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Micro-PET imaging and small animal models of disease
Abstract
Positron emission tomography (PET) has been used clinically to measure enzyme reactions, ligand-receptor interactions, cellular metabolism and cell proliferation. Until recently, however, PET has not been suitable for small animal models because of resolution limitations. Development of micro-PET instrumentation for small animal imaging and the availability of positron-emitting tracers has made this technology accessible for the non-invasive, quantitative and repetitive imaging of biological function in living animals. The development of new probes and positron-imaging based reporter genes has extended micro-PET applications to investigations of metabolism, enzyme activity, receptor-ligand interactions, protein-protein interactions, gene expression, adoptive cell therapy and somatic gene therapy. Because small animal PET is immediately extrapolatable to the clinic, laboratory advances should rapidly be translated to clinical practice.
... The PET provides virtually unlimited possibilities regarding the study and diagnosis of epilepsy since it can trace large numbers of molecules. Its applications include the investigation of receptor-ligand/protein-protein interactions, enzymatic activities, gene expression studies, and cell and gene therapy [28]. ...
... Disadvantages: Radiolabeled probes are used to detect brain functions, imposing a risk of radiation hazards. Some probes are trapped inside the cell after metabolism [28]. PET images often capture poor analytical details and thus integration of MRI is needed. ...
Epilepsy is one of the most common neurological disorders, characterized by recurrent seizures, resulting from abnormally synchronized episodic neuronal discharges. Around 70 million people worldwide are suffering from epilepsy. The available antiepileptic medications are capable of controlling seizures in around 60–70% of patients, while the rest remain refractory. Poor seizure control is often associated with neuro-psychiatric comorbidities, mainly including memory impairment, depression, psychosis, neurodegeneration, motor impairment, neuroendocrine dysfunction, etc., resulting in poor prognosis. Effective treatment relies on early and correct detection of epileptic foci. Although there are currently a few well-established diagnostic techniques for epilepsy, they lack accuracy and cannot be applied to patients who are unsupportive or harbor metallic implants. Since a single test result from one of these techniques does not provide complete information about the epileptic foci, it is necessary to develop novel diagnostic tools. Herein, we provide a comprehensive overview of the current diagnostic tools of epilepsy, including electroencephalography (EEG) as well as structural and functional neuroimaging. We further discuss recent trends and advances in the diagnosis of epilepsy that will enable more effective diagnosis and clinical management of patients.
... Positron Emission Tomography (micro-PET) technique is the valuable research tool for 3D viewing of brain cancers and metastases as well as for mapping normal brain function and support drug development in living systems [14][15][16]. Due to high penetrating radiation micro-PET has practically unlimited depth of imaging within the small animal. On the other hand γ-rays radiation is harmful and may affect tumor size and mimics radiotherapy. ...
... On the other hand γ-rays radiation is harmful and may affect tumor size and mimics radiotherapy. PET images have low resolution around 1 mm [14,15] and for micro-scale structural information a combination of micro-MRI and micro-CT is necessary. ...
High-resolution non-invasive 3D study of intact spine and spinal cord morphology on the level of complex vascular and neuronal organization is a crucial issue for the development of treatments for the injuries and pathologies of central nervous system (CNS). X-ray phase contrast tomography enables high quality 3D visualization in ex-vivo mouse model of both vascular and neuronal network of the soft spinal cord tissue at the scale from millimeters to hundreds of nanometers without any contrast agents and sectioning. Until now, 3D high resolution visualization of spinal cord mostly has been limited by imaging of organ extracted from vertebral column because high absorbing boney tissue drastically reduces the morphological details of soft tissue in image. However, the extremely destructive procedure of bones removal leads to sample deterioration and, therefore, to the lack of considerable part of information about the object. In this work we present the data analysis procedure to get high resolution and high contrast 3D images of intact mice spinal cord surrounded by vertebras, preserving all richness of micro-details of the spinal cord inhabiting inside. Our results are the first step forward to the difficult way toward the high- resolution investigation of in-vivo model central nervous system.
... Overall, PET is an extremely powerful technology and the in vivo receptor occupancy can help answer many vital questions in the MS research (Matthews et al., 2012;Bodini et al., 2015). Furthermore, PET offers an opportunity for the detection of enzyme reactions, ligandreceptor interactions, cellular metabolism, cell proliferation, protein-protein interactions, as well as gene and cell therapy (Herschman, 2003;Ono, 2009;Thorek et al., 2013). The development of new PET tracers is challenging because the binding affinity and selectivity of the tracer have to be high and the dissociation must be fast enough to obtain the binding equilibrium in time frame of scan (1-2 h) (Hicks, 2006;Sharma et al., 2010). ...
... Modern micro-PET instrumentation (resolution < 1 mm) is rapidly expanding the use of non-invasive PET imaging techniques in basic research. These advances have been progressively translated to human studies (Herschman, 2003;Liang et al., 2007;Lancelot and Zimmer, 2010). PET imaging offers tools to evaluate a great variety of molecular aspects of MS and neurodegeneration in animal models as well as in clinics (see Figures 3, 4). ...
Non-invasive molecular imaging techniques can enhance diagnosis to achieve successful treatment, as well as reveal underlying pathogenic mechanisms in disorders such as multiple sclerosis (MS). The cooperation of advanced multimodal imaging techniques and increased knowledge of the MS disease mechanism allows both monitoring of neuronal network and therapeutic outcome as well as the tools to discover novel therapeutic targets. Diverse imaging modalities provide reliable diagnostic and prognostic platforms to better achieve precision medicine. Traditionally, magnetic resonance imaging (MRI) has been considered the golden standard in MS research and diagnosis. However, positron emission tomography (PET) imaging can provide functional information of molecular biology in detail even prior to anatomic changes, allowing close follow up of disease progression and treatment response. The recent findings support three major neuroinflammation components in MS: astrogliosis, cytokine elevation, and significant changes in specific proteins, which offer a great variety of specific targets for imaging purposes. Regardless of the fact that imaging of astrocyte function is still a young field and in need for development of suitable imaging ligands, recent studies have shown that inflammation and astrocyte activation are related to progression of MS. MS is a complex disease, which requires understanding of disease mechanisms for successful treatment. PET is a precise non-invasive imaging method for biochemical functions and has potential to enhance early and accurate diagnosis for precision therapy of MS. In this review we focus on modulation of different receptor systems and inflammatory aspect of MS, especially on activation of glial cells, and summarize the recent findings of PET imaging in MS and present the most potent targets for new biomarkers with the main focus on experimental MS research.
... Micropositron emission tomography ( -PET) is one technique which can be used to monitor the protein molecules and their release/retention in vivo at the implant site. -PET is a noninvasive technique to measure the concentration of positron emitting isotopes in living entities [33]. With this ability it can be used to measure the concentration of a variety of molecules in vivo such as receptors, antigens, enzymes, and reporter gene products, [33], and therefore it can be used to study specific aspects of metabolism [34] and immunology [35]. ...
... -PET is a noninvasive technique to measure the concentration of positron emitting isotopes in living entities [33]. With this ability it can be used to measure the concentration of a variety of molecules in vivo such as receptors, antigens, enzymes, and reporter gene products, [33], and therefore it can be used to study specific aspects of metabolism [34] and immunology [35]. ...
Delivering growth factors (GFs) at bone/implant interface needs to be optimized to achieve faster osseointegration. Amorphous microporous silica (AMS) has a potential to be used as a carrier and delivery platform for GFs. In this work, adsorption (loading) and release (delivery) mechanism of a model protein, bovine serum albumin (BSA), from AMS was investigated in vitro as well as in vivo. In general, strong BSA adsorption to AMS was observed. The interaction was stronger at lower pH owing to favorable electrostatic interaction. In vitro evaluation of BSA release revealed a peculiar release profile, involving a burst release followed by a 6 h period without appreciable BSA release and a further slower release later. Experimental data supporting this observation are discussed. Apart from understanding protein/biomaterial (BSA/AMS) interaction, determination of in vivo protein release is an essential aspect of the evaluation of a protein delivery system. In this regard micropositron emission tomography (í µí¼-PET) was used in an exploratory experiment to determine in vivo BSA release profile from AMS. Results suggest stronger in vivo retention of BSA when adsorbed on AMS. This study highlights the possible use of AMS as a controlled protein delivery platform which may facilitate osseointegration.
... A imagem PET pode ser combinada com imagens obtidas usando outras modalidades de imagem, como a MRI, ajudando na análise da imagem. . Nos últimos anos foram publicados vários artigos descrevendo o design e as características das diferenças dos sistemas comerciais e não comerciais, e realçando o potencial das câmaras dedicadas aos pequenos animais, dos quais se destacam os seguintes: [Chatziioannou, 2002b], , [Myers, 2001], [Myers, 2002], [Weber, 2004], [Cherry, 2001], [Gambhir, 2000], [Gambhir, 2002], [Phelps, 2000], [Lecomte, 2004], [Rowland, 2002], [Herschman, 2003]. Contudo, enquanto que a avaliação do desempenho em sistemas para humanos segue padrões standard regulados pela NEMA, para os sistemas dedicados aos pequenos animais não existem normas reguladoras e portanto os parâmetros de desempenho de diferentes câmaras não são directamente comparáveis. ...
... Estes detectores têm uma resolução espacial intrínseca < 1 mm. 2004], [Weissleder, 2001], [Gambhir, 2002], [Herschman, 2003], [Herschman, 2004], [Lammertsma, 2004], [MacLaren, 2000], [Blasberg, 2002], [Blasberg, 2002b], [Schelbert, 2003]. Aqui será apresentada uma breve abordagem sobre as diversas áreas de aplicação PET aos pequenos animais: ...
... Micropositron emission tomography ( -PET) is one technique which can be used to monitor the protein molecules and their release/retention in vivo at the implant site. -PET is a noninvasive technique to measure the concentration of positron emitting isotopes in living entities [33]. With this ability it can be used to measure the concentration of a variety of molecules in vivo such as receptors, antigens, enzymes, and reporter gene products, [33], and therefore it can be used to study specific aspects of metabolism [34] and immunology [35]. ...
... -PET is a noninvasive technique to measure the concentration of positron emitting isotopes in living entities [33]. With this ability it can be used to measure the concentration of a variety of molecules in vivo such as receptors, antigens, enzymes, and reporter gene products, [33], and therefore it can be used to study specific aspects of metabolism [34] and immunology [35]. ...
Delivering growth factors (GFs) at bone/implant interface needs to be optimized to achieve faster osseointegration. Amorphous microporous silica (AMS) has a potential to be used as a carrier and delivery platform for GFs. In this work, adsorption (loading) and release (delivery) mechanism of a model protein, bovine serum albumin (BSA), from AMS was investigated in vitro as well as in vivo. In general, strong BSA adsorption to AMS was observed. The interaction was stronger at lower pH owing to favorable electrostatic interaction. In vitro evaluation of BSA release revealed a peculiar release profile, involving a burst release followed by a 6 h period without appreciable BSA release and a further slower release later. Experimental data supporting this observation are discussed. Apart from understanding protein/biomaterial (BSA/AMS) interaction, determination of in vivo protein release is an essential aspect of the evaluation of a protein delivery system. In this regard micropositron emission tomography ( μ -PET) was used in an exploratory experiment to determine in vivo BSA release profile from AMS. Results suggest stronger in vivo retention of BSA when adsorbed on AMS. This study highlights the possible use of AMS as a controlled protein delivery platform which may facilitate osseointegration.
... Molecular imaging via positron emission tomography (PET) [1][2][3][4][5][6][7] continues to improve our understanding of the in vivo molecular mechanisms of various diseases [3] owing to ever-evolving animal models of disease [7]. This trend highlights the need for robust methodology and validated analysis procedures for accurate quanti cation of PET outcome variables. ...
Purpose
Combined micro-PET/CT scanners are widely employed to investigate models of brain disorders in rodents using PET-based coregistration. We examined if CT-based coregistration could improve estimates of brain dimensions and consequently estimates of nondisplaceable binding potential (BPND) in rodent PET studies.
Procedures
PET and CT scans were acquired on 5 female and 5 male CD-1 mice with PET and CT scans were acquired on 5 female and 5 male CD-1 mice with 3-[¹⁸F]fluoro-5-(2-pyridinylethynyl)benzonitrile ([¹⁸F]FPEB), a radiotracer for the metabotropic glutamate receptor subtype 5 (mGluR5). In the proposed PET/CT (PTCT) approach, the tracer-specific standard volume was dimension-customized to each animal using the scaling factors from CT-to-standard CT coregistration to simplify PET-to-standard PET coregistration (i.e., 3 CT- and 6 PET-derived parameters). For comparison, conventional PET-based coregistration was performed with 9 (PT9) or 12 (PT12) parameters. PET frames were transferred to the standard space by the three approaches (PTCT, PT9, and PT12) to obtain regional time-activity curves (TACs) and BPND in 14 standard volumes of interest (VOIs). Lastly, CT images of the animals were transferred to the standard space by CT-based parameters from PTCT and with the scaling factors replaced with those from PET-based PT9 to evaluate agreement of the skull to the standard CT.
Results
The PET-based approaches showed various degrees of underestimations of scaling factors in the posterior-anterior-direction compared to PTCT, which resulted in negatively proportional overestimation of radioactivity in the cerebellum (reference region) up to 20%, and proportional, more prominent underestimation of BPND in target regions down to -50%. The skulls of individual animals agreed with the standard skull for scaling factors from PTCT but not for the scaling factors from PT9, which suggested inaccuracy of the latter.
Conclusions
The results indicated that conventional PET-based coregistration approaches could yield biased estimates of BPND due to erroneous estimates of brain dimensions when applied to tracers for which the cerebellum serves as reference region. The proposed PTCT provides evidence of a quantitative improvement over PET-based approaches for brain studies using micro-PET/CT scanners.
... Some imaging agents have also been developed in the eye [10][11][12]. Among them, the 18 kDa translocator protein (TSPO) ligand as the imaging agent of PET imaging technology plays an important role in the diagnosis and treatment of neurodegenerative diseases, such as Alzheimer's disease and cerebral ischemia [13][14][15][16]. TSPO (translocator protein) is an evolutionarily conserved outer mitochondrial membrane (OMM) protein highly expressed in activated microglia, and TSPO-targeting PET imaging is used for the evaluation of neuroinflammation. ...
Purpose:
Positron emission tomography (PET) is widely used in high-precision imaging, which may provide a simple and noninvasive method for the detection of pathology and therapeutic effects. [18F]-DPA-714 is a second-generation translocator protein (TSPO) positron emission tomography radiotracer that shows great promise in a model of neuroinflammation. In this study, [18F]-DPA-714 micro-PET imaging was used to evaluate retinal inflammation in mice exposed to blue light, a well-established model of age-related macular degeneration (AMD) for molecular mechanism research and drug screening.
Methods:
C57BL/6J melanized mice were subjected to 10,000, 15,000, and 20,000 lux blue light for 5 days (8 h/day) to develop the retinal injury model, and the structure and function of the retina were assessed using hematoxylin-eosin (HE) staining, electroretinography (ERG), and terminal-deoxynucleotidyl transferase (TdT)-mediated nick-end labeling (TUNEL) immunostaining. Then, [18F]-DPA-714 was injected approximately 100 μCi through each tail vein, and static imaging was performed 1 h after injection. Finally, the mice eyeballs were collected for biodistribution and immune analysis.
Results:
The blue light exposure significantly destroyed the structure and function of the retina, and the uptake of [18F]-DPA-714 in the retinas of the mice exposed to blue light were the most significantly upregulated, which was consistent with the biodistribution data. In addition, the immunohistochemical, western blot, and immunofluorescence data showed an increase in microglial TSPO expression.
Conclusions:
[18F]-DPA-714 micro-PET imaging might be a good method for evaluating early inflammatory status during retinal pathology.
... These two obstacles make Ca 2+ and NO possible targets for ED diagnostic tools through simultaneous sensing of both analytes. Unfortunately, in vivo imaging has multiple limitations such as biocompatibility, cell bilayer penetration by molecules, intracellular targeting, light penetration, and signal-tonoise ratio, so new molecular probes and sensing methods must be developed 29,30 . This review provides an overview of recent developments in the modelling of ED, in the sensing of NO and calcium cations in biologically relevant conditions, and it gives an outlook for future research. ...
