Biomedical subjects

Ralph Weissleder

Publications and source records attributed to Ralph Weissleder.

At least 181 records · Page 10Linked to original sources

PubMed2002

Detection of dysplastic intestinal adenomas using enzyme-sensing molecular beacons in mice.

BACKGROUND & AIMS: Proteases play key roles in the pathogenesis of tumor growth and invasion. This study assesses the expression of cathepsin B in dysplastic adenomatous polyps. METHODS: Aged Apc(Min/+) mice served as an experimental model for familial adenomatous polyposis. The 4 experimental groups consisted of (a) animals injected with a novel activatable, cathepsin B sensing near infrared fluorescence (NIRF) imaging probe; (b) animals injected with a nonspecific NIRF; (c) uninjected control animals; and (d) non-APC(Min/+) mice injected with the cathepsin B probe. Lesions were analyzed by immunohistochemistry, Western blotting, reverse transcription-polymerase chain reaction, and optical imaging. RESULTS: Cathepsin B was consistently overexpressed in adenomatous polyps. When mice were injected intravenously with the cathepsin reporter probe, intestinal adenomas became highly fluorescent indicative of high cathepsin B enzyme activity. Even microscopic adenomas were readily detectable by fluorescence, but not light, imaging. The smallest lesion detectable measured 50 microm in diameter. Adenomas in the indocyanine green and/or noninjected group were only barely detectable above the background. CONCLUSIONS: The current experimental study shows that cathepsin B is up-regulated in a mouse model of adenomatous polyposis. Cathepsin B activity can be used as a biomarker to readily identify such lesions, particularly when contrasted against normal adjacent mucosa. This detection technology can be adapted to endoscopy or tomographic optical imaging methods for screening of suspicious lesions and potentially for molecular profiling in vivo.

Adenomatous Polyps↗

PubMed2002

Charge-coupled-device based scanner for tomography of fluorescent near-infrared probes in turbid media.

We present a novel tomographer for three-dimensional reconstructions of fluorochromes in diffuse media. Photon detection is based on charge-coupled device technology that allows the implementation of a large parallel array of detection channels with high sensitivity. Using this instrument we studied the response and detection limits of near-infrared fluorochromes in diffuse media as a function of light intensity and for a wide range of biologically relevant concentrations. We further examined the resolution of the scanner and the reconstruction linearity achieved. We demonstrate that the instrument attains better than 3 mm resolution, is linear within more than two orders of magnitude of fluorochrome concentration, and can detect fluorescent objects at femto-mole quantities in small animal-like geometries. These measurements delineate detection and reconstruction characteristics associated with imaging of novel classes of fluorescent probes developed for in vivo molecular and functional probing of tissues.

Biophysical Phenomena↗

PubMed2002

Imaging of differential protease expression in breast cancers for detection of aggressive tumor phenotypes.

PURPOSE: To determine if different expression levels of tumor cathepsin-B activity in well differentiated and undifferentiated breast cancers could be revealed in vivo with optical imaging. MATERIALS AND METHODS: A well differentiated human breast cancer (BT20, n = 8) and a highly invasive metastatic human breast cancer (DU4475, n = 8) were implanted orthotopically in athymic nude mice. Tumor-bearing animals were examined in vivo with near-infrared fluorescence (NIRF) imaging 24 hours after intravenous injection of an enzyme-sensing imaging probe. Immunohistochemistry, Western blotting (on cells and whole tumor samples), and correlative fluorescence microscopy were performed. RESULTS: Both types of breast cancers activated the NIRF probe so that tumors became readily detectable. However, in tumors of equal size, there was a 1.5-fold higher fluorescence signal in the highly invasive breast cancer (861 arbitrary units +/- 88) compared with the well differentiated lesion (566 arbitrary units +/- 36, P <.01). Western blotting confirmed a higher cathepsin-B protein content in the highly invasive breast cancer (DU4475) of about 1.4-fold (whole tumor samples) to 1.7-fold (cells). Immunohistochemistry and fluorescence microscopy findings confirmed the imaging findings. CONCLUSION: Cathepsin-B enzyme activity can be determined in vivo with NIRF optical imaging, while differences in tumoral expression may correlate with tumor aggressiveness.

