Showing papers in "Molecular Imaging in 2009"

Toxicity of organic fluorophores used in molecular imaging: literature review.

Abstract: Fluorophores are potentially useful for in vivo cancer diagnosis. Using relatively inexpensive and portable equipment, optical imaging with fluorophores permits real-time detection of cancer. However, fluorophores can be toxic and must be investigated before they can be administered safely to patients. A review of published literature on the toxicity of 19 widely used fluorophores was conducted by searching 26 comprehensive biomedical and chemical literature databases and analyzing the retrieved material. These fluorophores included Alexa Fluor 488 and 514, BODIPY FL, BODIPY R6G, Cy 5.5, Cy 7, cypate, fluorescein, indocyanine green, Oregon green, 8-phenyl BODIPY, rhodamine 110, rhodamine 6G, rhodamine X, rhodol, TAMRA, Texas red, and Tokyo green. Information regarding cytotoxicity, tissue toxicity, in vivo toxicity, and mutagenicity was included. Considerable toxicity-related information was available for the Food and Drug Administration (FDA)-approved compounds indocyanine green and fluorescein, but published information on many of the non-FDA-approved fluorophores was limited. The information located was encouraging because the amounts of fluorophore used in molecular imaging probes are typically much lower than the toxic doses described in the literature. Ultimately, the most effective and appropriate probes for use in patients will be determined by their fluorescent characteristics and the safety of the conjugates.

Dual-Modality Probes for In Vivo Molecular Imaging

Abstract: Various imaging modalities have been exploited and are successfully employed to investigate the anatomic or functional dissemination of tissues in the body. However, no single imaging modality allo...

Comparative analysis of nanoparticle-antibody conjugations: carbodiimide versus click chemistry.

Abstract: The ability to modify the physical, chemical, and biologic properties of nanoparticles has led to their use as multifunctional platforms for drug delivery and diagnostic imaging applications. Typic...

Real-time monitoring of nuclear factor kappaB activity in cultured cells and in animal models.

Abstract: Nuclear factor kappaB (NF-kappaB) is a transcription factor that plays a major role in many human disorders, including immune diseases and cancer. We designed a reporter system based on NF-kappaB responsive promoter elements driving expression of the secreted Gaussia princeps luciferase (Gluc). We show that this bioluminescent reporter is a highly sensitive tool for noninvasive monitoring of the kinetics of NF-kappaB activation and inhibition over time, both in conditioned medium of cultured cells and in the blood and urine of animals. NF-kappaB activation was successfully monitored in real time in endothelial cells in response to tumor angiogenic signaling, as well as in monocytes in response to inflammation. Further, we demonstrated dual blood monitoring of both NF-kappaB activation during tumor development as correlated to tumor formation using the NF-kappaB Gluc reporter, as well as the secreted alkaline phosphatase reporter. This NF-kappaB reporter system provides a powerful tool for monitoring NF-kappaB activity in real time in vitro and in vivo.

Optical imaging of cellular immunotherapy against prostate cancer.

Abstract: The purpose of this study was to track fluorophore-labeled, tumor-targeted natural killer (NK) cells to human prostate cancer xenografts with optical imaging (OI). NK-92-scFv(MOC31)-zeta cells targeted to the epithelial cell adhesion molecule (EpCAM) antigen on prostate cancer cells and nontargeted NK-92 parental cells were labeled with the near-infrared dye DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine). The fluorescence, viability, and cytotoxicity of the labeled cells were evaluated. Subsequently, 12 athymic rats with prostate cancer xenografts underwent OI scans before and up to 24 hours postinjection of DiD-labeled parental NK-92 cells or NK-92-scFv(MOC31)-zeta cells. The tumor fluorescence intensity was measured and compared between pre- and postinjection scans and between both groups using t-tests. OI data were confirmed with fluorescence microscopy. In vitro studies demonstrated a significant increase in the fluorescence of labeled cells compared with unlabeled controls, which persisted over a period of 24 hours without any significant change in the viability. In vivo studies demonstrated a significant increase in tumor fluorescence at 24 hours postinjection of tumor-targeted NK-92-scFv(MOC31)-zeta cells but not parental NK cells. Ex vivo OI scans and fluorescence microscopy confirmed a specific accumulation of NK-92-scFv(MOC31)-zeta cells but not parental NK cells in the tumors. Tumor-targeted NK-92-scFv(MOC31)-zeta cells could be tracked to prostate cancer xenografts with OI.

