RESUMO
Ferritin, the iron storage protein, was recently suggested to be a candidate reporter for the detection of gene expression by magnetic resonance imaging (MRI). Here we report the generation of TET:EGFP-HAferritin (tet-hfer) transgenic mice, in which tissue-specific inducible transcriptional regulation of expression of the heavy chain of ferritin could be detected in vivo by MRI. We show organ specificity by mating the tet-hfer mice with transgenic mice expressing tetracycline transactivator (tTA) in liver hepatocytes and in vascular endothelial cells. Tetracycline-regulated overexpression of ferritin resulted in specific alterations of the transverse relaxation rate (R(2)) of water. Transgene-dependent changes in R(2) were detectable by MRI in adult mice, and we also found fetal developmental induction of transgene expression in utero. Thus, the tet-hfer MRI reporter mice provide a new transgenic mouse platform for in vivo molecular imaging of reporter gene expression by MRI during both embryonic and adult life.
Assuntos
Ferritinas/metabolismo , Regulação da Expressão Gênica/fisiologia , Imageamento por Ressonância Magnética/métodos , Animais , Sequência de Bases , Western Blotting , Embrião de Mamíferos/metabolismo , Células Endoteliais/metabolismo , Ferritinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hepatócitos/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Tetraciclina/metabolismo , Tetraciclina/farmacologiaRESUMO
PURPOSE: To quantitatively monitor the dynamic perivascular recruitment of ferritin heavy chain (FHC)-overexpressing fibroblasts to ovarian carcinoma xenografts by using R2 mapping and biexponential magnetic resonance (MR) relaxometry. MATERIALS AND METHODS: In vivo studies of female mice were approved by the institutional animal care and use committee. In vitro analysis included MR-based R2 relaxation measurements of monkey kidney cell line (CV1) fibroblasts that overexpress FHC, followed by inductively coupled plasma mass spectrometry to assess cellular iron content. For in vivo analysis, CV1-FHC fibroblasts were either mixed with fluorescent human ovarian carcinoma cells before subcutaneous implantation (coinjection) or injected intraperitoneally 4 days after the cancer cells were injected (remote recruitment). Dynamic changes in tumor R2 were used to derive CV1-FHC cell fraction in both models. In coinjection tumors, dynamic contrast material-enhanced MR imaging was used to measure tumor fractional blood volume. Whole-body fluorescence imaging and immunohistochemical staining were performed to validate MR results. One-way repeated measures analysis of variance was used to assess MR and fluorescence imaging results and tumor volume, and one-way analysis of variance was used to assess spectrometric results, fractional blood volume, and immunohistochemical evaluation. RESULTS: CV1-FHC fibroblasts (vs CV1 fibroblasts) showed enhanced iron uptake (1.8 mmol ± 0.5 × 10(-8) vs 0.9 mmol ± 0.5 × 10(-8); P < .05), retention (1.6 mmol ± 0.5 × 10(-8) vs 0.5 mmol ± 0.5 × 10(-8), P < .05), and cell density-dependent R2 contrast. R2 mapping in vivo revealed preferential recruitment of CV1-FHC cells to the tumor rim in both models. Measurement of fractional blood volume was similar in all tumors (2.6 AU ± 0.5 × 10(-3) for CV1, 2.3 AU ± 0.3 × 10(-3) for CV1-FHC, 2.9 ± 0.3 × 10(-3) for CV1-FHC-ferric citrate). Dynamic changes in CV1-FHC cell fraction determined at MR relaxometry in both models were confirmed at immunohistochemical analysis. CONCLUSION: FHC overexpression, when combined with R2 mapping and MR relaxometry, enabled in vivo detection of the dynamic recruitment of exogenously administered fibroblasts to the vasculature of solid tumors.
