Molecular Imaging

New Developments in Medicine

Molecular Imaging: An Overview1

Martin G. Pomper, MD, PhD

Concurrent advances in imaging research, molecular biology, chemistry, and computing have enabled the recent emergence of molecular imaging. This overview discusses the current and near-term future state of this field and its relevance to clinical radiology. In the context of radiology, molecular imaging can be briefly defined as the remote sensing of cellular processes at the molecular level in vivo. Context is important, as molecular imaging has a different connotation for x-ray crystallographers, who decipher the three-dimensional structure of molecules, and to chemical physicists, who may attempt to image single molecules or even atoms on the surface of an especially reactive material. Molecular imaging enables cellular processes to be sensed remotely, without any perturbation of the system under study. In the strictest sense of the term, nothing touches the sample of interest, not even a very thin fiberoptic wire. Although many in vitro and ex vivo assays are critical to the development of a sound molecular imaging program, molecular imaging typically involves living animals, generally rodents and specifically mice. Molecular imaging is often described as “revolutionary,” but radiologists, especially nuclear medicine physicians, have been familiar with it for years, since the first use of iodine-131 to test for recurrent thyroid carcinoma. Fluorine-18 –labeled fluorodeoxyglucose positron emission tomography (PET), the standard procedure used to assess for mediastinal lymphadenopathy, is actually a sophisticated molecular imaging technique that can serve to quantify a specific Acad Radiol 2001; 8:1141–1153 1From the Department of Radiology, In Vivo Cellular and Molecular Imaging Center, Johns Hopkins University School of Medicine, 600 N Wolfe St, Phipps B-126A, Baltimore, MD 21287-2182. Received May 21; accepted May 24. Address correspondence to M.G.P.

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cellular process, namely, phosphorylation of 2-deoxyglucose by hexokinase within malignant tissue. The concept of molecular imaging, then, is not new or foreign. The new excitement reflects (a) the recent marriage of imaging techniques with molecular biology, (b) the expansion of molecular imaging to a variety of modalities, and (c) recent (since 1995) advances in several key scientific disciplines germane to highly specific and sensitive imaging. There is now a sense that molecular imaging research may lead to new ways of diagnosing and correcting many causes of human infirmity. Supporting this optimism is the convergence of advances in several seemingly unrelated fields. In the field of imaging, devices are now available to interrogate function and metabolism at extremely high resolution. Functional changes occur earlier than anatomic ones, suggesting that molecular imaging promises earlier diagnosis of disease than currently possible and may even aid in predicting disease. Anatomic techniques for diagnosis and therapeutic monitoring will increasingly become secondary to those based on function and may eventually become obsolete. One new high-resolution imaging device is the microPET scanner (1), produced by Concorde Microsystems (Knoxville, Tenn), which is capable of resolution (1.8 mm3) up to two orders of magnitude superior to that of clinical PET devices. High-field-strength magnets are proliferating for research with magnetic resonance (MR) imaging and spectroscopy in small animals. The new field of ultrasound (US) biomicroscopy promises real-time imaging of microvasculature (2). In molecular biology, the manipulation of DNA (ie, genetic information) has become facile and in many cases automated because of discoveries in amplification mechanisms, such as the polymerase chain reaction (PCR), and automated techniques of studying gene expression in

