Christoph Bremer

Fluorescence molecular tomography resolves protease activity in vivo.

Systematic efforts are under way to develop novel technologies that would allow molecular sensing in intact organisms in vivo. Using near-infrared fluorescent molecular beacons and inversion techniques that take into account the diffuse nature of photon propagation in tissue, we were able to obtain three-dimensional in vivo images of a protease in orthopic gliomas. We demonstrate that enzyme-activatable fluorochromes can be detected with high positional accuracy in deep tissues, that molecular specificities of different beacons towards enzymes can be resolved and that tomography of beacon activation is linearly related to enzyme concentration. The tomographic imaging method offers a range of new capabilities for studying biological function; for example, identifying molecular-expression patterns by multispectral imaging or continuously monitoring the efficacy of therapeutic drugs.

Fluorescence imaging with near-infrared light: new technological advances that enable in vivo molecular imaging.

Abstract. A recent development in biomedical imaging is the non-invasive mapping of molecular events in intact tissues using fluorescence. Underpinning to this development is the discovery of bio-compatible, specific fluorescent probes and proteins and the development of highly sensitive imaging technologies for in vivo fluorescent detection. Of particular interest are fluorochromes that emit in the near infrared (NIR), a spectral window, whereas hemoglobin and water absorb minimally so as to allow photons to penetrate for several centimetres in tissue. In this review article we concentrate on optical imaging technologies used for non-invasive imaging of the distribution of such probes. We illuminate the advantages and limitations of simple photographic methods and turn our attention to fluorescence-mediated molecular tomography (FMT), a technique that can three-dimensionally image gene expression by resolving fluorescence activation in deep tissues. We describe theoretical specifics, and we provide insight into its in vivo capacity and the sensitivity achieved. Finally, we discuss its clinical feasibility.

In vivo molecular target assessment of matrix metalloproteinase inhibition

A number of different matrix metalloproteinase (MMP) inhibitors have been developed as cytostatic and anti-angiogenic agents and are currently in clinical testing. One major hurdle in assessing the efficacy of such drugs has been the inability to sense or image anti-proteinase activity directly and non-invasively in vivo. We show here that novel, biocompatible near-infrared fluorogenic MMP substrates can be used as activatable reporter probes to sense MMP activity in intact tumors in nude mice. Moreover, we show for the first time that the effect of MMP inhibition can be directly imaged using this approach within hours after initiation of treatment using the potent MMP inhibitor, prinomastat (AG3340). The developed probes, together with novel near-infrared fluorescence imaging technology will enable the detailed analysis of a number of proteinases critical for advancing the therapeutic use of clinical proteinase inhibitors.

In vivo tomographic imaging of near-infrared fluorescent probes.

Fluorescence imaging is increasingly used to probe protein function and gene expression in live animals. This technology could enhance the study of pathogenesis, drug development, and therapeutic intervention. In this article, we focus on three-dimensional fluorescence observations using fluorescence-mediated molecular tomography (FMT), a novel imaging technique that can resolve molecular function in deep tissues by reconstructing fluorescent probe distributions in vivo. We have compared FMT findings with conventional fluorescence reflectance imaging (FRI) to study protease function in nude mice with subsurface implanted tumors. This validation of FMT with FRI demonstrated the spatial congruence of fluorochrome activation as determined by the two techniques.

Oligomerization of Paramagnetic Substrates Result in Signal Amplification and can be Used for MR Imaging of Molecular Targets

Magnetic resonance imaging (MRI) has evolved into a sophisticated, noninvasive imaging modality capable of high-resolution anatomical and functional characterization of transgenic animals. To expan...

Optical imaging of spontaneous breast tumors using protease sensing 'smart' optical probes.

Objective:The objective of this study was to determine if spontaneous breast cancer lesions can be detected by fluorescence reflectance imaging (FRI) and fluorescence mediated tomography (FMT) using protease-sensing optical probes. Materials and Methods:Transgenic (FVB/N-TgN (WapHRAS)69Lin YSJL)) mice, which spontaneously develop breast cancer, were injected intravenously with a cathepsin-sensing fluorescent imaging probe. FRI and FMT were performed 24 hours after probe injection and region of interest (ROI) analysis was performed. Magnetic resonance images were acquired for anatomic coregistration with the FMT data. Moreover, correlative immunohistochemistry and fluorescence microscopy were performed. Results:All tumor nodules were clearly delineated by FRI showing an average signal intensity of 380 ± 106 AU. Similarly, tumors were clearly detected by FMT imaging. Immunohistochemistry confirmed cathepsin-B expression of primary tumors and fluorescence microscopy revealed a strong Cy 5.5 deposition in the tissue. Conclusions:FRI and FMT using “smart” protease sensing probes permits detection of experimental spontaneous breast cancers. Because the expression levels of various proteases correlate with patient outcome, this technique may not only help to detect, but also to differentiate breast cancers noninvasively.