Assuntos
Ferritinas/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Animais , Linhagem Celular Tumoral , Rastreamento de Células/métodos , Feminino , Imageamento por Ressonância Magnética/métodos , Camundongos , Camundongos NusRESUMO
Molecular imaging strives to detect molecular events at the level of the whole organism. In some cases, the molecule of interest can be detected either directly or with targeted contrast media. However many genes and proteins and particularly those located in intracellular compartments are not accessible for targeted agents. The transcriptional regulation of these genes can nevertheless be detected, although indirectly, using reporter gene encoding for readily detectable proteins. Such reporter proteins can be expressed in the tissue of interest by genetically introducing the reporter gene in the target cells. Imaging of reporter genes has become a powerful tool in modern biomedical research. Typically, expression of fluorescent and bioluminescent proteins and the reaction product of expressed enzymes and exogenous substrates were examined using in vitro histological methods and in vivo whole body imaging methods. Recent advances in MRI reporter gene methods raised the possibility that MRI could become a powerful tool for concomitant high-resolution anatomical and functional imaging and for imaging of reporter gene activity. An immediate application of MRI reporter gene methods was by monitoring gene expression patterns in gene therapy and in vivo imaging of the survival, proliferation, migration and differentiation of pluripotent and multipotent cells used in cell-based regenerative therapies for cancer, myocardial infarction and neural degeneration. In this review, we characterized a variety of MRI reporter gene methods based on their applicability to report cell survival/proliferation, migration and differentiation. In particular, we discussed which methods were best suited for translation to clinical use in regenerative therapies.
Assuntos
Diferenciação Celular , Movimento Celular , Genes Reporter , Imageamento por Ressonância Magnética/métodos , Animais , Proliferação de Células , Sobrevivência Celular , HumanosRESUMO
Tumors emerge as a result of the sequential acquisition of genetic, epigenetic and somatic alterations promoting cell proliferation and survival. The maintenance and expansion of tumor cells rely on their ability to adapt to changes in their microenvironment, together with the acquisition of the ability to remodel their surroundings. Tumor cells interact with two types of interconnected microenvironments: the metabolic cell autonomous microenvironment and the nonautonomous cellular-molecular microenvironment comprising interactions between tumor cells and the surrounding stroma. Hypoxia is a central player in cancer progression, affecting not only tumor cell autonomous functions, such as cell division and invasion, resistance to therapy and genetic instability, but also nonautonomous processes, such as angiogenesis, lymphangiogenesis and inflammation, all contributing to metastasis. Closely related microenvironmental stressors affecting cancer progression include, in addition to hypoxia, elevated interstitial pressure and oxidative stress. Noninvasive imaging offers multiple means to monitor the tumor microenvironment and its consequences, and can thus assist in the understanding of the biological basis of hypoxia and microenvironmental stress in cancer progression, and in the development of strategies to monitor therapies targeted at stress-induced tumor progression.
Assuntos
Imageamento por Ressonância Magnética/métodos , Neoplasias/diagnóstico , Neoplasias/patologia , Estresse Fisiológico , Animais , Hipóxia Celular , Humanos , Metástase Neoplásica/patologia , Neoplasias/irrigação sanguínea , Neoplasias/metabolismo , Neovascularização Patológica/diagnósticoRESUMO
Angiogenesis and lymphangiogenesis are key processes during embryogenesis as well as under physiological and pathological conditions. Vascular endothelial growth factor C (VEGFC), the ligand for both VEGFR2 and VEGFR3, is a central lymphangiogenic regulator that also drives angiogenesis. Here, we report that members of the highly conserved BACH (BTB and CNC homology) family of transcription factors regulate VEGFC expression, through direct binding to its promoter. Accordingly, down-regulation of bach2a hinders blood vessel formation and impairs lymphatic sprouting in a Vegfc-dependent manner during zebrafish embryonic development. In contrast, BACH1 overexpression enhances intratumoral blood vessel density and peritumoral lymphatic vessel diameter in ovarian and lung mouse tumor models. The effects on the vascular compartment correlate spatially and temporally with BACH1 transcriptional regulation of VEGFC expression. Altogether, our results uncover a novel role for the BACH/VEGFC signaling axis in lymphatic formation during embryogenesis and cancer, providing a novel potential target for therapeutic interventions.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Neovascularização Fisiológica/fisiologia , Fator C de Crescimento do Endotélio Vascular/genética , Proteínas de Peixe-Zebra/genética , Moduladores da Angiogênese/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linhagem Celular Tumoral , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Linfangiogênese/fisiologia , Vasos Linfáticos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Morfogênese , Transdução de Sinais , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismoRESUMO
Ferritin has gained significant attention as a potential reporter gene for in vivo imaging by magnetic resonance imaging (MRI). However, due to the ferritin ferrihydrite core, the relaxivity and sensitivity for detection of native ferritin is relatively low. We report here on a novel chimeric magneto-ferritin reporter gene - ferritin-M6A - in which the magnetite binding peptide from the magnetotactic bacteria magnetosome-associated Mms6 protein was fused to the C-terminal of murine h-ferritin. Biophysical experiments showed that purified ferritin-M6A assembled into a stable protein cage with the M6A protruding into the cage core, enabling magnetite biomineralisation. Ferritin-M6A-expressing C6-glioma cells showed enhanced (per iron) r2 relaxivity. MRI in vivo studies of ferritin-M6A-expressing tumour xenografts showed enhanced R2 relaxation rate in the central hypoxic region of the tumours. Such enhanced relaxivity would increase the sensitivity of ferritin as a reporter gene for non-invasive in vivo MRI-monitoring of cell delivery and differentiation in cellular or gene-based therapies.