Cardiovascular diseases are the leading cause of death worldwide and are not typically diagnosed until the disease has manifested. Endothelial dysfunction is an early, reversible precursor in the irreversible development of cardiovascular diseases and is characterized by a decrease in nitric oxide production. We believe that more reliable and reproducible methods are necessary for the detection of endothelial dysfunction. Both nitric oxide and calcium play important roles in the endothelial function. Here we review different types of molecular sensors used in biological settings. Next, we review the current nitric oxide and calcium sensors available. Finally, we review methods for using both sensors for the detection of endothelial dysfunction.
... Some imaging agents have also been developed in the eye [10][11][12]. Among them, the 18 kDa translocator protein (TSPO) ligand as the imaging agent of PET imaging technology plays an important role in the diagnosis and treatment of neurodegenerative diseases, such as Alzheimer's disease and cerebral ischemia [13][14][15][16]. TSPO (translocator protein) is an evolutionarily conserved outer mitochondrial membrane (OMM) protein highly expressed in activated microglia, and TSPO-targeting PET imaging is used for the evaluation of neuroinflammation. ...
Purpose:
Positron emission tomography (PET) is widely used in high-precision imaging, which may provide a simple and noninvasive method for the detection of pathology and therapeutic effects. [18F]-DPA-714 is a second-generation translocator protein (TSPO) positron emission tomography radiotracer that shows great promise in a model of neuroinflammation. In this study, [18F]-DPA-714 micro-PET imaging was used to evaluate retinal inflammation in mice exposed to blue light, a well-established model of age-related macular degeneration (AMD) for molecular mechanism research and drug screening.
Methods:
C57BL/6J melanized mice were subjected to 10,000, 15,000, and 20,000 lux blue light for 5 days (8 h/day) to develop the retinal injury model, and the structure and function of the retina were assessed using hematoxylin-eosin (HE) staining, electroretinography (ERG), and terminal-deoxynucleotidyl transferase (TdT)-mediated nick-end labeling (TUNEL) immunostaining. Then, [18F]-DPA-714 was injected approximately 100 μCi through each tail vein, and static imaging was performed 1 h after injection. Finally, the mice eyeballs were collected for biodistribution and immune analysis.
Results:
The blue light exposure significantly destroyed the structure and function of the retina, and the uptake of [18F]-DPA-714 in the retinas of the mice exposed to blue light were the most significantly upregulated, which was consistent with the biodistribution data. In addition, the immunohistochemical, western blot, and immunofluorescence data showed an increase in microglial TSPO expression.
Conclusions:
[18F]-DPA-714 micro-PET imaging might be a good method for evaluating early inflammatory status during retinal pathology.
... In more recent years, the development of micro-PET technology [20] has enabled the study of small animal models of human disease, allowing for translational investigation of underlying pathology and supporting the development of novel therapies. Mice and rats in particular offer the advantage of being experimentally tractable models, including the generation of phenotypes by pharmacological treatment, genetic models using protein knockout or overexpression, and functional manipulation using optogenetic or chemogenetic regulation [21][22][23]. ...
Positron emission tomography (PET) using the radiotracer [¹⁸F]-FDOPA provides a tool for studying brain dopamine synthesis capacity in animals and humans. We have previously standardised a micro-PET methodology in mice by intravenously administering [¹⁸F]-FDOPA via jugular vein cannulation and assessment of striatal dopamine synthesis capacity, indexed as the influx rate constant KiMod of [¹⁸F]-FDOPA, using an extended graphical Patlak analysis with the cerebellum as a reference region. This enables a direct comparison between preclinical and clinical output values. However, chronic intravenous catheters are technically difficult to maintain for longitudinal studies. Hence, in this study, intraperitoneal administration of [¹⁸F]-FDOPA was evaluated as a less-invasive alternative that facilitates longitudinal imaging. Our experiments comprised the following assessments: (i) comparison of [¹⁸F]-FDOPA uptake between intravenous and intraperitoneal radiotracer administration and optimisation of the time window used for extended Patlak analysis, (ii) comparison of KiMod in a within-subject design of both administration routes, (iii) test-retest evaluation of KiMod in a within-subject design of intraperitoneal radiotracer administration, and (iv) validation of KiMod estimates by comparing the two administration routes in a mouse model of hyperdopaminergia induced by subchronic ketamine. Our results demonstrate that intraperitoneal [¹⁸F]-FDOPA administration resulted in good brain uptake, with no significant effect of administration route on KiMod estimates (intraperitoneal: 0.024±0.0047 min−1, intravenous: 0.022±0.0041 min−1, p=0.42) and similar coefficient of variation (intraperitoneal: 19.6%; intravenous: 18.4%). The technique had a moderate test-retest validity (intraclass correlation coefficient ICC=0.52, N=6) and thus supports longitudinal studies. Following subchronic ketamine administration, elevated KiMod as compared to control condition was measured with a large effect size for both methods (intraperitoneal: Cohen’s d=1.3; intravenous: Cohen’s d=0.9), providing further evidence that ketamine has lasting effects on the dopamine system, which could contribute to its therapeutic actions and/or abuse liability.
... In vivo molecular imaging is an advanced imaging approach that combines biochemical and physiological changes occurring in living animals at the molecular level [1,2]. Fluorescence [3,4], luminescence [5,6], singlephoton emission computed tomography (SPECT) [7], positron emission tomography (PET) [8,9], computed tomography (CT) [10,11], and magnetic resonance imaging (MRI) are techniques used to obtain images [12,13]. Each imaging method has advantages and disadvantages. ...
In vivo molecular imaging is a research field in which molecular biology and advanced imaging techniques are combined for imaging molecular-level biochemical and physiological changes that occur in a living body. For biomolecular imaging, the knowledge of molecular biology, cell biology, biochemistry, and physiology must be applied. Imaging techniques such as fluorescence, luminescence, single-photon emission computed tomography (SPECT), positron emission tomography (PET), computed tomography (CT), and magnetic resonance imaging (MRI) are used for biomolecular imaging. These imaging techniques are used in various fields, i.e., diagnosis of various diseases, development of new drugs, development of treatments, and evaluation of effects. Moreover, as biomolecular imaging can repeatedly acquire images without damaging biological tissues or sacrificing the integrity of objects, changes over time can be evaluated.
Phenotypes or diseases in a living body are caused by the accumulation of various biological phenomena. Genetic differences cause biochemical and physiological differences, which accumulate and cause anatomical or structural changes. Biomolecular imaging techniques are suitable for each step. In evaluating anatomical or structural changes, MRI, CT, and ultrasound have advantages in obtaining high-resolution images. SPECT and MRI are advantageous for the evaluation of various physiological phenomena. PET and magnetic resonance spectroscopy can be used to image biochemical phenomena in vivo. Although various biomolecular imaging techniques can be used to evaluate various biological phenomena, it is important to use imaging techniques suitable for each purpose.
... PET combines several crucial features like absolute quantitation, allows repetitive and longitudinal examination before/after interventions with the help of short-lived PET radionuclides. Very high resolution (< 2.5 mm) can be achieved at any part of the body with a dedicated PET scanner [6,7]. Most importantly, procedures developed in preclinical research can be applied to clinical studies. ...
Positron emission tomography (PET) offers the study of biochemical, physiological, and pharmacological functions at a cellular and molecular level. The performance of a PET study mostly depends on the used radiotracer of interest. However, the development of a novel PET tracer is very difficult, as it is required to fulfill a lot of important criteria. PET radiotracers usually encounter different chemical modifications including redox reaction, hydrolysis, decarboxylation, and various conjugation processes within living organisms. Due to this biotransformation, different chemical entities are produced, and the amount of the parent radiotracer is declined. Consequently, the signal measured by the PET scanner indicates the entire amount of radioactivity deposited in the tissue; however, it does not offer any indication about the chemical disposition of the parent radiotracer itself. From a radiopharmaceutical perspective, it is necessary to quantify the parent radiotracer’s fraction present in the tissue. Hence, the identification of radiometabolites of the radiotracers is vital for PET imaging. There are mainly two reasons for the chemical identification of PET radiometabolites: firstly, to determine the amount of parent radiotracers in plasma, and secondly, to rule out (if a radiometabolite enters the brain) or correct any radiometabolite accumulation in peripheral tissue. Besides, radiometabolite formations of the tracer might be of concern for the PET study, as the radiometabolic products may display considerably contrasting distribution patterns inside the body when compared with the radiotracer itself. Therefore, necessary information is needed about these biochemical transformations to understand the distribution of radioactivity throughout the body. Various published review articles on PET radiometabolites mainly focus on the sample preparation techniques and recently available technology to improve the radiometabolite analysis process. This article essentially summarizes the chemical and structural identity of the radiometabolites of various radiotracers including [¹¹C]PBB3, [¹¹C]flumazenil, [¹⁸F]FEPE2I, [¹¹C]PBR28, [¹¹C]MADAM, and (+)[¹⁸F]flubatine. Besides, the importance of radiometabolite analysis in PET imaging is also briefly summarized. Moreover, this review also highlights how a slight chemical modification could reduce the formation of radiometabolites, which could interfere with the results of PET imaging.
Graphical abstract
... Studies using saPET can not only use this technology to define metabolic characteristics of epileptic networks, which can then be adapted for defining foci in human imaging, but also enable screening of the effects of antiepileptic drugs and novel compounds on normal and abnormal brain activity. 95,96 Animal studies allow for correlation with histology and immunohistochemistry at different seizure states, providing a mechanism to understand the cellular and molecular changes that underlie epileptogenesis and opening the possibility of developing new targeted therapies. ...
Technical limitations and clinical challenges have historically limited the diagnostic tools and treatment methods available for surgical approaches to the management of epilepsy. By contrast, recent technological innovations in several areas hold significant promise in improving outcomes and decreasing morbidity. We review innovations in the neurosurgical management of epilepsy in several areas, including wireless recording and stimulation systems (particularly responsive neurostimulation [NeuroPace]), conformal electrodes for high-resolution electrocorticography, robot-assisted stereotactic surgery, optogenetics and optical imaging methods, novel positron emission tomography ligands, and new applications of focused ultrasonography. Investigation into genetic causes of and susceptibilities to epilepsy has introduced a new era of precision medicine, enabling the understanding of cell signaling mechanisms underlying epileptic activity as well as patient-specific molecularly targeted treatment options. We discuss the emerging path to individualized treatment plans, predicted outcomes, and improved selection of effective interventions, on the basis of these developments.
... 37 Micro-PET is accessible for the non-invasive, quantitative and repetitive imaging of biological function in living animals. 38 Therefore, we used micro-PET to observe the changes in the level of glucose metabolism in the whole brain (cortex, frontal lobe and hippocampus). According to our results, the 18 F-FDG uptake rate was increased in both acupuncture-treated groups relative to the SAMP8 group but did not differ significantly between SAMP8+D, SAMP8+MA and SAMP8+D+MA groups. ...
Objectives::
To explore whether combined therapy with donepezil and acupuncture is better than treatment with donepezil or acupuncture individually in a rat model of Alzheimer's disease.
Methods::
In this study, we randomly divided 40 7.5-month-old senescence-accelerated mouse prone 8 (SAMP8) male mice into four groups: SAMP8, SAMP8+D, SAMP8+MA and SAMP8+D+MA. An additional 10 7.5-month-old SAMR1 male mice were included as a healthy control group (SAMR1). Mice in the SAMP8+D group were given donepezil at a dose of 0.65 µg/g/day; mice in the SAMP8+MA group underwent manual acupuncture at GV20, GV26 and Yintang for 20 min per day; mice in the SAMP8+D+MA received both donepezil and manual acupuncture; and mice in the SAMR1 and SAMP8 groups underwent restraint only to control for the effects of handling. After the 15-day treatment, the Morris water maze test, micro-PET(positron-emission tomography), H&E (haematoxylin and eosin) staining, and immunohistochemistry were used to study the differences between donepezil (SAMP8+D), acupuncture (SAMP8+MA), and donepezil combined with acupuncture (SAMP8+D+MA) therapy for the treatment of Alzheimer's disease.
Results::
We found that, compared with the untreated SAMP8 group, donepezil, manual acupuncture, and combined therapy with donepezil and manual acupuncture all improved spatial learning and memory ability, the level of glucose metabolism in the brain, and the content of Aβ amyloid in the cortex. Moreover, combined therapy outperformed treatment with donepezil or acupuncture individually in the SAMP8 mice.
Conclusion::
This study shows that the combination of manual acupuncture and donepezil in an Alzheimer's disease animal model is superior to acupuncture and donezepil alone. However, randomised controlled trials should be undertaken to clarify the clinical efficacy of combination therapy.
... Technetium-99m, Thallium-8 and Iodine-131/125) (Chrastina and Schnitzer, 2010;Liu et al., 2012), contrast agents (Lusic and Grinstaff, 2013) and bioluminescent markers/reporters (luciferins, proluciferins etc.) (Patrick et al., 2014) are commonly used in these imaging studies (Li et al., 2013;Meikle et al., 2005;Sato et al., 2004, Zhu andShim, 2011). The overview of major characteristics of common imaging technologies is listed in state detector materials (Herschman, 2003). Although, radiation dose to various vital organs in humans is a major limiting factor in selection of dosage, in animals, an optimal radioactivity dose is required for quality images (Kung and Kung, 2005). ...
Cancer research is primarily dependent on rodent models. There has been a huge advancement in anticancer drug discovery in the last two decades, which is mainly due to improved animal models and introduction of non-invasive imaging technologies. Non-invasive imaging technology plays an important role in cancer and other biomedical research areas including neurology, cardiology, nanotechnology and stem cell biology. The intricate pathways in cancer initiation, progression and metastasis can be visualized and the processes can be analyzed quantitatively using these techniques. Robust preclinical data is the backbone of anticancer drug discovery, which can be obtained precisely using imaging technology. This review is focused on the dedicated preclinical imaging modalities such as Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), X-ray Computed Tomography (CT), Magnetic Resonance Imaging (MRI), Optical imaging and Cerenkov imaging. This paper discusses their present roles in basic and translational research towards evaluation of suitability of target molecules for further clinical trials.The unique features of the imaging modalities enable them to provide information necessary for understanding structural, functional and molecular processes involved in various stages of cancer development thus bridging the gap between bench and bedside.
... specific probes. The introduction of microPET for small-animal imaging has similarly made PET amenable to preclinical studies (11). Ongoing activity of ACT against both on-and off-tumor sites can therefore be monitored in vivo by quantitative, radiotracer-based imaging of T cell distribution and expansion upon interaction with target antigen-expressing cells (2,10,12). ...
Clinical monitoring of adoptive T cell transfer (ACT) utilizes serial blood analyses to discern T cell activity. While useful, these data are 1-dimensional and lack spatiotemporal information related to treatment efficacy or toxicity. We utilized a human genetic reporter, somatostatin receptor 2 (SSTR2), and PET, to quantitatively and longitudinally visualize whole-body T cell distribution and antitumor dynamics using a clinically approved radiotracer. Initial evaluations determined that SSTR2-expressing T cells were detectable at low densities with high sensitivity and specificity. SSTR2-based PET was applied to ACT of chimeric antigen receptor (CAR) T cells targeting intercellular adhesion molecule-1, which is overexpressed in anaplastic thyroid tumors. Timely CAR T cell infusions resulted in survival of tumor-bearing mice, while later infusions led to uniform death. Real-time PET imaging revealed biphasic T cell expansion and contraction at tumor sites among survivors, with peak tumor burden preceding peak T cell burden by several days. In contrast, nonsurvivors displayed unrelenting increases in tumor and T cell burden, indicating that tumor growth was outpacing T cell killing. Thus, longitudinal PET imaging of SSTR2-positive ACT dynamics enables prognostic, spatiotemporal monitoring with unprecedented clarity and detail to facilitate comprehensive therapy evaluation with potential for clinical translation.
... measurement. PET/CT uniting metabolism imaging and anatomy imaging [61] displays high sensibility for osteopathy and great capacity to quantitatively analyze and compare those two images [62,63] with the developing agent 18 F-NaF. The scanister technically for rodent experiments with high resolution and sensibility [64,65] mPET/CT helps to acquire comprehensive information on characteristics of animal. ...
... specific probes. The introduction of microPET for small-animal imaging has similarly made PET amenable to preclinical studies (11). Ongoing activity of ACT against both on-and off-tumor sites can therefore be monitored in vivo by quantitative, radiotracer-based imaging of T cell distribution and expansion upon interaction with target antigen-expressing cells (2,10,12). ...
... Development of micro-PET instrumentation for small animal imaging and the availability of positron-emitting tracers have made this technology accessible for the noninvasive, quantitative, and repetitive imaging of biological function in living animals. The development of new probes and positron-imaging based reporter genes has extended micro-PET applications to investigations of metabolism, enzyme activity, receptor-ligand interactions, protein-protein interactions, gene expression, adoptive cell therapy, and somatic gene therapy [47]. ...