Adenocarcinoma↗

PubMed2002

Feasibility of in vivo multichannel optical imaging of gene expression: experimental study in mice.

PURPOSE: To develop and test a multichannel reflectance imaging system for small animals on the basis of a previously developed single-channel setup. MATERIALS AND METHODS: The imaging system was composed of modular parts, including a light source, excitation filters, emission filters, and a charged-coupled device for recording images. On the basis of generated excitation and absorption spectra of green fluorescent protein (GFP), tricarbocyanine 5.5 (Cy5.5), and indocyanine green (ICG), filters were selected to allow spectral separation and optimize resultant recorded signal. The system was tested by using a combination of the fluorochromes to confirm spectral separation. In vivo tests were performed in nude mice with tumors that expressed cathepsin B, which could be evaluated by using a Cy5.5-based activatable probe and GFP. For each in vivo tumor type and channel, statistical analysis was performed on the basis of signal intensity in the region of interest. RESULTS: The different fluorochromes were readily distinguished with the system; characteristics such as power were determined for all wavelengths. The system demonstrated a linear response for GFP, a monotonic response for Cy5.5 over a range of more than three orders of magnitude of concentration, and a more complex response for ICG. In vivo analysis demonstrated the ability to image GFP expression and cathepsin B expression separately in tumors: As expected, marked differences were observed in GFP-expression imaging between tumor types (1,363 arbitrary units [AU] +/- 236 [SD] vs 110 AU +/- 11 for GFP-positive and GFP-negative tumors, respectively; P <.001), whereas similar cathepsin B expression (1,070 AU +/- 285 vs 1,168 AU +/- 367; P >.5) was observed. Histologic analysis confirmed in vivo findings. CONCLUSION: Imaging multiple gene expressions simultaneously in vivo by using optical imaging is feasible.

Animals↗

PubMed2002

Coded aperture nuclear scintigraphy: a novel small animal imaging technique.

We introduce and demonstrate the utility of coded aperture (CA) nuclear scintigraphy for imaging small animals. CA imaging uses multiple pinholes in a carefully designed mask pattern, mounted on a conventional gamma camera. System performance was assessed using point sources and phantoms, while several animal experiments were performed to test the usefulness of the imaging system in vivo, with commonly used radiopharmaceuticals. The sensitivity of the CA system for 99mTc was 4.2 x 10(3) cps/Bq (9400 cpm/microCi), compared to 4.4 x 10(4) cps/Bq (990 cpm/microCi) for a conventional collimator system. The system resolution was 1.7 mm, as compared to 4-6 mm for the conventional imaging system (using a high-sensitivity low-energy collimator). Animal imaging demonstrated artifact-free imaging with superior resolution and image quality compared to conventional collimator images in several mouse and rat models. We conclude that: (a) CA imaging is a useful nuclear imaging technique for small animal imaging. The advantage in signal-to-noise can be traded to achieve higher resolution, decreased dose or reduced imaging time. (b) CA imaging works best for images where activity is concentrated in small volumes; a low count outline may be better demonstrated using conventional collimator imaging. Thus, CA imaging should be viewed as a technique to complement rather than replace traditional nuclear imaging methods. (c) CA hardware and software can be readily adapted to existing gamma cameras, making their implementation a relatively inexpensive retrofit to most systems.

Animals↗

PubMed2002

Steady-state and dynamic contrast MR imaging of human prostate cancer xenograft tumors: a comparative study.

Understanding tumor vascular physiology is critically important for developing non-invasive, molecularly targeted diagnostic agents and therapies. In this study, using three different human prostate cancer xenografts (MDA PCa 2b, PC3, and LnCap), structural and physiological parameters of neoplastic vasculature and interstitum were explored with a widely available magnetic resonance imaging (MRI) pulse sequence (3D SPGR: spoiled gradient echo). Using dual injection technique employing two T1 contrast agents of different molecular masses (Weissleder, R., Cheng, H. C., Marecos, E., Kwong, K. K., Bogdanov, A., Jr. Eur. J. Cancer 34, 1448-1454 (1998).), steady state (SS) MRI measurements and dynamic contrast agent enhancement (DCE) MRI measurements were simultaneously acquired and analyzed using a two-compartment model for calculating parameters reflecting tumoral architecture and physiology. In particular, interstitial volume and vascular permeability were independently quantified using these two different MRI techniques. Relative vascular water exchange rate, calculated by the flip angle (FA) dependence of measured blood volume using SS technique, and vascular permeability of contrast agent, extrapolated from DCE MRI, were compared. It was found that the SS and DCE techniques were comparable and yielded similar qualitative results for extravascular compartment (interstitial volume). However, the permeability (water exchange rate and contrast agent vascular permeability) values were in disagreement. The results of MR studies are important for interpreting optical imaging results obtained using long-circulating of tumor-associated enzymatic activity.

