Biomedical subjects

Ralph Weissleder

Publications and source records attributed to Ralph Weissleder.

At least 73 records · Page 4Linked to original sources

PubMed2005

Volumetric tomography of fluorescent proteins through small animals in vivo.

Volumetric detection and accurate quantification of fluorescent proteins in entire animals would greatly enhance our ability to monitor biological processes in vivo. Here we present a quantitative tomographic technique for visualization of superficial and deep-seated (>2-3 mm) fluorescent protein activity in vivo. We demonstrate noninvasive imaging of lung tumor progression in a murine model, as well as imaging of gene delivery using a herpes virus vector. This technology can significantly improve imaging capacity over the current state of the art and should find wide in vivo imaging applications in drug discovery, immunology, and cancer research.

Animals↗

PubMed2005

Mapping of nodal disease in locally advanced prostate cancer: rethinking the clinical target volume for pelvic nodal irradiation based on vascular rather than bony anatomy.

PURPOSE: Toxicity from pelvic irradiation could be reduced if fields were limited to likely areas of nodal involvement rather than using the standard "four-field box." We employed a novel magnetic resonance lymphangiographic technique to highlight the likely sites of occult nodal metastasis from prostate cancer. METHODS AND MATERIALS: Eighteen prostate cancer patients with pathologically confirmed node-positive disease had a total of 69 pathologic nodes identifiable by lymphotropic nanoparticle-enhanced MRI and semiquantitative nodal analysis. Fourteen of these nodes were in the para-aortic region, and 55 were in the pelvis. The position of each of these malignant nodes was mapped to a common template based on its relation to skeletal or vascular anatomy. RESULTS: Relative to skeletal anatomy, nodes covered a diffuse volume from the mid lumbar spine to the superior pubic ramus and along the sacrum and pelvic side walls. In contrast, the nodal metastases mapped much more tightly relative to the large pelvic vessels. A proposed pelvic clinical target volume to encompass the region at greatest risk of containing occult nodal metastases would include a 2.0-cm radial expansion volume around the distal common iliac and proximal external and internal iliac vessels that would encompass 94.5% of the pelvic nodes at risk as defined by our node-positive prostate cancer patient cohort. CONCLUSIONS: Nodal metastases from prostate cancer are largely localized along the major pelvic vasculature. Defining nodal radiation treatment portals based on vascular rather than bony anatomy may allow for a significant decrease in normal pelvic tissue irradiation and its associated toxicities.

Aged↗

PubMed2005

The progress and promise of molecular imaging probes in oncologic drug development.

As addressed by the recent Food and Drug Administration Critical Path Initiative, tools are urgently needed to increase the speed, efficiency, and cost-effectiveness of drug development for cancer and other diseases. Molecular imaging probes developed based on recent scientific advances have great potential as oncologic drug development tools. Basic science studies using molecular imaging probes can help to identify and characterize disease-specific targets for oncologic drug therapy. Imaging end points, based on these disease-specific biomarkers, hold great promise to better define, stratify, and enrich study groups and to provide direct biological measures of response. Imaging-based biomarkers also have promise for speeding drug evaluation by supplementing or replacing preclinical and clinical pharmacokinetic and pharmacodynamic evaluations, including target interaction and modulation. Such analyses may be particularly valuable in early comparative studies among candidates designed to interact with the same molecular target. Finally, as response biomarkers, imaging end points that characterize tumor vitality, growth, or apoptosis can also serve as early surrogates of therapy success. This article outlines the scientific basis of oncology imaging probes and presents examples of probes that could facilitate progress. The current regulatory opportunities for new and existing probe development and testing are also reviewed, with a focus on recent Food and Drug Administration guidance to facilitate early clinical development of promising probes.

Antineoplastic Agents↗

PubMed2005

A novel mouse model for segmental orthotopic colon cancer.

