Imagerie de la néoangiogenèse en médecine nucléaire

The formation of new vessels, a process referred to as neoangiogenesis, is one of the key pathophysiological mechanisms in the development and progression of cancer. It contributes to tumour growth and dissemination of neoplastic cells and can determine response or resistance to anticancer therapies. It involves different signaling pathways including the vascular endothelial growth factor (VEGF) pathway and integrins, which are also preferred targets for the development of antiangiogenic therapies. Changes in the microvasculature induced by antiangiogenic treatments occur before morphological changes can be detected with conventional imaging approaches. The development of molecular tools enabling an assessment of these targets before initiating therapy, or early detection of response or recurrence during or following treatment is essential for the close monitoring of antiangiogenic treatments. These outstanding needs call for the development of specific probes enabling the characterization of the molecules and pathways involved. This review summarizes the major signaling pathway involved in promoting tumor neoangiogenes is, the different radiotracers recently developed in preclinical and clinical settings, as well as their potential use in humans in order to improve the management of patients treated with antiangiogenic treatments.     

Volumetric computed tomography (VCT): a new technology for noninvasive, high-resolution monitoring of tumor angiogenesis

Volumetric computed tomography (VCT) is a technology in which area detectors are used for imaging large volumes of a subject with isotropic imaging resolution. We are experimenting with a prototype VCT scanner that uses flat-panel X-ray detectors and is designed for high-resolution three-dimensional (3D) imaging. Using this technique, we have demonstrated microangiography of xeno-transplanted skin squamous cell carcinomas in nude mice. VCT shows the vessel architecture of tumors and animals with greater detail and plasticity than has previously been achieved, and is superior to contrast-enhanced magnetic resonance (MR) angiography. VCT and MR images correlate well for larger tumor vessels, which are tracked from their origin on 3D reconstructions of VCT images. When compared with histology, small tumor vessels with a diameter as small as 50 microm were clearly visualized. Furthermore, imaging small vessel networks inside the tumor tissue improved discrimination of vital and necrotic regions. Thus, VCT substantially improves imaging of vascularization in tumors and offers a promising tool for preclinical studies of tumor angiogenesis and antiangiogenic therapies.     

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.     

In vivo bioluminescence imaging

In vivo bioluminescent imaging (BLI) is a versatile and sensitive tool that is based on detection of light emission from cells or tissues. Bioluminescence, the biochemical generation of light by a living organism, is a naturally occurring phenomenon. Luciferase enzymes, such as that from the North American firefly (Photinus pyralis), catalyze the oxidation of a substrate (luciferin), and photons of light are a product of the reaction. Optical imaging by bioluminescence allows a low-cost, noninvasive, and real-time analysis of disease processes at the molecular level in living organisms. Bioluminescence has been used to track tumor cells, bacterial and viral infections, gene expression, and treatment response. Bioluminescence in vivo imaging allows longitudinal monitoring of a disease course in the same animal, a desirable alternative to analyzing a number of animals at many time points during the course of the disease. We provide a brief introduction to BLI technology, specific examples of in vivo BLI studies investigating bacterial/viral pathogenesis and tumor growth in animal models, and highlight some future perspectives of BLI as a molecular imaging tool.     

In Vivo Bioluminescent Imaging of Influenza A Virus Infection and Characterization of Novel Cross-Protective Monoclonal Antibodies

Influenza A virus is a major human pathogen responsible for seasonal epidemics as well as pandemic outbreaks. Due to the continuing burden on human health, the need for new tools to study influenza virus pathogenesis as well as to evaluate new therapeutics is paramount. We report the development of a stable, replication-competent luciferase reporter influenza A virus that can be used for in vivo imaging of viral replication. This imaging is noninvasive and allows for the longitudinal monitoring of infection in living animals. We used this tool to characterize novel monoclonal antibodies that bind the conserved stalk domain of the viral hemagglutinin of H1 and H5 subtypes and protect mice from lethal disease. The use of luciferase reporter influenza viruses allows for new mechanistic studies to expand our knowledge of virus-induced disease and provides a new quantitative method to evaluate future antiviral therapies.     