Contrast Media↗

PubMedDate not supplied

Myeloperoxidase activity imaging using (67)Ga labeled substrate.

PURPOSE: The aim of the study is to assess the feasibility of imaging specific activity of myeloperoxidase (MPO), a leukocyte enzyme with important roles in inflammation and atherosclerosis, by single photon emission computed tomography (SPECT) using a novel (67)Ga-labeled radiotracer obtained by conjugating desferrioxamine (DF) and hydroxyindolyl acetic acid in vivo. MATERIALS AND METHODS: A reducing substrate of MPO (I) was synthesized by reacting commercially available DF with 2-(5-hydroxy-1H-indol-3-yl) acetic acid in the presence of a coupling agent [dicyclohexyl carbodiimide (DCC)]. The chelating unit was labeled with (67)Ga, and its interaction with MPO was characterized using MALDI-TOF and UV-vis. Mice with Matrigel implants containing human MPO were used to model diseased tissues rich in MPO. Three hours after the injection of (67)Ga-I, SPECT/computed tomography (CT) imaging was performed on a high-resolution Gamma Medica X-SPECT system. Biodistribution studies were performed six hours after the injection of the radiotracer. RESULTS: The feasibility of compound I oligomerization in the presence of MPO and MPO-mediated cross-linking with proteins was initially confirmed in vitro. In vivo, a 2.7-fold increase in target-to-muscle ratio could be measured in MPO-containing Matrigel implants in mice. Biodistribution experiments demonstrated a 60% increase of radioactivity in MPO-containing vs. control (contralateral) Matrigel implants. CONCLUSION: (67)Ga-I can be used to image MPO activity in a model system. The accumulation mechanism is based on a differential pharmacokinetics because of the size increase resulting from (67)Ga-I interaction with the target enzyme.

Animals↗

PubMedDate not supplied

In vivo phage display selection yields atherosclerotic plaque targeted peptides for imaging.

PURPOSE: Atherosclerosis is a leading cause of morbidity and mortality in the Western world, yet specific imaging agents to detect and map inflammatory plaques are still lacking. PROCEDURES: We used in vivo phage display to interrogate early atherosclerotic lesions present in ApoE-/- mice with the goal of identifying plaque-associated endothelial cell internalized affinity ligands. RESULTS: We identified 30 phage families with some of these families exhibiting homology to known atherosclerotic proteins, namely, leukemia inhibitory factor, transferrin, and VLA-4. VLA-4 homologous peptides [termed vascular cellular adhesion molecule-1 (VCAM-1) internalizing peptide-28 (VINP28)] bound to and were internalized by VCAM-1-expressing cells and were inhibited by soluble VCAM-1. In addition, a VINP28 modified multimodal nanoparticle showed high affinity for endothelial cells expressing VCAM-1 but low affinity for macrophages or smooth muscle cells. CONCLUSION: The identified peptides represent a set of probes to interrogate the cell surface repertoire and potentially allow early detection of atherosclerosis.

Affinity Labels↗

PubMedDate not supplied

The transferrin receptor: a potential molecular imaging marker for human cancer.

