Targeted imaging of colonic tumors in smad3-/- mice discriminates cancer and inflammation

The peripheral benzodiazepine receptor (PBR) is a trans-mitochondrial membrane protein that modulates steroid biosynthesis. Recently, up-regulation and nuclear localization of PBR has been shown to be associated with colon, prostate, and breast cancer. PBR has been targeted by the exogenous synthetic ligand, PK11195, for various purposes including imaging. To capitalize on these observations, we developed a high-throughput, noninvasive, in vivo imaging approach to detect spontaneously arising colonic tumors in mice using a novel PBR-targeted molecular imaging agent (NIR-conPK11195). NIR-conPK11195 localized and was retained in colonic adenomas and carcinomas in Smad3(-/-) mice but not in non-neoplastic hamartomas or chronically inflamed colonic tissue. Using a fluorescence signal-to-noise ratio of > or =4-fold 13 h after injection of the agent, we detected colonic tumors with a sensitivity of 67% and a specificity of 86% in a cohort of 37 Smad3(-/-) mice and control littermates. Furthermore, using oral administration of dextran sulfate to induce colonic inflammation, we showed that the clearance profile of NIR-conPK11195 distinguished transient uptake in inflammatory tissue from longer term retention in tumors. Taken together, these results indicate that NIR-conPK11195 is a promising optical molecular imaging tool to rapidly screen for colonic tumors in mice and to discriminate inflammation from cancer.     

A peripheral benzodiazepine receptor targeted agent for in vitro imaging and screening

We developed a molecular imaging agent (MIA), a conjugable form of PK11195 (conPK11195) coupled to a lissamine dye (Liss-ConPK11195), which targets the peripheral benzodiazepine receptor (PBR). To determine that our compound specifically binds to this 18 kDa protein, primarily expressed on the mitochondria, we performed classic binding studies on live MDA-MB-231 breast cancer cells and measured fluorescence in cell fractions of C6 glioma cells. We found that conPK11195 conjugated to the fluorophore retained significant binding to its target. Here we demonstrate the utility of the agent for in vitro imaging of live cells by specific binding to the protein of interest.     

Multiple target-specific molecular imaging agents detect liver cancer in a preclinical model

Liver cancer is the fifth most common cause of cancer deaths worldwide. Noninvasive diagnosis is difficult and the disease heterogeneity reduces the accuracy of pathological assays. Improvement in diagnostic imaging of specific molecular disease markers has provided hope for accurate and early noninvasive detection of liver cancer. However, all current imaging technologies, including ultrasonography, computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging, are not specific targets for detection of liver cancer. The aim of this study was to test the feasibility of injecting a cocktail of specific molecular imaging agents to noninvasively image liver cancer. The target-specific cocktail contained agents for imaging the neovasculature (RGD peptide), matrix metalloproteinase (MMP), and glucose transport (18F-fluorodeoxyglucose [18F-FDG]). Imaging studies were performed in liver cancer cells and xenograft models. The distribution of MMP at the intracellular level was imaged by confocal microscopy. RGD, MMP, and 18F-FDG were imaged on tumor-bearing mice using PET, CT, X-ray, and multi-wavelength optical imaging modalities. Image data demonstrated that each agent bound to a specific disease target component. The same liver cancer xenograft contained multiple disease markers. Those disease markers were heterogenetically distributed in the same tumor nodule. The molecular imaging agents had different distributions in the whole body and inside the tumor nodule. All target-specific agents yielded high tumor-to-background ratios after injection. In conclusion, target-specific molecular imaging agents can be used to study liver cancer in vitro and in vivo. Noninvasive multimodal/multi-target-specific molecular imaging agents could provide tools to simultaneously study multiple liver cancer components.     