Multimodal Imaging of Integrin Receptor-Positive Tumors by Bioluminescence, Fluorescence, Gamma Scintigraphy, and Single-Photon Emission Computed Tomography Using a Cyclic RGD Peptide Labeled with a Near-Infrared Fluorescent Dye and a Radionuclide

Abstract: Integrins, particularly the α v β 3 heterodimers, play important roles in tumor-induced angiogenesis and invasiveness. To image the expression pattern of the α v β 3 integrin in tumors through a multimodality imaging paradigm, we prepared a cyclic RGDyK peptide analogue (LS308) bearing a tetraazamacrocycle 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) and a lipophilic near-infrared (NIR) fluorescent dye cypate. The α v β 3 integrin binding affinity and the internalization properties of LS308 mediated by the α v β 3 integrin in 4t1luc cells were investigated by receptor binding assay and fluorescence microscopy, respectively. The in vivo distribution of 111 In-labeled LS308 in a 4t1luc tumor‐bearing mouse model was studied by fluorescence, bioluminescence, planar gamma, and single-photon emission computed tomography (SPECT). The results show that LS308 has high affinity for α v β 3 integrin and internalized preferentially via the α v β 3 integrin-mediated endocytosis in 4t1luc cells. We also found that LS308 selectively accumulated in α v β 3 -positve tumors in a receptor-specific manner and was visualized by the four imaging methods. Whereas the endogenous bioluminescence imaging identified the ensemble of the tumor tissue, the fluorescence and SPECT methods with the exogenous contrast agent LS308 reported the local expression of α v β 3 integrin. Thus, the multimodal imaging approach could provide important complementary diagnostic information for monitoring the efficacy of new antiangiogenic drugs.

Magnetic resonance imaging of cells overexpressing MagA, an endogenous contrast agent for live cell imaging.

Abstract: Molecular imaging with magnetic resonance imaging (MRI) may benefit from the ferrimagnetic properties of magnetosomes, membrane-enclosed iron biominerals whose formation in magnetotactic bacteria is encoded by multiple genes. One such gene is MagA, a putative iron transporter. We have examined expression of MagA in mouse neuroblastoma N2A cells and characterized their response to iron loading and cellular imaging by MRI. MagA expression augmented both Prussian blue staining and the elemental iron content of N2A cells, without altering cell proliferation, in cultures grown in the presence of iron supplements. Despite evidence for iron incorporation in both MagA and a variant, MagAE137V, only MagA expression produced intracellular contrast detectable by MRI at 11 Tesla. We used this stable expression system to model a new sequence for cellular imaging with MRI, using the difference between gradient and spin echo images to distinguish cells from artifacts in the field of view. Our results show that MagA activity in mammalian cells responds to iron supplementation and functions as a contrast agent that can be deactivated by a single point mutation. We conclude that MagA is a candidate MRI reporter gene that can exploit more fully the superior resolution of MRI in noninvasive medical imaging.

High-power, computer-controlled, light-emitting diode-based light sources for fluorescence imaging and image-guided surgery.

Abstract: Optical imaging requires appropriate light sources. For image-guided surgery, in particular fluorescence-guided surgery, a high fluence rate, a long working distance, computer control, and precise control of wavelength are required. In this article, we describe the development of light-emitting diode (LED)-based light sources that meet these criteria. These light sources are enabled by a compact LED module that includes an integrated linear driver, heat dissipation technology, and real-time temperature monitoring. Measuring only 27 mm wide by 29 mm high and weighing only 14.7 g, each module provides up to 6,500 lx of white (400-650 nm) light and up to 157 mW of filtered fluorescence excitation light while maintaining an operating temperature < or = 50 degrees C. We also describe software that can be used to design multimodule light housings and an embedded processor that permits computer control and temperature monitoring. With these tools, we constructed a 76-module, sterilizable, three-wavelength surgical light source capable of providing up to 40,000 lx of white light, 4.0 mW/cm2 of 670 nm near-infrared (NIR) fluorescence excitation light, and 14.0 mW/cm2 of 760 nm NIR fluorescence excitation light over a 15 cm diameter field of view. Using this light source, we demonstrated NIR fluorescence-guided surgery in a large-animal model.

Evaluation of [64Cu]Cu-DOTA and [64Cu]Cu-CB-TE2A chelates for targeted positron emission tomography with an alphavbeta6-specific peptide.