Noninvasive imaging of differences between the molecular properties of cancer and normal tissue has the potential to enhance the detection of tumors. Because overexpression of endogenous transferrin receptor (TfR) has been qualitatively described for various cancers and is presumably due to malignant transformation of cells, TfR may represent a suitable target for application of molecular imaging technologies to increase detection of smaller tumors. In the work reported here, investigation into the biology of this receptor using electron microscopy has demonstrated that iron oxide particles targeted to TfR are internalized and accumulate in lysosomal vesicles within cells. Biochemical analysis of the interaction of imaging probes with cells overexpressing the TfR demonstrated that the extent of accumulation, and therefore probe efficacy, is dependent on the nature of the chemical cross-link between transferrin and the iron oxide particle. These data were utilized to design and synthesize an improved imaging probe. Experiments demonstrate that the novel magnetic resonance imaging (MRI) probe is sensitive enough to detect small differences in endogenous TfR expression in human cancer cell lines. Quantitative measurement of TfR overexpression in a panel of 27 human breast cancer patients demonstrated that 74% of patient cancer tissues overexpressed the TfR and that the sensitivity of the new imaging agent was suitable to detect TfR overexpression in greater than 40% of these cases. Based on a biochemical and cell biological approach, these studies have resulted in the synthesis and development of an improved MRI probe with the best in vitro and in vivo imaging properties reported to date.

Animals↗

PubMedDate not supplied

Optical imaging of apoptosis as a biomarker of tumor response to chemotherapy.

A rapid and accurate assessment of the antitumor efficacy of new therapeutic drugs could speed up drug discovery and improve clinical decision making. Based on the hypothesis that most effective antitumor agents induce apoptosis, we developed a near-infrared fluorescent (NIRF) annexin V to be used for optical sensing of tumor environments. To demonstrate probe specificity, we developed both an active (i.e., apoptosis-recognizing) and an inactive form of annexin V with very similar properties (to account for nonspecific tumor accumulation), and tested the agents in nude mice each bearing a cyclophosphamide (CPA) chemosensitive (LLC) and a chemoresistant LLC (CR-LLC). After injection with active annexin V, the tumor-annexin V ratio (TAR; tumor NIRF/background NIRF) for untreated mice was 1.22+/-0.34 for LLC and 1.43+/-0.53 for CR-LLC (n=4). The LLC of CPA-treated mice had significant elevations of TAR (2.56+/-0.29, P=.001, n=4), but only a moderate increase was obtained for the CR-LLC (TAR=1.89+/-0.19, P=.183). The in vivo measurements correlated well with terminal deoxyribosyl transferase-mediated dUTP nick end labeling indexes. When inactive Cy-annexin V was used, with or without CPA treatment and in both CCL and CR-CCL tumors, tumor NIRF values ranged from 0.91 to 1.17 (i.e., tumor were equal to background). We conclude that active Cy-annexin V and surface reflectance fluorescence imaging provide a nonradioactive, semiquantitative method of determining chemosensitivity in LLC xenografts. The method maybe used to image pharmacologic responses in other animal models and, potentially, may permit the clinical imaging of apoptosis with noninvasive or minimally invasive instrumentation.

Animals↗

PubMedDate not supplied

Murine Lewis lung carcinoma-derived endothelium expresses markers of endothelial activation and requires tumor-specific extracellular matrix in vitro.

The purpose of the study was to identify characteristics specific to tumor-derived endothelium that may be important in tumor biology, or for the development of targeted therapeutics or imaging agents. Normal C57Bl/6 murine heart or lung endothelium, or C57Bl/6 murine Lewis lung carcinoma tumor-derived endothelium was isolated from excised tissues using specific antibodies. The endothelium was cultured using either native fibronectin, or the oncofetal form of fibronectin. Cell surface adhesion molecule expression was analyzed by flow cytometry, and the cellular distribution of specific molecules was examined using indirect immunofluorescence staining. Oncofetal fibronectin was critical for maintaining the phenotype of tumor-derived endothelium, which demonstrated an elongated morphology in vitro, with few cell-cell contacts. They expressed high levels of CD31, CD102, and vascular endothelial cadherin, and constitutively expressed CD62E, CD54, and CD106, indicating an "activated" phenotype. Moreover, they expressed significantly greater levels of Sca-1 and Flk-1 than normal murine endothelium. Cellular distribution of CD31, beta-catenin, and CD106 in tumor-derived endothelium was not continuous at cell borders, as observed in cultures of murine heart endothelium. In conclusion, Lewis lung carcinoma-derived tumor endothelium exhibits a specific phenotype in vitro, distinct from normal endothelium, and could be used as an in vitro tool for developing targeted agents.

Animals↗

PubMedDate not supplied

High throughput magnetic resonance imaging for evaluating targeted nanoparticle probes.