Biomarkers predicting tumor response and evasion to anti-angiogenic therapy

No fully validated biological markers currently exist to predict responsiveness to or the development of evasion to anti-angiogenic therapy of cancer. The identification of such biomarkers is vital to move these therapies forward, as failure to respond to these treatments is often associated with rapid tumor progression that could have been averted had the intrinsic or acquired evasion to anti-angiogenic therapy been identified in a timely fashion. Furthermore, the high cost of antiangiogenic therapies makes it important to avoid utilizing them in the setting of lack of response or developing evasion, making the identification of biomarkers even more important. A number of potential physiologic, circulating, tissue, and imaging biomarkers have emerged from recently completed preclinical animal studies and clinical trials. In this review, we define 5 different types of biomarkers (physiologic, circulating, intratumoral, genetic polymorphisms, and radiographic); discuss the challenges in establishing biomarkers of antiangiogenic therapy in animal models and in clinical trials; and discuss future strategies to identify and validate biomarkers of anti-angiogenic therapy.     

Functional imaging of angiogenesis in an orthotopic model of pancreatic cancer

Pancreatic cancer is a major unsolved health problem. The estimated overall 5-year survival rate of only 1-4% is due to aggressiveness of the disease and the lack of effective systemic therapies. Most pancreatic cancer-related deaths are due to the development of metastases, which represents the culmination of a complex interaction between the host organism and neoplastic cells within the primary tumor. Therefore, the study of tumor-host interaction in the context of the whole organism is necessary to evaluate the pathogenesis of tumor growth and metastasis so that effective therapies can be developed. Recent advances in functional imaging combined with animal models that faithfully recreate the biology of human tumors have elevated our ability to examine these complex interactions. In this review, we will use the example of orthotopic mouse models of pancreatic cancer as a tool to survey the challenges and possibilities of functional imaging of angiogenesis, a critical determinant of metastasis.     

Biomarkers predicting tumor response and evasion to anti-angiogenic therapy

No fully validated biological markers currently exist to predict responsiveness to or the development of evasion to anti-angiogenic therapy of cancer. The identification of such biomarkers is vital to move these therapies forward, as failure to respond to these treatments is often associated with rapid tumor progression that could have been averted had the intrinsic or acquired evasion to anti-angiogenic therapy been identified in a timely fashion. Furthermore, the high cost of antiangiogenic therapies makes it important to avoid utilizing them in the setting of lack of response or developing evasion, making the identification of biomarkers even more important. A number of potential physiologic, circulating, tissue, and imaging biomarkers have emerged from recently completed preclinical animal studies and clinical trials. In this review, we define 5 different types of biomarkers (physiologic, circulating, intratumoral, genetic polymorphisms, and radiographic); discuss the challenges in establishing biomarkers of antiangiogenic therapy in animal models and in clinical trials; and discuss future strategies to identify and validate biomarkers of anti-angiogenic therapy.     

Regulation of tumor angiogenesis by the microtubule-binding protein CLIP-170

Angiogenesis, the expansion of preexisting blood vessels, is a complex process required for tumor growth and metastasis. Although current antiangiogenic strategies have shown promising results in several cancer types, identifi-cation of additional antiangiogenic targets is required to improve the therapeutic response. Herein, we show that the microtubule-binding protein CLIP-170 (cytoplasmic linker protein of 170 kDa) is highly expressed in breast tumor samples and correlates positively with blood vessel density. Depletion of CLIP-170 significantly impaired vascular endothelial tube formation and sprouting in vitro and inhibited breast tumor growth in mice by decreasing tumor vascularization. Our data further show that CLIP-170 is important for the migration but not the proliferation of vascular endothelial cells. In addition, CLIP-170 promotes the polarization of endothelial cells in response to the angiogenic stimulus. These findings thus demonstrate a critical role for CLIP-170 in tumor angiogenesis and suggest its potential as a novel antiangiogenic target     

Viral nanoparticles as tools for intravital vascular imaging

A significant impediment to the widespread use of noninvasive in vivo vascular imaging techniques is the current lack of suitable intravital imaging probes. We describe here a new strategy to use viral nanoparticles as a platform for the multivalent display of fluorescent dyes to image tissues deep inside living organisms. The bioavailable cowpea mosaic virus (CPMV) can be fluorescently labeled to high densities with no measurable quenching, resulting in exceptionally bright particles with in vivo dispersion properties that allow high-resolution intravital imaging of vascular endothelium for periods of at least 72 h. We show that CPMV nanoparticles can be used to visualize the vasculature and blood flow in living mouse and chick embryos to a depth of up to 500 microm. Furthermore, we show that the intravital visualization of human fibrosarcoma-mediated tumor angiogenesis using fluorescent CPMV provides a means to identify arterial and venous vessels and to monitor the neovascularization of the tumor microenvironment.     