Abstract: Significant upregulation of the integrin alpha(v)beta(6) has been described as a prognostic indicator in several cancers, making it an attractive target for tumor imaging This study compares variants of a PEGylated alpha(v)beta(6)-targeting peptide, bearing either an [(18)F]fluorobenzoyl prosthetic group ([(18)F]FBA-PEG-A20FMDV2) or different [(64)Cu]copper chelators (DOTA-PEG-A20FMDV2, CB-TE2A-PEG-A20FMDV2) The compounds were evaluated in vitro by enzyme-linked immunosorbent assay (against the integrin alpha(v)beta(6) and the closely related integrin alpha(v)beta(3)) and by cell labeling (alpha(v)beta(6)-positive DX3purobeta6/alpha(v)beta(6)-negative DX3puro) and in vivo using micro-positron emission tomography in a mouse model bearing paired DX3purobeta6/Dx3puro xenografts In vitro, all three compounds showed excellent alpha(v)beta(6)-specific binding (50% inhibitory concentration [IC(50)](alpha(v)beta(6)) = 3 to 6 nmol/L; IC(50)(alpha(v)beta(3)) > 10 micromol/L) In vivo, they displayed comparable, preferential uptake for the alpha(v)beta(6)-expressing xenograft over the alpha(v)beta(6)-negative control (> 4:1 ratio at 4 hours postinjection) Whereas [(64)Cu]Cu-DOTA-PEG-A20FMDV2 resulted in increased levels of radioactivity in the liver, [(64)Cu]Cu-CB-TE2A-PEG-A20FMDV2 did not Significantly, both (64)Cu-labeled tracers showed unexpectedly high and persistent levels of radioactivity in the kidneys (> 40% injected dose/g at 4 and 12 hours postinjection) The findings underscore the potential influence of the prosthetic group on targeted in vivo imaging of clinically relevant markers such as alpha(v)beta(6) Despite identical targeting peptide moiety and largely equal in vitro behavior, both (64)Cu-labeled tracers displayed inferior pharmacokinetics, making them in their present form less suitable candidates than the (18)F-labeled tracer for in vivo imaging of alpha(v)beta(6)

Measuring Brain Tumor Growth: Combined Bioluminescence Imaging–Magnetic Resonance Imaging Strategy:

Abstract: Small-animal tumor models are essential for developing translational therapeutic strategies in oncology research, with imaging having an increasingly important role. Magnetic resonance imaging (MRI) offers tumor localization, volumetric measurement, and the potential for advanced physiologic imaging but is less well suited to high-throughput studies and has limited capacity to assess early tumor growth. Bioluminescence imaging (BLI) identifies tumors early, monitors tumor growth, and efficiently measures response to therapeutic intervention. Generally, BLI signals have been found to correlate well with magnetic resonance measurements of tumor volume. However, in our studies of small-animal models of malignant brain tumors, we have observed specific instances in which BLI data do not correlate with corresponding MRIs. These observations led us to hypothesize that use of BLI and MRI together, rather than in isolation, would allow more effective and efficient measures of tumor growth in preclinical studies. Herein we describe combining BLI and MRI studies to characterize tumor growth in a mouse model of glioblastoma. The results led us to suggest a cost-effective, multimodality strategy for selecting cohorts of animals with similar tumor growth patterns that improves the accuracy of longitudinal in vivo measurements of tumor growth and treatment response in preclinical therapeutic studies.

RGD-Human Serum Albumin Conjugates as Efficient Tumor Targeting Probes:

Abstract: Cyclic arginine-glycine-aspartate (RGD) peptides and their derivatives have been intensively studied as tumor targeting probes. One major drawback, however, is their short blood circulation half-lives, which greatly compromises their targeting efficacy. To address this issue, a cyclic peptide, c(RGDyK), and an organic dye (IRDye800 or Cy5.5) were covalently conjugated onto human serum albumin (HSA). The conjugates were subjected to in vitro cell staining, in vivo near-infrared fluorescence (NIRF) imaging, ex vivo NIRF imaging, and histologic studies to evaluate their feasibility as tumor imaging probes. As a control, RAD peptide was also coupled with HSA and labeled with IRDye800 for in vivo imaging. The HSA-RGDIRDye800 exhibited integrin α v β 3 ‐specific binding in cell staining experiment. In vivo NIRF imaging showed higher tumor accumulation and tumor to background contrast of HSA-RGD-IRDye800 over RGD-IRDye800. The integrin specificity of HSA-RGD-IRDye800 is confirmed by both successful inhibition of tumor uptake in the presence of c(RGDyK) and the inability to accumulate in integrin-positive tumors by RAD-HSA-IRDye800. Histologic examination revealed initial tumor vascular binding and eventually both tumor vasculature and tumor cell integrin binding in vivo. In summary, we successfully developed an RGD-based protein conjugate with prolonged circulation half-life for NIRF imaging of tumor integrin α v β 3 expression. The success of this study may be generalizable for other peptide-based probes to be conjugated with HSA for prolonged tumor contrast and improved pharmacokinetics.

