Abdominal Imaging

, Volume 30, Issue 3, pp 343–355

MR and optical approaches to molecular imaging

Authors

  • T. Persigehl
    • Department of Clinical RadiologyUniversity Hospital Muenster
  • W. Heindel
    • Department of Clinical RadiologyUniversity Hospital Muenster
    • Department of Clinical RadiologyUniversity Hospital Muenster
    • Interdisciplinary Center for Clinical Research Muenster (IZKF Muenster)
Invited update

DOI: 10.1007/s00261-004-0230-3

Cite this article as:
Persigehl, T., Heindel, W. & Bremer, C. Abdom Imaging (2005) 30: 343. doi:10.1007/s00261-004-0230-3

Abstract

With an increasing understanding of the molecular basis of disease, various new imaging targets have recently been defined that potentially allow for an early, sensitive, and specific diagnosis of disease or monitoring of treatment response. Different approaches to depict these molecular structures in vivo are currently being explored by the molecular imaging community. We briefly review methodologies for molecular imaging by magnetic resonance imaging and optical methods. Special emphasis is put on different contrast agent designs (e.g., targeted and smart probes). New technical developments in optical imaging are briefly discussed. In addition, current research results are put into a clinical perspective to elucidate the potential merits one might expect from this new research field.

Keywords

Molecular imagingMagnetic resonance imagingOptical imagingContrast mediaAmplification strategySmart probesAbdominal imagingReview

Molecular imaging is a rapidly evolving field. New findings in molecular research will increase our understanding of the cellular and molecular pathogenesis of many diseases. New disease-specific targets will be unraveled by modern molecular high-throughput techniques such as gene chip technology, genomics, and proteomics. Research efforts by the imaging community combined with those by basic scientists are underway to translate this knowledge into highly specific, noninvasive imaging techniques, commonly referred to as molecular imaging. A comprehensive definition of this research field was given by Weissleder et al. in 2001 [1]: “Molecular imaging is a growing research discipline aimed at developing and testing novel tools, reagents, and methods to image specific molecular pathways in vivo, particularly those that are key targets in disease processes.” A broader understanding of the term molecular imaging also would comprise approaches to visualize physiologic tissue parameters such as tumor vascularization, which is also referred to as parametric imaging.

This review presents an overview on magnetic resonance (MR) and optical techniques for molecular imaging. Further, the potential of future applications for abdominal imaging are discussed.

Contrast agents

MR and optical contrast agents can basically be divided into nonspecific, targeted, and smart probes (Fig. 1) [2]. Nonspecific contrast agents such as gadolinium chelates and iodinated contrast media are widely available for clinical use. They show a nonspecific distribution pattern that allows measurement of tissue perfusion, vascular permeability, or vascular volume in a given voxel. These parameters can be extracted by fast imaging techniques and pharmacologic modeling or steady-state imaging techniques [3, 4]. However, true molecular targets can not be displayed with this approach.
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Figure 1

Design of different optical contrast agents. Whereas nonspecific MRI and optical contrast agents (A) show simple perfusion and/or permeability properties of the tissue, (B) targeted probes bind by specific ligands to protein structures on the cell surface (e.g., tumor associated receptors). C Smart probes are activated by a specific target interaction (e.g., enzymatic conversion). Optical smart probes show very little to no signal in their native state (left) and become brightly fluorescent after enzymatic cleavage (right). Tumor-associated proteases can be sensitively detected with this technology. For MRI smart probes have been described which significantly alter their T1 or T2 relaxation properties upon target interaction. (Reprinted with permission from Bremer et al. [2])

Targeted contrast agents are linked to specific affinity ligands such as peptides, antibody fragments, or small molecules imparting molecular specificity to the probe. In the early phase after injection, a high level of unbound circulating contrast medium contributes to the background signal, resulting in a slight decrease of the signal-to-noise ratio (SNR). Combining efficient targeting strategies with sensitive imaging techniques such as optical imaging helps to resolve molecular targets in the nanomolar range in vivo. Thus, the detection of cell surface proteins (e.g., tumor associated receptors) is feasible with this approach (Fig. 1).