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vitro, such as DNA microarray technology (3) (gene chips) and differential display (4). These advances have enabled the recent sequencing of the human genome (5), providing a multitude of new targets for drug discovery and other therapies. Recombinant DNA technology enables the design of clever imaging probes and experiments. The proliferation of mouse models of human disease, including transgenic knock-in and knock-out mice, enables the efficient study of many illnesses that were previously difficult if not impossible to study under controlled conditions. In the field of chemistry, specific small molecule and peptide probes of cellular (physiologic) processes can be generated and screened with increasing efficiency by means of combinatorial chemistry (6) and phage display (7). New ways to generate lead compounds in drug discovery, such as identification of structure-activity relationships with nuclear MR (8), are constantly being developed. Computing—the ability to manipulate vast quantities of biologic data, or bioinformatics (9)—is enabled by more readily available high-speed computers. High computing power has also driven microarray technology with gene chips fashioned, literally, after computer chips. It is in the spirit of “molecular medicine” that we pursue molecular imaging research, driven by the assumption that human illness has a molecular basis. Just as the use of taxonomy to describe evolutionary biology is being overtaken by molecular genetics, so too are disorders such as cancer being redefined from organ system– based diseases to aberrations in molecular structure and function traceable to the genetic (DNA) level. Soon cancers will no longer be categorized as colon or prostate cancer but will be diagnosed and treated according to the predominant underlying genetic abnormality, for example, a mutation in the tumor suppressor gene p53. In this fashion the diagnosis and staging of cancer will be tailored to each patient. Tumor characterization will consequently become more accurate and treatment more effective. More specifically, the current goals of molecular imaging research are as follows: (a) to image gene delivery and expression, (b) to understand cellular processes in their intact microenvironments, (c) to develop new imaging technologies, (d) to facilitate new drug development and methods for therapeutic monitoring, and (e) to promote an interdisciplinary approach to biomedical imaging issues through education and the fostering of appropriate collaborations. By demonstrating the utility of molecular imaging to those outside the imaging community (eg, mo-

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lecular biologists), we hope to help them accept imaging as a standard technique. Presently, much molecular imaging research attempts to bring established in vitro processes for studying cellular phenomena in vivo, where more relevant information resides. Key elements to enable molecular imaging include (a) highly specific imaging probes, (b) suitable amplification strategies, particularly for the less sensitive modalities such as MR imaging, and (c) sensitive imaging systems (10). Although many imaging probes are based on drugs, particularly in nuclear imaging, it is more difficult to develop a successful imaging probe than a successful drug. The imaging probe must clear from all irrelevant sites within the time frame of an imaging study, whereas a drug may bind many sites as long as it carries out its intended action without major toxic effects. Like drugs, however, imaging probes must traverse physiologic barriers, such as the vascular endothelium or cell membrane, to get to their target sites of action. Probes must be designed with those barriers in mind. Imaging probes other than those for PET are often bulky and carry metal chelating moieties, so that the barrier issue becomes important. One advantage of a nuclear imaging probe is that it is usually administered in tracer quantities, that is, in concentrations that will not alter the system under study and therefore will have no pharmacologic effect, enabling relatively easy approval for use in humans. RECENT INITIATIVES AND SCOPE There are many scientific and technical reasons for the recent upsurge in molecular imaging research. Also accounting for that upsurge is the recent support for such work through various funding mechanisms sponsored by the National Institutes of Health (NIH). Interdisciplinary scientists, such as chemists who focus on imaging, have often found the traditional R01 mechanism for funding their work inhospitable, largely due to the lack of familiarity with their approaches on the part of the study sections evaluating them. Recognizing this problem, the National Cancer Institute (NCI) has taken the lead by sponsoring a series of workshops and symposia that have led to new programs to fund imaging research. Since 1999 there has been a 30% increase in funding for imaging research at the NCI, including at least nine new requests for applications (RFAs). Two of those RFAs emanated from a workshop held in early 1998 by the In Vivo Molecular/Functional

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MOLECULAR IMAGING OVERVIEW

OPERATION OF A MOLECULAR IMAGING CENTER: ONE EXAMPLE

Figure 1. Structure of a molecular imaging center.