The ability to image specific molecular targets in vivo would have significant impact in allowing earlier disease detection and in tailoring molecular therapies. One of the rate-limiting steps in the development of novel compounds as reporter probes has been the lack of cell-based, biologically relevant, high throughput screening methods. Here we describe the development and validation of magnetic resonance imaging (MRI) as a technique to rapidly screen compounds that are potential MR reporter agents for their interaction with specific cellular targets. We show that MR imaging can (1) evaluate thousands of samples simultaneously and rapidly, (2) provide exceedingly accurate measurements, and (3) provide receptor binding/internalization data as validated by radioactive assays. The technique allows the screening of libraries of peptide-nanoparticle conjugates against target cells and the identification of conjugates that may be subsequently used as reporter agents in vivo. The technology should greatly accelerate the development of target-specific or cell-specific MR contrast agents.

Cell Line↗

PubMedDate not supplied

Magnetic resonance imaging of inducible E-selectin expression in human endothelial cell culture.

Covalent conjugates of the cross-linked iron oxide nanoparticles (CLIO) and high-affinity (K(d)(app) = 8.5 nM) anti-human E-selectin (CD62E) F(ab')(2) fragments were prepared and tested in vitro to establish feasibility of endothelial proinflammatory marker magnetic resonance (MR) imaging. The conjugates were obtained by using thiol-disulfide exchange reaction between 3-(2-pyridyl)propionyl-CLIO and S-acetylthioacetate-modified F(ab')(2) fragments. The purified CLIO-F(ab')(2) conjugates (average hydrodynamic diameter 40.6 nm) were used in experiments with the live human endothelial umbilical vein cells (HUVEC). Cells treated with IL-1 beta expressed E-selectin and showed a 100-200 times higher binding of CLIO particles (83-104 ng iron/million cells) than control cells. The binding resulted in a high superparamagnetism of HUVEC with the transverse water proton relaxation time (T2) decrease to 30-40 ms in cell precipitates. Cells did not bind/internalize CLIO-F(ab')(2) conjugates prepared using a control fragment or nonconjugated iron oxide particles before or after treatment with IL-1 beta. MR imaging of cells showed a highly specific T2-weighted signal darkening associated with cells treated with IL-1 beta and incubated with anti-E selectin. Demonstration of MR imaging of E-selectin expression justifies further development of MR-targeted agents for monitoring tumor vascular endothelial proliferation, angiogenesis, and atherosclerosis.

Cells, Cultured↗

PubMedDate not supplied

Near-infrared fluorescent nanoparticles as combined MR/optical imaging probes.

A number of quantitative three-dimensional tomographic near-infrared fluorescence imaging techniques have recently been developed and combined with MR imaging to yield highly detailed anatomic and molecular information in living organisms (1, 2). Here we describe magnetic nanoparticle based MR contrast agents that have a near-infrared fluorescence (NIRF) that is activated by certain enzymes. The probes are prepared by conjugation of arginyl peptides to cross-linked iron oxide amine (amino-CLIO), either by a disulfide linkage or a thioether linker, followed by the attachment of the indocyanine dye Cy5.5. The NIRF of disulfide-linked conjugate was activated by DTT, while the NIRF of thioether-linked conjugate was activated by trypsin. Fluorescent quenching of the attached fluorochrome occurs in part due to the interaction with iron oxide, as evident by the activation of fluorescence with DTT when nanoparticles that have less than one dye attached per particle. With a SC injection of the probe, axillary and brachial lymph nodes were darkened on MR images and easily delineated by NIRF imaging. The probes may provide the basis for a new class of so-called smart nanoparticles, capable of pinpointing their position through their magnetic properties, while providing information on their environment by optical imaging techniques.

Animals↗

PubMedDate not supplied

Tat peptide directs enhanced clearance and hepatic permeability of magnetic nanoparticles.