Spontaneous colon tumor mouse strains offer numerous advantages in modeling disease. However, the wide temporal window in which lesions form and the stochastic nature of lesion location require larger cohorts for assessment of disease modulation. Reliable, reproducible and inexpensive mouse models of early-stage and invasive cancer would add to existing transgenic models. We show a new method for the creation of orthotopic murine tumors centered in the mucosal and submucosal layers anywhere in the colon, allowing creation of lesions of known age, location and extent. The system overcomes the disadvantages of heterotopic implantation and allows evaluation of lesions distally in the colon as well as proximally, thereby providing an additional method to study the effects of regionality. Invasion, host vascularization and application to disparate cell lines are demonstrated. Noninvasive imaging with magnetic resonance and colonoscopy, allowed in part by the tumor location, show potential applications of this approach.

Animals↗

PubMed2005

Cell-specific targeting of nanoparticles by multivalent attachment of small molecules.

Nanomaterials with precise biological functions have considerable potential for use in biomedical applications. Here we investigate whether multivalent attachment of small molecules can increase specific binding affinity and reveal new biological properties of such nanomaterials. We describe the parallel synthesis of a library comprising 146 nanoparticles decorated with different synthetic small molecules. Using fluorescent magnetic nanoparticles, we rapidly screened the library against different cell lines and discovered a series of nanoparticles with high specificity for endothelial cells, activated human macrophages or pancreatic cancer cells. Hits from the last-mentioned screen were shown to target pancreatic cancer in vivo. The method and described materials could facilitate development of functional nanomaterials for applications such as differentiating cell lines, detecting distinct cellular states and targeting specific cell types.

Animals↗

PubMed2005

Detection of early antiangiogenic effects in human colon adenocarcinoma xenografts: in vivo changes of tumor blood volume in response to experimental VEGFR tyrosine kinase inhibitor.

Antiangiogenesis is emerging as efficient strategy for targeting and potentially eliminating neoplastic tumor vessels. The main goal of this study was to establish whether absolute tumor blood volume (V(b)) change could be used as an early predictor of antiangiogenesis in ectopic and orthotopic colon carcinomas. To assess therapy-induced changes of V(b), we did comparative analysis of signal intensities in tumors and muscle using steady-state magnetic resonance imaging (MRI) assisted with an intravascular paramagnetic contrast agent [gadolinium-labeled protected graft copolymer (PGC-Gd)]. Athymic mice with implanted human MV522 tumors were treated with vascular endothelial growth factor type 2 receptor tyrosine kinase inhibitor (VEGFR2-TKI) that has been shown to inhibit VEGFR2 phosphorylation and tumor growth in vivo. Animals were imaged either after a single day or after a 1-week course of treatments. The measured V(b) in ectopic tumors was 2.5 +/- 1.5% of total tissue volume 1 week after the implantation (n = 8). Two doses of VEGFR2-TKI (25 mg/kg, p.o., b.i.d.) resulted in a decrease of V(b) to 1.3 +/- 0.3%. In orthotopic tumors, the measured V(b) was initially higher (11.9 +/- 2.0%); however, VEGFR2-TKI treatment also resulted in a statistically significant decrease of V(b). The absolute V(b) was not affected in the muscle as a result of treatments. MRI measurements were corroborated by using isotope and correlative histology experiments. Our results show that steady-state MRI is highly sensitive to early antiangiogenic effects caused by small molecule drugs.

Adenocarcinoma↗

PubMed2005

Use of gene expression profiling to direct in vivo molecular imaging of lung cancer.

Using gene expression profiling, we identified cathepsin cysteine proteases as highly up-regulated genes in a mouse model of human lung adenocarcinoma. Overexpression of cathepsin proteases in these lung tumors was confirmed by immunohistochemistry and Western blotting. Therefore, an optical probe activated by cathepsin proteases was selected to detect murine lung tumors in vivo as small as 1 mm in diameter and spatially separated. We generated 3D maps of the fluorescence signal and fused them with anatomical computed tomography images to show a close correlation between fluorescence signal and tumor burden. By serially imaging the same mouse, optical imaging was used to follow tumor progression. This study demonstrates the capability for molecular imaging of a primary lung tumor by using endogenous proteases expressed by a tumor. It also highlights the feasibility of using gene expression profiling to identify molecular targets for imaging lung cancer.