Smart contrast agents change their signal characteristics upon interaction with the specific target. Ideally, they exhibit a strong signal alteration upon target interaction, resulting in an “off” and “on” status. This makes them ideal candidates for molecular imaging because they provide the highest SNR for molecular target identification. However, in general, probe design and synthesis are more complex, so that human applications of these probes may less likely be expected within the nearer future.

MR imaging

MR imaging (MRI) is one of the most versatile imaging techniques for clinical and experimental in vivo imaging. MRI offers high three-dimensional spatial resolution down to the 10-μm range [5, 6], complete body coverage, and the opportunity to determine additional physiologic parameters noninvasively (e.g., blood flow, perfusion, and diffusion). MRI has considerable potential for molecular imaging. However, sensitivity of MRI to depict small (or “molecular”) substrates is constrained by the omnipresence of protons in the body, resulting in a high background and, hence, lower SNR. Currently, MRI is limited to detecting approximately micromolar concentrations of an imaging reporter within a given voxel (Fig. 2). This is about three to six orders of magnitude lower than the sensitivity of optical imaging for detection of fluorochromes in vivo (Fig. 2). Strong amplification strategies using smart contrast agents are therefore required to yield a higher sensitivity [7]. More recently, other compounds such as carbon or fluorine have been explored for MR molecular imaging, which may exhibit significantly higher SNR [8, 9].
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Figure 2

Potential of different imaging techniques for molecular imaging. Due to their high SNRs, optical imaging and nuclear imaging techniques can detect molecular structures in picomolar (10−12) concentrations within a given voxel. This is about three to six orders of magnitude more sensitive than currently available MRI techniques. However, innovative MR contrast agents may change MRI into a truly molecular imaging modality. (Modified from Weissleder and Mahmood [1])

Nonspecific MR contrast agents for parametric imaging

Nonspecific MR contrast agents, based on their specific distribution patterns in vivo, allow measurement of physiologic tissue parameters (e.g., vascular permeability, blood flow, and blood volume) by MRI. Small, extracellular contrast agents are clinically approved and have been explored for fast kinetic analysis in vivo to measure vascular permeability [10, 11]. However, low-molecular-weight contrast agents are poorly suited to characterize tumor microvessels because the fraction of the contrast medium that diffuses transendothelially, even in normal tissues, at each circulatory pass varies widely, from 20% to 80% [12, 13]. High transendothelial diffusion makes the estimation of vascular volume problematic; more importantly, high transendothelial permeability of the agents can be observed in normal and tumorous microvessels. Thus, differentiation of benign from malignant tissues is problematic.

Macromolecular “blood pool” contrast agents with a lower first-pass extravasation in normal vessels have been shown in experimental studies to be advantageous for the determination of vascular permeability in vivo [12, 13]. However, macromolecular T1-based MR blood pool contrast agents are not clinically available. Ultrasmall particles of iron oxide (USPIOs) represent one class of clinically approved (or in phase III clinical trials) contrast media that exhibit a long blood half-life and, hence, significant blood pool effect. USPIOs recently have been explored successfully for measuring vascular permeability in vivo by kinetic analysis of dynamic T1-weighted fast gradient echo sequence [14]. A different approach has been explored by using USPIOs and steady-state imaging techniques [4]. Assuming a mainly intravascular distribution during the MR measurement, high-resolution maps of contrast-induced susceptibility changes can be obtained, which closely correlate with surrogate markers of tumor angiogenesis such as microvessel density or vascular endothelial growth factor expression (Fig. 3) [4].
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Figure 3

Nonspecific MR contrast agents for steady-state imaging of tumor vascularization. MR images of four tumors (9L, DU4475, HT 1080, and EOMA) with significantly different microvessel density as a surrogate parameter for tumor angiogenesis. Top Grey scale-coded vascular volume fraction (VVF) maps of contrast-induced susceptibility changes (ΔR2*) using a USPIO. Note the heterogeneity of the VVF among the different tumor models. Bottom Different tumors in T1-weighted spin-echo MR images. MR-derived VVF maps correlated well with different microvessel densities and vascular endothelial growth factor expression of different tumor types. (Reprinted with permission from Bremer et al. [4])