Imaging Subgroup of the NCI Biomedical Imaging Program. Those RFAs, each issued twice to date, included calls for the development of in vivo cellular and molecular imaging centers (ICMICs) and pre- (or planning) ICMICs and for the initiation of small-animal imaging resource programs (SAIRPs). For reasons to be discussed, molecular imaging and small-animal imaging go hand in hand. There are currently three ICMICs, nine preICMICs, and five SAIRPs from the first round of RFAs, with an equal number of each from the second round to be announced shortly. The intention of those RFAs was to create “centers of excellence” in molecular imaging research, but in an effort to promote regional centers, each center is expected to collaborate with smaller institutions and educate potential molecular imaging scientists, considerably broadening the effect of the original RFAs. This is only part of the story, however, as the current ICMICs and SAIRPs are designed to support only cancer research. Other institutes at the NIH are beginning to promote molecular imaging research, supporting, for example, an upcoming HiRes meeting, which is dedicated solely to small-animal imaging with a variety of modalities (http://www.ornl.gov /HiRes2001/). A summary of additional NIH programs that support molecular imaging research is beyond the scope of this review but can be found elsewhere (11–13). In addition to new NIH initiatives, interest in molecular imaging in the biomedical research community can be measured by the new emphasis in small-animal imaging expressed at meetings of the Society of Nuclear Medicine and the Society of Nuclear Imaging in Drug Development. Furthermore, industry is showing increasing interest, not only by sending representatives to molecular imaging workshops but by sponsoring their own workshops on the topic, as did General Electric in February 2001. Increasing collaboration between industry, academics, and government agencies is anticipated to further molecular imaging research.

Molecular imaging is the most interdisciplinary field within radiology research, requiring molecular and cell biologists, synthetic chemists, biochemists, radiopharmaceutical chemists, physicists, statisticians, computer engineers, biomedical engineers, veterinarians, animal handling technologists, and geneticists. To keep molecular imaging research relevant to important medical issues, input from clinicians is essential. Unlike many radiology departments, in which expertise is aligned within imaging modalities, a molecular imaging center is a biology-driven enterprise in which the modalities and imaging probes are suited to the questions of interest. Molecular imaging centers are often organized according to core resource centers, each core contributing to the ultimate goal of imaging in vivo (Fig 1). The core resource centers make up the framework of the center and are interdependent and interdisciplinary by necessity. Information and feedback continually flow between the cores. For example, probes synthesized in the chemistry core are evaluated in the imaging core, with the information provided by imaging used to optimize probes further. Although members of the center are assigned to a specific core, many of their activities overlap between the cores in accordance with their expertise. Technology Development Technology development is an important part of a molecular imaging center, which must remain renewable. Examples of technology development include the design and implementation of planar devices for x-ray/gamma imaging, the building of new gradient coils for MR imaging, and the development of new pulse sequences for MR imaging and multinuclear spectroscopic imaging. Molecular Biology This core consists of groups that isolate and synthesize DNA and perform standard molecular biologic techniques, such as cloning and polymerase chain reaction (manipulation of DNA). Groups involved in gene therapy are also included. They determine the genes and associated regulatory enzymes that may provide imaging targets. Groups that contribute to this core will also work with tissue cultures, rodent models, and xenografts.

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Chemistry Groups involved in chemical synthesis form the heart of this core. They are responsible for radiopharmaceutical synthesis and bioconjugate chemistry, including, for example, modification of antisense oligodeoxynucleotides and monoclonal antibodies. They design and synthesize imaging probes and contrast agents. Researchers versed in cell biology and microscopy may use fluorescence or electron microscopy to study probe access to cellular targets. This core also includes ex vivo nuclear MR experiments, autoradiography, histology, and immunohistochemistry for probe validation. Imaging Information from all other cores flows toward the imaging core. Imaging techniques include well-established modalities, such as computed tomography (CT), MR imaging, MR spectroscopy, and PET, and newer modalities under development, such as US biomicroscopy. Pharmacokinetic and biodistribution studies are performed in this core, as is most in vivo work with animals (eg, tumor induction and xenograft transplantation). Maintenance and upgrading of scanning devices are undertaken by a team of engineers and physicists supported in part by resources directed toward this core. Quantitative Subcore of Imaging Core In addition to tracer kinetic modeling and mathematical modeling of drug biodistribution, molecular modeling of small molecules, peptides, and proteins is performed in this subcore. Computer operations are undertaken, ranging from simple transfer of image files, archiving, and retrieval to data mining and manipulation of the large amounts of data accumulated in imaging studies. That manipulation includes image processing, fusion, and analysis. Also included are statistical analyses and computer networking (via the Internet, for example) to public databases such as the Protein Data Bank and the emerging cancer gene data banks (eg, the Cancer Genome Anatomy Project Library of the NCI). Because mathematical and computer techniques are ubiquitous in biomedical and imaging research, this subcore supports all of the others. MODALITIES When confronted with an important biologic question—for example, whether invasiveness is a sufficient condition to ensure cancer metastasis or, more practically, which concentration a drug achieves at its target site—

