and MMP-14 and by the decrease in relative blood volume. MMP blockade also increased the fraction of mature vessels, as demonstrated by an increased mean tumor vessel diameter and a higher ratio of Ng2-positive vessels. Thus, this study highlights the importance of targeting the tumor stroma to defeat cancer.
Assuntos
Metaloproteinases da Matriz Secretadas/antagonistas & inibidores , Fator de Crescimento Derivado de Plaquetas/biossíntese , Neoplasias Cutâneas/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Becaplermina , Carcinoma de Células Escamosas/irrigação sanguínea , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Colágeno/metabolismo , Fibroblastos/enzimologia , Fibroblastos/patologia , Humanos , Metaloproteinases da Matriz Secretadas/biossíntese , Metaloproteinases da Matriz Secretadas/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica , Compostos Orgânicos/farmacologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-sis , Pirimidinas/farmacologia , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Inibidor Tecidual de Metaloproteinase-1/biossíntese , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/biossíntese , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Vascular endothelial growth factor (VEGF), which is a key regulator of angiogenesis, often induces formation of immature vessels with increased permeability and reduced vessel functionality. Here, we demonstrate that de novo expression of murine (m)VEGF-164 induces malignant and invasive tumor growth of HaCaT keratinocytes. However, the mVEGF-164-induced tumors are ulcerated with a disorganized epithelium that is interrupted by lacunae with limited basement membrane and endothelial cell coverage. Vessel maturation is strongly impaired. Tumor and vessel micromorphology are markedly improved by the combined expression of human platelet-derived growth factor (hPDGF)-B and mVEGF-164. Although tumor size and malignancy are comparable with either mVEGF-164 alone or combined human PDGF-B and mVEGF-164 expression, combined hPDGF-B and mVEGF-164 expression leads to a more solid and compact tumor tissue with a mature functional tumor vasculature and a higher microvessel density, as demonstrated histologically and by dynamic contrast-enhanced magnetic resonance imaging. Treatment of the hPDGF-B- and mVEGF-164-expressing tumors with imatinib mesylate to block PDGF-B signaling reverses this effect. In addition, tumor cell invasion of mVEGF-164 transfectants and mVEGF-164 plus hPDGF-B transfectants in vivo is associated with a marked induction of tumor-derived matrix metalloproteinase-1 and stromal matrix metalloproteinase-9 and -13, as was confirmed in three-dimensional organotypic co-cultures with fibroblasts in vitro. These data clearly demonstrate the need for a concerted action of different growth factors in the establishment of solid tumors with functional vasculature and emphasize the need for a multifactorial therapy.
Assuntos
Carcinoma de Células Escamosas/patologia , Neovascularização Patológica/fisiopatologia , Proteínas Proto-Oncogênicas c-sis/fisiologia , Neoplasias Cutâneas/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Benzamidas , Carcinoma de Células Escamosas/irrigação sanguínea , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/ultraestrutura , Proliferação de Células , Células Cultivadas , Humanos , Mesilato de Imatinib , Camundongos , Camundongos Nus , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-sis/genética , Pirimidinas/farmacologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/ultraestrutura , Transfecção , Transplante Heterólogo , Carga Tumoral/genéticaRESUMO
PURPOSE: To investigate the biologic effect of arginine-glycine-aspartic acid (RGD)-labeled ultrasmall superparamagnetic iron oxide (USPIO) (referred to as RGD-USPIO) on human umbilical vein endothelial cells (HUVECs), ovarian carcinoma (MLS) cells, and glioblastoma (U87MG) cells and on U87MG xenografts in vivo. MATERIALS AND METHODS: All experiments were approved by the governmental review committee on animal care.USPIOs were coated with integrin-specific (RGD) or unspecific (arginine-alanine-aspartic acid [RAD]) peptides. USPIO uptake in HUVECs, MLS cells, and U87MG cells and in U87MG tumor xenografts was determined with T2 magnetic resonance (MR) relaxometry in 16 nude mice. Cells and tumors were characterized by using immunofluorescence microscopy. Trypan blue staining and lactate dehydrogenase assay were used to assess cytotoxicity. Statistical evaluation was performed by using a Mann-Whitney test or a linear mixed model with random intercept for the comparison of data from different experiments. Post hoc pairwise comparisons were adjusted according to a Tukey test. RESULTS: HUVECs and MLS cells internalized RGD-USPIOs significantly more than unspecific probes. Controversially, U87MG cells accumulated RGD-USPIOs to a lesser extent than USPIO. Furthermore, only in U87MG cells, free RGD and alpha(v)beta(3) integrin-blocking antibodies strongly reduced endocytosis of nonspecific USPIOs. This was accompanied by a loss of cadherin-dependent intercellular contacts, which could not be attributed to cell damage. In U87MG tumors, RGD-USPIO accumulated exclusively at the neovasculature but not within tumor cells. The vascular accumulation of RGD-USPIO caused significantly higher changes of the R2 relaxation rate of tumors than observed for USPIO. CONCLUSION: In glioma cells with unstable intercellular contacts, inhibition of alpha(v)beta(3) integrins by antibodies and RGD and RGD-USPIO disintegrated intercellular contacts and reduced endocytotic activity, illustrating the risk of inducing biologic effects by using molecular MR probes.