The ultimate goal of developing these parametric imaging methods is to provide a clinical tool for tumor characterization and treatment monitoring. Measurements of vascular volume and vascular permeability after, for example, antiangiogenic therapy, may serve as an early indicator for evaluation of treatment effectiveness [15, 16, 17, 18]. Treatment with a vascular targeting agent that induces selective thrombosis in tumor neovasculature has resulted in a significant reduction of the vascular volume fractions when using USPIOs and steady-state imaging approaches as soon as 4 h after initiation of treatment, long before conventional phenotypic changes (size regression) occurred (unpublished data).

For more in-depth reading on angiogenesis imaging by MRI, the reader is referred to some excellent review articles [3, 12, 19, 20, 21].

Targeted MR contrast agents

Many molecular targets are overexpressed in tumors and can be targeted by attaching an affinity ligand to the MR reporter. For example, in apoptotic cells, the inner leaflet of the cell membrane, which contains negatively charged phosphatidylserines, is everted to the cell surface. Phosphatidylserines can be targeted with annexin V, which has a high binding affinity to these membrane compounds. After attaching annexin V to a supraparamagnetic iron oxide nanoparticle (cross-linked iron oxide), Schellenberger et al. were able to selectively visualize cells that undergo apoptosis [22]. This targeted contrast agent might be a strategy to detect apoptosis in vivo by providing an early surrogate marker for tumor response to chemotherapy. By using a cross-linked iron oxide attached to a high-affinity antibody, E-selectin expression on human endothelial cells could be visualized by MRI [23]. E-selectin is a key molecule that is overexpressed in tumor vascular proliferation, inflammation, angiogenesis, and atherosclerosis. Noninvasive detection of E-selectin expression might enhance the early diagnosis of these diseases.

Approaches to increase the sensitivity of MR with targeted contrast agents were presented by Wickline et al. who significantly amplified the number of reporter molecule per nanoparticle (up to 90,000 gadolinium chelates per nanoparticle). The nanoparticles can be targeted with high affinity to specific molecules via antibodies. This approach might be exploited for visualization of fibrin in vascular plaques and vascular expression of integrins (e.g., αv β3) in tumors (Fig. 4) [9, 24, 25, 26, 27, 28]. Other studies have reported targeted contrast agents for imaging Alzheimer amyloid plaques, the human transferrin receptor, and the secretin receptor or the endothelial integrin αv β3 [25, 29]. More recently, an antibody-conjugated gadolinium chelate was targeted to tumor-associated Her-2/neu receptors in a mouse xenograft model [30, 31].
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Figure 4

Fibrin-targeted MRI contrast agent. Fibrin-targeted and control carotid endarterectomy specimens in enhanced MRI show contrast enhancement (white areas) of a small fibrin deposit on symptomatic ruptured plaque. Targeting of the contrast agent was done by a specific antibody linked to a perfluorocarbon nanoparticle, which is loaded with multiple gadolinium chelates. (Reprinted with permission from Flacke et al. [24])

Smart MR contrast agents

Smart MR contrast agents (i.e., agents that can be activated) undergo conformational changes upon target interaction, which significantly alter their signal properties (e.g., shortening of T1 relaxation time).