In vivo pharmacodynamic imaging of proteasome inhibition.

Abstract: Inhibiting the proteolytic activity of the 26S proteasome has been shown to have selective apoptotic effects on cancer cells and to be clinically efficacious in certain malignancies. There is an unmet medical need for additional proteasome inhibitors, and their development will be facilitated by surrogate markers of proteasome function. Toward this end, ectopic fusion of the destruction domain from ornithine decarboxylase (ODC) to reporter proteins is often used for assessing proteasome function. For luciferase-based reporters, we hypothesized that the oxygen-dependent destruction domain (ODD) from hypoxia-inducible factor 1 alpha (HIF-1 alpha) may provide improved sensitivity over luciferase-ODC, owing to its extremely rapid turnover by the proteasome (HIF-1 alpha has a half-life of less than 5 minutes). In the current study, we show that ODD-luciferase affords a greater dynamic range and faster kinetics than luciferase-ODC in sensing proteasome inhibition in vitro. Importantly, ODD-luciferase also serves as an effective in vivo marker of proteasome function in xenograft tumor models, with inhibition being detected by noninvasive imaging within 3 hours of bortezomib administration. These data establish ODD-luciferase as a surrogate marker of proteasome function that can be used both in vitro and in vivo for the development of novel proteasome inhibitors.

The problem is background, not signal.

Abstract: and reading articles where the tumor signal, usually a subcutaneous mass on the periphery, is barely discernable above background from the abdomen, viscera, and/or blood? This is not progress. The same is true for single-photon emission computed tomography (SPECT) radiotracers and optical imaging agents. For example, virtually every near-infrared (NIR) fl uorophore described to date has enormously high nonspecifi c tissue retention or biliary clearance, both of which lead to high background in the body, especially in the gut. Thus, although one can demonstrate receptor-specifi c uptake in artifi cial model systems such as xenograft tumors, fi nding small tumors in the abdomen or liver under realistic clinical scenarios would be nearly impossible with most optical agents. Fluorescence “quenching” until a binding or metabolic event occurs is a unique, potential background reduction strategy available to optical imaging. Conversely, autofl uorescence from endogenous fl uorophores is a unique source of background for optical imaging, but can be minimized by working with NIR wavelengths. At the Lasers in Medicine Gordon Conference in 2008, Brian Pogue from Dartmouth College showed a sobering slide, which reminded the audience that a signal to backgroundratio (SBR) ≥ 5:1 was still rare for intravenously injected exogenous contrast agents. This is abysmal. PET scanners and SPECT scanners, as well as optical imaging systems, have more than adequate sensitivity and dynamic range to achieve SBRs ≥ 100. The problem is background, not signal. So, what do we do about it? In my opinion, each fi eld within the umbrella of molecular imaging should start developing strategies to reduce nontarget tissue uptake and to maximize the elimination from the body (ie clearance) of nontarget bound contrast agents and radiotracers after intravenous injection. This is an enormously diffi cult challenge because it requires synergistic collaboration among chemists, physiologists, and imaging scientists. The fi eld has yet to devise a set of general purpose “chemical rules” to predict, a priori , which agents will work well and which will fail, or to devise coinjected agents that decrease nonspecifi c uptake and/or accelerate elimination from the body. All articles describing new diagnostic agents should also report the total W e have a lot to learn from the elder statesmen of molecular imaging. Fifty years ago, long before the term “molecular imaging” became chic, they wrestled with the problem of “background” from intravenously injected diagnostic agents, especially radiotracers. If you pull them aside at a meeting and ask them what they think the paramount problem is in the fi eld today, I suspect that most will still respond “Background.” This problem is not modality specifi c. It underlies the inherent failure of every modality with which we now work. If molecular imaging is ever to make a major clinical impact in cancer, heart disease, and diabetes, and I would argue that we have yet to do so, it will all come down to “background reduction.” Consider positron emission tomography (PET), for example. Commercially available clinical scanners can detect ≈ 700 Bq/voxel, which is equivalent to ≈ 0.01 radioatoms per cell. Yet, fi nding small tumors with 18 F-fl uorodeoxyglucose (FDG) and most other radiotracers remains diffi cult. The problem is neither the signal strength nor the sensitivity of the scanner; the problem is background. After intravenous injection of virtually all radiotracers described to date, including 18 FDG, the percent injected dose (%ID) taken up by nontarget tissue and remaining in the body after a few hours of clearance is extremely high. Persistent background retention (PBR) is inherent to the physicochemical properties of the diagnostic agent and can be caused by physiologic or non-physiologic binding to nontarget tissue, physiologic or non-physiologic cellular uptake in nontarget tissue, inadequate clearance from nontarget tissue, and/or metabolism in nontarget tissue. Survey the literature or posters at a meeting and you will fi nd that the order of 10%ID to 30%ID is commonly retained after intravenous injection, even when using small molecules and peptides. Are we not all weary of hearing talks The Problem is Background, not Signal