Detection of lymph node metastases by contrast‐enhanced MRI in an experimental model

Lymph node size, the accumulation of a nodal lymphotrophic contrast agent (LCDIO), and MRI were compared as methods for detecting nodal metastases in an experimental murine model. Lymph node metastases (B16‐F1 melanoma expressing green fluorescent protein (GFP) and C57BL/6 mice) were generated to obtain a wide spectrum of nodes, including normal nodes and nodes bearing micrometastases, small metastases, or large metastases. Nodal uptake of LCDIO was measured using 111Indium‐labeled LCDIO and was found to be lower in micrometastastic nodes (4.20 ± 1.4%ID/gm) than in normal nodes (8.60 ± 0.22% ID dose/gram, P < 0.005). Nodal tumor burden was quantified from the amount of GFP present in nodes measured using the Western blot method, and was found to correlate with the decrease of LCDIO uptake. By MRI, nodes bearing small and large metastases contained regions of high signal intensity (SI) that corresponded to the visual pattern of tumor in nodes. Micrometastatic nodes were distinguishable from normal nodes based on a diffuse pattern of inhomogeneous SI. The signal‐to‐background ratio (SBR) of normal nodes (0.0112 ± 0.0061) was different from micrometastatic nodes (0.179 ± 0.080, P < 0.00046) and nodes bearing small metastases (0.723 ± 0.269, P < 0.00013), with high degrees of significance. Magn Reson Med 47:292–297, 2002.

In Vivo Imaging of Gene Expression

With the ability to readily engineer genes, create knock-in and knock-out models of human disease, and replace and insert genes in clinical trials of gene therapy, it has become clear that imaging will play a critical role in these fields. Imaging is particularly helpful in recording temporal and spatial resolution of gene expression in vivo, determining vector distribution, and, ultimately, understanding endogenous gene expression during disease development. While endeavors are under way to image targets ranging from DNA to entire phenotypes in vivo, this short review focuses on in vivo imaging of gene expression with magnetic resonance and optical techniques.

Molecular imaging of MMP expression and therapeutic MMP inhibition.

Matrix metalloproteinase (MMP), a family of proteolytic enzymes, are critically involved in tumor progression and angiogenesis (1). A number of MMP-inhibitors have been developed as cytostatic and antiangiogenic agents. Direct imaging of enzyme activity has only recently been proposed using near infrared fluorescence (NIRF) imaging and enzyme-sensitive optical probes (2). The purpose of this study was to: (a) develop an imaging approach to visualize matrix metalloproteinase-2 (MMP-2) activity in vivo and (b) apply this approach to monitor therapeutic MMP-inhibition in vivo.

Near-infrared fluorescence imaging of lymph nodes using a new enzyme sensing activatable macromolecular optical probe

The aim of this study was to validate the use of near infrared fluorescence imaging (NIRF) using enzyme-sensitive optical probes for lymph node detection. An optical contrast probe that is activated by cystein proteases, such as cathepsin B, was used to visualize lymph nodes by NIRF reflectance imaging. In order to quantitate the uptake of the optical probe in lymphatic tissue, the biodistribution was assessed using the Indium-111 labeled optical probe. Sixteen Balb-c mice were injected either intravenously (i.v.) or subcutaneously (s.c.) with the NIRF-probe (2xa0μmol cyanine (Cy)/animal; i.v., n=10; s.c., n=6) and imaged 24xa0h after injection. Signal intensities and target-to-background ratios of various lymph nodes were measured by manual regions of interest (ROIs). Additional signal intensity measurements were performed of excised lymph nodes (n=21) from i.v. injected mice (24xa0h after injection) and compared with excised lymph nodes (n=8) of non-injected mice. The probe employed in this study was lymphotropic with approximately 3–4% accumulation in lymph nodes (3.4±0.8% ID/g). Measurements of the excised lymph nodes (after i.v. injection) confirmed a significant increase in lymph node fluorescence signal from baseline 26±7.6 arbitary units (AU) to 146±10.9xa0AU (p<0.0001). A significant increase in lymph node fluorescence signal was also seen in vivo throughout the body after i.v. injection (96±7.8xa0AU) and/or regionally after s.c. injection (141±11.5xa0AU) in comparison with baseline autofluorescence (26±7.6xa0AU). Target-to-background ratio was significantly higher after s.c. injection (6.6%±0.81) compared with i.v. injection (4.8±0.67%). Detection and visualization of lymph nodes is feasible by NIRF imaging using a cystein-protease sensitive optical probe.