Introduction. Alzheimer's disease (AD) causes progressive hippocampus dysfunctions leading to the impairment of learning and memory ability and low level of uptake rate of glucose in hippocampus. What is more, there is no effective treatment for AD. In this study, we evaluated the beneficial and protective effects of electroacupuncture in senescence-accelerated mouse prone 8 (SAMP8). Method. In the electroacupuncture paradigm, electroacupuncture treatment was performed once a day for 15 days on 7.5-month-old SAMP8 male mice. In the normal control paradigm and AD control group, 7.5-month-old SAMR1 male mice and SAMP8 male mice were grabbed and bandaged while electroacupuncture group therapy, in order to ensure the same treatment conditions, once a day, 15 days. Results. From the Morris water maze (MWM) test, we found that the treatment of electroacupuncture can improve the spatial learning and memory ability of SAMP8 mouse, and from the micro-PET test, we proved that after the electroacupuncture treatment the level of uptake rate of glucose in hippocampus was higher than normal control group. Conclusion. These results suggest that the treatment of electroacupuncture may provide a viable treatment option for AD.
... Several excellent review articles including extensive reference lists that give a more detailed presentation of each subfield of small animal PET imaging: [Kornblum et al. , 2000;Phelps, 2000;Pomper, 2001;Weissleder & Mahmood, 2001;Cherry & Gambhir, 2001;Myers, 2001;Rowland, 2002;Schelbert, 2003;Herschman, 2003;Cherry, 2004;Laforest, 2005;Rowland & R., 2008;Lewis et al. , 2002;Riemann et al. , 2008;Lammertsma, 2004;Blasberg, 2002;Blasberg & Tjuvajev, 2002;MacLaren et al. , 2000;Vassaux & Wassink, 2003;Gambhir et al. , 1999Gambhir et al. , , 2000Hume & Jones, 1998;Chatziioannou, 2005] Sequential microPET imaging studies of a nude mouse carrying four tumours. Four tumour cell lines -two positive controls (constitutive reporter-gene expression), one negative control and one inducible line (reporter-gene expression induced by doxycycline -were injected subcutaneously into four separate sites in a single mouse. ...
Organ motion has become of great concern in medical imaging only recently. Respiratory motion is one source of degradation of PET images. Respiratory motion may lead to image blurring, which may result in reduced contrast and quantitative accuracy in terms of recovered activity concentration and functional volumes. Consequently, the motion of lungs hinders the localization, detection, and the quantification of tracer uptake in lung lesions. There is, therefore, a need to better understand the effects of this motion on PET data outcome.
Medical imaging methods and devices are commonly evaluated through computer simulation. Computer generated phantoms are used to model patient anatomy and physiology, as well as the imaging process itself. A major advantage of using computer generated phantoms in simulation studies is that the anatomy and physiological functions of the phantom are known, thus providing a gold standard from which to evaluate and improve medical imaging devices and techniques.
In this thesis, are presented the results of a research studied the combined effects of lesion size, lesion-to-background activity concentration ratio and respiratory motion on signal recovery of spherical lesions in small animal PET images using Monte Carlo simulation. Moreover, background activity is unavoidable and it causes significant noise and contrast loss in PET images. For these purposes, has been used the Geant4 Application for Tomographic Emission (GATE) Monte Carlo platform to model the microPET^{\text{\textregistered}}
FOCUS 220 system. Additionaly, was implemented the digital 4D Mouse Whole-Body (MOBY) phantom into GATE. A physiological “stress breathing” condition was created for MOBY in order to reproduce the respiratory mouse motion during a typical PET examination. A spherical lung lesion was implemented within this phantom and its motion also modelled. Over a complete respiratory cycle of 0.37 s was retrieved a set of 10 temporal frames (including the lesion movement) generated in addition to a non-gated data set. Sets of static (non-gated data) and dynamic (gated data) 2-Deoxy-[^{\text{\emph{18}}}
F]fluoro-D-glucose (FDG) simulations were performed considering different lesion sizes and different activity uptakes. Image noise and temporal resolution were determined on 3D and 4D images. Signal-to-Noise Ratio (SNR), Contrast-to-Noise Ratio (CNR), Target-to-Background activity concentration Ratio (TBR), Contrast Recovery (CR) and Volume Recovery (VR) were also evaluated as a function of lesion size and activity uptake.
Globally, the results obtained show that signal loss depends both on lesion size and lesion activity uptake. In the non-gated data, where was no motion included (perfect motion correction), the recovery coefficients were influenced by the partial volume effect for the smallest lesion size. Moreover, the increased lesion contrast produces a significant increase on the standard deviation of the mean signal recover. This led to a decrease in CNR and SNR. In addition, respiratory motion significantly deteriorates signal recovery and this loss depends mainly of the lesion size. Best temporal resolution (volume recovery) and spatial resolution was given by the non-gated data, where no motion is involved.
The simulated results show that the partial volume effect is dominant for small objects due to limited FOCUS system resolution in both 3D and 4D PET images. In addition, lower activity concentrations significantly deteriorates the lesion signal recovery compromising quantitative analysis.
Keywords: Positron Emission Tomography (PET); Small Animal PET; 4D Mouse Whole-Body (MOBY) Phantom; Geant4 Application for Tomographic Emission (GATE); Lung Lesion Detectability; Image Quantification.
... Positron emission tomography (PET) is used extensively in the clinic as a non-invasive imaging modality, with much importance in the staging of cancer. This technology has been successfully used to image the endogenous expression of reporter genes thanks to the development of complimentary radiolabelled probes and micro-PET scanners for use with small animals (Blasberg, 2002;Herschman, 2003). However, despite the relatively high sensitivity of nuclear imaging methods, resolution limitations restrict the detail of the anatomical information yielded from such a technique, and so reduce its suitability as a candidate for a bacterial tracking imaging modality (Blasberg, 2002;Ray et al., 2004;Cohen et al., 2007). ...
S. aureus is an opportunistic pathogen with an extensive host range, and is the etiologic agent of a wide variety of human diseases, with the ability to promote disease in most human tissues. A repertoire of virulence factors expressed during colonization and infection all contribute to the success of S. aureus as a pathogen. S. aureus is not classically considered as an intracellular pathogen, yet accumulating scientific evidence has demonstrated that this organism has intracellular survival strategies, with the ability to become internalised by, and persist within, a wide range of non-professional eukaryotic phagocytes. This phenomenon has been shown to be linked to the expression of genes under the regulation of the S. aureus quorum sensing system, agr. Specifically, S. aureus has been shown to breach the host-membrane bound endosome within which it is situated upon internalisation, a process which has been hypothesised to rely upon a switch in gene expression due to the accumulation of the agr signalling peptide within the endosome.
Many in vitro imaging modalities have been designed to investigate further the mechanisms behind S. aureus internalisation. However, in vivo systems such as bioluminescence and fluorescence reporter genes are limited by various factors including light extinction, restricting investigations to those using small animals. Additionally, as fundamentally two-dimensional modalities, useful anatomical information is minimal in these imaging techniques. At present few imaging modalities exist which enable the non-invasive in vivo detection of bacterial reporter gene expression while offering detailed anatomical information. This study aimed to address the need to develop reporter systems for the non-invasive in vivo tracking systems for the monitoring of infectious disease processes, in the context of using such a technology to investigate further the genes involved in the process of S. aureus internalisation, and more specifically, endosomal escape.
Although it is clear that agr regulated genes have a role in endosomal escape of internalised S. aureus, it is not fully clear which genes specifically are important. A number of haemolysin genes are under the control of the agr system, all of which have the ability to disrupt eukaryotic cell membranes. A β-haemolysin mutant was constructed in this study to use in conjunction with existing α-, δ- and γ-haemolysin mutants in in vitro internalisation assays. Initial experiments indicated a role for α-haemolysin in the endosomal escape of internalised S. aureus. The use of an in vivo reporter gene system would allow for further analysis of the role of haemolysins in endosomal escape, in addition to providing detailed anatomical information.
MRI bacterioferritin (BFR) reporter genes with inducible or constitutive promoters, the bfr gene and the lux operon, were constructed and evaluated in S. aureus. The reporter gene was shown to be translated in vitro, with the expression of functional BFR which collected iron. However, ICP-MS data revealed relatively low levels of BFR. Pilot in vivo studies were carried out to confirm the potential of the reporter gene for studying specific aspects of staphylococcal disease. Experiments tracking bioluminescence in a mouse tumour model demonstrated expression of the MRI reporter genes, suggesting that bacterioferritin should be successfully synthesised in vivo. However, due to plasmid instability in vivo and the relatively low levels of iron present in S. aureus samples as determined by ICP-MS, the tumours in this study were not scanned by MRI.
... One potential solution to many aspects of the post-transplantation safety concerns is the "transgenic modification/post-transplantation safety" hypothesis which proposes that through specific transgenic modifications of iPS cells it should be possible to generate iPS cell derivatives that can be safely used post-transplantation. The top candidate for this approach is the Herpres Simplex Virus 1 thymidine kinase sr39 mutated transgene (HSV1-sr39tk) which, remarkably, permits both positron emission tomography (PET)-based in vivo imaging (using the PET probe FHBG) of the transplanted iPS cell derivatives (Phelps, 2000;Herschman, 2003) as well as the ability to induce cell death (using the drug ganciclovir) in the event the transplanted cells result in tumor formation or other negative outcomes (Cao et al., 2007). One limitation of this approach is the blood-brain-barrier (BBB) is not permeable to either the PET probe FHBG or the suicide induction agent ganciclovir. ...
John Gurdon's discovery that somatic cells could be reprogrammed back into a pluripotent state has immense implications across multiple different fields, including the future potential for autologous cellular therapies. This review briefly examines the history of nuclear reprogramming, from Gurdon's original work in amphibia, through the generation of oocyte-reprogrammed pluripotent stem cells in the non-human primate and recent defined factor-based reprogramming approaches to generate human induced pluripotent stem (iPS) cells. This review also examines the five principle challenges towards safely advancing pluripotent stem cell derivatives into personalized human therapeutics, specifically: genetic stability, epigenetic memory, post-transplantation efficacy, post-transplantation safety and feasibility, and additionally discusses various hypotheses that may play a role in resolving the aforementioned challenges. Focused on iPS cells and derivatives, these hypotheses essentially deal with aging research, genomic stability and culture conditions, immunogenicity and epigenetic memory, epigenetic memory elimination by chromatin modifying chemicals or by developmental competence factors and/or by candidate oocyte reprogramming factors (CORFs), and small molecules acting on the blood-brain-barrier. In light of these hypotheses, progress in nuclear reprogramming mechanistic are discussed in terms of securing the therapeutic promise of autologous reprogrammed personalized stem cell derivatives in the foreseeable future, thereby opening a probable new era for anti-aging control and regenerative medicine.
... Imaging angiogenesis in small animals is extremely challenging due to the size of the tumor vessels and requires dedicated small animals systems. Tumor imaging in rodents is currently performed using several modalities, including MRI [3] , optical [4] , nuclear techniques [5] and micro-CT [6] [7] [8] [9] [10] . ...
Imaging tumor angiogenesis in small animals is extremely challenging due
to the size of the tumor vessels. Consequently, both dedicated small
animal imaging systems and specialized intravascular contrast agents are
required. The goal of this study was to investigate the use of a
liposomal contrast agent for high-resolution micro-CT imaging of breast
tumors in small animals. A liposomal blood pool agent encapsulating
iodine with a concentration of 65.5 mg/ml was used with a Duke Center
for In Vivo Microscopy (CIVM) prototype micro-computed tomography
(micro-CT) system to image the R3230AC mammary carcinoma implanted in
rats. The animals were injected with equivalent volume doses (0.02
ml/kg) of contrast agent. Micro-CT with the liposomal blood pool
contrast agent ensured a signal difference between the blood and the
muscle higher than 450 HU allowing the visualization of the tumors 3D
vascular architecture in exquisite detail at 100-micron resolution. The
micro-CT data correlated well with the histological examination of tumor
tissue. We also studied the ability to detect vascular enhancement with
limited angle based reconstruction, i.e. tomosynthesis. Tumor volumes
and their regional vascular percentage were estimated. This imaging
approach could be used to better understand tumor angiogenesis and be
the basis for evaluating anti-angiogenic therapies.
... Therefore, imaging tumors in rodents requires dedicated small animal systems. Tumor imaging in rodents is currently performed using several modalities, including MRI (5), micro-CT (6-9), optical (10) and nuclear techniques (11). In some cases, combinations of two modalities are used, for example micro-CT/micro-PET to provide complementary information related to the morphology (micro-CT) and function, i.e. metabolism (micro-PET) (12). ...
X-ray based micro-computed tomography (CT) and micro-digital subtraction angiography (DSA) are important non-invasive imaging modalities for following tumorogenesis in small animals. To exploit these imaging capabilities further, the two modalities were combined into a single system to provide both morphological and functional data from the same tumor in a single imaging session. The system is described and examples are given of imaging implanted fibrosarcoma tumors in rats using two types of contrast media: (a) a new generation of blood pool contrast agent containing iodine with a concentration of 130 mg/mL (Fenestra TM VC, Alerion Biomedical, San Diego, CA, USA) for micro-CT and (b) a conventional iodinated contrast agent (Isovue 1-370 mg/mL iodine, trademark of Bracco Diagnostics, Princeton, NJ, USA) for micro-DSA. With the blood pool contrast agent, the 3D vascular architecture is revealed in exquisite detail at 100 mm resolution. Micro-DSA images, in perfect registration with the 3D micro-CT datasets, provide complementary functional information such as mean transit times and relative blood flow through the tumor. This imaging approach could be used to understand tumor angiogenesis better and be the basis for evaluating anti-angiogenic therapies.
... More recently, small pilot studies have been conducted to determine whether this method might be feasible in humans undergoing TB treatment (14)(15)(16), although validation data are limited. PET-CT has also been used in experimental animal models (e.g., mice and rabbits) to identify tuberculous disease and monitor the treatment response (17)(18)(19). ...
Trials to test new drugs currently in development against tuberculosis in humans are impractical. All animal models to prioritize new regimens are imperfect but non-human primates (NHP) infected with Mtb develop active tuberculosis (TB) disease with a full spectrum of lesion types seen in humans. Serial 2-deoxy -2-[(18)F]-D-deoxyglucose (FDG) positron emission tomography (PET) with computed tomography (CT) imaging was performed on cynomolgus macaques during infection and chemotherapy with individual agents or the four-drug combination therapy most widely used globally. Size and metabolic activity of lung granulomas varied among animals, and even within a single animal, during development of disease. Individual granulomas within untreated animals had highly local and independent outcomes, some progressing in size and FDG uptake while others waned, illustrating the highly dynamic nature of active TB. At necropsy even untreated animals were found to have a proportion of sterile lesions consistent with the dynamics of this infection. A more marked reduction in overall metabolic activity in the lungs (decreased FDG uptake) was associated with effective treatment. Reduction in size of individual lesions correlated with lower bacterial burden at necropsy. Isoniazid treatment was associated with a transient increase in metabolic activity in individual lesions whereas a net reduction occurred in most lesions from rifampin-treated animals. Quadruple-drug therapy resulted in the highest decrease in FDG uptake. PET/CT may be an important early correlate of efficacy of novel combinations of new drugs that can be directly translated to human clinical trials.
... PET imaging was chosen since it is a quantitative, highly sensitive, tomographic clinical imaging modality without limitation of penetration depth. 15 In addition, 18-fluoro-2-deoxyglucose (FDG) PET probes have been used to detect persistent foreign body reactions induced by mesh prostheses. 16 Given the fact that activated MΦs possess a large number of folic acid (FA) receptors, 14 we chose FA as the targeting ligand for the PET imaging probe design. ...
To image implant-surrounding activated macrophages, a macrophage-specific PET probe was prepared by conjugating folic acid (FA) and 2,2',2″,2‴-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetracetic acid (DOTA) to polyethylene glycol (PEG) and then labeling the conjugate with Ga-68. In vivo PET imaging evaluations demonstrate that the probe is able to detect foreign body reactions, and more importantly, quantify the degree of inflammatory responses to an implanted medical device. These results were further validated by histological analysis.
Background
The development of less expensive and pivotal methodologies, capable to support the researchers in the radiopharmaceutical pre-clinical investigations could provide a crucial incentive for developing biomedical research involved in the realization of tailored target therapies.