Molecular imaging of cathepsin B proteolytic enzyme activity reflects the inflammatory component of atherosclerotic pathology and can quantitatively demonstrate the antiatherosclerotic therapeutic effects of atorvastatin and glucosamine

Abstract: Inflammation in atherosclerotic plaques causes plaque vulnerability and rupture, leading to thromboembolic complications. Cathepsin B (CatB) proteases secreted by macrophages play a major role in plaque inflammation. We used a CatB-activatable near-infrared fluorescence (NIRF) imaging agent to demonstrate the inflammatory component in mice atheromata and the atherosclerosis- modulating effects of atorvastatin or glucosamine treatments. Apolipoprotein E knockout mice (n = 35) were fed normal chow, a Western diet, a Western diet + atorvastatin, a Western diet + glucosamine, or a Western diet + atorvastatin + glucosamine for 14 weeks. Twenty-four hours after the intravenous injection of a CatB-activatable probe, ex vivo NIRF imaging of the aortas and brains was performed, followed by histology. The CatB-related signal, observed in the aortas but not in the cerebral arteries, correlated very well with protease activity and the presence of macrophages on histology. Animals on Western diets could be distinguished from animals on a normal diet. The antiatherosclerotic effects of atorvastatin and glucosamine could be demonstrated, with reduced CatB-related signal compared with untreated animals. Plaque populations were heterogeneous within individuals, with some plaques showing a high and others a lower CatB-related signal. These differences in signal intensity could not be predicted by visual inspection of the plaques but did correlate with histologic evidence of inflammation in every case. This suggests that vulnerable inflamed plaques can be identified by optical molecular imaging.

Novel electro-optical coupling technique for magnetic resonance-compatible positron emission tomography detectors.

Abstract: A new magnetic resonance imaging (MRI)-compatible positron emission tomography (PET) detector design is being developed that uses electro-optical coupling to bring the amplitude and arrival time information of high-speed PET detector scintillation pulses out of an MRI system. The electro-optical coupling technology consists of a magnetically insensitive photodetector output signal connected to a nonmagnetic vertical cavity surface emitting laser (VCSEL) diode that is coupled to a multimode optical fiber. This scheme essentially acts as an optical wire with no influence on the MRI system. To test the feasibility of this approach, a lutetium-yttrium oxyorthosilicate crystal coupled to a single pixel of a solid-state photomultiplier array was placed in coincidence with a lutetium oxyorthosilicate crystal coupled to a fast photomultiplier tube with both the new nonmagnetic VCSEL coupling and the standard coaxial cable signal transmission scheme. No significant change was observed in 511 keV photopeak energy resolution and coincidence time resolution. This electro-optical coupling technology enables an MRI-compatible PET block detector to have a reduced electromagnetic footprint compared with the signal transmission schemes deployed in the current MRI/PET designs.

Near-Infrared Imaging of Injured Tissue in Living Subjects Using IR-820:

Abstract: The unprecedented increase in preclinical studies necessitates high-throughput, inexpensive, and straightforward methods for evaluating diseased tissues. Near-infrared imaging of live subjects is a versatile, cost-effective technology that can be effectively used in a variety of pathologic conditions. We have characterized an inexpensive optoelectronic chemical, IR-820, as an infrared blood pool contrast agent to detect and quantify diseased tissue in live animals. IR-820 has maximal excitation and emission wavelengths of 710 and 820 nm, respectively. IR-820 emission is significantly improved in vivo on serum binding to albumin, and elimination occurs predominantly via the gastrointestinal tract. We demonstrate the utility of this contrast agent for serially imaging of traumatized tissue (muscle), tissue following reperfusion (eg, stroke), and tumors. IR-820 can also be employed to map regional lymph nodes. This novel contrast agent is anticipated to be a useful and an inexpensive tool for screening a wide variety of preclinical models of human diseases.

Repetitive noninvasive monitoring of HSV1-tk-expressing T cells intravenously infused into nonhuman primates using positron emission tomography and computed tomography with 18F-FEAU.