Assuntos
Apoferritinas/metabolismo , Neoplasias Encefálicas/diagnóstico por imagem , Compostos Férricos/metabolismo , Óxido Ferroso-Férrico/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Apoferritinas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Genes Reporter , Engenharia Genética , Imageamento por Ressonância Magnética , Camundongos , Modelos Moleculares , Transplante de Neoplasias , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
The heavy chain of murine ferritin, an iron storage molecule with ferroxidase activity, was developed as a novel endogenous reporter for the detection of gene expression by magnetic resonance imaging (MRI). Expression of both enhanced green fluorescent protein (EGFP) and influenza hemagglutinin (HA)-tagged ferritin were tightly coregulated by tetracycline (TET), using a bidirectional expression vector. C6 cells stably expressing a TET-EGFP-HA-ferritin construct enabled the dynamic detection of TET-regulated gene expression by MRI, followed by independent validation using fluorescence microscopy and histology. MR relaxation rates were significantly elevated both in vitro and in vivo on TET withdrawal, and were consistent with induced expression of ferritin and increase in intracellular iron content. Hence, overexpression of ferritin was sufficient to trigger cellular response, augmenting iron uptake to a degree detectable by MRI. Application of this novel MR reporter gene that generates significant contrast in the absence of exogenously administered substrates opens new possibilities for noninvasive molecular imaging of gene expression by MRI.
Assuntos
Ferritinas/genética , Expressão Gênica , Genes Reporter , Glioma/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Imageamento por Ressonância Magnética , Animais , Antibacterianos/farmacologia , Neoplasias Encefálicas/metabolismo , Proliferação de Células , Feminino , Proteínas de Fluorescência Verde/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Técnicas In Vitro , Ferro/metabolismo , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Tetraciclina/farmacologia , Transfecção , Células Tumorais CultivadasRESUMO
Heparanase expression has been linked to increased tumor invasion, metastasis, and angiogenesis and with poor prognosis. The aim of the study was to monitor the effect of heparanase expression on lymph node metastasis, in heparanase-overexpressing subcutaneous Eb mouse T-lymphoma tumors, and their draining lymph node. Dynamic contrast-enhanced magnetic resonance imaging (MRI) using biotin-BSA-GdDTPA-FAM/ROX was applied for analysis of blood volume, vascular permeability, and interstitial convection, and for detection of very early stages of such metastatic dissemination. Eb tumors increased extravasation, interstitial convection, and lymphatic drain of the contrast material. Interstitial flow directions were mapped by showing radial outflow interrupted in some tumors by directional flow toward the popliteal lymph node. Heparanase expression significantly increased contrast enhancement of the popliteal lymph node but not of the primary tumor. Changes in MR contrast enhancement preceded the formation of pathologically detectable metastases, and were detectable when only a few enhanced green fluorescent protein (EGFP)-expressing Eb cells were found near and within the nodes. These results demonstrate very early, heparanase-dependent vascular changes in lymph nodes that were visible by MRI following administration of biotin-BSA-GdDTPA-FAM/ROX, and can be used for studying the initial stages of lymph node infiltration.