Bogdanov et al. developed a MR signal amplification strategy based on oxidoreductase-mediated polymerization of paramagnetic monomer into oligomers of higher magnetic relaxivity [7]. The substrates consist of chelated gadolinium covalently bound to phenols, which serve as electron donators during enzymatic hydrogen peroxidase reduction of peroxidase. The converted monomers undergo rapid condensation into paramagnetic oligomers, leading to an increased T1 relaxation of about threefold (300%) signal increase. In vitro, this amplification strategy allows a sensitive detection of E-selectin expression, which is a marker for vascular endothelial proliferation, inflammation, angiogenesis, and atherosclerosis (see above). Other enzyme systems such as β-galactosidase, a frequently used marker enzyme in molecular biology, has been explored for smart MR contrasting [32]. In this system, water access to the chelated gadolinium is blocked by an enzymatically cleavable substrate (galactopyranoside). β-Galactosidase activity results in a decrease of T1 relaxation and thus a signal increase in T1-weighted images. The feasibility of detecting β-galactosidase expression in Xenopus laevis embryos could be demonstrated with this approach. Josephson et al. recently explored oligonucleotide-labeled iron oxides, which demonstrated a significant decrease in T2 relaxation time upon hybridization with a complementary oligonucleotide (magnetic relaxation switches) [33, 34, 35]. This system might be exploited to detect specific RNA fragments and to determine telomerase activity, which is frequently elevated in malignant tissue, in different human and murine samples [36].

Cell labeling

Due to its high spatial resolution capabilities and excellent soft tissue contrast, MRI is excellently suited for cell-tracking applications. Different labeling methods have been developed for various cell types using para- and supraparamagnetic contrast agents. In an experimental setting, as few as two cells per voxel in vitro and approximate three cells per voxel in vivo might be identified using MRI [37]. By labeling lymphocytes with paramagnetic iron oxide nanoparticles (cross-linked iron oxide), Moore et al. visualized autoimmune destruction of the insulin-producing pancreatic β-cells by insulin-dependent diabetes mellitus (type 1 diabetes) [38]. Monitoring of cellular autoimmune response in diabetes formation as an early parameter of disease progression may help to evaluate novel therapies. Visualization of macrophages with small particles of iron oxide has shown the feasibility of early detection of chronic allograft kidney rejection or active inflammatory reaction in arthritis [39, 40]. Labeling of antigen-specific cytotoxic T lymphocytes has been exploited by Kircher et al. to monitor treatment effectiveness of novel cell-based therapies (Fig. 5) [37]. In this study, MRI noninvasively depicted different homing sites of various lymphocyte populations after repetitive cell injections.
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Figure 5

High-resolution cell tracking in vivo using MRI. The homing of labeled, antigen-specific, cytotoxic T lymphocytes can be visualized by MRI. Time course of cross-linked iron oxide (CLIO) HD labeled CD8+ T cells selectively homing to B16-OVA tumors. Serial MRI was performed after adoptive transfer into a mouse carrying B16F0 (left) and B16-OVA (right) melanomas. AD Axial slices through the mouse thighs shown in A before adoptive transfer of CLIO-HD–labeled OT-I CD8+ T cells. EI Three-dimensional grey-scaled reconstructions of the melanomas (E) 0 h, (F) 12 h, (G) 16 h, and (H, I) 36 h after adoptive transfer. Numbers of cells per voxel are black and white coded as shown in the scale. (J) Axial, (K) sagittal, and (L) coronal plane slices through the three-dimensional reconstruction. (Reprinted with permission from Kircher et al. [37])

In addition, cell labeling is an excellent tool to monitor noninvasively homing of stem cell populations in vivo. Hematopoietic stem cell homing has been labeled by various para- and superparamagnetic contrast agents [41, 42]. Even single CD34+ cells were visualized homing into the bone marrow when using this approach [43]. Hill et al. visualized mesenchymal stem cell infiltration into fresh infarcted myocardium [44]. The currently available tagging methods usually result in an endosomal label deposition, which allows stable label retention over time. Thus, longitudinal studies over several days can be performed with magnetically labeled cells. The interhemispheric migration of oligodendroglial progenitor cells (neuronal stem cell), for instance, was followed for 6 weeks after transplantation in the ventricle into brain parenchyma in dysmyelinated rats [45, 46]. Cell tracking could become an important tool for monitoring clinical stem cell transplantation and localization of specific cell populations (lymphocytes and macrophages) within the next few years.