Abstract: Adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTLs) has been successfully used to treat patients with different types of cancer However, the long-term spatial-temporal dynamics of the distribution of systemically infused CTLs remains largely unknown Noninvasive imaging of adoptively transferred CTLs using molecular-genetic reporter imaging with positron emission tomography and computed tomography (PET-CT) represents an innovative approach to understanding the long-term migratory patterns and therapeutic potential of adoptively transferred T cells Here we report the application of repetitive PET-CT imaging with [18F]fluoro-5-ethyl-1-beta-D-arabinofuranosyluracil (18F-FEAU) in two nonhuman primates demonstrating that autologous polyclonal macaque T lymphocytes activated and transduced with a retroviral vector encoding for the sr39 mutant herpes simplex virus 1 thymidine kinase (sr39HSV1-tk) reporter gene can be detected after intravenous infusion in discrete lymphoid organs and in sites of inflammation This study represents a proof of principle and supports the application of 18F-FEAU PET-CT imaging for monitoring the distribution of intravenously administered sr39HSV1-tk gene-transduced CTLs in humans

Impact of magnetic labeling on human and mouse stem cells and their long-term magnetic resonance tracking in a rat model of Parkinson disease.

Abstract: Magnetic resonance imaging (MRI) of magnetically labeled stem cells has become a valuable tool in the understanding and evaluation of experimental stem cell–based therapies of degenerative central ...

Semiautomatic landmark-based two-dimensional-three-dimensional image fusion in living mice: correlation of near-infrared fluorescence imaging of Cy5.5-labeled antibodies with flat-panel volume computed tomography.

Abstract: Connecting fluorescence signals with anatomic structures enhances our ability to monitor biologic processes in mice Here, we present a semiautomated approach to correlate two-dimensional (2D) noni

Molecular Imaging of Prostate Cancer: A Concise Synopsis

Abstract: Prostate cancer is the most common malignancy in men and continues to be a major public health problem. Imaging of prostate cancer remains particularly challenging owing to disease heterogeneity. Molecular imaging can provide unprecedented opportunities for deciphering the molecular mechanisms that are involved in the development and natural progression of prostate cancer from a localized process to the hormone-refractory metastatic disease. Such understanding will be the key for targeted imaging and therapy and for predicting and evaluating treatment response and prognosis. In this article, we review briefly the contribution of multimodality molecular imaging methods for the in vivo characterization of the pathophysiology of prostate cancer.

Automated segmentation of in vivo and ex vivo mouse brain magnetic resonance images.

Abstract: Segmentation of magnetic resonance imaging (MRI) data is required for many applications, such as the comparison of different structures or time points, and for annotation purposes. Currently, the g...

Comparison of superparamagnetic and ultrasmall superparamagnetic iron oxide cell labeling for tracking green fluorescent protein gene marker with negative and positive contrast magnetic resonance imaging.

Abstract: The objectives of this study were to investigate the feasibility of imaging green fluorescent protein (GFP)-expressing cells labeled with iron oxide nanoparticles with the fast low-angle positive c...

Molecular targeted enhanced ultrasound imaging of flk1 reveals diagnosis and prognosis potential in a genetically engineered mouse prostate cancer model.

Abstract: Molecular imaging techniques used to detect the initiation of disease have the potential to provide the best opportunity for early treatment and cure. This report aimed at testing the possibility that Flk1 + (vascular endothelial growth factor receptor 2), a crucial angiogenesis factor of most tumor cells, could be a molecular targeted imaging marker for the diagnosis and prognosis of cancer. We performed Flk1-targeted microbubble-enhanced ultrasound (US) imaging of prostate cancer in a genetically engineered mouse model with normal-appearing intact US (negative) prostates and with three different tumor sizes (small, medium, and large). Higher levels of Flk1 + molecular signals were identifi ed in the intact US (negative) prostate group by US-targeted imaging and immunohistochemical analysis. The increase in Flk1 + expression occurred prior to the angiogenesis switch-on phase and vascularity peak. After this peak accumulation stage of Flk1 + molecules, lower and stabilized levels of Flk1 + signals were maintained together with tumor growth from small, to medium, to large size. In a longitudinal observation in a subset ( n = 5) of mice with established tumors, elevated Flk1 + signals were observed in tissues surrounding the prostate cancer, for example, the ipsilateral boundary zones between two developing tumor lobes, new tumor blood vessel recruits, the urethra border, and the pelvic node basin. The potential of Flk1targeted US imaging as a predictive imaging tool was confi rmed by correlation studies of three-dimensional US B-mode imaging, gross pathology, and histology analyses. The results of the application in a genetically engineered mouse model with prostate cancer of molecular Flk1-targeted US imaging support the contention that Flk1 can be used as a molecular imaging marker for small tumors undetectable by microimaging and as a molecular diagnostic and prognosis marker for tumor metastasis and progression.