Assuntos
Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/fisiologia , Meios de Contraste/farmacologia , Dextranos/farmacologia , Endocitose/efeitos dos fármacos , Endotélio Vascular/metabolismo , Óxido Ferroso-Férrico/farmacologia , Glioma/metabolismo , Integrina alfaVbeta3/metabolismo , Oligopeptídeos/farmacologia , Animais , Sobrevivência Celular , Células Cultivadas , Meios de Contraste/farmacocinética , Dextranos/farmacocinética , Feminino , Óxido Ferroso-Férrico/farmacocinética , Imunofluorescência , Glioblastoma , Glioma/irrigação sanguínea , Glioma/fisiopatologia , Humanos , Imageamento por Ressonância Magnética , Nanopartículas de Magnetita , Camundongos , Camundongos Nus , Transplante de Neoplasias , Oligopeptídeos/farmacocinética , Neoplasias Ovarianas , Veias UmbilicaisRESUMO
PURPOSE: To measure longitudinal relaxation rate (R1) changes during contrast agent studies using a driven equilibrium single pulse observation of T1 (DESPOT) method with a sliding window (sw) acquisition. MATERIALS AND METHODS: A sw-DESPOT technique was implemented that uses several three-dimensional (3D) image data sets to calculate R1 with a temporal resolution of only a single data set. Different sources of systematic errors were studied in simulations, and the technique was tested in a tumor-bearing mouse using an intravascular contrast agent. RESULTS: Consistent concentration distributions of the CA were calculated with a temporal resolution of 10 s. CONCLUSION: Sw-DESPOT offers a precise and fast method to monitor the CA dynamics in 3D volumes.
Assuntos
Meios de Contraste/farmacologia , Imageamento por Ressonância Magnética/métodos , Algoritmos , Animais , Humanos , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Camundongos , Modelos Estatísticos , Imagens de Fantasmas , Software , Fatores de TempoRESUMO
PURPOSE: To assess vascular remodeling in tumors during two different antiangiogenic therapies with dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and vessel size imaging and to evaluate the vessel size index (VSI) as a novel biomarker of therapy response. MATERIALS AND METHODS: In two independent experiments, nude mice bearing human skin squamous cell carcinoma xenografts were treated with a vascular endothelial growth factor (VEGF) inhibitor (bevacizumab) or a multitargeted tyrosine kinase inhibitor (SU11248). Changes in tumor vascularity were assessed by DCE-MRI and vessel size imaging. DCE-MRI data were analyzed applying a two-compartment model (Brix), calculating the parameters Amplitude and k(ep). RESULTS: For both experiments Amplitude decreased significantly in treated tumors while k(ep) did not change significantly. VSI showed controversial results. VSI was significantly increased in SU11248-treated A431 tumors, whereas no changes were found in bevacizumab-treated HaCaT-ras-A-5RT3 tumors. Immunohistology confirmed these results and suggest differences in the maturation of tumor vascularization as a possible explanation. CONCLUSION: DCE-MRI and vessel size imaging provide reliable and supplementing biomarkers of antiangiogenic therapy response. The results of both methods are in excellent agreement with histology. Nevertheless, our results also indicate that vascular remodeling is complex and that a uniform response cannot be expected for different tumors and therapies.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Vasos Sanguíneos/patologia , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Imageamento por Ressonância Magnética/métodos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Animais , Antineoplásicos/administração & dosagem , Vasos Sanguíneos/efeitos dos fármacos , Carcinoma de Células Escamosas/irrigação sanguínea , Meios de Contraste , Feminino , Indóis/administração & dosagem , Camundongos , Camundongos Nus , Pirróis/administração & dosagem , Sunitinibe , Resultado do TratamentoRESUMO
OBJECTIVES: To assess the pharmacodynamic behavior of cyanoacrylate, streptavidin-coated microbubbles (MBs) and to investigate their suitability for molecular ultrasound imaging. MATERIALS AND METHODS: Biodistribution of MBs was analyzed in tumor-bearing mice using gamma-counting, immunohistochemistry, flow cytometry, and ultrasound. Further, vascular endothelial growth factor receptor 2-antibody coupled MBs were used to image tumor neovasculature. RESULTS: After 1 minute >90% of MBs were cleared from the blood and pooled in the lungs, liver, and spleen. Subsequently, within 1 hour a decent reincrease of MB-concentration was observed in the blood. The remaining MBs were removed by liver and spleen macrophages. About 30% of the phagocytosed MBs were intact after 48 hours. Shell fragments were found in the kidneys only. No relevant MB-accumulation was observed in tumors. In contrast, vascular endothelial growth factor receptor 2-specific MBs accumulated significantly within the tumor vasculature (P < 0.05). CONCLUSIONS: The pharmacokinetic behavior of streptavidin-coated cyanoacrylate MBs has been studied. In this context, the low amount of MBs in tumors after >5 minutes is beneficial for specific targeting of angiogenesis.