Optical imaging

With strong advances in laser technology, mathematical modeling of photon propagation in tissue, and the development of new optical contrast agents operating in the near infrared range (NIR; 650 to 900 nm), molecular optical imaging is a new, emerging imaging tool. In the NIR, very little tissue autofluorescence is present, which offers excellent SNRs and thus very high sensitivity to detect molecular structures (such as cell receptors). Optical imaging techniques allow delineation of structures in the picomolar (10−12) range, which is comparable to conventional nuclear imaging techniques and is about three to six orders of magnitude more sensitive than MRI (Fig. 2). Aside from high SNRs, imaging in the NIR shows very efficient tissue penetration because the absorption by water and hemoglobin is relatively low (diagnostic window; Fig. 6). Moreover, different optical analytic techniques have been developed in molecular biology to probe tissue samples, cells, or cell compounds (e.g., fluorescent proteins and fluorescently labeled antibodies). Some of these approaches can be modified for in vivo applications.
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Figure 6

Photon absorption and tissue autofluorescence in the visible and NIR light spectrum. In the NIR, tissue autofluorescence is minimal, whereas tissue penetration of the photons is optimal (diagnostic window). Low tissue autofluorescence yields high SNRs in optical imaging techniques. Cyanine dyes are frequently used optical contrast agents that can be tuned to absorb and emit in the NIR. Note the diagnostic window in the NIR of 650 to 950 nm. (Reprinted with the permission from Weissleder and Ntziachristos [52])

Optical imaging techniques

Different optical imaging techniques have been developed for experimental applications. Simple reflectance imaging methods (fluorescence reflecting imaging) deliver surface-weighted images of emitted photons from superficial tissue structures. The advantages of this method are rapid data acquisition and no time-consuming data reconstruction. However, reflectance imaging applications are confined to superficially located structures (≤10 mm depth). New hardware developments, including sensitive charge couple device cameras and efficient laser sources combined with novel mathematic modeling, have paved to way to three-dimensional optical imaging techniques such as fluorescence-mediated tomography, which allows three-dimensional quantitative imaging of photon absorption of tissue fluorescence in vivo (Fig. 7) [47, 48, 49, 50]. This technique has been up scaled for human use, and the feasibility of detecting optical signatures in a clinical setting has been shown [51].
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Figure 7

Fluorescence-mediated tomography (FMT) of cathepsin-B expression levels in a 9L gliosarcoma. (A) Axial and (B) sagittal contrast-enhanced MR sections of a nude mouse implanted with a 9L gliosarcoma. FMT was acquired after intravenous injection of a cathepsin-B–sensitive smart contrast agent. CF Consecutive FMT slices obtained from the top to bottom from the volume of interest shown in B by thin white horizontal lines. Superposition (D) of the MR axial slice passing through the tumor (A) onto the corresponding FMT slice (C). Whereas MR provides detailed anatomic information, FMT shows molecular information of the tumor nodule (i.e., cathepsin-B expression levels). (Reprinted with permission from Nztiachristos et al. [50])

Optical contrast agents

Parallel to these technical advances, a whole range of novel optical contrasting strategies are currently under development. As for MRI, various contrasting strategies can be pursued to obtain optical information from the tissue. Optical contrast agents for in vivo purposes are ideally flourochromes with excitation and emission maxima in the NIR of 650 to 950 nm (see above). In that regard, cyanine dyes represent one of the most prominent classes of optical contrast agents for in vivo imaging [52]. More recently, inorganic quantum dots (fluorescent semiconductor nanocrystals) have been introduced as a novel class of optical contrast agents that can be exactly tuned to a specific wavelength by size variation [53, 54, 55]. The unique property of quantum dots is a broad absorption band, relatively narrow and symmetric luminescence band, and high resistance to photo degeneration. Biological applications of the novel contrast agents are currently under investigation.

Nonspecific contrast agents

As outlined for unspecific MR-contrast media, nonspecific optical contrast agents such as indocyanine green (ICG) can depict tumor physiology such as perfusion, vessel permeability, and tissue blood volume. ICG was successfully applied in a clinical study examining various breast lesions by gadolinium-enhanced MRI and ICG-enhanced optical tomography. In this patient population, ICG accumulation within the tumors was successfully resolved by optical tomography [51]. In another study by Intes et al., differentiation between benign and malignant breast tumors was shown with ICG [56]. A second generation of nonspecific optical contrast agents is currently under development. The contrasting mechanism of these substances is based on the permeability of tumor vessels that leads to an accumulation of fluorescent dyes in the tumor interstitium, which can be detected in absorption and fluorescence modes of novel optical tomography systems [57].