Role of receptor-mediated endocytosis in the antiangiogenic effects of human T lymphoblastic cell-derived microparticles

Microparticles possess therapeutic potential regarding angiogenesis. We have demonstrated the contribution of apoptotic human CEM T lymphocyte-derived microparticles (LMPs) as inhibitors of angiogenic responses in animal models of inflammation and tumor growth. In the present study, we characterized the antivascular endothelial growth factor (VEGF) effects of LMPs on pathological angiogenesis in an animal model of oxygen-induced retinopathy and explored the role of receptor-mediated endocytosis in the effects of LMPs on human retinal endothelial cells (HRECs). LMPs dramatically inhibited cell growth of HRECs, suppressed VEGF-induced cell migration in vitro experiments, and attenuated VEGF-induced retinal vascular leakage in vivo. Intravitreal injections of fluorescently labeled LMPs revealed accumulation of LMPs in retinal tissue, with more than 60% reductions of the vascular density in retinas of rats with oxygen-induced neovascularization. LMP uptake experiments demonstrated that the interaction between LMPs and HRECs is dependent on temperature. In addition, endocytosis is partially dependent on extracellular calcium. RNAi-mediated knockdown of low-density lipoprotein receptor (LDLR) reduced the uptake of LMPs and attenuated the inhibitory effects of LMPs on VEGF-A protein expression and HRECs cell growth. Intravitreal injection of lentivirus-mediated RNA interference reduced LDLR protein expression in retina by 53% and significantly blocked the antiangiogenic effects of LMPs on pathological vascularization. In summary, the potent antiangiogenic LMPs lead to a significant reduction of pathological retinal angiogenesis through modulation of VEGF signaling, whereas LDLR-mediated endocytosis plays a partial, but pivotal, role in the uptake of LMPs in HRECs.     

Cranial MRI of small rodents using a clinical MR scanner

Increasing numbers of small animal models are in use in the field of neuroscience research. Magnetic resonance imaging (MRI) provides an excellent method for non-invasive imaging of the brain. Using three-dimensional (3D) MR sequences allows lesion volumetry, e.g. for the quantification of tumor size. Specialized small-bore animal MRI scanners are available for high-resolution MRI of small rodents' brain, but major drawbacks of this dedicated equipment are its high costs and thus its limited availability. Therefore, more and more research groups use clinical MR scanners for imaging small animal models. But to achieve a reasonable spatial resolution at an acceptable signal-to-noise ratio with these scanners, some requirements concerning sequence parameters have to be matched. Thus, the aim of this paper was to present in detail a method how to perform MRI of small rodents brain using a standard clinical 1.5 T scanner and clinically available radio frequency coils to keep material costs low and to circumvent the development of custom-made coils.     

Rat C6 glioma as experimental model system for the study of glioblastoma growth and invasion

Infiltration of the central nervous system by neoplastic cells in patients with glioblastoma multiforme (GBM) leads to neurological dysfunction and eventually to death. The elucidation of the mechanisms underlying the aggressive nature of GBM aims at improving radio-, chemo- and gene therapy. This review is focused on the use of rat C6 glioma as an experimental model system for GBM and provides an overview of the experimental data published in the literature using this cell line in elucidating the mechanism of tumor growth, angiogenesis and invasion, and in the design and evaluation of anticancer therapies. Understanding the stages of malignant brain tumor progression requires a series of experimental approaches with a varying degree of complexity. Implantation of malignant cells into animal brain tissue closely resembles in vivo tumor growth and has the advantage over simplified models that inflammatory and vascular mechanisms are activated. However, the complexity of these models makes it difficult to identify the individual processes involved in sustained tumor growth, angiogenesis and invasion. In cell culture models, the effect of growth factors, extracellular matrix components, proteases and adhesion molecules can be investigated. The secretion of tumor-derived factors into the medium can also be analyzed when simplified models are used. This review is a compilation of experimental data focused on the characterization of tumor-related processes and on the evaluation of new therapies for the treatment of malignant glial neoplasms using rat C6 glioma as a model system.     