Molecular-functional imaging of cancer: to image and imagine

The integration of advances in molecular biology, synthetic chemistry and visualization techniques has catapulted imaging into a molecular-functional realm, so that imaging is finding basic-research, preclinical and translational applications in cancer. Currently, molecular-imaging capabilities include the ability to image gene expression, receptors, signaling pathways, apoptosis, multidrug resistance and the extracellular matrix (ECM). Functional-imaging capabilities include the ability to assess angiogenesis, hypoxia and metabolism. Traditionally, imaging has played an important role in cancer diagnosing and determining response to treatment. However, it is the realization of the goal of noninvasively visualizing molecules and molecular pathways and relating these to function that makes multi-modality imaging such an exciting and powerful means for studying a multifaceted disease such as cancer.     

Molecular imaging: Bridging the gap between neuroradiology and neurohistology

Historically, in vivo imaging methods have largely relied on imaging gross anatomy. More recently it has become possible to depict biological processes at the cellular and molecular level. These new research methods use magnetic resonance imaging (MRI), positron emission tomography (PET), near-infrared optical imaging, scintigraphy, and autoradiography in vivo and in vitro. Of primary interest is the development of methods using MRI and PET with which the progress of gene therapy in glioblastoma (herpes simplex virus-thymidine kinase) and Parkinson's disease can be monitored and graphically displayed. The distribution of serotonin receptors in the human brain and the duration of serotonin-receptor antagonist binding can be assessed by PET. With PET, it is possible to localize neurofibrillary tangles (NFTs) and beta-amyloid senile plaques (APs) in the brains of living Alzheimer disease (AD) patients. MR tracking of transplanted oligodendrocyte progenitors is feasible for determining the extent of remyelinization in myelin-deficient rats. Stroke therapy in adult rats with subventricular zone cells can be monitored by MRI. Transgene expression (beta-galactosidase, tyrosinase, engineered transferrin receptor) can also be visualized using MRI. Macrophages can be marked with certain iron-containing contrast agents which, through accumulation at the margins of glioblastomas, ameliorate the visual demarcation in MRI. The use of near-infrared optical imaging techniques to visualize matrix-metalloproteinases and cathepsin B can improve the assessment of tumor aggressiveness and angiogenesis-inhibitory therapy. Apoptosis could be detected using near-infrared optical imaging representation of caspase 3 activity and annexin B. This review demonstrates the need for neurohistological research if further progress is to be made in the emerging but burgeoning field of molecular imaging.     

In vivo imaging of peripheral benzodiazepine receptors in mouse lungs: a biomarker of inflammation

The ability to visualize the immune response with radioligands targeted to immune cells will enhance our understanding of cellular responses in inflammatory diseases. Peripheral benzodiazepine receptors (PBR) are present in monocytes and neutrophils as well as in lung tissue. We used lipopolysaccharide (LPS) as a model of inflammation to assess whether the PBR could be used as a noninvasive marker of inflammation in the lungs. Planar imaging of mice administrated 10 or 30 mg/kg LPS showed increased [(123)I]-(R)-PK11195 radioactivity in the thorax 2 days after LPS treatment relative to control. Following imaging, lungs from control and LPS-treated mice were harvested for ex vivo gamma counting and showed significantly increased radioactivity above control levels. The specificity of the PBR response was determined using a blocking dose of nonradioactive PK11195 given 30 min prior to radiotracer injection. Static planar images of the thorax of nonradioactive PK11195 pretreated animals showed a significantly lower level of radiotracer accumulation in control and in LPS-treated animals (p < .05). These data show that LPS induces specific increases in PBR ligand binding in the lungs. We also used in vivo small-animal PET studies to demonstrate increased [(11)C]-(R)-PK11195 accumulation in the lungs of LPS-treated mice. This study suggests that measuring PBR expression using in vivo imaging techniques may be a useful biomarker to image lung inflammation.     