Targeted optical contrast agents

The approaches to modify optical contrast agents for tissue specificity are similar to those described for MRI and include linking large molecules such as antibodies or antibody fragments or small peptide derivatives to the fluorochrome. Targeted optical contrast agents have successfully been developed for receptors that are overexpressed on the extracellular matrix of tumor cells (e.g., bombesin and somatostatin). By conjugation of fluorescein and carbocyanine dyes to somatostatin and bombesin receptor-avid peptides, selective visualization of somatostatin- and bombesin-positive tumors in rodents can be performed (Fig. 8 [58, 59]). Similarly, fluorochromes can be tagged with vasoactive intestinal peptide or folic acid for the detection of tumors [60, 61]. Affinity ligands such as annexin V, a protein used for the detection of apoptosis based on binding affinity to phosphatidylserine (see above), could be conjugated to a near-infrared fluorescent (NIRF) dye (Cy-5.5). The annexin V conjugate showed a high affinity to apoptotic cells, resulting in a 41-fold higher fluorescence signal for apoptotic as opposed to nonapoptotic cells in flow cytometry. Because apoptosis is frequently one of the earliest events in tumor response to therapy, this dye may allow evaluation of therapy response in vivo sooner than conventional parameters such as tumor perfusion or size would show any significant effects [62]. In addition to tumor-selective targets, imaging of markers associated with inflammation has been described. For example, E-selectin is an early proinflammatory marker. Targeting of polyamino acid-based protected copolymers as a carrier of an NIRF-indocyanine covalently with a monoclonal antibody fragment allowed specific detection of E-selectin expression on human endothelial cells activated with interleukin-1β (20- to 30-fold over nonspecific uptake) [63].
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Figure 8

Somatostatin receptor-targeted optical contrast agent. In vivo fluorescence images of tumor-bearing mice (RIN38; pancreatic carcinoma expressing the somatostatin receptor subtype 2) (A, B) before and (C, D) 6 h after injection of a dye-peptide conjugate. C The receptor-binding indotricarbocyanine-octreotate conjugate leads to a significantly elevated tumor signal. D A control conjugate with no receptor affinity does not generate contrast. (Reprinted with the permission from Becker et al. [59])

Synthesis of fluorescent bisphosphonate derivatives (e.g., PAM 78) is a different strategy for specification of optical contrast agents. PAM 78 exhibits specific binding to hydroxylapatite, which allows detection of osteoblastic activity in vivo with an optical imager [64]. This imaging approach might be useful for measuring osteoblastic activity or to monitor skeletal development in vivo (Fig. 9). Ke et al. presented an epidermal growth factor and Cy-5.5 conjugate that selectively visualized breast cancers positive for epidermal growth factor in vivo [65].
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Figure 9

Optical imaging of osteoblastic activity. In vivo NIR fluorescence imaging of osteoblastic activity using a fluorescent bisphosphonate derivative (PAM 78) shows a specific binding to hydroxylapatite. NIR fluorescent imaging of mice imaged 6 h after injection of PAM 78. Left There is greater definition of all bony structures identified in the dorsal image. Right The ventral image permits visualization of the maxilla (Mx), mandible (Ma), sternum (St), bladder (B), and the knee (Kn). Middle Same animal, immediately after death, with the skin removed. The sinuses (Si), scapula (Sc), and the elbow (E) are now visible, and all other bony structures have better resolution. (Reprinted with permission from Zaheer et al. [64])