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Table 1 Relative Attributes of Molecular Imaging Modalities Resolution Modality

Sensitivity

Spatial

Temporal

Contrast

CT MR imaging Nuclear medicine Optical imaging US

⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹

⫹⫹ ⫹⫹⫹ ⫹ ⫹ ⫹⫹⫹

⫹ ⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹⫹

⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹

Note.—⫹ ⫽ low resolution, ⫹⫹ ⫽ intermediate resolution, ⫹⫹⫹ ⫽ high resolution.

one must decide which imaging modality best suits the problem at hand. The available modalities have their unique advantages and disadvantages, but they are applied with the same goal in mind: high specificity. They differ in terms of sensitivity and resolution, whether spatial, temporal, or due to contrast. They also differ in complexity, the degree of expertise required to interpret the images, and the cost of performing the studies. Currently available molecular imaging modalities include CT, MR, PET, single photon emission CT (SPECT), optical imaging techniques, and US. Variations and subcategories of these modalities are also available, including optical coherence tomography, fluorescence or luminescence imaging, MR microscopy, photoacoustic US, and US biomicroscopy. Efforts are under way to perform dual x-ray /gamma imaging, CT/PET, MR/PET, and other combinations of modalities. In recent years these modalities have been adapted for imaging small animals that can be studied under controlled conditions. The biologic community has focused on the mouse in developing transgenic and other models of human disease; consequently, devices for small-animal molecular imaging are designed with mice in mind. The ultimate goal of small-animal imaging, irrespective of modality, is to achieve the same quality of quantitative, high-resolution images in mice as can be achieved in humans. There are many problems related to scaling and quantification; their details are beyond the scope of this review but have been addressed elsewhere (14 –17). Table 1 summarizes the relative attributes of the various modalities. CT Scanning CT is the two- or three-dimensional reconstruction of x-ray data. High-resolution CT imaging in small ani-

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Figure 2. Sagittal, coronal, and axial CT/fluorodeoxyglucose PET images of a rat brain. (Images courtesy of Juan Vaquero, PhD, NIH Clinical Center, Bethesda, Md.)

mals—that is, micro CT, has been a recent advance in molecular imaging research (18). Image resolution is on the order of 50 –100 ␮m, with data acquisition taking between 5 and 30 minutes. Micro CT can be used in screening programs for phenotyping new transgenic animals. It can also be used in therapeutic monitoring (eg, longitudinal studies of new anticancer chemotherapy) and in the development of new contrast agents. One important application is the merger of micro CT with other modalities, particularly nuclear imaging modalities, which have lower spatial resolution. With the use of coregistration algorithms, precise anatomic localization of physiologic data is possible, particularly for the brain (Fig 2). For other applications, combinations of CT or MR imaging with nuclear imaging devices will likely be necessary. MR Imaging MR tissue characterization relies on the detection of water protons through the application of radiofrequency pulses to the sample in a magnetic field. Differences in the microenvironment of those protons from one tissue to the next will determine the appearance of the image. The concentration of water, the longitudinal (T1) and transverse (T2) relaxation times of the tissue, and, in certain cases, the diffusion of water are detected. Other nuclei besides protons, including sodium-23 and phosphorus-31, are also of physiologic importance and can be studied in vivo. MR imaging is capable of extracting anatomic and physiologic information concurrently, so that no coregistration or other mathematical manipulation of the images