An alteration in the lateral geniculate nucleus of experimental glaucoma monkeys: in vivo positron emission tomography imaging of glial activation

We examined lateral geniculate nucleus (LGN) degeneration as an indicator for possible diagnosis of glaucoma in experimental glaucoma monkeys using positron emission tomography (PET). Chronic intraocular pressure (IOP) elevation was induced by laser trabeculoplasty in the left eyes of 5 cynomolgus monkeys. Glial cell activation was detected by PET imaging with [(11)C]PK11195, a PET ligand for peripheral-type benzodiazepine receptor (PBR), before and at 4 weeks after laser treatment (moderate glaucoma stage). At mild, moderate, and advanced experimental glaucoma stages (classified by histological changes based on the extent of axonal loss), brains were stained with cresyl violet, or antibodies against PBR, Iba-1 (a microglial marker), and GFAP (an activated astrocyte marker). In laser-treated eyes, IOP was persistently elevated throughout all observation periods. PET imaging showed increased [(11)C]PK11195 binding potential in the bilateral LGN at 4 weeks after laser treatment; the increase in the ipsilateral LGN was statistically significant (P<0.05, n = 4). Immunostaining showed bilateral activations of microglia and astrocytes in LGN layers receiving input from the laser-treated eye. PBR-positive cells were observed in LGN layers receiving input from laser-treated eye at all experimental glaucoma stages including the mild glaucoma stage and their localization coincided with Iba-1 positive microglia and GFAP-positive astrocytes. These data suggest that glial activation occurs in the LGN at a mild glaucoma stage, and that the LGN degeneration could be detected by a PET imaging with [(11)C]PK11195 during the moderate experimental glaucoma stage after unilateral ocular hypertension. Therefore, activated glial markers such as PBR in the LGN may be useful in noninvasive molecular imaging for diagnosis of glaucoma.     

In vivo molecular and single cell imaging

Molecular imaging is used to improve the disease diagnosis, prognosis, monitoring of treatment in living subjects. Numerous molecular targets have been developed for various cellular and molecular processes in genetic, metabolic, proteomic, and cellular biologic level. Molecular imaging modalities such as Optical Imaging, Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), and Computed Tomography (CT) can be used to visualize anatomic, genetic, biochemical, and physiologic changes in vivo. For in vivo cell imaging, certain cells such as cancer cells, immune cells, stem cells could be labeled by direct and indirect labeling methods to monitor cell migration, cell activity, and cell effects in cell-based therapy. In case of cancer, it could be used to investigate biological processes such as cancer metastasis and to analyze the drug treatment process. In addition, transplanted stem cells and immune cells in cell-based therapy could be visualized and tracked to confirm the fate, activity, and function of cells. In conventional molecular imaging, cells can be monitored in vivo in bulk non-invasively with optical imaging, MRI, PET, and SPECT imaging. However, single cell imaging in vivo has been a great challenge due to an extremely high sensitive detection of single cell. Recently, there has been great attention for in vivo single cell imaging due to the development of single cell study. In vivo single imaging could analyze the survival or death, movement direction, and characteristics of a single cell in live subjects. In this article, we reviewed basic principle of in vivo molecular imaging and introduced recent studies for in vivo single cell imaging based on the concept of in vivo molecular imaging.     

Preclinical TSPO Ligand PET to Visualize Human Glioma Xenotransplants: A Preliminary Study

Current positron emission tomography (PET) imaging biomarkers for detection of infiltrating gliomas are limited. Translocator protein (TSPO) is a novel and promising biomarker for glioma PET imaging. To validate TSPO as a potential target for molecular imaging of glioma, TSPO expression was assayed in a tumor microarray containing 37 high-grade (III, IV) gliomas. TSPO staining was detected in all tumor specimens. Subsequently, PET imaging was performed with an aryloxyanilide-based TSPO ligand, [¹⁸F]PBR06, in primary orthotopic xenograft models of WHO grade III and IV gliomas. Selective uptake of [¹⁸F]PBR06 in engrafted tumor was measured. Furthermore, PET imaging with [¹⁸F]PBR06 demonstrated infiltrative glioma growth that was undetectable by traditional magnetic resonance imaging (MRI). Preliminary PET with [¹⁸F]PBR06 demonstrated a preferential tumor-to-normal background ratio in comparison to 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG). These results suggest that TSPO PET imaging with such high-affinity radiotracers may represent a novel strategy to characterize distinct molecular features of glioma growth, as well as better define the extent of glioma infiltration for therapeutic purposes.     