Assuntos
Carcinoma de Células Escamosas/diagnóstico por imagem , Carcinoma de Células Escamosas/metabolismo , Meios de Contraste/farmacocinética , Cianoacrilatos/química , Microbolhas , Estreptavidina/farmacocinética , Animais , Materiais Revestidos Biocompatíveis/química , Taxa de Depuração Metabólica , Camundongos , Camundongos Nus , Especificidade de Órgãos , Estreptavidina/química , Distribuição Tecidual , UltrassonografiaRESUMO
Molecular ultrasound is capable of elucidating the expression of angiogenic markers in vivo. However, the capability of the method for volumetric "multitarget quantification" and for the assessment of antiangiogenic therapy response has rather been investigated. Therefore, we generated cyanoacrylate microbubbles linked to vascular endothelial growth factor receptor 2 (VEGFR2) and alphavbeta3 integrin binding ligands and quantified their accumulation in squamous cell carcinoma xenografts (HaCaT-ras-A-5RT3) in mice with the quantitative volumetric ultrasound scanning technique, sensitive particle acoustic quantification. Specificity of VEGFR2 and alphavbeta3 integrin binding microbubbles was shown, and changes in marker expression during matrix metalloproteinase inhibitor treatment were investigated. In tumors, accumulation of targeted microbubbles was significantly higher compared with nonspecific ones and could be inhibited competitively by addition of the free ligand in excess. Also, multimarker imaging could successfully be done during the same imaging session. Molecular ultrasound further indicated a significant increase of VEGFR2 and alphavbeta3 integrin expression during tumor growth and a considerable decrease in both marker densities after matrix metalloproteinase inhibitor treatment. Histologic data suggested that the increasing VEGFR2 and alphavbeta3 integrin concentrations in tumors during growth are related to an up-regulation of its expression by the endothelial cells, whereas its decrease under therapy is more related to the decreasing relative vessel density. In conclusion, targeted ultrasound appears feasible for the longitudinal molecular profiling of tumor angiogenesis and for the sensitive assessment of therapy effects in vivo.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Neoplasias/irrigação sanguínea , Neoplasias/diagnóstico por imagem , Neovascularização Patológica/diagnóstico por imagem , Neovascularização Patológica/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Integrina alfaVbeta3/metabolismo , Inibidores de Metaloproteinases de Matriz , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Nus , Microtúbulos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Ultrassonografia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The sensitivity of Doppler ultrasound below 10 MHz to assess antiangiogenic therapy effects in tumor xenografts has been shown to be limited. Thus, our aim was to evaluate high-frequency volumetric power-Doppler ultrasound (HF-VPDU) for monitoring antiangiogenic treatments. Squamous cell carcinoma xenografts grown in nude mice were scanned with HF-VPDU at a center frequency of 30 MHz. Images with 200-microm slice thicknesses were recorded and merged into a three-dimensional dataset, of which the relative color pixel density was determined. Animals received either VEGFR2 antibodies or 0.9% NaCl and were examined at days 0, 3 and 6 of treatment. After the last examination, tumors were resected and their vascularization characterized by immunohistology. At day 6, the volumes of treated and untreated tumors were not significantly different yet. In contrast, mean tumor vascularization in treated animals had decreased to 44%, while in the control group it had increased to 152% (P < 0.01). In correspondence, vessel density, as determined by CD31 staining, was 0.19 +/- 0.10% in treated and 0.92 +/- 0.41% in untreated tumors (P < 0.01). Additionally, the fraction of mature (SMA-positive) vessels increased under therapy. Thus, HF-VPDU can be considered as an easily applicable and fast method to screen high animal numbers for antiangiogenic therapy effects.