Superparamagnetic nanoparticles have a number of important biomedical applications, serving as MR contrast agents for imaging specific molecular targets, as reagents for cell labeling and cell tracking, and for the isolation of specific classes of cells. We have determined the physical and biological properties of MION-47 and amino-CLIO, nanoparticles which serve as precursors for the synthesis of targeted MR contrast agents, and Tat-CLIO, a nanoparticle used as a cell labeling reagent. Blood half-lives for MION-47 and amino-CLIO were 682 +/- 34 and 655 +/- 37 min, respectively. The attachment of 9.7 tat peptides per crystal to amino-CLIO resulted in a reduction in blood half-life to 47 +/- 6 min. MION-47, amino-CLIO, and Tat-CLIO were present in highest concentrations in liver and spleen and lymph nodes, where concentrations for all three nanoparticles ranged from 8.80 to 6.11% of injected dose per gram. Twenty-four hours after the intravenous injection of amino-CLIO, the nanoparticle was concentrated in cells surrounding hepatic blood vessels (endothelial and Kupffer cells), in a fashion similar to that obtained with other nanoparticle preparations. In contrast, Tat-CLIO was present as numerous discrete foci of intense fluorescence throughout the parenchyma. Using the peptide as a component of future nanoparticles, it might be possible to design sensors for the detection of macromolecules present in intracellular compartments.

Amino Acid Sequence↗

PubMedDate not supplied

Novel near-infrared cyanine fluorochromes: synthesis, properties, and bioconjugation.

Recently, near-infrared (NIR) fluorescence light has been applied to image various biological events in vivo, because it penetrates tissue more efficiently than light in the visible spectrum. Compounds exhibiting fluorescent properties in the NIR range are key elements for this upcoming optical imaging technology. In this paper, we report the synthesis of four new, water-soluble NIR cyanine fluorochromes which have superior chemical stability and optical properties. Each fluorochrome was designed with a monoreactive carboxyl group for labeling purposes. When multiple fluorochromes were attached to a single macromolecule, fluorescence quenching was observed. On the basis of this property, a novel autoquenched enzyme sensitive NIR fluorescence probe was prepared.

Amides↗

PubMedDate not supplied

Differential conjugation of tat peptide to superparamagnetic nanoparticles and its effect on cellular uptake.

Surface modification of superparamagnetic contrast agents with HIV-1 tat peptide has emerged as a promising means for intracellular magnetic labeling and noninvasive tracking of a large number of cell types with MRI. To achieve efficient intracellular delivery of the nanoparticles, we investigated the effect on cellular uptake of superparamagnetic iron oxide particles by varying the number of attached tat peptides. First, we report here a modified P2T method in measuring the numbers of surface attachments per particle through disulfide linkage. The method was shown to have desirable simplicity and reproducibility. With the P2T method as a tool, conjugates with progressively higher ratios of peptide-to-particle were synthesized. We were able to demonstrate that higher numbers of tat peptide facilitate the cellular uptake of iron oxide nanoparticles in a nonlinear fashion. Cells labeled with these optimized preparations were readily detectable by MR imaging. The increase in sensitivity could allow in vivo tracking of 100-fold lower cell concentration than currently described.

Animals↗

PubMedDate not supplied

Enhanced tumor detection using a folate receptor-targeted near-infrared fluorochrome conjugate.

Fluorescence optical imaging technologies are currently being developed to image specific molecular targets in vivo. Detection technologies range from those providing microscopic detail to whole body imaging systems with potential clinical use. A number of target-specific near-infrared imaging probes have recently been developed to image receptors, antigens, and enzymes. The goal of the current study was to evaluate a new near-infrared (NIR) folate receptor (FR)-targeted imaging probe for its ability to improve detection of FR-positive cancers. We hypothesized that modification of folate would retain receptor affinity in vivo, despite the bulkier NIR fluorochrome, NIR2 (em = 682 nm). Cellular uptake of the NIR conjugates was significantly higher in FR-positive nasopharyngeal epidermoid carcinoma, KB cells, compared to FR-negative human fibrosarcoma, HT1080 cells. When tumors were implanted in vivo, equal-sized KB tumors showed a 2.4-fold higher signal intensity compared to HT1080 tumors (24 h). The maximum signal-to-background ratio (3-fold) was observed at 24 h in KB tumor. Injection of the unmodified NIR2 fluorochrome did not result in persistent contrast increases under similar conditions. Furthermore, tumor enhancement with the NIR2-folate probe persisted over 48 h and was inhibitable in vivo by administration of unlabeled folate. These results indicate that folate-modified NIR fluorochrome conjugate can be used for improved detection of FR-positive tumors.

Animals↗