Molecular imaging in prostate cancer

Prostate cancer (PCa) is the most common non-cutaneous malignancy in men. New ways to diagnose this cancer in its early stages are needed. Unique genetic and biochemical changes in the cell pave the way for tumors to grow and metastasize. Novel imaging approaches attempt to detect pathological processes in cancer cells at the molecular level. This has led to the establishment and development of the field of molecular imaging. Positron emission tomography (PET), magnetic resonance spectroscopic imaging (MRSI), magnetic resonance imaging (MRI), and radiolabeled antibodies are a few of the modalities that can detect abnormal tumor metabolic processes in the clinical setting. Other imaging techniques are still in their early phase of development but hold promise for the future, including bioluminescence imaging (BLI), measurement of tumor oxygenation, and measurement of uptake of iodine by tumors. These techniques are non-invasive and can spare the patient undue morbidity, while potentially providing early diagnosis, accurate follow-up and, finally, valuable prognostic information.     

Peptide-based molecular beacons for cancer imaging and therapy

Peptide-based molecular beacons are Förster resonance energy transfer-based target-activatable probes. They offer control of fluorescence emission in response to specific cancer targets and thus are useful tools for in vivo cancer imaging. With our increasing knowledge about human genome in health and disease, peptide-based “smart” probes are continually developed for in vivo optical imaging of specific molecular targets, biological pathways and cancer progression and diagnosis. A class of fluorescent photosensitizers further extends the application of peptide beacons to cancer therapeutics. This review highlights the applications of peptide beacons in cancer imaging, the simultaneous treatment and response monitoring and smart therapeutics with a focus on recent improvements in the design of these probes.     

Increased expression of peripheral benzodiazepine receptor (PBR) in dimethylbenz[a]anthracene-induced mammary tumors in rats

Expression of peripheral benzodiazepine receptors (PBR) has been found in every tissue examined; however, it is most abundant in steroid-producing tissues. Although the primary function of PBR is the regulation of steroidogenesis, its existence in nonsteroidogenic tissues as well as in other cellular compartments including the nucleus suggests that there may be other roles for PBR. Our laboratory reported earlier a significant increase of PBR density in the nucleus of DMBA-induced malignant submandibular glands of rats, suggesting a role of PBR in nuclear events of peripheral tissues. Since then numerous studies have demonstrated the abundance of PBR in tumors. Numerous studies implicate a role for cholesterol in the mechanisms underlying cell proliferation and cancer progression. Based on studies with a battery of human breast cancer cell lines and several human tissue biopsies, Hardwick et al. suggested that PBR expression, nuclear localization, and PBR-mediated cholesterol transport into the nucleus are involved in human breast cancer cell proliferation and aggressive phenotype expression. The purpose of the present study is to confirm this hypothesis by developing an animal breast cancer model and correlating the above events with the breast cancer. Weanling rats were maintained on a diet containing animal protein (casein) for 30 days and then a single dose of DMBA in sesame oil (80 mg/kg) was administered by gavage to the animals. Control animals received the vehicle only. After 122 days of DMBA administration, the animals were sacrificed. All tumors were detected by palpation. B_max of PBRs was 52.6% and 128.4% higher in the non-aggressive and aggressive cancer tissues, respectively, than that in normal tissues. Cholesterol uptake into isolated nuclei was found to be higher in both non-aggressive and aggressive tumor breast tissue than that in control tissue. There was also corresponding increase in B_max of PBRs in the nucleus of cancer tissues. Furthermore, the nuclear nucleoside triphosphatase (NTPase) activity was found to be higher in aggressive tumor tissues than that in non-aggressive tumor tissues. In conclusion, these data suggest that PBR ligand binding, and PBR-mediated cholesterol transport into the nucleus may be involved in the development of mammary gland adenocarcinoma, thus participating in the advancement of the disease.     