generated is necessary. Standard clinical imagers (1.5-T magnetic field strength) provide imaging at about 1-mm resolution, but when special coils and/or probes are employed, MR microscopy is capable of 10-␮m resolution. Intravascular and intracavitary MR imaging is also now possible (19). MR imaging does not require ionizing radiation, but its sensitivity is relatively low, about three to six orders of magnitude less than that of nuclear techniques. Milli- to micromolar concentrations of metabolites or other molecules of interest are required to detect an MR signal. MR therefore often requires amplification mechanisms to enable its use in molecular imaging (see below). MR imaging experiments can be done reasonably quickly, so that the temporal resolution is considered intermediate. Functional imaging with MR (diffusionweighted, perfusion, and functional MR imaging) has seen remarkable growth in recent years, clinically as well as in small-animal studies (20,21). MR has a wide array of applications to molecular imaging, including clear anatomic depiction, the study of blood flow changes in tissues with pharmacologic or other functional activation, spectroscopic quantification of metabolite concentrations, the generation of pH maps, studies of vascular volume or permeability, pharmacokinetic studies of chemotherapeutic agents, the denoting of gene expression, and the imaging of probes that are activated only when they come into contact with tissues of interest. One example of a specialized MR probe is a “smart” imaging agent in which a gadolinium atom remains caged (within a molecule covered by a galactopyranose) and

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unavailable for performing its enhancement function until brought into contact with an enzyme (␤-galactosidase) that “opens” the cage (22). One can imagine a multitude of different cages, opened by different enzymes of medical interest. Other smart or mechanism-based contrast agents, which act not only by getting across leaky borders (the mechanism of the current clinical agents) but also by being phagocytized, have been developed to study tumors in patients (23). Nuclear Medicine As mentioned above, molecular imaging at the organ level, with nuclear techniques, has been around for many years. What is new is the application of nuclear techniques to study phenomena at the tissue, cellular, and genetic levels. Nuclear imaging techniques—PET, SPECT, and gamma scintigraphy—are uniquely capable of such study due to their high sensitivities, in the nano- to picomolar range. It has been estimated that approximately 10,000 targets per cell can be detected with PET, at a minimum. PET works by detecting the simultaneous arrival of two photons that travel away from each other at an angle of approximately 180°, after the collision of a positron (positive electron) and an electron within tissue. Radiopharmaceuticals for PET are therefore imbued with a positron-emitting radionuclide, such as oxygen-15, carbon-11, nitrogen-13, or F-18. SPECT involves the detection of gamma ray photons emitted from tissue that sequesters a gamma-emitting agent. Benefits of PET over SPECT include higher resolution and the use of physiologic tracers, which means that chelation chemistry is not required to incorporate the radionuclide within the tracer. Copper-64 and iodine-124 have recently been used for PET imaging of hypoxia and gene expression, respectively (24,25). Nuclear techniques suffer from relatively poor spatial and temporal resolution. PET (and to a lesser extent SPECT) also requires a team of scientists and technicians to generate radiotracers that are often short-lived. The use of PET with tracers other than fluorodeoxyglucose is consequently limited to relatively large centers. Nevertheless, nuclear techniques have been applied creatively in the imaging of gene expression. The judicious use of resolution-recovery algorithms promises to enable imaging at resolutions of 1 mm or less for small-animal PET. Many small-animal PET systems are being constructed, and two are currently commercially available (Oxford Positron

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Systems, Oxfordshire, England; and Concorde Microsystems). Optical Techniques Optical techniques for molecular imaging include optical coherence tomography, fluorescence or luminescence imaging, and infrared imaging. Optical coherence tomography is a high-resolution cross-sectional imaging technique (26). Image resolutions are on the order of 1–15 ␮m. Images can be obtained in real time, which attests to the high temporal resolution of this technique. As with other optical techniques, optical coherence tomography is limited to a 2–3-mm depth of penetration. Its applications have therefore been limited to the eye, cervical mucosa, and other superficial structures. Perhaps more along the lines of true molecular imaging, fluorescence and luminescence imaging in vivo are gaining increasing application. Although no tomographic device for optical imaging is currently available, homemade systems, which employ a charge-coupled device camera, have enabled the study of interesting biologic phenomena (27). With fluorescence imaging, the tissue of interest must contain a substance capable of absorbing and emitting light. The light emitted must be in the nearinfrared (NIR) range (700 –900 nm), because light of that wavelength has greater depth of penetration (several centimeters) than other wavelengths, due to the lack of interference by endogenous substrates (28). Consequently, there is currently a tremendous interest in tagging biomolecules of interest with dyes that fluoresce in the NIR range. For bioluminescence, a substrate (luciferin) is administered to an animal that has been designed to carry the luciferase enzyme, such that when the substrate and enzyme meet, the luciferase is oxidized, emitting light (29). Because the animal has no endogenous substrate for luciferase, there is essentially no background noise from this technique, a relative advantage over fluorescence imaging. As few as 1,000 cells have been visualized in vivo with this technique. Luminescence imaging is particularly well suited to drug development applications. Taking advantage of abnormal scattering of light due to tumor angiogenesis (the generation by a tumor of its own blood supply to import nutrients and oxygen), NIR mammography can already be performed in human subjects (30). The development of new NIR contrast agents, including possible smart agents analogous to those for MR imaging, is a very active field. Infrared imaging is