Adenocarcinoma, Bronchiolo-Alveolar↗

PubMed2005

In vivo assessment of RAS-dependent maintenance of tumor angiogenesis by real-time magnetic resonance imaging.

New blood vessel formation is a prominent feature of human cancers and tumor progression and is frequently accompanied by the acquisition of an angiogenic phenotype associated with a switch in the balance of proangiogenic and antiangiogenic molecules. This study was designed to investigate the role of activated H-RAS on the angiogenic phenotype of melanoma that arises in the inducible Tyr/Tet-RAS Ink4a/Arf(-/-) model using in vivo imaging with histopathologic correlation. We show that loss of RAS activity in fully established melanomas led to a reduction in tumor volume, which was preceded by impairment of vascular function as determined by in vivo magnetic resonance imaging. This correlated with activation of apoptosis in host-derived endothelial cells as well as in tumor cells. Thus, real-time in vivo imaging provided evidence that maintenance of tumor angiogenesis requires activated RAS in this model system, and that loss of vascular integrity upon inactivation of RAS is an active process rather than a consequence of loss of tumor cell viability.

Animals↗

PubMed2005

Noninvasive imaging of pancreatic inflammation and its reversal in type 1 diabetes.

A major stumbling block for research on and treatment of type 1 diabetes is the inability to directly, but noninvasively, visualize the lymphocytic/inflammatory lesions in the pancreatic islets. One potential approach to surmounting this impediment is to exploit MRI of magnetic nanoparticles (MNP) to visualize changes in the microvasculature that invariably accompany inflammation. MNP-MRI did indeed detect vascular leakage in association with insulitis in murine models of type 1 diabetes, permitting noninvasive visualization of the inflammatory lesions in vivo in real time. We demonstrate, in proof-of-principle experiments, that this strategy allows one to predict, within 3 days of completing treatment with an anti-CD3 monoclonal antibody, which NOD mice with recent-onset diabetes are responding to therapy and may eventually be cured. Importantly, an essentially identical MNP-MRI strategy has previously been used with great success to image lymph node metastases in prostate cancer patients. This success strongly argues for rapid translation of these preclinical observations to prediction and/or stratification of type 1 diabetes and treatment of individuals with the disease; this would provide a crucially needed early predictor of response to therapy.

Animals↗

PubMed2005

Tomographic fluorescence mapping of tumor targets.

Methods that allow robust imaging of specific molecular targets and biological processes in vivo should have widespread applications in biology and clinical medicine. Here we use a quantitative, three-dimensional fluorescence-mediated tomographic technique (FMT) that enables rapid measurements of fluorochrome-based affinity tags in live xenograft models. We validate the method by showing its sensitivity in quantitating tumor angiogenesis and therapeutic modulation using an anti-vascular endothelial growth factor antibody. Furthermore, we show the feasibility of simultaneous multichannel measurements of distinct biological phenomena such as receptor tyrosine kinase expression and angiogenesis. FMT measurements can be done serially, with short imaging times and within the same live animal. The described method should be valuable for rapidly profiling biological phenomena in vivo.

Animals↗

PubMed2005

Detection of peroxidase/H2O2-mediated oxidation with enhanced yellow fluorescent protein.

The ability to sense oxidative stress in live cells and organisms would have far-reaching implications for biotechnology, drug discovery, and potentially medical imaging. We hypothesized that tyrosine-containing fluorescent proteins could be used as switches for sensing oxidative stress, based on their sensitivity to environmental and structural variations. We therefore tested purified EGFP, EYFP, ECFP, and DsRed proteins against the heme-peroxidase/H(2)O(2) reaction. We found that peroxidase-mediated oxidation resulted in up to 99.5% quenching of EYFP fluorescence (but not that of other fluorescent proteins) in a dose-dependent manner. Western blotting revealed inter- and intramolecular cross-linking. The observed detection limit for hydrogen peroxide was approximately 100 nM, well below the extracellular levels previously reported to occur in mammalian tissue during signaling. Combined expression of EYFP (quenchable) and ECFP or EGFP (nonquenchable) is expected to allow sensitive monitoring of oxidative stress.