Smart optical contrast agents

In 1999, the first autoquench fluorescent probe was developed by Weissleder et al., which is converted from a nonfluorescent to a fluorescent state by proteolytic activation [66]. The first generation of this smart contrast agent consists of a long, circulating, carrier molecule conjugated to 12 to 14 cyanine dyes, resulting in a signal quench due to fluorescence resonance energy transfer. Thus, in its native state, the molecule exhibits very little to no fluorescence; after enzymatic cleavage, a strong fluorescence signal increase (up to several 100-fold) can be detected. Inhibition experiments have shown that this first generation of protease-sensing optical probes is activated mainly by lysosomal cysteine or serine proteases such as cathepsin-B. After injection of a cathepsin reporter probe, intestinal adenomas became highly fluorescent as a indication of high cathepsin-B enzyme activity (Fig. 10) [66]. By insertion of enzyme-specific peptide stalks between the carrier and the fluorochromes, the specificity could be changed to other enzymes such as cathepsin-D, matrix metalloproteinase-2, or thrombin [66, 67, 68]. Smart contrast probes have since been applied to study cancer progression, tumor metastasis, atherosclerosis, thrombosis, and inflammation [66, 67, 68, 69, 70]. The major advantage of these probes is the high SNR, which is achieved by background suppression (quenching) and high local activation (repetitive enzymatic cleavage) by the targeted enzyme system.
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Figure 10

Detection of dysplastic jejunal adenomas with NIRF imaging using protease-sensing probes. (A, B) Light images and (C, D) NIRF images of jejunal adenomas from 6-month-old ApcMin+ mice serve as an experimental model for familial adenomatosis polyposis. Noninjected ApcMin+ mouse (A, C) and a 6-month-old ApcMin+ mouse injected with the NIRF probe activated by cathepsin-B (B, D). In the noninjected mouse, multiple adenomas can be seen on the light image (A) but show essentially no autofluorescence (C). With injection of the NIRF probe, the adenomas are clearly identified by NIRF imaging (D scale bar = 5 mm). Even adenomas as small as 50 μm can be visualized with this technique. (Reprinted with permission from Marten et al. [69])

Optical tools for imaging gene expression

Optical imaging marker genes (IMGs) have for many years been used in different in vitro assays for studying, for instance, cellular gene regulation. Generally speaking, IMGs encode for a transcriptional product (fluorescent protein or luciferase) that can be detected by optical methods (e.g., microscopy, reflectance imaging, and optical tomography) [71, 72]. IMGs can be linked to genes of interest so that the coexpression of both genes occurs and the gene regulation of the gene of interest can be visualized. A prominent example of an IMG is the green fluorescent protein, which has been cloned from various organisms such as the jellyfish Aequoria victoria (Fig. 6) [73, 74]. HcRed, a protein with a right-shift absorption and emission spectrum and, hence, better tissue penetration (see above), was isolated from the reef coral Heteractis crispa (588-nm absorption and 618-nm emission). In addition to monitoring of gene expression, stably transfected cell lines can be visualized in vivo and can be used to monitor cell migration (e.g., metastases, stem cell therapy, and inflammation), tumor progression, and tumor treatment [75, 76, 77, 78].

Bioluminescence represents another class of image marker genes that is based on light emission by luciferase-mediated oxidation of luciferin. The emitted light can be detected by sensitive charge-coupled device cameras for real-time monitoring of molecular events in vivo. Different luciferase genes have been isolated from various organisms (e.g., firefly) [79]. The different luciferase types show similar bioluminescence pathways resulting in emission spectra between 400 and 620 nm. Because luciferase is not found in mammalian cells, bioluminescence operates without background noise, thus yielding extremely high SNRs [79, 80].

Clinical perspectives for abdominal imaging

Molecular imaging will have a significant impact on diagnostic procedures within the next few years. MRI provides full body coverage and has, with increasing scanning speed, become an excellent tool for abdominal imaging studies, including dynamic liver imaging. Parametric MRI will provide quantitative tools for tissue evaluation. New surrogate markers for microvessel density will be available and provide high-resolution maps of tumors. This will be beneficial for noninvasive tumor grading and monitoring of tumor therapy. Early imaging markers for tumor response to therapy will emerge and provide imaging paradigms, which allow for treatment evaluation before phenotypic changes (e.g., regression of tumor size or perfusion) occur.