Extra Domain B Fibronectin as a Target for Near-Infrared Fluorescence Imaging of Rheumatoid Arthritis Affected Joints In Vivo

Abstract: We investigated a molecular imaging approach for the detection of collagen-induced arthritis in rats by targeting the extra domain B (ED-B) of the extracellular matrix protein fi bronectin. ED-B is a highly conserved domain (identical in human and rats) that is produced by alternative splicing during embryonic development and during vascular remodeling such as angiogenesis. The hallmark of rheumatoid arthritis is synovitis leading to both angiogenesis in the synovium and the promotion of cartilage and bone disruption. For in vivo diagnostics, the ED-B-binding single-chain antibody fragment AP39 was used as a targeting probe. It was covalently linked to the near-infrared dye tetrasulfocyanine (TSC) to be visualized by near-infrared fl uorescence imaging. The resulting AP39TSC conjugate was intravenously administered to rats with collagen-induced arthritis and the respective controls. Ovalbumin-TSC was used as control conjugate. Optical imaging over a time period of 24 hours using a planar imaging setup resulted in a clear enhancement of fl uorescence intensity in joints with moderate to severe arthritis compared with control joints between 3 and 8 hours postinjection. Given that AP39 is a fully human antibody fragment, this molecular imaging approach for arthritis detection might be translated to humans.

Fluorescence imaging of fast retrograde axonal transport in living animals.

Abstract: Our purpose was to enable an in vivo imaging technology that can assess the anatomy and function of peripheral nerve tissue (neurography). To do this, we designed and tested a fluorescently labeled molecular probe based on the nontoxic C fragment of tetanus toxin (TTc). TTc was purified, labeled, and subjected to immunoassays and cell uptake assays. The compound was then injected into C57BL/6 mice (N = 60) for in vivo imaging and histologic studies. Image analysis and immunohistochemistry were performed. We found that TTc could be labeled with fluorescent moieties without loss of immunoreactivity or biologic potency in cell uptake assays. In vivo fluorescent imaging experiments demonstrated uptake and retrograde transport of the compound along the course of the sciatic nerve and in the spinal cord. Ex vivo imaging and immunohistochemical studies confirmed the presence of TTc in the sciatic nerve and spinal cord, whereas control animals injected with human serum albumin did not exhibit these features. We have demonstrated neurography with a fluorescently labeled molecular imaging contrast agent based on the TTc.

Novel infectivity-enhanced oncolytic adenovirus with a capsid-incorporated dual-imaging moiety for monitoring virotherapy in ovarian cancer.

Abstract: We sought to develop a cancer-targeted, infectivity-enhanced oncolytic adenovirus that embodies a capsid-labeling fusion for noninvasive dual-modality imaging of ovarian cancer virotherapy. A functional fusion protein composed of fluorescent and nuclear imaging tags was genetically incorporated into the capsid of an infectivity-enhanced conditionally replicative adenovirus. Incorporation of herpes simplex virus thymidine kinase (HSV-tk) and monomeric red fluorescent protein 1 (mRFP1) into the viral capsid and its genomic stability were verified by molecular analyses. Replication and oncolysis were evaluated in ovarian cancer cells. Fusion functionality was confirmed by in vitro gamma camera and fluorescent microscopy imaging. Comparison of tk-mRFP virus to single-modality controls revealed similar replication efficiency and oncolytic potency. Molecular fusion did not abolish enzymatic activity of HSV-tk as the virus effectively phosphorylated thymidine both ex vivo and in vitro. In vitro fluorescence imaging demonstrated a strong correlation between the intensity of fluorescent signal and cytopathic effect in infected ovarian cancer cells, suggesting that fluorescence can be used to monitor viral replication. We have in vitro validated a new infectivity-enhanced oncolytic adenovirus with a dual-imaging modality-labeled capsid, optimized for ovarian cancer virotherapy. The new agent could provide incremental gains toward climbing the barriers for achieving conditionally replicated adenovirus efficacy in human trials.

Biodistribution of a nonpeptidic fluorescent endothelin A receptor imaging probe.