Bacterial Proteins↗

PubMed2005

DTPA-bisamide-based MR sensor agents for peroxidase imaging.

[reaction: see text] The synthesis and some properties of two novel DTPA-bisamides are reported. These derivatives were designed as enzyme-activated contrast agents (CA) for magnetic resonance imaging. Both derivatives bear tyramido or 5-hydroxytryptamido groups that could be oligomerized in situ in the presence of peroxidase/H(2)O(2) pair resulting in a net increase in longitudinal (R1) relaxivity.

Contrast Media↗

PubMed2005

Near-infrared fluorescent imaging of matrix metalloproteinase activity after myocardial infarction.

BACKGROUND: We used a molecular probe activated by protease cleavage to image expression of matrix metalloproteinases (MMPs) in the heart after myocardial infarction. METHODS AND RESULTS: We synthesized and characterized a near-infrared fluorescent (NIRF) probe that is activated by proteolytic cleavage by MMP2 and MMP9. The NIRF probe was injected into mice at various time points up to 4 weeks after myocardial infarction induced by ligation of the left anterior descending coronary artery. NIRF imaging of MMP activity increased in the infarct region, with maximal expression at 1 to 2 weeks, persisting to 4 weeks. Zymography and real-time polymerase chain reaction analysis showed that MMP9 expression is increased at 2 to 4 days, and MMP2 expression is increased at 1 to 2 weeks. Dual-label confocal microscopy showed colocalization of NIRF imaging with neutrophils on day 2, and flow cytometric analysis confirmed that NIRF signal is associated with leukocytes in the infarct zone. CONCLUSIONS: This study demonstrates that the activity of MMPs in the myocardium may be imaged by use of specific activity-dependent molecular probes.

Animals↗

PubMed2005

Optical zymography for specific detection of urokinase plasminogen activator activity in biological samples.

Zymography techniques are routinely used to quantify proteolytic activity. In the current study, we describe an optical zymographic procedure that specifically detects urokinase-type plasminogen activator (uPA) activity in biological samples. The method employs a synthetic polymeric uPA fluorescent probe, which is copolymerized in sodium dodecyl sulfate (SDS)-polyacrylamide gel. Following electrophoresis and renaturation, enzymatic digestions of the substrate in 50 mM of Tris buffer at pH 7.4 generates fluorescence emission at 695 nm. The enzymatic activities can be analyzed directly by conventional gel imaging systems with a detection limit of 40 pg. This protocol is fast (hours) and does not require staining and destaining steps. The procedure is independent of plasminogen and, therefore, can efficiently distinguish the active two-chain uPA from its proenzyme. Densitometry analysis demonstrated a highly correlative relationship (r2=0.999) between the amount of uPA (over the range of 0.1-8.0 ng) and the average intensity of the fluorescent band. We were able to directly measure uPA activities in different cancer cell lines. This newly developed technique could be expanded to nearly all proteases, including the ones that cannot be analyzed by traditional zymography.

Caseins↗

PubMed2005

Molecular imaging in the clinical arena.

Molecular imaging is an emerging field that aims to integrate patient-specific and disease-specific molecular information with traditional anatomical imaging readouts. The information provided by this field may ultimately allow for noninvasive or minimally invasive molecular diagnostic capabilities, better clinical risk stratification, more optimal selection of disease therapy, and improved assessment of treatment efficacy. In this update, we first provide an overview of clinically relevant molecular imaging technologies and imaging agents. Next, their applications to disease detection, drug discovery, and biomedical research are discussed. To specifically demonstrate the potential of molecular imaging, we highlight recent advances in clinical and preclinical molecular imaging of cancer and atherosclerosis.

Biotechnology↗

PubMed2005

Method of determining nanoparticle core weight.