Further, MRI will have its place in high-resolution applications such as in vivo cell tracking. In addition to monitoring stem cell migration cell-labeling techniques might be exploited to visualize inflammatory processes. Early detection of organ rejection after tissue transplantation and grading of autoimmune disease can thus be envisioned. Moreover, localization of active inflammatory processes may be realized with this approach.

However, the transformation of MRI into a truly “molecular’’ imaging modality is not straight forward because SNRs are limited compared with radioisotopic or optical methods. The detection of cellular proteins thus will require more efficient signal amplification strategies than possible with conventional contrasting strategies. High relaxivity compounds labeled with multiple paramagnetic reporters are one example that may provide sufficient SNR for in vivo molecular MRI [24]. Smart MR contrast agents should be even more suitable for molecular MRI. However, the currently available contrast medium technology does not seem to yield sufficient signal amplification for detecting molecular structures in a human being. Promising approaches such as imaging hyperpolarized C13 or imaging fluorine as opposed to hydrogen protons have been proposed to overcome the SNR burden.

Whereas optical imaging is a true molecular imaging modality, methods of imaging with light are constrained by their limited depth penetration. However, different applications for optical imaging will be seen in the near future to supplement the diagnostic tools in abdominal imaging. Surface-weighted reflection techniques can easily be integrated into endoscopic or laparoscopic imaging techniques [81]. Moreover, hand-held devices comparable to ultrasound transducers should be available soon, thus offering considerable tissue penetration. Combining these sensors with specific (molecular) optical contrast agents will greatly enhance the diagnostic possibilities for abdominal imaging. Moreover, perioperative imaging can be envisioned using dual-labeled (i.e., labeled with MR and optical contrast agents) probes [82]. A combination of preoperative MRI and intraoperative fluorescence reflectance imaging could facilitate preoperative surgical planning and intraoperative quality control [83]. This imaging paradigm could also be applied to preoperative nodal staging and intraoperative metastatic node detection in diseases such as prostate, testicular, and renal cancers, which show frequent abdominal nodal involvement [83, 84].

Many molecular structures have been targeted by optical contrast agents. The feasibility of targeting neuroendocrine tumors with modified cyanine dyes has been demonstrated and might be combined with endoscopic or tomographic optical imaging methods. The design of other fluorochromes with tumor-specific affinity ligands should be straightforward and provide a wide range of specific agents for noninvasive tissue characterization. In this context, a multi-wavelength imaging approach that covers different fluorochromes (and thus molecular targets) simultaneously can be envisioned [85].

Smart protease-sensing probes can be exploited to detect tumor associated enzymes, which are very relevant for local tumor progression and metastatic tumor spread [86, 87, 88, 89, 90, 91]. Clinical data have suggested that tumoral protease expression correlates with tumor aggressiveness and clinical outcome [92, 93, 94]. In colon cancer, for example, cathepsin-B and matrix metalloproteinase have been found to be upregulated within the invasive tumor margins [95, 96, 97, 98]. In line with this observation is the fact that even premalignant gastrointestinal lesions can be detected by enzyme-sensing molecular beacons [69]. Clinical and experimental studies have demonstrated that highly invasive cancers express higher levels of proteases compared with less invasive tumor types [92, 93, 94, 99, 100, 101]. Tumor invasiveness was correlated with protease-related fluorescence signal in one study [70]. A grading of tumor type according to protease load can be envisioned with protease-sensing optical probes. Moreover, protease imaging would be an efficient tool for imaging inflammatory processes such as autoimmune disease. A detection of atherosclerotic plaques by cathepsin-B imaging and visualization of inflammatory activity in a rheumatoid arthritis model could be described with these approaches [102, 103].

In conclusion, different molecular imaging approaches are being explored. Some imaging techniques have been adapted for clinical use (e.g., parametric MRI), and new molecular contrasting strategies will soon push the envelope to include relevant molecular information into routine clinical imaging. In this regard, optical imaging emerges as a novel imaging tool with an excellent sensitivity profile for molecular probing.

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© Springer Science+Business Media, Inc. 2005