Objective
The aim of this pilot study was to evaluate the capability of a digital autoradiography system equipped with a laser scanning device to perform [18F]choline biodistribution evaluation in a xenograft mouse model of prostate cancer.
Methods
PC3 prostate cancer cells were used to develop xenografts in NOD/SCID mice. The biodistribution of the radiopharmaceutical was evaluated at 30,60 and 120 min after injection in excised organs by using a digital autoradiography system equipped with super resolution laser screen. Histological and immunohistochemical analysis were performed to correlate the [18F]choline uptake with morphological and molecular tumours characteristics.
Results
Data here reported clearly indicate the possibility to perform accurate biodistribution studies by using the digital autoradiographic system equipped with a super resolution screen. Specifically, a significant increase in the [18F]choline inhibitor uptake in PC3 tumours as compared to heart, bowel, liver and kidney at both 30 and 60 min was observed. More important, the digital autoradiographic system showed signal uptake almost exclusively in the PC3 tumors at 60 min post-injection. Noteworthy, immunohistochemical analysis demonstrated a strong overlapping between the [18F]choline uptake and the proliferation index (Ki67 expression).
Conclusions
The use of autoradiography system in pre-clinical investigations could shed new light on the molecular mechanisms that orchestrate the tissues damage induced by therapeutical radiopharmaceuticals.
Laboratory animals play an important role in biomedical research and act as a backbone of new drug research and development. Biomedical research is primarily dependent on laboratory rodents. There is a steady shift of therapeutic strategies for diseases from physiological to cellular and presently molecular targets. There is a huge advancement in personalized medicine and ways of drug research in the recent past that is primarily due to the implementation of 3Rs in animal study design and looking into molecular insights of the disease processes with the help of precise tools such as noninvasive imaging. Imaging technologies proved extremely important in diagnosis and evaluating human diseases in clinics. Besides, it has an important role in biomedical research in oncology and other research areas including stem cell research, bone metabolism, endocrinology, neurology, cardiology, and nanotechnology. The complex pathways involved in the initiation and progression of the disease can be visualized and quantitatively monitored using noninvasive imaging technologies. The strong preclinical data is the backbone of biomedical research, and new drug development and imaging technologies provide relevant accurate information. This chapter is focused on the dedicated noninvasive preclinical imaging modalities for their present role in laboratory animal science. This chapter also deals with protocols for positron emission tomography (PET), single-photon emission computed tomography (SPECT), and X-ray computed tomography (CT) for rodent imaging.
Applicability of optoacoustic imaging in biology and medicine is largely determined by a number of key performance characteristics. In particular, an inherent trade-off exists between the acquired field-of-view (FOV) and temporal resolution of the measurements, which may hinder studies looking at rapid biodynamics at the whole-body level. Here, we report on a single-sweep volumetric optoacoustic tomography (sSVOT) system that attains whole body 3D mouse scans with better than 200µm spatial resolution and an unprecedented scanning speed of only 1.8 seconds. The system employs a spherical matric array transducer in conjunction with uniform multi-beam illumination delivery, the latter playing a critical role in facilitating the optimal FOV and imaging speed performance. The system further takes advantage of the spatial response of the individual ultrasound detection elements in order to mitigate common image artifacts related to limited-view acquisitions, thus enabling ultrafast acquisitions without compromising image quality and contrast. We compare performance metrics to the previous spiral volumetric optoacoustic tomography implementations and alternative image compounding and reconstruction strategies. It is anticipated that sSVOT opens new venues for studying large-scale biodynamics, such as accumulation and clearance of molecular agents and drugs across multiple organs, circulation of cells, as well as functional responses to stimuli.
Background:
Cellular imagery using histology sections is one of the most common techniques used in Neuroscience. However, this inescapable technique has severe limitations due to the need to delineate regions of interest on each brain, which is time consuming and variable across experimenters.
New method:
We developed algorithms based on a vectors field elastic registration allowing fast, automatic realignment of experimental brain sections and associated labeling in a brain atlas with high accuracy and in a streamlined way. Thereby, brain areas of interest can be finely identified without outlining them and different experimental groups can be easily analyzed using conventional tools. This method directly readjusts labeling in the brain atlas without any intermediate manipulation of images.
Results:
We mapped the expression of cFos, in the mouse brain (C57Bl/6J) after olfactory stimulation or a non-stimulated control condition and found an increased density of cFos-positive cells in the primary olfactory cortex but not in non-olfactory areas of the odor-stimulated animals compared to the controls.
Comparison with existing method(s):
Existing methods of matching are based on image registration which often requires expensive material (two-photon tomography mapping or imaging with iDISCO) or are less accurate since they are based on mutual information contained in the images. Our new method is non-imaged based and relies only on the positions of detected labeling and the external contours of sections.
Conclusions:
We thus provide a new method that permits automated matching of histology sections of experimental brains with a brain reference atlas.
The development of a fast and novel methodology to generate carbon-11 carbon monoxide ([11C]CO) from cyclotron-produced carbon-11 carbon dioxide ([11C]CO2) mediated by fluoride-activated disilane species is described. This methodology allows up to 74% conversion of [11C]CO2 to [11C]CO using commercially available reagents, readily available labware and mild reaction conditions (room temperature). As proof of utility, radiochemically pure [carbonyl-11C]N-benzylbenzamide was successfully synthesized from produced [11C]CO in up to 74% radiochemical yield (RCY) and > 99% radiochemical purity (RCP) in ≤ 10 min from end of [11C]CO2 delivery.
IntroductionMarket Needs and Changing Disease PatternsHistorical AspectsImpact of New Technology on Drug DiscoveryExamples of Drug DiscoveryEnzymes and Enzyme InhibitorsProtein-Protein Interaction InhibitorsProtein and Antibody TherapeuticsExamples of Currently Available Drugs for Major DiseasesSummary
Significance statement:
The present work provides the first successful demonstration of in vivo positron emission tomographic (PET) visualization of a chemogenetic designer receptor (designer receptor exclusively activated by designer drugs, DREADD) expressed in living brains. This technology has been applied to longitudinal PET reporter imaging of neuronal grafts differentiated from induced pluripotent stem cells. Differentiated from currently used reporter genes for neuroimaging, DREADD has also been available for functional manipulation of target cells, which could be visualized by functional magnetic resonance imaging (fMRI) in a real-time manner. Multimodal imaging with PET/fMRI enables the visualization of the differentiation of iPSC-derived neural progenitors into mature neurons and DREADD-mediated functional manipulation along the time course of the graft and is accordingly capable of fortifying the utility of stem cells in cell replacement therapies.
In vivo molecular imaging includes a range of techniques that are aimed at macroscopically and in vivo visualising molecular events at a cellular level.
Small animal positron emission tomography (SA-PET), small animal single-photon emission tomography (SA-SPECT), small animal magnetic resonance imaging and optical imaging are the most interesting and are based on the use of targeted probes binding to specific molecules (receptors or ligands) or those that are included in specific metabolic processes.
Those techniques are very expensive but meet a wider and wider employment since they have the advantage of analysing the same subject over time.
The application of those preclinical imaging techniques for the basic study of kidney diseases is still an emerging field. This chapter will approach the use of preclinical molecular imaging and tumoral antigen targeting in kidney diseases.
This chapter deals with preclinical studies involving animal model systems and non-invasive methodologies toward understanding the role of proteases in apoptotic pathways and their potential as therapeutic targets. Several critical diseases are associated with imbalance in the apoptotic machinery including neurodegeneration and cancer, and hence an effective strategy to target these molecules might be a tractable solution for combating these ailments. Proapoptotic proteases and their binding partners therefore have always been of special interest for designing and evaluating the efficacy of several drug-like molecules (both activators and inhibitors). This chapter is focused on discussing the development of few such molecules with specific examples. It also vividly describes the different in vivo model systems that are essential for rigorous screening of these molecules at different stages of drug development. Current role and future prospects of preclinical translational imaging platforms (PET, SPECT, CT and MRI) and their utility in clinical trials are also outlined in this chapter.
Small-animal positron emission tomography (PET) and single photon emission tomography (SPECT) are invaluable tools in drug discovery and in assessing novel therapeutic agents. In a typical small-animal PET or SPECT imaging session, a radiopharmaceutical is administered and the kinetics of its distribution tracked. Whereas the PET and SPECT protocols for the radiotraceras used in clinical practice are well established, at least for the radiotracers that are used in clinical practice, the acquisition, reconstruction and data analysis procedures applied in small rodents are heterogeneous among centres, often being determined by the scanner characteristics and by the animal models and anaesthesia procedures used in the laboratory. This review, intended to serve as a guide to preclinical PET and SPECT experiments, aims to familiarise the reader with the aspects of PET and SPECT that pertain to in vivo preclinical acquisitions and, in particular, with the technical and methodological know-how that is needed for better application of radionuclide imaging techniques in preclinical cancer research.
Samuel E DePrimo received a BSc in biology from King's College in Wilkes-Barre, PA, and in 1996 received his PhD from the University of Cincinnati College of Medicine, where he developed and studied transgenic mouse models for the detection of somatic mutations in the laboratory of James Stringer. This was followed by postdoctoral work in George Stark's laboratory at the Cleveland Clinic Foundation's Lerner Institute, studying p53 signaling pathways and forward genetics applications, and with James Brooks at the Stanford University School of Medicine, in collaboration with Patrick Brown, applying gene expression profiling in studying the biology of prostate cancer cells. Dr DePrimo joined SUGEN, Inc. in 2001, as a scientist in the Biomarkers group, and focused on translational research and analysis of molecular biomarkers in oncology clinical trials of receptor tyrosine kinase inhibitors. He joined Pfizer Global Research and Development at the La Jolla campus in 2003, where as a scientist in the Translational Medicine group he continues to focus on biomarker applications and translational biology, primarily in oncology drug development.
Objective: To investigate the feasibility of labeling mice spleen lymphocytes with superparamagnetic iron oxide (SPIO) and in vitro MR imaging of the labeled cells. Methods: Spleen lymphocytes of 5 mice were isolated and then labeled with SPIO of 100, 50, 25, 15, 10, 5 μg/ml, which was previously prepared with PLL. Prussian blue staining was performed to show the intracellular iron. Cell viability was compared among fresh, labeled and unlabeled cells. Different concentrations of mice spleen lymphocytes were screened using 3.0 T MR on T 2WI, T 2* WI and SWI sequences in vitro. Cell viability was compared using independent-sample t test between groups. The MRI values among different groups were compared using one-way ANOVA. Results: SPIO prepared with PLL could successfully label mice spleen lymphocytes, the optimum concentration of SPIO was 5 μg/ml. The Prussian blue staining showed intracellular blue spots and a labeling efficiency of (93.6 ± 2.1)%. Three groups of fresh, labeled and unlabeled cells showed a Trypan blue staining result of (94.8 ± 3.1)%, (88.7 ± 2.7)%, and (88.9 ± 3.2)%, respectively; no statistically significant difference was found in cell viability between labeled and unlabeled lymphocytes (t = 0.281, P > 0.05); however, the cell viability of fresh cells were statistically significant higher than the labeled and unlabeled lymphocytes (t = 8.125 and 7.253 respectively, P < 0.05 for all). Among the T 2WI, T 2* WI and SWI sequences under the same concentrations of cells, the SWI sequence was the most sensitive. Conclusions: The mice spleen lymphocytes can be effectively labeled with SPIO with no impact on cell viability, and MR can be used to track these labeled cells in vitro. The SWI sequence is the most sensitive.
While long-recognized as vital components of cancer research, mouse models largely remained a “black box” until the development of advanced tools for preclinical imaging (Edinger et al. 2002; Van Dyke and Jacks 2002; Hirst and Balmain 2004; Lyons 2005). The dynamics of cancer progression, metastatic spread, and therapeutic response were difficult to study without noninvasive access to real-time information in living animals. Histology and other ex vivo analyses provided some insight into the molecular features of malignancy, but required biopsy and invasive acquisition, or were limited to terminal samples at necropsy. Moreover, such measurements offered only a static snapshot of disease and therapeutic outcome, and did not capture the active nature of malignancy or the dynamic changes associated with treatment. Overcoming these limitations required a set of tools that could probe tumor cells in their native habitat, and track molecular and biochemical changes accompanying tumor growth and regression in real time.
This chapter provides the reader with an introductory look into the significance of preclinical imaging relative to human medicine. The concept of translating preclinical research to the clinical realm is presented with a focus on the primary human disease model, the mouse. Here, the term Small Animal Imaging (SAI) is used to describe preclinical imaging of mice. The fundamental operating principles of the various SAI technologies and primary differences with their clinical counterparts are described. First generation and state-of-the-art instruments are reviewed as well as the utility of combining these technologies into multimodality instruments. Considerations in small animal use, such as anesthesia and radiation dose are followed by a brief look at SAI center design. The application of SAI in the areas of cardiology, neurology, and oncology are reviewed and finally, a perspective on the future of SAI is given. This is not meant to be a comprehensive review, but rather a primer for the biomedical student or researcher to become familiarized with the overall field of preclinical imaging. For further information the reader is referred to external sources of literature.
Imaging modalities for small rodents are mostly derived from clinical imaging such as ultrasound (US) imaging, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and single photon emission computed tomography (SPECT) but require some technical adaptations due mainly to the small size of mice. Optical imaging (bioluminescence, BLI, and fluorescence, FLI) is an additional in vivo technique only performed on small animals, originating from in vitro studies. The objective of this chapter is to provide an overview of these small-animal imaging modalities, their physical basis, and their main applications in mice.
F-Fluorodeoxyglucose (F-FDG), F-fluoro-3'-deoxy-3'-L-fluorothymidine (F-FLT), F-fluoromisonidazole (F-FMISO), and F-AlF-NOTA-PRGD2 (F-RGD) are all commonly used PET tracers for tumor diagnosis based on different mechanisms of tissue uptake. This study compared the ex-vivo biodistribution and PET/computed tomography (CT) imaging studies of these four PET tracers in a xenograft prostate tumor-bearing mouse model.
Nude mice were inoculated with 5×10 PC-3 cells in the right armpit. The ex-vivo biodistribution of F-FDG, F-FLT, F-FMISO, and F-RGD at 30, 60, 90, and 120 min after injection was compared. Micro-PET/CT images of F-FDG, F-FLT, and F-RGD were acquired at 60 min, whereas F-FMISO images were acquired at 90 min after injection.
The tumors were clearly visualized by micro-PET/CT using all four PET tracers. Ex-vivo biodistribution results showed highest tumor accumulation and tumor-to-muscle ratio of F-FDG at each time point, accompanied by physiologically high uptakes in the brain, heart, and intestinal tract. Modest uptake of F-FLT and F-FMISO in tumors was observed at 60 and 90 min after injection, with less interference from other tissues compared with F-FDG. Besides, F-RGD also exhibited high tumor specificity; however, relatively low uptake was observed in the tumor.
Our results demonstrated the potential of F-FMISO and F-FLT in the diagnosis of xenograft prostate cancer.
Due to its unique structure, parallel panel configuration may lead to
novel applications for positron emission tomography (PET). The major
challenge of panel PET imaging is the limited angle problem, to which
the time-of-flight (TOF) information seems to be a promising solution.
This work investigated the required TOF capability of a panel PET
design, which has a feasible size of field of view (FOV) for human
torso. Such a system's corresponding angular coverage can vary from 70
to 102 degrees. The recovery ability of small lesions was assessed, and
the contrast recovery coefficient (CRC) and signal-to-noise ratio (SNR)
were analyzed, with a full ring PET as the benchmark system. We also
varied the timing resolution and the distance between panels, to
evaluate their impacts on image quality. Encouraging results were
obtained in simulation study. Distortions and artifacts caused by the
limited angular coverage were greatly reduced with timing resolutions
better than 300 ps. The recovery ability of small lesions in most part
of FOV was desirable. Meanwhile, varying panel distance in the range of
25 to 45 cm seemed to have trivial influence, when timing resolution was
fixed at 300 ps. That means that such changes of panel distance might
not affect the requirement on TOF capability, allowing more flexibility
in panel PET's design and applications.