Polymer-coated metal or metal oxide nanoparticles have a variety of uses in industry, biological research, and medicine. Characterization of nanoparticles often includes determination of the dimensions of the electron-dense core by transmission electron microscopy (TEM), with the weight of the core determined from core volume and core density. However, TEM is labor intensive, has a long turnaround time, and uses equipment that is sometimes not readily available. Here we present an alternative method for determining the weight of nanoparticle cores termed the viscosity/light scattering method, which uses (i) measurements of viscosity over a wide concentration range to obtain the partial specific volume, (ii) measurements of particle diameter by light scattering, to obtain the volume of an individual particle, and (iii) the concentration of nanoparticles (w/v). We have applied this method to determine the weights of nanoparticle cores (iron of amino-CLIO and ferritin), the weights of globular proteins (molecular weight of IgG and albumin), and the weight of polystyrene microspheres. The viscosity/light scattering method is nondestructive of the sample and can be performed with a variety of materials on a routine basis.

Albumins↗

PubMed2005

Arthritis imaging using a near-infrared fluorescence folate-targeted probe.

A recently developed near-infrared fluorescence-labeled folate probe (NIR2-folate) was tested for in vivo imaging of arthritis using a lipopolysaccharide intra-articular injection model and a KRN transgenic mice serum induction mouse model. In the lipopolysaccharide injection model, the fluorescence signal intensity of NIR2-folate (n = 12) and of free NIR2 (n = 5) was compared between lipopolysaccharide-treated and control joints. The fluorescence signal intensity of the NIR2-folate probe at the inflammatory joints was found to be significantly higher than the control normal joints (up to 2.3-fold, P < 0.001). The NIR2-free dye injection group showed a persistent lower enhancement ratio than the NIR2-folate probe injection group. Excessive folic acid was also given to demonstrate a competitive effect with the NIR2-folate. In the KRN serum transfer model (n = 4), NIR2-folate was applied at different time points after serum transfer, and the inflamed joints could be detected as early as 30 hours after arthritogenic antibody transfer (1.8-fold increase in signal intensity). Fluorescence microscopy, histology, and immunohistochemistry validated the optical imaging results. We conclude that in vivo arthritis detection was feasible using a folate-targeted near-infrared fluorescence probe. This receptor-targeted imaging method may facilitate improved arthritis diagnosis and early assessment of the disease progress by providing an in vivo characterization of active macrophage status in inflammatory joint diseases.

Animals↗

PubMed2005

Detection of vascular adhesion molecule-1 expression using a novel multimodal nanoparticle.

Endothelial vascular adhesion molecule-1 (VCAM-1) is a critical component of the leukocyte-endothelial adhesion cascade, and its strict temporal and spatial regulation make it an ideal target for imaging and therapy. The goal of this study was to develop novel VCAM-1-targeted imaging agents detectable by MRI and fluorescence imaging using phage display-derived peptide sequences and multimodal nanoparticles (NPs). We hypothesized that VCAM-1-mediated cell internalization of phage display-selected peptides could be harnessed as an amplification strategy to chaperone and trap imaging agents inside VCAM-1-expressing cells, thus improving target-to-background ratios. To accomplish our goal, iterative phage display was performed on murine endothelium under physiological flow conditions to identify a family of VCAM-1-mediated cell-internalizing peptides. One specific sequence, containing the VHSPNKK motif that has homology to the alpha-chain of very late antigen (a known ligand for VCAM-1), was shown to bind VCAM-1 and block leukocyte-endothelial interactions. Compared with VCAM-1 monoclonal antibody, the peptide showed 12-fold higher target-to-background ratios. A VHSPNKK-modified magnetofluorescent NP (VNP) showed high affinity for endothelial cells expressing VCAM-1 but surprisingly low affinity for macrophages. In contrast, a control NP without VCAM-1-targeting sequences showed no affinity for endothelial cells. In vivo, VNP successfully identified VCAM-1-expressing endothelial cells in a murine tumor necrosis factor-alpha-induced inflammatory model and colocalized with VCAM-1-expressing cells in atherosclerotic lesions present in cholesterol-fed apolipoprotein E apoE-/- mice. These results indicate that: (1) small peptide sequences can significantly alter targeting of NPs, (2) the used amplification strategy of internalization results in high target-to-background ratios, and (3) this technology is useful for in vivo imaging of endothelial markers.

Animals↗