Abstract: Biodistribution studies are essential for understanding the biologic behavior of novel fluorochrome-based molecular imaging agents. In this study, the biodistribution of a recently developed fluorescent imaging probe with high affinity to the endothelin A (ETA) receptor was evaluated by fluorescence reflectance imaging (FRI). CD-1 mice were injected with 2 nmol of the probe intravenously and sacrificed at various time points. Tissue samples of the heart, spleen, lung, kidneys, liver, brain, and muscle were removed and imaged by FRI. Initially, the signal intensity (SI) was highest in lung, kidney, and liver tissue, followed by the heart, whereas spleen, muscle, and brain showed the lowest SI. In the kidneys, the SI decreased rapidly. In the heart, an initial SI increase was observed, followed by SI attenuation, whereas in the lung, the SI steadily increased. Competition experiments showed a significant (p , .005) degree of specific binding in the heart, with a reduction in SI of . 50%. In conclusion, FRI allows us to perform biodistribution studies of novel fluorescent tracers. The developed imaging probe can be exploited to image ETA receptor expression ideally 30 minutes to 3 hours after injection.

Coregistration of ultrasonography and magnetic resonance imaging with a preliminary investigation of the spatial colocalization of vascular endothelial growth factor receptor 2 expression and tumor perfusion in a murine tumor model.

Abstract: We present an ultrasonography (US)-magnetic resonance imaging (MRI) coregistration technique and examine its application in a preliminary multimodal, multiparametric study in a preclinical model of breast cancer. Nine mice were injected with 67NR breast cancer cells and imaged 6 and 9 days later with 4.7 T MRI and high-frequency US. Tumor volumes from each data set were segmented independently by two investigators and coregistered using an iterative closest point algorithm. In addition to anatomic images, vascular endothelial growth factor receptor 2 (VEGFR2) distribution images from the central tumor slice using VEGFR2-targeted ultrasound contrast agent (UCA) and measurements of perfusion and extravascular-extracellular volume fraction using dynamic contrast-enhanced MRI were acquired from five mice for multiparametric coregistration. Parametric maps from each modality were coregistered and examined for spatial correlation. Average registration root mean square (RMS) error was 0.36 +/- 0.11 mm, less than approximately two voxels. Segmented volumes were compared between investigators to minimize interobserver variability; the average RMS error was 0.23 +/- 0.09 mm. In the preliminary study, VEGFR2-targeted UCA data did not demonstrate direct spatial correlation with magnetic resonance measures of vascular properties. In summary, a method for accurately coregistering small animal US and MRI has been presented that allows for comparison of quantitative metrics provided by the two modalities.

Metabolism of orthotopic mouse brain tumor models.

Abstract: We used magnetic resonance spectroscopy to determine whether orthotopic mouse brain tumors grown as xenografts in immunocompromised mice either from human brain tumor cells implanted immediately after surgery or from cultured human tumor lines show metabolic profiles comparable to those of the original tumors. Using a 7 T scanner, spectra were acquired from mice with a human atypical teratoid/rhabdoid tumor (AT/RT) either implanted directly from the surgical specimen or first grown in culture, directly implanted choroid plexus carcinoma (CPC), and two medulloblastoma cell lines. The results were compared with spectra from these same tumors or tumor types in patients and with controls. Metabolic variability of tumors from a single cell line was also evaluated using the medulloblastoma lines. The main metabolic features of human tumors were qualitatively replicated in xenografts. AT/RTs in mice exhibited choline, creatine, and myo-inositol levels comparable to those observed in the patient. As in patients, choline was prominent in experimental CPC. Tumors from a single cell line were comparable. Significant correlations were found with key metabolites in humans and mice; however, differences including lower lipids in the implanted AT/RTs than in patient spectra and taurine observed in all animal spectra were also noted. The causes of these dissimilarities warrant further investigation.

Dual-contrast cellular magnetic resonance imaging.

Abstract: Negative contrast magnetic resonance imaging (MRI) methods using magnetic susceptibility shifting agents have become one of the most important approaches in cellular imaging research. However, visualizing and tracking labeled cells on the basis of negative contrast is often met with limited specifi city and sensitivity. Here we report on a MRI method for cellular imaging that generates a new contrast with a distinct topology for identifying labeled cells that has the potential to signifi cantly improve both the sensitivity and the specifi city. Specifi cally, we show that low fl ip-angle steady-state free precession MRI can be used to generate fast threedimensional images of tissue that can be rapidly processed to generate quantitative metrics enabling color overlays indicative of regions containing labeled cells. The technique substantially improves the ability of MRI for detecting labeled cells by overcoming the fundamental limits that currently plague negative contrast methods.