In this paper, 99mTc-TRODAT-1 Micro-SPECT (single-photon emission computed tomography) was used for imaging dopamine transporter (DAT) in retinas and to investigate the changes of DAT in retinas of guinea pigs with form deprivation myopia. Pigmented guinea pigs aged 3 weeks were devided into form deprivation myopia (FDM) group (n=6) and normal control group (n=6). The test group wore translucent goggles randomly for 4 weeks, and both groups underwent biometric measurement (refraction and axial length) before and after the experiment. Micro-SPECT retinas imaging was performed at the 4th week after injection of 99mTc-TRODAT-1. The retinas were clearly resolved in the images. The ratio of 99mTc-TRODAT-1 uptake in the myopic retinas (11.55±2.80) was 3.64±1.40 lower than that in the control eye (15.20±1.98), and 2.35±1.05 lower than that in the fellow eyes (13.90±2.04). The results showed that 99mTc-TRODAT-1 Micro-SPECT eye imaging can be used to trace the distribution and changes of DAT in retina, and DAT in the myopic retinas were lower than that in the normal control eyes and fellow eyes. Micro-SPECT may provide a new approach for further studies on the role of dopamine system in the experimental myopia.
In vivo molecular imaging includes a range of techniques that are aimed at macroscopically and in vivo visualising molecular events at a cellular level.
Small animal positron emission tomography (SA-PET), small animal single photon emission tomography (SA-SPET), small animal magnetic resonance imaging and optical imaging are the most interesting and are based on the use of targeted probes binding to specific molecules (receptors or ligands) or those that are included in specific metabolic processes.
Those techniques are very expensive, but meet a wider and wider employment since they have the advantage of analysing the same subject over time.
The application of those pre-clinical imaging techniques for the basic study of kidney diseases is still an emerging field. This chapter will approach the use of pre-clinical molecular imaging and tumoural antigen targeting in kidney diseases.
The synthesis and preliminary biological evaluation of a lipophilic, fluorine-18-labeled 5-ethynyl-2′-deoxyuridine derivative [18F]-3 is described. Initially, 5-ethynyl-2′-deoxyuridine 5 was synthesized by coupling trimethylsilyl protected acetylene to 5-iodo-2′-deoxyuridine 4, followed by deprotection in alkaline conditions. Compound 5 was then reacted with 4-(4′-iodophenyl)phenol to give 5-[4(4′-hydroxyphenyl)phenyl]ethynyl-2′-deoxyuridine 6. Compound 6 was reacted with BrCH2CHF as alkylating agent to give stable or radiolabeled 3. The crude products were purified using reversed phase-high performance liquid chromatography to obtain compound 3 and [18F]-3 in 33 and 7.4% yield (decay corrected), respectively. The synthesis time to obtain pure [18F]-3 was about 60 min (starting from BrCH2CHF). The specific radioactivity of the tracer was between 74 and 222 GBq/µmol. The log P7.4 of [18F]-3 was found to be 2.4. However, biodistribution study in normal mice showed low uptake of the tracer in the brain. The affinity of compounds 6 and 3 for varicella-zoster virus thymidine kinase enzyme (VZV-TK) was examined in vitro and the results revealed that the fluorinated analog 3 has a poor affinity for the enzyme in contrast to the phenol precursor 6. Copyright © 2007 John Wiley & Sons, Ltd.
Because no effective cures are available for cutaneous malignant melanoma, early diagnosis and accurate staging are of the utmost importance in increasing patient survival. Fluorodeoxyglucose positron emission tomography (PET)/computed tomography is a functional imaging technique that has contributed to ameliorating surveillance of patients with melanoma. New PET probes are under evaluation, and many have been tried in in vivo imaging protocols based on the use of small animal PET and animal models of cutaneous melanoma. Those compounds are targeted to a-melanocyte-stimulating hormone receptor and to the intracellular biosynthesis of melanin, and all of them showed promising results.
A noninvasive method for molecular imaging of T-cell activity in vivo would be of considerable value. It would aid in understanding the role of specific genes and signal transduction pathways in the course of normal and pathologic immune responses, could elucidate temporal dynamics and immune regulation at different stages of disease and following therapy. We developed and assessed a novel method for monitoring the T-cell receptor (TCR) -dependent nuclear factor of activated T cells (NFAT) -mediated activation of T cells by optical fluorescence imaging (OFI) and positron emission tomography (PET). The herpes simplex virus type 1 thymidine kinase/green fluorescent protein [HSV1-tk/GFP (TKGFP) ] dual reporter gene was used to monitor NFAT-mediated transcriptional activation in human Jurkat cells. A recombinant retrovirus bearing the NFAT-TKGFP reporter system was constructed in which the TKGFP reporter gene was placed under control of an artificial cis-acting NFAT-specific enhancer. Transduced Jurkat cells were used to establish subcutaneous infiltrates in nude rats. We demonstrated that noninvasive OR and nuclear imaging of T-cell activation is feasible using the NFAT-TKGFP reporter system. PET imaging with [124]FIAU using the NFAT-TKGFP reporter system is sufficiently sensitive to detect T-cell activation in vivo. PET images were confirmed by independent measurements of T-cell activation (e.g., CD69) and induction of GFP fluorescence. PET imaging of TCR-induced NFAT-dependent transcriptional activity may be useful in the assessment of T cell responses, T-cell-based adoptive therapies, vaccination strategies and immunosuppressive drugs.
Recent developments in in vivo imaging using optical, radionuclide and paramagnetic reporter probes now enables continuous measurements of gene expression in living animals. In vivo bioluminescence imaging (BLI) is a sensitive, versatile and accessible imaging strategy that has been applied to a variety of small-animal models of human biology and disease. We discuss current strategies in BLI and the potential of combining BLI with other in vivo and ex vivo techniques. BLI will have a significant role in in vivo cellular and molecular imaging, a field that will help reveal the molecular basis of biology and disease.
Herpes simplex virus type 1 (HSV-1) thymidine kinase is currently used as a suicide agent in the gene therapy of cancer. This therapy is based on the preferential phosphorylation of nucleoside analogs by tumor cells expressing HSV-1 thymidine kinase. However, the use of HSV-1 thymidine kinase is limited in part by the toxicity of the nucleoside analogs. We have used random sequence mutagenesis to create new HSV-1 thymidine kinases that, compared with wild-type thymidine kinase, render cells much more sensitive to specific nucleoside analogs. A segment of the HSV-1 thymidine kinase gene at the putative nucleoside binding site was substituted with random nucleotide sequences. Mutant enzymes that demonstrate preferential phosphorylation of the nucleoside analogs, ganciclovir or acyclovir, were selected from more than one million Escherichia coli transformants. Among the 426 active mutants we have isolated, 26 demonstrated enhanced sensitivity to ganciclovir, and 54 were more sensitive to acyclovir. Only 6 mutant enzymes displayed sensitivity to both ganciclovir and acyclovir when expressed in E. coli. Analysis of 3 drug-sensitive enzymes demonstrated that 1 produced stable mammalian cell transfectants that are 43-fold more sensitive to ganciclovir and 20-fold more sensitive to acyclovir.
We report a series of studies that assess the feasibility and sensitivity of imaging of herpes virus type one thymidine kinase (HSV1-tk) gene transfer and expression with [124I]-5-iodo-2'-fluoro-1-beta-D-arabinofuranosyluracil ([124I]-FIAU) and positron emission tomography (PET) and the ability of [124I]-FIAU-PET imaging to discriminate different levels of HSV1-tk gene expression. Studies were performed in rats bearing multiple s.c. tumors derived from W256 rat carcinoma and RG2 rat glioma cells. In the first set, we tested the sensitivity of [124I]-FIAU-PET imaging to detect low levels of HSV1-tk gene expression after retroviral-mediated gene transfer. HSV1-tk gene transduction of one of preestablished wild-type W256 tumor in each animal was accomplished by direct intratumoral injection of retroviral vector-producer cells (W256-->W256TK* tumors). Tumors produced from W256 and W256TK+ cells served as the negative and positive control in each animal. Highly specific images of [124I]-FIAU-derived radioactivity were obtained in W256TK* tumors (that were transduced in vivo) and in W256TK+ tumors but not in nontransduced wild-type W256 tumors. The level of "specific" incorporated radioactivity in transduced portions of both W256TK* and W256TK+ tumors was relatively constant between 4 and 50 h. In the second set, we tested whether [124I]-FIAU and PET imaging can measure and discriminate between different levels of HSV1-tk gene expression. Multiple s.c. tumors were produced from wild-type RG2 cells and stably transduced RG2TK cell lines that express different levels of HSV1-tk. A highly significant relationship between the level of [124I]-FIAU accumulation [% injected dose/g and incorporation constant (Ki)] and an independent measure of HSV1-tk expression (sensitivity of the transduced tumor cells to ganciclovir, IC50) was demonstrated, and the slope of this relationship was defined as a sensitivity index. We have demonstrated for the first time that highly specific noninvasive images of HSV1-tk expression in experimental animal tumors can be obtained using radiolabeled 2'-fluoro-nucleoside [124I]-FIAU and a clinical PET system. The ability to image the location (distribution) of gene expression and the level of expression over time provides new and useful information for monitoring clinical gene therapy protocols in the future.
We are developing quantitative assays to repeatedly and noninvasively image expression of reporter genes in living animals, using positron emission tomography (PET). We synthesized positron-emitting 8-[18F]fluoroganciclovir (FGCV) and demonstrated that this compound is a substrate for the herpes simplex virus 1 thymidine kinase enzyme (HSV1-TK). Using positron-emitting FGCV as a PET reporter probe, we imaged adenovirus-directed hepatic expression of the HSV1-tk reporter gene in living mice. There is a significant positive correlation between the percent injected dose of FGCV retained per gram of liver and the levels of hepatic HSV1-tk reporter gene expression (r2 > 0.80). Over a similar range of HSV1-tk expression in vivo, the percent injected dose retained per gram of liver was 0-23% for ganciclovir and 0-3% for FGCV. Repeated, noninvasive, and quantitative imaging of PET reporter gene expression should be a valuable tool for studies of human gene therapy, of organ/cell transplantation, and of both environmental and behavioral modulation of gene expression in transgenic mice.
Reporter genes (e.g. beta-galactosidase, chloramphenicol-acetyltransferase, green fluorescent protein, luciferase) play critical roles in investigating mechanisms of gene expression in transgenic animals and in developing gene delivery systems for gene therapy. However, measuring expression of these reporter genes requires biopsy or death. We now report a procedure to image reporter gene expression repetitively and non-invasively in living animals with positron emission tomography (PET), using the dopamine type 2 receptor (D2R) as a reporter gene and 3-(2'-[18F]fluoroethyl)spiperone (FESP) as a reporter probe. We use a viral delivery system to demonstrate the ability of this PET reporter gene/PET reporter probe system to image reporter gene expression following somatic gene transfer. In mice injected intravenously with replication-deficient adenovirus carrying a D2R reporter gene, PET in vivo measures of hepatic [18F] retention are proportional to in vitro measures of hepatic FESP retention, D2R ligand binding and D2R mRNA. We use tumor-forming cells carrying a stably transfected D2R gene to demonstrate imaging of this PET reporter gene/PET reporter probe system in 'tissues'. Tumors expressing the transfected D2R reporter gene retain substantially more FESP than control tumors. The D2R/FESP reporter gene/reporter probe system should be a valuable technique to monitor, in vivo, expression from both gene therapy vectors and transgenes.
We are developing assays for noninvasive, quantitative imaging of reporter genes with positron emission tomography (PET), for application both in animal models and in human gene therapy. We report here a method to improve the detection of lower levels of PET reporter gene expression by utilizing a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) as a PET reporter gene. The HSV1-sr39tk mutant was identified from a library of site-directed mutants. Accumulation (net uptake) of the radioactively labeled substrates [8-(3)H]penciclovir ([8-(3)H]PCV), and 8-[(18)F]fluoropenciclovir (FPCV) in C6 rat glioma cells expressing HSV1-sr39tk is increased by a factor of approximately 2.0 when compared with C6 cells expressing wild-type HSV1-tk. The increased imaging sensitivity of HSV1-sr39tk when FPCV is used is also demonstrated in vivo both with tumor cells stably transfected with either HSV1-tk or HSV1-sr39tk, and after hepatic delivery of HSV1-tk or HSV1-sr39tk by using adenoviral vectors. The use of HSV1-sr39tk as a PET reporter gene and FPCV as a PET reporter probe results in significantly enhanced sensitivity for imaging reporter gene expression in vivo.
Rapid imaging by antitumor antibodies has been limited by the prolonged targeting kinetics and clearance of labeled whole antibodies. Genetically engineered fragments with rapid access and high retention in tumor tissue combined with rapid blood clearance are suitable for labeling with short-lived radionuclides, including positron-emitting isotopes for positron-emission tomography (PET). An engineered fragment was developed from the high-affinity anticarcinoembryonic antigen (CEA) monoclonal antibody T84.66. This single-chain variable fragment (Fv)-C(H)3, or minibody, was produced as a bivalent 80 kDa dimer. The macrocyclic chelating agent 1,4,7, 10-tetraazacyclododecane-N,N',N", N"'-tetraacetic acid (DOTA) was conjugated to the anti-CEA minibody for labeling with copper-64, a positron-emitting radionuclide (t(1/2) = 12.7 h). In vivo distribution was evaluated in athymic mice bearing paired LS174T human colon carcinoma (CEA positive) and C6 rat glioma (CEA negative) xenografts. Five hours after injection with (64)Cu-DOTA-minibody, microPET imaging showed high uptake in CEA-positive tumor (17.9% injected dose per gram +/- 3.79) compared with control tumor (6.0% injected dose per gram +/- 1.0). In addition, significant uptake was seen in liver, with low uptake in other tissues. Average target/background ratios relative to neighboring tissue were 3-4:1. Engineered antibody fragments labeled with positron-emitting isotopes such as copper-64 provide a new class of agents for PET imaging of tumors.
A variety of imaging technologies are being investigated as tools for studying gene expression in living subjects. Noninvasive, repetitive and quantitative imaging of gene expression will help both to facilitate human gene therapy trials and to allow for the study of animal models of molecular and cellular therapy. Radionuclide approaches using single photon emission computed tomography (SPECT) and positron emission tomography (PET) are the most mature of the current imaging technologies and offer many advantages for imaging gene expression compared to optical and magnetic resonance imaging (MRI)-based approaches. These advantages include relatively high sensitivity, full quantitative capability (for PET), and the ability to extend small animal assays directly into clinical human applications. We describe a PET scanner (microPET) designed specifically for studies of small animals. We review "marker/reporter gene" imaging approaches using the herpes simplex type 1 virus thymidine kinase (HSV1-tk) and the dopamine type 2 receptor (D2R) genes. We describe and contrast several radiolabeled probes that can be used with the HSV1-tk reporter gene both for SPECT and for PET imaging. We also describe the advantages/disadvantages of each of the assays developed and discuss future animal and human applications.
Non-invasive imaging of gene expression opens new prospects for the study of transgenic animals and the implementation of genetically based therapies in patients. We have sought to establish a general paradigm to enable whole body non-invasive imaging of any transgene. We show that the expression and imaging of HSV1-tk (a marker gene) can be used to monitor the expression of the LacZ gene (a second gene) under the transcriptional control of a single promoter within a bicistronic unit that includes a type II internal ribosomal entry site. In cells bearing a single copy of the vector, the expression of the two genes is proportional and constant, both in vitro and in vivo. We demonstrate that non-invasive imaging of HSV1-tk gene accurately reflects the topology and activity of the other cis-linked transgene.
Various radiotracers based on uracil nucleosides (e.g. [124I]2'-fluoro-2'-deoxy-5-iodo-1-beta-D-arabinofuranosyluracil, [124I]FIAU) and acycloguanosine derivatives (e.g. [18F]9-[(3-fluoro-1-hydroxy-2-propoxy) methyl] guanine, [18F]FHPG) have been proposed for the non-invasive imaging of herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene expression. However, these radiotracers have been evaluated in different in vitro and in vivo models, precluding a direct comparison. Therefore, we directly compared [18F]FHPG and radioiodinated FIAU to assess their potential for PET imaging of transgene expression. The uptake of [125I]FIAU, [18F]FHPG and [3H]acyclovir was determined in vitro using four different HSV1-tk expressing cell lines and their respective negative controls. The in vitro tracer uptake was generally low in non-transduced parental cell lines. In HSV1-tk expressing cells, [3H]acyclovir showed approximately a twofold higher tracer accumulation, the [18F]FHPG uptake increased by about sixfold and the [125I]FIAU accumulation increased by about 28-fold after 120-min incubation of T1115 human glioblastoma cells. Similar results were found in the other cell lines. In addition, biodistribution and positron emission tomography (PET) studies with [18F]FHPG and [124/125I]FIAU were carried out in tumour-bearing BALB/c mice. Significantly higher specific accumulation of radioactivity was found for [125I]FIAU compared with [18F]FHPG. The ratio of specific tracer accumulation between [125I]FIAU and [18F]FHPG increased from 21 (30 min p.i.) to 119 (4 h p.i.). PET imaging, using [124I]FIAU, clearly visualised and delineated HSV1-tk expressing tumours, whereas only a negligible uptake of [18F]FHPG was observed. This study demonstrated that in vitro and in vivo, the radioiodinated uracil nucleoside FIAU has a significantly higher specific accumulation than the acycloguanosine derivative [18F]FHPG. This suggests that [124I]FIAU should be the preferred reporter probe for PET imaging of HSV1-tk gene expression. Thus, further attempts to develop suitable PET tracers for the assessment of HSV1-tk gene expression should also focus on 18F-labelled uracil derivatives.