Dynamic contrast-enhanced magnetic resonance imaging of sunitinib-induced vascular changes to schedule chemotherapy in renal cell carcinoma xenograft tumors

In an attempt to develop better therapeutic approaches for metastatic renal cell carcinoma (RCC), the combination of the antiangiogenic drug sunitinib with gemcitabine was studied. Using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), we have previously determined that a sunitinib dosage of 20 mg/kg per day increased kidney tumor perfusion and decreased vascular permeability in a preclinical murine RCC model. This sunitinib dosage causing regularization of tumor vessels was selected to improve delivery of gemcitabine to the tumor. DCE-MRI was used to monitor regularization of vasculature with sunitinib in kidney tumors to schedule gemcitabine. We established an effective and nontoxic schedule of sunitinib combined with gemcitabine consisting of pretreatment with sunitinib for 3 days followed by four treatments of gemcitabine at 20 mg/kg given 3 days apart while continuing daily sunitinib treatment. This treatment caused significant tumor growth inhibition resulting in small residual tumor nodules exhibiting giant tumor cells with degenerative changes, which were observed both in kidney tumors and in spontaneous lung metastases, suggesting a systemic antitumor response. The combined therapy caused a significant increase in mouse survival. DCE-MRI monitoring of vascular changes induced by sunitinib, gemcitabine, and both combined showed increased tumor perfusion and decreased vascular permeability in kidney tumors. These findings, confirmed histologically by thinning of tumor blood vessels, suggest that both sunitinib and gemcitabine exert antiangiogenic effects in addition to cytotoxic antitumor activity. These studies show that DCE-MRI can be used to select the dose and schedule of antiangiogenic drugs to schedule chemotherapy and improve its efficacy.     

Micro-MRI at 11.7 T of a murine brain tumor model using delayed contrast enhancement

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.     

HER2 targeted molecular MR imaging using a de novo designed protein contrast agent

The application of magnetic resonance imaging (MRI) to non-invasively assess disease biomarkers has been hampered by the lack of desired contrast agents with high relaxivity, targeting capability, and optimized pharmacokinetics. We have developed a novel MR imaging probe targeting to HER2, a biomarker for various cancer types and a drug target for anti-cancer therapies. This multimodal HER20targeted MR imaging probe integrates a de novo designed protein contrast agent with a high affinity HER2 affibody and a near IR fluorescent dye. Our probe can differentially monitor tumors with different expression levels of HER2 in both human cell lines and xenograft mice models. In addition to its 100-fold higher dose efficiency compared to clinically approved non-targeting contrast agent DTPA, our developed agent also exhibits advantages in crossing the endothelial boundary, tissue distribution, and tumor tissue retention over reported contrast agents as demonstrated by even distribution of the imaging probe across the entire tumor mass. This contrast agent will provide a powerful tool for quantitative assessment of molecular markers, and improved resolution for diagnosis, prognosis and drug discovery.     

Evolutionarily repurposed networks reveal the well-known antifungal drug thiabendazole to be a novel vascular disrupting agent

Studies in diverse organisms have revealed a surprising depth to the evolutionary conservation of genetic modules. For example, a systematic analysis of such conserved modules has recently shown that genes in yeast that maintain cell walls have been repurposed in vertebrates to regulate vein and artery growth. We reasoned that by analyzing this particular module, we might identify small molecules targeting the yeast pathway that also act as angiogenesis inhibitors suitable for chemotherapy. This insight led to the finding that thiabendazole, an orally available antifungal drug in clinical use for 40 years, also potently inhibits angiogenesis in animal models and in human cells. Moreover, in vivo time-lapse imaging revealed that thiabendazole reversibly disassembles newly established blood vessels, marking it as vascular disrupting agent (VDA) and thus as a potential complementary therapeutic for use in combination with current anti-angiogenic therapies. Importantly, we also show that thiabendazole slows tumor growth and decreases vascular density in preclinical fibrosarcoma xenografts. Thus, an exploration of the evolutionary repurposing of gene networks has led directly to the identification of a potential new therapeutic application for an inexpensive drug that is already approved for clinical use in humans.     