Mouse models in neurological disorders: Applications of non-invasive imaging

Neuroimaging techniques represent powerful tools to assess disease-specific cellular, biochemical and molecular processes non-invasively in vivo. Besides providing precise anatomical localisation and quantification, the most exciting advantage of non-invasive imaging techniques is the opportunity to investigate the spatial and temporal dynamics of disease-specific functional and molecular events longitudinally in intact living organisms, so called molecular imaging (MI). Combining neuroimaging technologies with in vivo models of neurological disorders provides unique opportunities to understand the aetiology and pathophysiology of human neurological disorders. In this way, neuroimaging in mouse models of neurological disorders not only can be used for phenotyping specific diseases and monitoring disease progression but also plays an essential role in the development and evaluation of disease-specific treatment approaches. In this way MI is a key technology in translational research, helping to design improved disease models as well as experimental treatment protocols that may afterwards be implemented into clinical routine. The most widely used imaging modalities in animal models to assess in vivo anatomical, functional and molecular events are positron emission tomography (PET), magnetic resonance imaging (MRI) and optical imaging (OI). Here, we review the application of neuroimaging in mouse models of neurodegeneration (Parkinson's disease, PD, and Alzheimer's disease, AD) and brain cancer (glioma).     

Molecular-genetic imaging: a nuclear medicine-based perspective

Molecular imaging is a relatively new discipline, which developed over the past decade, initially driven by in situ reporter imaging technology. Noninvasive in vivo molecular-genetic imaging developed more recently and is based on nuclear (positron emission tomography [PET], gamma camera, autoradiography) imaging as well as magnetic resonance (MR) and in vivo optical imaging. Molecular-genetic imaging has its roots in both molecular biology and cell biology, as well as in new imaging technologies. The focus of this presentation will be nuclear-based molecular-genetic imaging, but it will comment on the value and utility of combining different imaging modalities. Nuclear-based molecular imaging can be viewed in terms of three different imaging strategies: (1) "indirect" reporter gene imaging; (2) "direct" imaging of endogenous molecules; or (3) "surrogate" or "bio-marker" imaging. Examples of each imaging strategy will be presented and discussed. The rapid growth of in vivo molecular imaging is due to the established base of in vivo imaging technologies, the established programs in molecular and cell biology, and the convergence of these disciplines. The development of versatile and sensitive assays that do not require tissue samples will be of considerable value for monitoring molecular-genetic and cellular processes in animal models of human disease, as well as for studies in human subjects in the future. Noninvasive imaging of molecular-genetic and cellular processes will complement established ex vivo molecular-biological assays that require tissue sampling, and will provide a spatial as well as a temporal dimension to our understanding of various diseases and disease processes.     

Tumor-specific imaging through progression elevated gene-3 promoter-driven gene expression

Molecular-genetic imaging is advancing from a valuable preclinical tool to a guide for patient management. The strategy involves pairing an imaging reporter gene with a complementary imaging agent in a system that can be used to measure gene expression or protein interaction or track gene-tagged cells in vivo. Tissue-specific promoters can be used to delineate gene expression in certain tissues, particularly when coupled with an appropriate amplification mechanism. Here we show that the progression elevated gene-3 (PEG-3) promoter, derived from a rodent gene mediating tumor progression and metastatic phenotypes, can be used to drive imaging reporters selectively to enable detection of micrometastatic disease in mouse models of human melanoma and breast cancer using bioluminescence and radionuclide-based molecular imaging techniques. Because of its strong promoter activity, tumor specificity and capacity for clinical translation, PEG-3 promoter-driven gene expression may represent a practical, new system for facilitating cancer imaging and therapy.     