Biodistribution, magnitude and duration of a therapeutic transgene's expression may be assessed by linking it to the expression of a positron emission tomography (PET) reporter gene (PRG) and then imaging the PRG's expression by a PET reporter probe (PRP) in living animals. We validate the simple approach of co-administering two distinct but otherwise identical adenoviruses, one expressing a therapeutic transgene and the other expressing the PRG, to track the therapeutic gene's expression. Two PET reporter genes, a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) and dopamine-2 receptor (D(2)R), each regulated by the same cytomegalovirus (CMV) promoter, have been inserted into separate adenoviral vectors (Ad). We demonstrate that cells co-infected with equivalent titers of Ad-CMV-HSV1-sr39tk and Ad-CMV-D(2)R express both reporter genes with good correlation (r(2) = 0.93). Similarly, a high correlation (r(2) = 0.97) was observed between the expression of both PRGs in the livers of mice co-infected via tail-vein injection with equivalent titers of these two adenoviruses. Finally, microPET imaging of HSV1-sr39tk and D(2)R expression with 9-(4-[(18)F]fluoro-3-hydroxymethylbutyl) guanine ([(18)F]FHBG) and 3-(2-[(18)F]fluoroethyl)spiperone ([(18)F]FESP), utilizing several adenovirus-mediated delivery routes, illustrates the feasibility of evaluating relative levels of transgene expression in living animals, using this approach.
The dopamine D2 receptor (D2R) has been used in adenoviral delivery systems and in tumor cell xenografts as an in vivo reporter gene. D2R reporter gene expression has been non-invasively, repetitively and quantitatively imaged by positron emission tomography (PET), following systemic injection of a positron-labeled ligand (3-(2'-[18F]-fluoroethyl)-spiperone; FESP) and subsequent D2R-dependent sequestration. However, dopamine binding to the D2R can modulate cyclic AMP levels. For optimal utilization of D2R as a reporter gene, it is important to uncouple ligand-binding from Gi-protein-mediated inhibition of cAMP production. Mutation of Asp80 or Ser194 produces D2Rs that still bind [3H]spiperone in transfected cells. The D2R80A mutation completely eliminates the ability of the D2R to suppress forskolin-stimulated cAMP accumulation in response to dopamine, in cells transfected with a D2R80A expression plasmid and in cells infected with replication-defective adenovirus expressing D2R80A. The D2R194A mutation substantially reduces, but does not completely eliminate, dopamine modulation of cAMP levels. Cultured cells infected with adenoviruses expressing D2R and D2R80A demonstrated equivalent [3H]spiperone binding activity. Moreover, hepatic FESP sequestration is equivalent, following intravenous injection of adenoviruses expressing D2R and D2R80A. The D2R80A mutant, which can no longer modulate cAMP levels following ligand binding, has full capability as a PET reporter gene.
Methods to repeatedly, non-invasively, and quantitatively image gene expression in living animals are rapidly emerging and should fundamentally change studies of gene expression in vivo. We previously developed assays utilizing positron emission tomography (PET) to image reporter gene expression. In this paper we: (1) describe a new bi-directional, tetracycline-inducible system that can be used to pharmacologically induce target gene expression and to quantitatively image induced expression by using a PET reporter gene; (2) demonstrate the potential of this system in transient and stable cell transfection assays; and (3) demonstrate the ability to repetitively and quantitatively image tetracycline and tetracycline analog induction of gene expression in living animals. We utilize the dopamine type-2 receptor (D(2)R) and the mutant herpes-simplex virus type 1 thymidine kinase (HSV1-sr39tk) reporter genes to validate this system. We utilize microPET technology to show that quantitative tomographic imaging of gene induction is possible. We find a high correlation (r(2) = 0.98) between 'target' and reporter gene expression. This work establishes a new technique for imaging time-dependent variation of gene expression both from vectors with inducible promoters and in transgenic animals in which pharmacologic induction of gene expression must be monitored. These techniques may be applied both in gene therapy and for the study of gene expression in transgenic animals.
A noninvasive method for molecular imaging of T-cell activity in vivo would be of considerable value. It would aid in understanding the role of specific genes and signal transduction pathways in the course of normal and pathologic immune responses, and could elucidate temporal dynamics and immune regulation at different stages of disease and following therapy. We developed and assessed a novel method for monitoring the T-cell receptor (TCR)-dependent nuclear factor of activated T cells (NFAT)-mediated activation of T cells by optical fluorescence imaging (OFI) and positron emission tomography (PET). The herpes simplex virus type 1 thymidine kinase/green fluorescent protein [HSV1-tk/GFP (TKGFP)] dual reporter gene was used to monitor NFAT-mediated transcriptional activation in human Jurkat cells. A recombinant retrovirus bearing the NFAT-TKGFP reporter system was constructed in which the TKGFP reporter gene was placed under control of an artificial cis-acting NFAT-specific enhancer. Transduced Jurkat cells were used to establish subcutaneous infiltrates in nude rats. We demonstrated that noninvasive OFI and nuclear imaging of T-cell activation is feasible using the NFAT-TKGFP reporter system. PET imaging with [(124)I]FIAU using the NFAT-TKGFP reporter system is sufficiently sensitive to detect T-cell activation in vivo. PET images were confirmed by independent measurements of T-cell activation (e.g., CD69) and induction of GFP fluorescence. PET imaging of TCR-induced NFAT-dependent transcriptional activity may be useful in the assessment of T cell responses, T-cell-based adoptive therapies, vaccination strategies and immunosuppressive drugs.
In this article, we describe the radiosynthesis and evaluation of 18F-labeled cyclooxygenase (COX) inhibitors. 18F-SC63217 is selective to COX-1 and has a COX-1 inhibitory concentration of 50% (IC(50)) < 10 nmol/L and a COX-2 IC(50) > 100 micromol/L. 18F-SC58125 has IC(50) values of >100 micromol/L (COX-1) and <86 nmol/L (COX-2).
SC63217 and SC58125 were both labeled with 18F by nucleophilic displacement of a trimethylammonium triflate salt using a dedicated microwave cavity. Each compound was evaluated in vitro using a murine macrophage cell line (J774). COX-2 was stimulated in these cells by treatment with lipopolysaccharide and interferon-gamma. Both radiotracers were further investigated in vivo using rat biodistribution techniques. Brain uptake of the COX-2 inhibitor, 18F-SC58125, was further investigated by brain PET of a baboon.
The in vitro studies showed that uptake of 18F-SC58125 was increased in stimulated cells and was totally inhibited by the addition of nonradioactive SC58125. In contrast, no increase in uptake was seen for 18F-SC63217. In the biodistribution experiments, 18F-SC63217 showed much higher uptake in the small intestine (an organ known to express high levels of COX-1) than did 18F-SC58125. Higher levels of 18F-SC58125 were observed in the kidney, an organ known to contain high levels of COX-2 rather than COX-1. 18F-SC58125 was retained in brain tissue. PET images of the baboon showed no regional distribution of the radiotracer in the brain.
We have developed a radiosynthetic route that can yield 18F-labeled selective inhibitors of COX-1 or COX-2. Both compounds have been fully characterized in vitro and in vivo. Our results indicate that 18F-SC58125 has potential as a marker of COX-2 activity but that, because of high nonspecific binding, 18F-SC63217 was not a good choice as a marker of COX-1.
We have used copper-64-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (64Cu-PTSM) to radiolabel cells ex vivo for in vivo positron-emission tomography (PET) imaging studies of cell trafficking in mice and for eventual application in patients. 2-[18F]-Fluoro-2-deoxy-d-glucose (FDG) cell labeling also was evaluated for comparison. 64Cu-PTSM uptake by C6 rat glioma (C6) cells increased for 180 min and then stabilized. The labeling efficiency was directly proportional to 64Cu-PTSM concentration and influenced negatively by serum. Label uptake per cell was greater with 64Cu-PTSM than with FDG. However, both 64Cu-PTSM- and FDG-labeled cells showed efflux of cell activity into supernatant. The 64Cu-PTSM labeling procedure did not interfere significantly with C6 cell viability and proliferation rate. MicroPET images of living mice indicate that tail-vein-injected labeled C6 cells traffic to the lungs and liver. In addition, transient splenic accumulation of radioactivity was clearly detectable in a mouse scanned at 3.33 h postinfusion of 64Cu-PTSM-labeled lymphocytes. In contrast, the liver was the principal organ of tracer localization after tail-vein administration of 64Cu-PTSM alone. These results indicate that in vivo imaging of cell trafficking is possible with 64Cu-PTSM-labeled cells. Given the longer t(1/2) of 64Cu (12.7 h) relative to 18F (110 min), longer cell-tracking periods (up to 24-36 h) should be possible now with PET.
In the K/BxN mouse model of rheumatoid arthritis, the transfer of autoantibodies specific for glucose-6-phosphate isomerase (GPI) into naïve mice rapidly induces joint-specific inflammation similar to that seen in human rheumatoid arthritis. The ubiquitous expression of GPI and the systemic circulation of anti-GPI immunoglobulin G (IgG) seem incongruous with the tissue specificity of this disease. By using PET (positron emission tomography), we show here that purified anti-GPI IgG localizes specifically to distal joints in the front and rear limbs within minutes of intravenous injection, reaches saturation by 20 min and remains localized for at least 24 h. In contrast, control IgG does not localize to joints or cause inflammation. The rapid kinetics of anti-GPI IgG joint localization supports a model in which autoantibodies bind directly to pre-existing extracellular GPI in normal healthy mouse joints.
Protein-protein interactions control transcription, cell division, and cell proliferation as well as mediate signal transduction, oncogenic transformation, and regulation of cell death. Although a variety of methods have been used to investigate protein interactions in vitro and in cultured cells, none can analyze these interactions in intact, living animals. To enable noninvasive molecular imaging of protein-protein interactions in vivo by positron-emission tomography and fluorescence imaging, we engineered a fusion reporter gene comprising a mutant herpes simplex virus 1 thymidine kinase and green fluorescent protein for readout of a tetracycline-inducible, two-hybrid system in vivo. By using micro-positron-emission tomography, interactions between p53 tumor suppressor and the large T antigen of simian virus 40 were visualized in tumor xenografts of HeLa cells stably transfected with the imaging constructs. Imaging protein-binding partners in vivo will enable functional proteomics in whole animals and provide a tool for screening compounds targeted to specific protein-protein interactions in living animals.
To compare two systems for assessing gene transfer to cancer cells and xenograft tumors with noninvasive gamma camera imaging.
A replication-incompetent adenovirus encoding the human type 2 somatostatin receptor (hSSTr2) and the herpes simplex virus thymidine kinase (TK) enzyme (Ad-hSSTr2-TK) was constructed. A-427 human lung cancer cells were infected in vitro and mixed with uninfected cells at different ratios. A-427 tumors in nude mice (n = 23) were injected with 1 x 10(6) to 5 x 10(8) plaque-forming units (pfu) of Ad-hSSTr2-TK. The expressed hSSTr2 and TK proteins were imaged owing to internally bound, or trapped, technetium 99m ((99m)Tc)-labeled hSSTr2-binding peptide (P2045) and radioiodinated 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodouracil (FIAU), respectively. Iodine 125 ((125)I)-labeled FIAU was used in vitro and iodine 131 ((131)I)-labeled FIAU, in vivo. The (99m)Tc-labeled P2045 and (125)I- or (131)I-labeled FIAU were imaged simultaneously with different window settings with an Anger gamma camera. Treatment effects were tested with analysis of variance.
Infected cells in culture trapped (125)I-labeled FIAU and (99m)Tc-labeled P2045; uptake correlated with the percentage of Ad-hSSTr2-TK-positive cells. For 100% of infected cells, 24% +/- 0.4 (mean +/- SD) of the added (99m)Tc-labeled P2045 was trapped, which is significantly lower (P <.05) than the 40% +/- 2 of (125)I-labeled FIAU that was trapped. For the highest Ad-hSSTr2-TK tumor dose (5 x 10(8) pfu), the uptake of (99m)Tc-labeled P2045 was 11.1% +/- 2.9 of injected dose per gram of tumor (thereafter, dose per gram), significantly higher (P <.05) than the uptake of (131)I-labeled FIAU at 1.6% +/- 0.4 dose per gram. (99m)Tc-labeled P2045 imaging consistently depicted hSSTr2 gene transfer in tumors at all adenovirus doses. Tumor uptake of (99m)Tc-labeled P2045 positively correlated with Ad-hSSTr2-TK dose; (131)I-labeled FIAU tumor uptake did not correlate with vector dose.
The hSSTr2 and TK proteins were simultaneously imaged following dual gene transfer with an adenovirus vector.
G2A is a lymphocyte-expressed G protein-coupled receptor whose genetic ablation results in the development of autoimmunity. Using HSV-TK reporter gene directed positron emission tomography (PET), we demonstrate that prior to any indication of the onset of illness, mice transplanted with BCR-ABL transduced G2A-deficient bone marrow harbor expanded populations of leukemic cells compared to recipients of wild-type bone marrow. The target cell type and anatomical locations of leukemia development are indistinguishable in animals transplanted with G2A+/+ or G2A-/- cells. Shorter disease latency in the G2A-deficient background is associated with an increased rate of cellular expansion. PET can be successfully applied to the temporal and spatial analysis of Bcr-Abl driven leukemic progression and should have utility for the study of other leukemias and lymphomas.
Gene therapy protocols are hampered by the inability to monitor the location, magnitude, and duration of ectopic gene expression following DNA delivery. Consequently, it is difficult to establish quantitative correlations and/or causal relationships between therapeutic gene expression and phenotypic responses in treated individuals. One approach to monitor "therapeutic gene" expression indirectly is to incorporate reporter genes that can be imaged in vivo into bicistronic transcription units, along with the therapeutic genes. Expression of the dopamine D2 receptor (D2R) and herpes simplex virus thymidine kinase (HSV1-TK) can both be monitored, in vivo, by positron-emission tomography (PET). We created ad.DTm, an adenovirus containing a cytomegalovirus (CMV) early promoter-driven transcription unit, in which the D2R gene is placed proximal to an encephalomyocarditis virus internal ribosomal entry site (IRES) and a modified HSV1-tk gene is placed distal to the IRES. Following intravenous ad.DTm injection into mice, correlated hepatic D2R and HSV1-sr39tk PET reporter gene expression was demonstrated. Repeated microPET scanning quantitated both D2R-dependent sequestration of a positron-emitting ligand and HSV1-TK-dependent sequestration of a positron-emitting product. It is possible, in living mice, to investigate noninvasively and to measure quantitatively and repeatedly correlated expression of two coding regions from a bicistronic transcription unit over a 3-month period following adenovirus delivery.
The efficacy of 3 radiolabeled probes of current interest for imaging herpes simplex virus type 1 thymidine kinase (HSV1-tk) expression in vivo with PET, including (124)I- or (131)I-labeled 2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil (FIAU), (18)F-labeled 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (FHBG), and (18)F-labeled 9-[3-fluoro-1-hydroxy-2-propoxymethyl]guanine (FHPG), was compared.
Two established rat glioma cell lines, stably transduced RG2TK+ and wild-type RG2, were used for paired comparisons of probe accumulation in vitro and for paired comparisons of subcutaneous xenografts produced from these cell lines in athymic rnu/rnu rats.