Assuntos
Meios de Contraste/farmacologia , Glucuronidase/fisiologia , Metástase Linfática , Imageamento por Ressonância Magnética/métodos , Animais , Biotina/farmacologia , Linhagem Celular Tumoral , Gadolínio DTPA/farmacologia , Glucuronidase/biossíntese , Proteínas de Fluorescência Verde/metabolismo , Linfonodos/patologia , Linfoma/patologia , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Invasividade Neoplásica , Metástase Neoplásica , Permeabilidade , Fatores de Tempo , TransfecçãoRESUMO
Dynamic imaging of gene expression in live animals is among the exciting challenges of molecular imaging. To achieve that, one of the approaches is to use reporter genes that encode for the synthesis of easily detectable products. Such reporter genes can be designed to be expressed under the control of the regulatory elements included in a promoter region of a gene of interest, thus allowing the use of the same reporter gene for the detection of multiple genes. The most commonly used reporter genes include the firefly light-generating enzyme luciferase and the green fluorescent protein detectable by bioluminescence and fluorescence optical imaging, respectively. Over the last years a number of studies demonstrated the ability to use the iron-binding protein ferritin as a reporter gene that allows the detection of gene expression by magnetic resonance imaging (MRI). MRI provides high spatial resolution and soft tissue contrast for deep tissues along with a large arsenal of functional and anatomical contrast mechanisms that can be correlated with gene expression, and can potentially be translated into clinical use.
Assuntos
Ferritinas/farmacocinética , Perfilação da Expressão Gênica/métodos , Genes Reporter , Distúrbios do Metabolismo do Ferro/diagnóstico , Imageamento por Ressonância Magnética/métodos , Nanopartículas , Animais , Ferritinas/genética , Humanos , Aumento da Imagem/métodos , Distúrbios do Metabolismo do Ferro/genética , Distúrbios do Metabolismo do Ferro/metabolismo , Técnicas de Sonda Molecular , Nanopartículas/ultraestruturaRESUMO
The risk and severity of ovarian carcinoma, the leading cause of gynecologic malignancy death, are significantly elevated in postmenopausal women. Ovarian failure at menopause, associated with a reduction in estrogen secretion, results in an increase of the gonadotropic luteinizing hormone (LH) and follicle-stimulating hormone (FSH), suggesting a role for these hormones in facilitating the progression of ovarian carcinoma. The current study examined the influence of hormonal stimulation on lymphangiogenesis in ovarian cancer cells. In vitro stimulation of ES2 ovarian carcinoma cells with LH and FSH induced expression of vascular endothelial growth factor (VEGF)-C. In vivo, ovariectomy of mice resulted in activation of the VEGF-C promoter in ovarian carcinoma xenografts, increased VEGF-C mRNA level, and enhanced tumor lymphangiogenesis and angiogenesis. Seeking the molecular mechanism, we examined the role of lens epithelium-derived growth factor (LEDGF/p75) and the possible contribution of its putative target, a conserved stress-response element identified in silico in the VEGF-C promoter. Using chromatin immunoprecipitation, we showed that LEDGF/p75 indeed binds the VEGF-C promoter, and binding is augmented by FSH. A corresponding hormonally regulated increase in the LEDGF/p75 mRNA and protein levels was observed. Suppression of LEDGF/p75 expression using small interfering RNA, suppression of LH and FSH production using the gonadotropin-releasing hormone antagonist cetrorelix, or mutation of the conserved stress-response element suppressed the hormonally induced expression of VEGF-C. Overall, our data suggest a possible role for elevated gonadotropins in augmenting ovarian tumor lymphangiogenesis in postmenopausal women.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Hormônio Foliculoestimulante/farmacologia , Hormônio Luteinizante/farmacologia , Neoplasias Ovarianas/irrigação sanguínea , Fatores de Transcrição/metabolismo , Fator C de Crescimento do Endotélio Vascular/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Animais , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Vasos Linfáticos/patologia , Camundongos , Camundongos Nus , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Ovariectomia , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fatores de Transcrição/biossíntese , Fator C de Crescimento do Endotélio Vascular/genéticaRESUMO
