MR and optical approaches to molecular imaging
- First Online:
- Cite this article as:
- Persigehl, T., Heindel, W. & Bremer, C. Abdom Imaging (2005) 30: 343. doi:10.1007/s00261-004-0230-3
- 116 Views
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.
KeywordsMolecular 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 : “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.
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.
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.
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).
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 . 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 . 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.
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 . 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 . 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 .
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 . Hill et al. visualized mesenchymal stem cell infiltration into fresh infarcted myocardium . 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 techniques
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 . 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 . In another study by Intes et al., differentiation between benign and malignant breast tumors was shown with ICG . 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 .
Targeted optical contrast agents
Smart optical contrast agents
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) . 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 . 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 . 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 . A combination of preoperative MRI and intraoperative fluorescence reflectance imaging could facilitate preoperative surgical planning and intraoperative quality control . 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 .
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 . 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 . 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.