Comparison of Optical and Power Doppler Ultrasound Imaging for Non-Invasive Evaluation of Arsenic Trioxide as a Vascular Disrupting Agent in Tumors

Small animal imaging provides diverse methods for evaluating tumor growth and acute response to therapy. This study compared the utility of non-invasive optical and ultrasound imaging to monitor growth of three diverse human tumor xenografts (brain U87-luc-mCherry, mammary MCF7-luc-mCherry, and prostate PC3-luc) growing in nude mice. Bioluminescence imaging (BLI), fluorescence imaging (FLI), and Power Doppler ultrasound (PD US) were then applied to examine acute vascular disruption following administration of arsenic trioxide (ATO).During initial tumor growth, strong correlations were found between manual caliper measured tumor volume and FLI intensity, BLI intensity following luciferin injection, and traditional B-mode US. Administration of ATO to established U87 tumors caused significant vascular shutdown within 2 hrs at all doses in the range 5 to 10 mg/kg in a dose dependant manner, as revealed by depressed bioluminescent light emission. At lower doses substantial recovery was seen within 4 hrs. At 8 mg/kg there was >85% reduction in tumor vascular perfusion, which remained depressed after 6 hrs, but showed some recovery after 24 hrs. Similar response was observed in MCF7 and PC3 tumors. Dynamic BLI and PD US each showed similar duration and percent reductions in tumor blood flow, but FLI showed no significant changes during the first 24 hrs.The results provide further evidence for comparable utility of optical and ultrasound imaging for monitoring tumor growth, More specifically, they confirm the utility of BLI and ultrasound imaging as facile assays of the vascular disruption in solid tumors based on ATO as a model agent.     

Antiangiogenic peptides and proteins: from experimental tools to clinical drugs

The formation of a 'tumor-associated vasculature', a process referred to as tumor angiogenesis, is a stromal reaction essential for tumor progression. Inhibition of tumor angiogenesis suppresses tumor growth in many experimental models, thereby indicating that tumor-associated vasculature may be a relevant target to inhibit tumor progression. Among the antiangiogenic molecules reported to date many are peptides and proteins. They include cytokines, chemokines, antibodies to vascular growth factors and growth factor receptors, soluble receptors, fragments derived from extracellular matrix proteins and small synthetic peptides. The polypeptide tumor necrosis factor (TNF, Beromun) was the first drug registered for the regional treatment of human cancer, whose mechanisms of action involved selective disruption of the tumor vasculature. More recently, bevacizumab (Avastin), an antibody against vascular endothelial growth factor (VEGF)-A, was approved as the first systemic antiangiogenic drug that had a significant impact on the survival of patients with advanced colorectal cancer, in combination with chemotherapy. Several additional peptides and antibodies with antiangiogenic activity are currently tested in clinical trials for their therapeutic efficacy. Thus, peptides, polypeptides and antibodies are emerging as leading molecules among the plethora of compounds with antiangiogenic activity. In this article, we will review some of these molecules and discuss their mechanism of action and their potential therapeutic use as anticancer agents in humans.     

A bright blue fluorescent dextran for two-photon in vivo imaging of blood vessels

Fluorescence-based in vivo imaging is one of the most important tools for monitoring of biological processes in cells and tissues of live animal models. Fluorescence imaging agents have also been used to monitor the microcirculation. Tracking microcirculation of the blood is vital to gain further insight into various vascular disease-related anomalies within the human body. As monitoring of vascular circulation is performed with visualization of both immune cells and pathogens, which are mainly labelled with red and green, the favorable color option for blood vessels could be blue. However, currently available blueish color-labeled agents for vascular monitoring is generally confronted with quick bleaching, because of its short excitation and emission wavelengths. Hereby, what we propose in this report is a newly generated bright blue fluorescent dextran, named HCD-70K that monitors the blood vessels using blue and inter-compatible typical fluorescent materials. DBCO-functionalized dextran-70K was fabricated with hydroxy-coumarin dye via metal-free bioorthogonal click chemistry, and generated HCD-70K, which can flow within the blood vessel and decipher the whole structure of the blood vessel successfully. The synthesis, spectroscopic analysis, and quantum chemical calculations were conducted. Using two-photon microscopy, efficient deep in vivo blood vessel imaging of a mouse model revealed exceptional bio-imaging capabilities of the HCD-70K and consequently it provided a promising opportunity for efficient vascular visualization in various research areas.