Vascular endothelial growth factor C (VEGF-C) plays a critical role in tumor lymphangiogenesis and lymph node metastasis. We report here that VEGF-C expression is regulated by microenvironmental stress including hyperthermia and oxidative stress. Furthermore, we show that this stress response is mediated by transcriptional activation mediated by lens epithelium-derived growth factor (LEDGF/p75). Ectopic expression of LEDGF/p75 in C6 rat glioma and in H1299 human non-small cell lung carcinoma induced VEGF-C expression in vitro, whereas in subcutaneous mouse tumor xenografts, LEDGF/p75 stimulated VEGF-C expression and augmented angiogenesis and lymphangiogenesis. Conversely, overexpression of a LEDGF/p75 native antisense or LEDGF/p75-targeted short interfering RNA downmodulated VEGF-C expression. LEDGF seemed to conferred this activity on binding to a conserved stress response element (STRE) located in the VEGF-C gene because mutating the STRE was sufficient for the suppression of basal and stress-induced activations of the VEGF-C promoter. Thus, the study reported here identified a role for LEDGF/p75 in stress-regulated transcriptional control of VEGF-C expression. These results provide a possible link for LEDGF/p75 in tumor lymphangiogenesis and cancer metastasis. Hence, our data suggest the LEDGF-VEGF-C axis as a putative biomarker for the detection of stress-induced lymphangiogenesis and LEDGF as a potential target for antimetastatic therapy.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Regulação da Expressão Gênica/fisiologia , Glioma/genética , Hipertermia Induzida , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Estresse Oxidativo , Fator C de Crescimento do Endotélio Vascular/genética , Animais , Sequência de Bases , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Imunoprecipitação da Cromatina , Glioma/metabolismo , Glioma/patologia , Humanos , Immunoblotting , Hibridização In Situ , Luciferases/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Dados de Sequência Molecular , Mutação/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Elementos de Resposta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator C de Crescimento do Endotélio Vascular/metabolismoRESUMO
Angiogenesis (the growth of new blood vessels) is a complex multistep process that involves multiple cell types, numerous growth factors, and complex regulatory checks and balances. Tight control of vascular remodeling evolved to ensure stability of the vasculature while maintaining the body's ability to rapidly mount an angiogenic response requiring a high degree of plasticity. Angiogenesis is critical not only for physiological development, but also for the progression of pathologies, and is thus a target for therapeutic intervention. The importance of the process coupled with the ease of access for delivery of contrast agents makes the vasculature at large, and angiogenesis in particular, a favorable target of functional and molecular imaging. Recent developments in molecular imaging tools have expanded our views to encompass many components of the process. Functional imaging of blood volume, vessel permeability, and vasoreactivity is complemented by novel contrast agents that reveal specific targets on endothelial cells. Methods have been developed to label vascular cells so as to track their recruitment to sites of angiogenesis, and new "smart" contrast agents have been designed to reveal the activity of enzymatic reactions in altering the extracellular matrix (ECM) during angiogenesis.
Assuntos
Neovascularização Patológica/diagnóstico , Neovascularização Fisiológica/fisiologia , Ressonância Magnética Nuclear Biomolecular/métodos , Indutores da Angiogênese/farmacologia , Inibidores da Angiogênese/fisiologia , Meios de Contraste , HumanosRESUMO
Maintaining homogeneous perfusion in tissues undergoing remodeling and vascular expansion requires tight orchestration of the signals leading to endothelial sprouting and subsequent recruitment of perivascular contractile cells and vascular maturation. This regulation, however, is frequently disrupted in tumors. We previously demonstrated the role of tumor-associated myofibroblasts in vascularization and exit from dormancy of human ovarian carcinoma xenografts in nude mice. The aim of this work was to determine the contribution of stroma- and tumor cell-derived angiogenic growth factors to the heterogeneity of vascular permeability and maturation in MLS human ovarian carcinoma tumors. We show by RT-PCR and by in situ hybridization that VEGF was expressed by the tumor cells, while angiopoietin-1 and -2 were expressed only by the infiltrating host stroma cells. Vascular maturation was detected in vivo by vasoreactivity to hypercapnia, measured by BOLD contrast MRI and validated by immunostaining of histologic sections to alpha-smooth muscle actin. Vascular permeability was measured in vivo by dynamic contrast-enhanced MRI using albumin-based contrast material and validated in histologic sections by fluorescent staining of the biotinylated contrast material. MRI as well as histologic correlation maps between vascular maturation and vascular permeability revealed a wide range of vascular phenotypes, in which the distribution of vascular maturation and vasoreactivity did not overlap spatially with reduced permeability. The large heterogeneity in the degree of vascular maturation and permeability is consistent with the differential expression pattern of VEGF and angiopoietins during tumor angiogenesis.