Selection of DNA Aptamers against Glioblastoma Cells with High Affinity and Specificity

Background

Glioblastoma is the most common and most lethal form of brain tumor in human. Unfortunately, there is still no effective therapy to this fatal disease and the median survival is generally less than one year from the time of diagnosis. Discovery of ligands that can bind specifically to this type of tumor cells will be of great significance to develop early molecular imaging, targeted delivery and guided surgery methods to battle this type of brain tumor.

Methodology/Principal Findings

We discovered two target-specific aptamers named GBM128 and GBM131 against cultured human glioblastoma cell line U118-MG after 30 rounds selection by a method called cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX). These two aptamers have high affinity and specificity against target glioblastoma cells. They neither recognize normal astraglial cells, nor do they recognize other normal and cancer cell lines tested. Clinical tissues were also tested and the results showed that these two aptamers can bind to different clinical glioma tissues but not normal brain tissues. More importantly, binding affinity and selectivity of these two aptamers were retained in complicated biological environment.

Conclusion/Significance

The selected aptamers could be used to identify specific glioblastoma biomarkers. Methods of molecular imaging, targeted drug delivery, ligand guided surgery can be further developed based on these ligands for early detection, targeted therapy, and guided surgery of glioblastoma leading to effective treatment of glioblastoma.     

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.     

Molecular imaging of proteolytic activity in cancer

The early detection of both primary tumors and metastatic disease continue to be significant challenges in the diagnosis and staging of cancer. The growing recognition of the role of proteinases and proteolytic cascades in both the growth and metastasis of tumors has led to the development not only of therapeutic strategies using proteinase inhibitors, but also of methods to detect and image tumors in vivo via tumor-associated proteolytic activities. These imaging strategies derive from the enhanced sensitivity afforded by amplification that can be obtained by enzymatic processing to increase the efficacy of imaging "contrast agents" coupled with the inherent substrate specificity and selectivity of proteinases. This review describes key proteinases important in cancer progression, the strategies that have been devised to detect and image proteolytic activity in vivo, and the potential for this kind of functional imaging to serve as a marker for targeted therapy. The intent is to draw attention to the developing methods of molecular imaging to facilitate not only cancer diagnosis, but also for devising strategies for individualized targeted therapy and non-invasive monitoring of therapeutic efficacy.     

Assessing responses to cancer therapy using molecular imaging

Tumor responses to therapy in the clinic are still evaluated primarily from non-invasive imaging measurements of reductions in tumor size. This approach, however, lacks sensitivity and can only give a delayed indication of a positive response to treatment. Major advances in our understanding of the molecular mechanisms responsible for cancer, combined with new targeted clinical imaging technologies designed to detect the molecular correlates of disease progression and response to treatment, are set to revolutionize our approach to the detection and treatment of the disease. We describe here the imaging technologies available to image tumor cell proliferation and migration, metabolism, receptor and gene expression, apoptosis and tumor angiogenesis and vascular function, and show how measurements of these parameters can be used to give early indications of positive responses to treatment or to detect drug resistance and/or disease recurrence. Special emphasis has been placed on those applications that are already used in the clinic and those that are likely to translate into clinical application in the near future or whose use in preclinical studies is likely to facilitate translation of new treatments into the clinic.     

Optical tomographic imaging of small animals

Diffuse optical tomography is emerging as a viable new biomedical imaging modality. Using visible and near-infrared light this technique can probe the absorption and scattering properties of biological tissues. The main applications are currently in brain, breast, limb and joint imaging; however, optical tomographic imaging of small animals is attracting increasing attention. This interest is fuelled by recent advances in the transgenic manipulation of small animals that has led to many models of human disease. In addition, an ever increasing number of optically reactive biochemical markers has become available, which allow diseases to be detected at the molecular level long before macroscopic symptoms appear. The past three years have seen an array of novel technological developments that have led to the first optical tomographic studies of small animals in the areas of cerebral ischemia and cancer.