Assuntos
Anticorpos Monoclonais/farmacologia , Carcinoma de Células Escamosas/diagnóstico por imagem , Neovascularização Patológica/diagnóstico por imagem , Neovascularização Patológica/tratamento farmacológico , Ultrassonografia Doppler/métodos , Animais , Imuno-Histoquímica , Camundongos , Camundongos Nus , Sensibilidade e Especificidade , Estatísticas não Paramétricas , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In prostate cancers (PCa), the formation of malignant stroma may substantially influence tumor phenotype and aggressiveness. Thus, the impact of the orthotopic and subcutaneous implantations of hormone-sensitive (H), hormone-insensitive (HI), and anaplastic (AT1) Dunning PCa in rats on growth, microcirculation, and metabolism was investigated. For this purpose, dynamic contrast-enhanced magnetic resonance imaging and (1)H magnetic resonance spectroscopy ([(1)H]MRS) were applied in combination with histology. Consistent observations revealed that orthotopic H tumors grew significantly slower compared to subcutaneous ones, whereas the growth of HI and AT1 tumors was comparable at both locations. Histologic analysis indicated that glandular differentiation and a close interaction of tumor cells and smooth muscle cells (SMC) were associated with slow tumor growth. Furthermore, there was a significantly lower SMC density in subcutaneous H tumors than in orthotopic H tumors. Perfusion was observed to be significantly lower in orthotopic H tumors than in subcutaneous H tumors. Regional blood volume and permeability-surface area product showed no significant differences between tumor models and their implantation sites. Differences in growth between subcutaneous and orthotopic H tumors can be attributed to tumor-stroma interaction and perfusion. Here, SMC, may stabilize glandular structures and contribute to the maintenance of differentiated phenotype.
Assuntos
Neoplasias da Próstata/patologia , Células Estromais/fisiologia , Animais , Proliferação de Células , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Masculino , Miócitos de Músculo Liso/patologia , Fenótipo , Neoplasias da Próstata/irrigação sanguínea , Neoplasias da Próstata/metabolismo , RatosRESUMO
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.
Assuntos
Carcinoma de Células Escamosas/irrigação sanguínea , Células Endoteliais/metabolismo , Compostos Férricos/farmacocinética , Integrina alfaVbeta3/metabolismo , Imageamento por Ressonância Magnética/métodos , Neoplasias Experimentais/irrigação sanguínea , Oligopeptídeos/farmacocinética , Animais , Carcinoma de Células Escamosas/metabolismo , Células Cultivadas , Humanos , Integrina alfaVbeta3/biossíntese , Camundongos , Camundongos Nus , Neoplasias Experimentais/metabolismo , Neovascularização Patológica/diagnóstico , Neovascularização Patológica/metabolismo , Tamanho da Partícula , Propilaminas/farmacocinética , Silanos/farmacocinéticaRESUMO
The switch to an angiogenic phenotype is an important precondition for tumor growth, invasion and spread. Since newly formed vessels are characterized by structural, functional and molecular abnormalities, they offer promising targets for tumor diagnosis and therapy. Previous studies indicate that MRI is valuable to assess vessel morphology and function. It can be used to distinguish between benign and malignant lesions and to improve delineation of proliferating areas within heterogeneous tumors. In addition, tracer kinetic analysis of contrast-enhanced image series allows the estimation of well-defined physiological parameters such as blood volume, blood flow and vessel permeability. Frequently, changes of these parameters during cytostatic, anti-angiogenic and radiation therapy precede tumor volume reduction. Moreover, target-specific MRI techniques can be used to elucidate the expression of angiogenic markers at the molecular level. This review summarizes strategies for non-invasive characterization of tumor vascularization by functional and molecular MRI, hereby introducing representative preclinical and clinical applications.