The in vitro paired probe uptake (0-3 h) comparisons in RG2TK+ cells showed that FIAU accumulation was 15-fold greater than that of FHBG and 41-fold greater than that of FHPG. The net accumulation rate values (+/-SD) calculated for RG2TK+ cells were 0.317 +/- 0.066, 0.022 +/- 0.001, and 0.0077 +/- 0.0003 mL/min/g cells for FIAU, FHBG, and FHPG, respectively. These results and similar uptake studies in RG2 wild-type cells suggest a possible cell membrane transport limitation for FHBG and FHPG. The paired 2-h in vivo uptake studies produced similar differences in RG2TK+ xenografts for FIAU and FHBG (1.22 +/- 0.21 vs. 0.074 +/- 0.49 %dose/g) and for FIAU and FHPG (1.27 +/- 0.14 vs. 0.023 +/- 0.008 %dose/g). These differences were clearly visible on the images. FIAU accumulation at 24 h was 1.53 +/- 0.40 %dose/g. Plasma clearance was FHBG > FHPG > FIAU. The FIAU images showed significant stomach and some intestinal background radioactivities, whereas hepatobiliary and intestinal background activities were very high for the guanosine analogs (FHBG > FHPG). Dynamic imaging showed early ( approximately 10 min) selective localization of FIAU in RG2TK+ xenografts, whereas FHBG and FHPG are being cleared from the HSV1-tk transduced and wild-type xenografts over the initial 2-h imaging period.
The in vitro and in vivo results (including the PET images) show that FIAU is a substantially more efficient probe than FHBG or FHPG for imaging HSV1-tk expression, with greater sensitivity and contrast as well as lower levels of abdominal background radioactivity at 2 and 24 h.
Diseases are biological processes, and molecular imaging with positron emission tomography (PET) is sensitive to and informative of these processes. This is illustrated by detection of biological abnormalities in neurological disorders with no computed tomography or MRI anatomic changes, as well as even before symptoms are expressed. PET whole body imaging in cancer provides the means to (i) identify early disease, (ii) differentiate benign from malignant lesions, (iii) examine all organs for metastases, and (iv) determine therapeutic effectiveness. Diagnostic accuracy of PET is 8-43% higher than conventional procedures and changes treatment in 20-40% of the patients, depending on the clinical question, in lung and colorectal cancers, melanoma, and lymphoma, with similar findings in breast, ovarian, head and neck, and renal cancers. A microPET scanner for mice, in concert with human PET systems, provides a novel technology for molecular imaging assays of metabolism and signal transduction to gene expression, from mice to patients: e.g., PET reporter gene assays are used to trace the location and temporal level of expression of therapeutic and endogenous genes. PET probes and drugs are being developed together-in low mass amounts, as molecular imaging probes to image the function of targets without disturbing them, and in mass amounts to modify the target's function as a drug. Molecular imaging by PET, optical technologies, magnetic resonance imaging, single photon emission tomography, and other technologies are assisting in moving research findings from in vitro biology to in vivo integrative mammalian biology of disease.
3-(2'-[18F]fluoroethyl)spiperone (FESP), a recently developed dopamine D2-receptor binding radiopharmaceutical, was used for dynamic characterization of dopamine-receptor binding in Macaca nemestrina monkeys and humans with positron emission tomography (PET). FESP in vitro binding properties to the dopamine receptor (IC50 = 1.5 nM) are similar to those of spiperone. Serial PET scans in monkeys after intravenous bolus injection of FESP revealed specific radioactivity accumulation in striatum (rich in dopamine D2-receptors), whereas radioactivity concentration declined after 20 min in frontal cortex (serotonin receptors) and more rapidly in cerebellum (nonspecific binding). Specific dopamine D2-receptor binding was saturated with increasing concentrations of radioligand (specific activity range: 1-10,000 Ci/mmol), was stereospecifically blocked with ( + )buta-clamol (0.5 mg/kg), and showed only partial displacement with spiperone (200 (xg/kg, i.v. administration 90 min after FESP injection). From PET experiments with FESP in humans, it is possible to visualize accumulation of radioactivity in striatum in a manner similar to that observed in monkeys and, ex vivo, in rodents (adult male Sprague-Dawley rats). Biochemical analyses in rat brain revealed that the activity (—90%) in striatum was unmodified FESP up to 4 h after injection. On the other hand, FESP was metabolized peripherally (rat > monkey > human), with only 11% of plasma radioactivity remaining as intact FESP in rodents and 54% in humans after 2 h. Based on these interspecies scaling pharmacokinetic data, it is unequivocal that FESP peripheral metabolites do not significantly contribute to the accumulated radioactivity in striatal tissue. Therefore, it is concluded that FESP is suitable for the quantitative estimation of dopamine D2-receptor sites using PET.Keywords: 3-(2'-[18F]fluo-roethyl)spiperone (FESP); Dopamine D2-receptors; Biochemical analysis; Kinetic characterization; Positron emission tomography
Gene therapists cannot track location, magnitude or duration of gene expression following somatic gene delivery. Consequently, they cannot correlate somatic gene transfer and phenotypic responses, since variations in gene delivery, gene expression levels and duration of gene expression cannot be monitored following gene administration. We developed two reporter genes whose expression can be non-invasively, quantitatively and repetitively monitored, in living individuals, by reporter protein-dependent sequestration of positron-labeled reporter probes, using positron emission tomography. We describe gene delivery systems from which therapeutic and PET reporter genes are co-expressed and demonstrated, using microPET in murine models, their ability to quantitate somatic gene transfer.
Various radiotracers based on uracil nucleosides (e.g. [124I]2'-fluoro-2'-deoxy-5-iodo-1-#-D-arabinofuranosyluracil, [124I]FIAU) and acycloguanosine derivatives (e.g. [18F]9-[(3-fluoro-1-hydroxy-2-propoxy)methyl]guanine, [18F]FHPG) have been proposed for the non-invasive imaging of herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene expression. However, these radiotracers have been evaluated in different in vitro and in vivo models, precluding a direct comparison. Therefore, we directly compared [18F]FHPG and radioiodinated FIAU to assess their potential for PET imaging of transgene expression. The uptake of [125I]FIAU, [18F]FHPG and [3H]acyclovir was determined in vitro using four different HSV1-tk expressing cell lines and their respective negative controls. The in vitro tracer uptake was generally low in non-transduced parental cell lines. In HSV1-tk expressing cells, [3H]acyclovir showed approximately a twofold higher tracer accumulation, the [18F]FHPG uptake increased by about sixfold and the [125I]FIAU accumulation increased by about 28-fold after 120-min incubation of T1115 human glioblastoma cells. Similar results were found in the other cell lines. In addition, biodistribution and positron emission tomography (PET) studies with [18F]FHPG and [124/125I]FIAU were carried out in tumour-bearing BALB/c mice. Significantly higher specific accumulation of radioactivity was found for [125I]FIAU compared with [18F]FHPG. The ratio of specific tracer accumulation between [125I]FIAU and [18F]FHPG increased from 21 (30 min p.i.) to 119 (4 h p.i.). PET imaging, using [124I]FIAU, clearly visualised and delineated HSV1-tk expressing tumours, whereas only a negligible uptake of [18F]FHPG was observed. This study demonstrated that in vitro and in vivo, the radioiodinated uracil nucleoside FIAU has a significantly higher specific accumulation than the acycloguanosine derivative [18F]FHPG. This suggests that [124I]FIAU should be the preferred reporter probe for PET imaging of HSV1-tk gene expression. Thus, further attempts to develop suitable PET tracers for the assessment of HSV1-tk gene expression should also focus on 18F-labelled uracil derivatives.
Four dopamine D2 receptor mutants were constructed, in each of which an alanine residue was substituted for one of four conserved serine residues, i.e., Ser-193, Ser-194, Ser-197, and Ser-391. Wild-type and mutant receptors were expressed transiently in COS-7 cells and stably in C6 glioma cells for analysis of ligand-receptor interactions. In radioligand binding assays, the affinity of D2 receptors for dopamine was decreased 50-fold by substitution of alanine for Ser-193, implicating this residue in the binding of dopamine. Each mutant had smaller decreases in affinity for one or more of the ligands tested, with no apparent relationship between the class of ligand and the pattern of mutation-induced changes in affinity, except that the potency of agonists was decreased by substitution for Ser-193. The potency of dopamine for inhibition of adenylyl cyclase was reduced substantially by substitution of alanine for Ser-193 or Ser-197. Mutation of Ser-194 led to a complete loss of efficacy for dopamine and p-tyramine, which would be consistent with an interaction between Ser-194 and the p-hydroxyl substituent of dopamine that is necessary for activation of the receptors to occur. Because mutation of the corresponding residues of beta 2-adrenergic receptors has very different consequences, we conclude that although the position of these serine residues is highly conserved among catecholamine receptors, and the residues as a group are important in ligand binding and activation of receptors by agonists, the function of each of the residues considered separately varies among catecholamine receptors.
An aspartate residue corresponding to aspartate-80 of dopamine D2 receptors is strictly conserved among receptors that couple to guanine nucleotide-binding proteins. Mutation of this residue alters the function of several classes of neurotransmitter receptors. Dopamine D2 receptors couple to the guanine nucleotide-binding protein Gi to inhibit adenylyl cyclase (ATP-pyrophosphate-lyase, cyclizing; EC 4.6.1.1). Like other Gi-coupled receptors, the binding of agonists and some antagonists to D2 receptors is sensitive to pH and sodium. In the present report, we demonstrate that substitution of an alanine or glutamate residue for aspartate-80 severely impairs inhibition of adenylyl cyclase by D2 receptors and also abolishes or decreases the regulation of the affinity of D2 receptors for agonists and substituted benzamide antagonists by sodium and pH. Our data support the hypothesis that the conformation of D2 receptors is maintained by interactions of monovalent cations with aspartate-80. The regulation of D2 receptors by this interaction has important consequences for the affinity of D2 receptors for ligands and for signal transduction by D2 receptors.
The synthesis of 3-(2'-[18F]fluoroethyl)spiperone (1c), a radiotracer useful for imaging the brain dopamine receptor system in vivo using positron emission tomography, is described. Precursors of 1c, the functional 3-N-alkyl derivatives of spiperone (4), were prepared by the alkylation of the amide group in spiperone (2a) by 1,2-disubstituted ethanes under phase transfer conditions. A comprehensive evaluation of the reaction of the derivatives 4a-h with no-carrier-added K18F/Kryptofix clearly indicated that the ketalized derivatives 4e-h were the choice of the precursors for 1c. The i.r., MS and NMR spectral data suggested that under phase transfer reaction conditions, the amide nitrogen was preferentially alkylated. To provide a firm basis for comparison with related analogues, an x-ray analysis was performed on a single crystal of 3-(2'-fluoroethyl)spiperone (1d). The tomographic behavior of 1c in human brain tissue was measured for more than 7 h and was consistent with the labeling of dopamine D-2 receptors.
A new dopamine receptor antagonist, Raclopride (S-(-)-3,5-dichloro-N-[(1-ethyl-2-pyrrolidinyl)]methyl-2-hydroxy- 6-methoxybenzamide, FLA 870) (1), has been labelled using [11C]ethyl iodide for alkylation of the nitrogen of the pyrrolidine ring in the corresponding secondary amine (5). The synthesis of 5 and an efficient method for the preparation of [11C]ethyl iodide are described. The 11C-labelled FLA 870 (1) was purified by HPLC and then used in positron emission tomography to visualize the dopamine receptor-rich areas of the monkey brain. The images obtained show selective accumulation of FLA 870 in striatum and a 10-fold separation between the binding to caudate vs cerebellum.
A no-carrier-added synthesis of 9-[(3-[18F]-fluoro-1-hydroxy-2-propoxy)methyl]-guanine ([18F]-FHPG) is reported. The 9-[(1,3-dihydroxy-2-propoxy)methyl)guanine (DHPG) was converted to 9-[N2,O-bis(methoxytrityl)-3-(tosyl)-2-propoxy-methyl]guanine by treatment with methoxytrityl chloride followed by tosylation. The tosylate was reacted with [18F]-KF in the presence of kryptofix 2.2.2. to produce the 3-fluoro-N2-O-bis-(methoxytrityl) derivative. Removal of the methoxytrityl protecting groups by acid hydrolysis produced [18F]-FHPG. The labeled product was purified by HPLC on a reverse-phase C18 column, and eluted in 9 min with a mobile phase of 5% acetonitrile in water. The radiochemical yield was 7-17%, with an average of 10% in 10 runs (corrected for decay to EOB). The radiochemical purity was > 99%, and specific activities with an average of 526 mCi/mumol were obtained. The synthesis time was 70-80 min, including HPLC purification and determination of radiochemical purity and specific activity.
Synthesis and preliminary biological evaluation of 9-(4-[18F]-fluoro-3-hydroxymethylbutyl)-guanine ([18F]FHBG) is reported. 9-(4-Hydroxy-3-hydroxymethylbutyl)-guanine (penciclovir) 4 was converted to 9-[N2, O-bis-(methoxytrityl)-3-(tosylmethybutyl)]guanine 7 by treatment with methoxytrityl chloride followed by tosylation. The tosylate 7 was reacted with either tetrabutylammonium fluoride or KF in the presence of kryptofix 2.2.2. to produce the 4-fluoro-N2-O-bis-(methoxytrityl) derivative 8. Removal of the methoxytrityl groups by acidic hydrolysis produced FHBG 5. Radiolabeled product [18F]FHBG was prepared by fluorination of the tosylate 7 with [18F]KF and kryptofix 2.2.2. The labeled product was isolated by HPLC purification on a reverse-phase C18 column, and eluted at 12 min with 15% acetonitrile in water at a flow rate of 2.25 mL/min. Radiochemical yield was 8.0-22.3% with an average of 12% in 7 runs (corrected for decay). Synthesis time was 90 to 100 min including HPLC purification with radiochemical purity >99%, and average specific activity of 320 mCi/micromol. In vitro studies of the compound in HT-29 colon cancer cells revealed 18.2-fold higher uptake into transduced cells compared to control in 3 h. The agent may be useful for imaging viral infection or transfected cells in gene therapy.
Positron emission tomography (PET) is now regularly used in the diagnosis and staging of cancer. These uses and its ability to monitor treatment response would be aided by the development of imaging agents that can be used to measure tissue and tumor proliferation. We have developed and tested [F-18]FLT (3'-deoxy-3'-fluorothymidine); it is resistant to degradation, is retained in proliferating tissues by the action of thymidine kinase 1 (TK), and produces high-contrast images of normal marrow and tumors in canine and human subjects.
Preparation of 9-[(3-18F-fluoro-1-hydroxy-2-propoxy)methyl]-guanine ([18F]-FHPG) for clinical use, and its evaluation as a positron emission tomography (PET) imaging agent for gene incorporation and expression in tumors are reported. In vitro studies in human colon cancer cells, HT-29, transduced with the retroviral vector G1Tk1SvNa and nontransduced (wild type) showed 4, 8, 12, and 15 times higher uptake of the probe in 1, 3, 5, and 7 h, respectively, in transduced cells compared with the controls. In vivo studies in tumor-bearing nude mice demonstrated that the tumor uptake of the radiotracer is three and six-fold higher in 2 and 5 h, respectively, in transduced cells compared with the control cells. These results suggest that [18F]-FHPG is a potential in vivo PET imaging agent for monitoring gene incorporation and expression in gene therapy of cancer.
A new dedicated PET scanner, microPET, was designed and developed at the University of California, Los Angeles, for imaging small laboratory animals. The goal was to provide a compact system with superior spatial resolution at a fraction of the cost of a clinical PET scanner.
The system uses fiberoptic readout of individually cut lutetium oxyorthosilicate (LSO) crystals to achieve high spatial resolution. Each microPET detector consists of an 8 x 8 array of 2 x 2 x 10-mm LSO scintillation crystals that are coupled to a 64-channel photomultiplier tube by optical fibers. The tomograph consists of 30 detectors in a continuous ring with a 17.2-cm diameter and fields of view (FOVs) of 11.25 cm in the transaxial direction and 1.8 cm in the axial direction. The system has eight crystal rings and no interplane septa. It operates exclusively in the three-dimensional mode and has an electronically controlled bed that is capable of wobbling with a radius of 300 microm. We describe the performance of the tomograph in terms of its spatial, energy and timing resolution, as well as its sensitivity and counting-rate performance. We also illustrate its overall imaging performance with phantom and animal studies that demonstrate the potential applications of this device to biomedical research.
Images reconstructed with three-dimensional filtered backprojection show a spatial resolution of 1.8 mm at the center of the FOV (CFOV), which remains <2.5 mm for the central 5 cm of the transaxial FOV. The resulting volumetric resolution of the system is <8 microL. The absolute system sensitivity measured with a 0.74 MBq (20 microCi) 68Ge point source at the CFOV is 5.62 Hz/kBq. The maximum noise equivalent counting rate obtained with a 6.4-cm diameter cylinder spanning the central 56% of the FOV is 10 kcps, whereas the scatter fraction is 37% at the CFOV for an energy window of 250-650 keV and the same diameter cylinder.
This is the first PET scanner to use the new scintillator LSO and uses a novel detector design to achieve high volumetric spatial resolution. The combination of imaging characteristics of this prototype system (resolution, sensitivity, counting-rate performance and scatter fraction) opens up new possibilities in the study of animal models with PET.
Next Section
The genes of all organisms have been shaped by selective pressures. The relationship between gene sequence and fitness has tremendous implications for understanding both evolutionary processes and functional constraints on the encoded proteins. Here, we have exploited deep sequencing technology to experimentally determine the fitness of all possible individual point mutants under controlled conditions for a nine-amino acid region of Hsp90. Over the past five decades, limited glimpses into the relationship between gene sequence and function have sparked a long debate regarding the distribution, relative proportion, and evolutionary significance of deleterious, neutral, and advantageous mutations. Our systematic experimental measurement of fitness effects of Hsp90 mutants in yeast, evaluated in the light of existing population genetic theory, are remarkably consistent with a nearly neutral model of molecular evolution.
At present, most methods to image gene expression in rodents, nonhuman primates and humans are limited to the use of fixed tissue obtained from biopsy or after death. Fixed tissue can be assayed for mRNA levels or a protein product using well-established techniques. Reporter genes sensitive to activation status of endogenous genes have also been used by molecular biologists. Endogenous gene expression is monitored through creation of a chimeric fusion gene in which the promoter of an endogenous gene is coupled to the reporter gene. If the endogenous gene promoter is 'turned off', neither the endogenous gene nor the reporter gene is transcribed. Similarly, if the promoter for the endogenous gene is activated, then the reporter gene is transcribed. Although introduction of a chimeric fusion gene into the target organism is required, this allows for monitoring of any endogenous gene of interest where the promoter is sequenced. However, conventional reporter gene methods are generally limited by their inability to determine the location(s), magnitude and time variation of gene expression in living animals.
We have synthesized and evaluated 8-[18F]fluoropenciclovir (FPCV) and compared it with 8-[18F]fluoroganciclovir (FGCV) for monitoring the expression of herpes simplex virus type 1 thymidine kinase (HSV1 -tk) reporter gene in cell culture and in vivo.
C6 rat glioma cells stably transfected with HSV1-tk (C6-stb-tk+) and control C6 cells were evaluated for their ability to accumulate FGCV versus FPCV. For in vivo studies, 15 mice were injected by tail vein with increasing levels of an adenoviral vector carrying HSV1-tk. Forty-eight hours later the mice were injected with FPCV and killed 3 h later. The percentage injected dose per gram (%ID/g) liver was then determined. Two additional mice were studied by microPET and autoradiography using FPCV to image adenoviral-mediated hepatic HSV1-tk reporter gene expression. A tumor-bearing mouse (C6 control and C6-stb-tk+) was imaged with FDG, FGCV, and FPCV. Two mice carrying tumors expressing two different reporter genes, HSV1-tk and dopamine type 2 receptor (D2R), were also imaged by microPET using FPCV (day 1) and 3-(2'-[18F]fluoroethyl)spiperone (FESP) (day 2).
FPCV shows a significantly greater accumulation in C6-stb-tk+ cells than does FGCV (P < 0.05). Over identical ranges of adenoviral administration, mouse liver shows a higher %ID/g liver for FPCV (0%-9%) compared with our previously reported results with FGCV (0%-3%). In C6 control and C6-stb-tk+ tumor-bearing mice, FPCV has a greater accumulation than does FGCV for equal levels of HSV1-tk gene expression. In mice carrying tumors expressing either HSV1-tk or D2R reporter genes, there is a corresponding retention of FPCV and FESP, respectively.
These results indicate that FPCV is a better reporter probe than is FGCV for imaging lower levels of HSV1 -tk gene expression in vivo. The results also reveal the ability to monitor the expression of two distinct reporter genes in the same animal using reporter probes specific for each gene.
Radiolabeling monoclonal antibodies (mAbs) allows the evaluation of biodistribution of constructs in vivo through gamma camera imaging and also permits quantitation of mAb uptake in tumors through biopsy-based counting techniques. The quantitation of radiolabeled mAb uptake in cancer patients is complicated by the attenuation of gamma emissions of routinely used isotopes (e.g., 131I and 111In) and the spatial resolution and sensitivity of gamma cameras.
We used the positron-emitting isotope 124I (half-life [T1/2] = 4.2 d) to label the recombinant humanized anti-colorectal cancer A33 antibody (huA33) and evaluated its biodistribution properties and PET imaging characteristics in BALB/c nude mice bearing SW1222 colorectal xenografts and control colon tumors.
The immunoreactivity of radioconjugate was 78% as determined using the cell-binding Lindmo assay. The apparent association constant was found to be 2.2 x 10(9) M(-1), and the number of antibody binding sites per cell was 371,000. The radioconjugate was found to be stable in serum obtained from mice at various times after injection. Assuming a two-compartment model with a four-parameter fit of mean blood levels, the T1/2alpha was 1.5 h and the T1/2beta was 38.2 h. Excellent tumor uptake was obtained, with maximal uptake reaching 50.0 +/- 7.0 percentage injected dose per gram of tumor by 4 d after injection. Specificity of localization was shown by lack of uptake in control tumor. PET imaging detected antigen-positive tumor by 4 h after injection, and high-resolution images were obtained by 24 h after injection.
In clinical trials using PET, huA33 labeled with 124I has potential for imaging and staging colon tumors and quantifying antibody uptake in colon tumors in vivo.
Although the biological actions of the cell membrane and serum lipid lysophosphatidylcholine (LPC) in atherosclerosis and
systemic autoimmune disease are well recognized, LPC has not been linked to a specific cell-surface receptor. We show that
LPC is a high-affinity ligand for G2A, a lymphocyte-expressed G protein–coupled receptor whose genetic ablation results in
the development of autoimmunity. Activation of G2A by LPC increased intracellular calcium concentration, induced receptor
internalization, activated ERK mitogen-activated protein kinase, and modified migratory responses of Jurkat T lymphocytes.
This finding implicates a role for LPC-G2A interaction in the etiology of inflammatory autoimmune disease and atherosclerosis.
The microPET Primate 4-ring system (P4) is an animal PET tomograph with a 7.8 cm axial extent, a 19 cm diameter transaxial field of view (FOV) and a 22 cm animal port. The system is composed of 168 detector modules, each with an 8 x 8 array of 2.2 x 2.2 x 10 mm3 lutetium oxyorthosilicate crystals, arranged as 32 crystal rings 26 cm in diameter. The detector crystals are coupled to a Hamamatsu R5900-C8 PS-PMT via a 10 cm long optical fibre bundle. The detectors have a timing resolution of 3.2 ns, an average energy resolution of 26%, and an average intrinsic spatial resolution of 1.75 mm. The system operates in 3D mode without inter-plane septa, acquiring data in list mode. The reconstructed image spatial resolution ranges from 1.8 mm at the centre to 3 mm at 4 cm radial offset. The tomograph has a peak system sensitivity of 2.25% at the centre of the FOV with a 250-750 keV energy window. The noise equivalent count rate peaks at 100-290 kcps for representative object sizes. Images from two phantoms and three different types of laboratory animal demonstrate the advantage of the P4 system over the original prototype microPET. including its threefold improvement in sensitivity and a large axial FOV sufficient to image an entire mouse in a single bed position.
Since cloning and characterization of the sodium iodide symporter (NIS) gene, several investigators explored the possibility of a novel cytoreductive gene therapy strategy based on NIS gene transfer into non-thyroidal tumor cells followed by radioiodine therapy. NIS gene transfer has been shown to be capable of inducing radioiodine accumulation in vitro and in vivo in several non-thyroidal cancer cell lines. Following PSA promoter-mediated NIS gene delivery we were able to demonstrate prostate-specific iodide accumulation in prostate cancer cells that was high enough to elicit a therapeutic response of 131-I in vitro and in vivo. This study clearly demonstrates the potential of NIS as a novel therapeutic gene for non-thyroidal cancers, in particular prostate cancer.
We are currently developing a small animal positron emission tomography (PET) scanner with a design goal of 1 microlitre (1 mm3) image resolution. The detectors consist of a 12 x 12 array of 1 x 1 x 10 mm lutetium oxyorthosilicate (LSO) scintillator crystals coupled to a 64-channel photomultiplier tube (PMT) via 5 cm long optical fibre bundles. The optical fibre connection allows a high detector packing fraction despite the dead space surrounding the active region of the PMT. Optical fibre bundles made from different types of glass were tested for light transmission, and also their effects on crystal identification and energy resolution, and compared to direct coupling of the LSO arrays to the PMTs. We also investigated the effects of extramural absorber (EMA) in the fibre bundles. Based on these results, fibre bundles manufactured from F2 glass were selected. We built three pairs of prototype detectors (directly coupled LSO array, fibre bundle without EMA and fibre bundle with EMA) and measured flood histograms, energy resolution, intrinsic spatial resolution and timing resolution. The results demonstrated an intrinsic spatial resolution (FWHM) of 1.12 mm (directly coupled), 1.23 mm (fibre bundle without EMA coupling) and 1.27 mm (fibre bundle with EMA coupling) using an approximately 500 microm diameter Na-22 point source. Using a 330 microm outer diameter steel needle line source filled with F-18, spatial resolution for the detector with the EMA optical fibre bundle improved to 1.05 mm. The respective timing and energy FWHM values were 1.96 ns, 21% (directly coupled), 2.20 ns, 23% (fibre bundle without EMA) and 2.99 ns, 30% (fibre bundle with EMA). The peak-to-valley ratio in the flood histograms was better with EMA (5:1) compared to the optical fibre bundle without EMA (2.5:1), due to the decreased optical cross-talk. In comparison to the detectors used in our current generation microPET scanner, these detectors substantially improve on the spatial resolution, preserve the timing resolution and provide adequate energy resolution for a modern high-resolution animal PET tomograph.
Background:
Novel approaches to increasing the therapeutic efficacy of targeted radiotherapy of cancer are required. One strategy to achieve this goal is to induce high-level expression of a receptor on the surface of tumor cells that can be targeted with a radiolabeled peptide. The objectives of this study were to 1) induce somatostatin receptor (SSTr2) expression in tumor cells using an adenovirus encoding the SSTr2 gene (AdSSTr2), 2) demonstrate tumor localization of [(111)In]-DTPA-D-Phe(1)-octreotide in AdSSTr2-injected tumors, and 3) show therapeutic efficacy with [(90)Y]-DOTA-D-Phe(1)-Tyr(3)-octreotide ([(90)Y]-SMT 487).
Methods:
SSTr2 expression was validated in vitro by the binding and subsequent internalization of [(111)In]-DTPA-D-Phe(1)-octreotide (21.3% per mg of total protein) in A-427 cells infected with AdSSTr2. In vivo imaging confirmed 5- to 10-fold greater uptake 5.5 hours after intravenous administration of [(111)In]-DTPA-D-Phe(1)-octreotide in AdSSTr2-injected tumors relative to control tumors. For therapy studies, mice bearing established subcutaneous A-427 tumors were given two intratumoral injections of AdSSTr2 1 week apart, followed by an intravenous injection of 400 microCi or 500 microCi [(90)Y]-SMT 487 at 2 and 4 days after each adenoviral administration. Control animals either were not treated or were administered 500 microCi [(90)Y]-SMT 487 with no AdSSTr2 injection.
Results:
These studies showed that untreated animals and animals treated with no virus and 500 microCi [(90)Y]-SMT 487 had median tumor quadrupling times of 16 and 25 days, respectively. Mice administered AdSSTr2 and either 400 microCi or 500 microCi of [(90)Y]-SMT 487 demonstrated significantly longer median tumor quadrupling times of 40 and 44 days, respectively (P < 0.02).
Conclusions:
These studies are the first to demonstrate in vivo therapeutic efficacy using a radiolabeled peptide targeted to a receptor expressed on the surface of tumor cells following gene transfer. Future studies will focus on the optimization of this approach.
The human somatostatin receptor subtype 2A (hSSTr2) is under evaluation as a reporter gene for molecular imaging applications. Two approved somatostatin analogues are already available for imaging expression of the reporter gene following delivery with adenoviral (Ad) vectors or other genetic targeting strategies. In animal models, Ad-mediated expression of hSSTr2 in subcutaneous and intraperitoneal tumors was detected by non-invasive gamma camera imaging. This review discusses the rationale and strategy for using the hSSTr2 reporter gene as a platform for imaging applications.
To advance our understanding of biological processes as they occur in living animals, imaging strategies have been developed and refined that reveal cellular and molecular features of biology and disease in real time. One rapid and accessible technology for in vivo analysis employs internal biological sources of light emitted from luminescent enzymes, luciferases, to label genes and cells. Combining this reporter system with the new generation of charge coupled device (CCD) cameras that detect the light transmitted through the animal's tissues has opened the door to sensitive in vivo measurements of mammalian gene expression in living animals. Here, we review the development and application of this imaging strategy, in vivo bioluminescence imaging (BLI), together with in vivo fluorescence imaging methods, which has enabled the real-time study of immune cell trafficking, of various genetic regulatory elements in transgenic mice, and of in vivo gene transfer. BLI has been combined with fluorescence methods that together offer access to in vivo measurements that were not previously available. Such studies will greatly facilitate the functional analysis of a wide range of genes for their roles in health and disease.
The imaging of specific molecular targets that are associated with cancer should allow earlier diagnosis and better management of oncology patients. Positron emission tomography (PET) is a highly sensitive non-invasive technology that is ideally suited for pre-clinical and clinical imaging of cancer biology, in contrast to anatomical approaches. By using radiolabelled tracers, which are injected in non-pharmacological doses, three-dimensional images can be reconstructed by a computer to show the concentration and location(s) of the tracer of interest. PET should become increasingly important in cancer imaging in the next decade.
Radiosynthesis of ML03 (N-[4-[(4,5-dichloro-2-fluorophenyl)amino]quinazolin-6-yl]acrylamide), an irreversible EGFr-TK inhibitor, was developed. Its in vitro and in vivo properties, its potential as PET biomarker in cancer and the feasibility of this type of compounds to be used as anticancer drug agents were evaluated. The compound was labeled with carbon-11 at the acryloyl amide group, via automated method with high yield, chemical and radiochemical purities. ELISA carried out with A431 lysate showed high potency of ML03 with an apparent IC(50) of 0.037 nM. The irreversible binding nature of ML03 was studied and 97.5% EGFr-TK autophosphorylation inhibition was observed in intact A431 cells 8 hr post incubation with the inhibitor. Specific binding (67%) of [(11)C]ML03 was obtained in cells. An A431 tumor-bearing rat model was developed and the validity of the model was tested. In biodistribution studies carried out with tumor-bearing rats, moderate uptake was observed in tumor and high uptake in liver, kidney and intestine. In metabolic studies, fast degradation of [(11)C]ML03 was observed in liver and blood indicating a short half-life of the compound in the body. PET scan with tumor-bearing rats confirmed the results obtained in the ex vivo biodistribution studies. Although in vitro experiments may indicate efficacy of ML03, non-specific binding, ligand delivery and degradation in vivo make ML03 ineffective as PET bioprobe. Derivatives of ML03 with lower metabolic clearance rate and higher bioavailability should be synthesized and their potential as anticancer drugs and PET bioprobes evaluated.
Thyroid iodide uptake is basic to the clinical applications of radioiodine. Iodide uptake occurs across the membrane of the thyroid follicular cells through an active transporter process mediated by the sodium iodide symporter (NIS). The recent cloning of the NIS gene enabled the better characterization of the molecular mechanisms underlying iodide transport, thus opening the way to the clarification and expansion of its role in nuclear medicine. In papillary and follicular carcinoma, NIS immunostaining was positive in only a few tumor cells, and no NIS protein expression was detected in anaplastic carcinomas. Decreased NIS expression levels account for the reduced iodide uptake in thyroid carcinomas. Thus, by targeting NIS expression in cancer cells, we could enable these cells to concentrate iodide from plasma and in so doing offer the possibility of radioiodine therapy. Several investigators have shown that gene transfer of NIS into a variety of cell types confers increased radioiodine uptake by up to several hundredfold that of controls in nonthyroid cancers as well as in thyroid cancer. In addition, my group proposes that NIS may serve as an alternative imaging reporter gene in addition to the HSVtk and dopaminergic receptor genes. The NIS has the potential to expand the role of nuclear medicine in the future, just as it has served as the base for the development of nuclear medicine in the past.