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Figure 3. (a) Visible light and (b) infrared images of an active Kaposi sarcoma lesion. (Images courtesy of Jerry Williams, PhD, Johns Hopkins University, Baltimore, Md.)

another light-based technique that relies on angiogenesis to generate image contrast (Fig 3). US Imaging Medical US relies on the emission and detection of sound waves, which are altered by traveling through tissue. Different tissues have different acoustic signatures. High-frequency US imaging (US biomicroscopy) is being used to study mouse embryonic development and tumor biology (31). US biomicroscopy is a very promising technique for the study of tumor angiogenesis (2). Clinical Doppler US is performed at frequencies of 2–10 MHz and has been used to map vessel morphology and estimate flow rate in vessels with diameters greater than 200 ␮m. US biomicroscopy operates in the 40 – 60-MHz range and can be used to image vessels 15– 40 ␮m in diameter. A tremendous advantage of US imaging is that the images are generated in real time (high temporal resolution) and are therefore very useful for functional imaging. Coupling US biomicroscopy with new US contrast agents (air or perfluorocarbon bubbles surrounded by a polymer shell), which remain entirely intravascular, will enable the quantification of microvascular blood flow, which can help in therapeutic monitoring (of antiangiogenic therapies, for example). SPECIFIC APPLICATIONS OF MOLECULAR IMAGING Molecular imaging research is taking many directions, but perhaps most compelling is the in vivo study of genetic events. Other goals of molecular imaging research include probing the tissue microenvironment and technology development.

Gene Expression Imaging Gene expression is the process by which a DNA molecule (ie, a gene) produces a protein. Proteins are the cellular workhorses. They may provide scaffolding for the cell (structural), catalyze intracellular reactions critical to survival (enzymes), serve as receptors on the cell surface, or act as signaling molecules themselves, as neurotransmitters in the more abbreviated (peptide) form. Protein structure is determined by the organizational pattern of the DNA that directs its synthesis. DNA is made up of nucleic acid bases that are complementary in structure (capable of hydrogen bonding with one another) and is organized in a double helix in three dimensions. Every triplet of bases along a DNA strand codes for one amino acid. To produce a protein, DNA is first transcribed to a complementary strand of messenger RNA (mRNA) in the nucleus. The mRNA then travels out to the cytoplasm, where ribosomes read the message, stringing together the amino acids (forming a protein) it decodes from the base triplets in a process called translation. Every cell within an organism has the same DNA, but the milieu of the cell determines which genes will be expressed. The regulation of gene expression in itself is a fascinating topic that is beginning to be explored with molecular imaging. When we speak of gene expression imaging, we may mean the imaging of transgene expression, expression from a gene specifically introduced into a tissue of interest (eg, a tumor or the basal ganglia) for diagnostic or therapeutic purposes. The transgene is introduced to the host cell either as naked DNA (plasmid) within a viral vector or within a synthetic delivery system, such as a liposome. Viral vectors are the preferred method, since they enable ready incorporation of the transgene into the host DNA, engendering long-standing or stable transfec-

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tion. Imaging transgene expression can be used as a step toward the validation of gene therapy. Earlier imagingrelated steps in that process include delivering the gene by using minimally invasive techniques and quantifying delivery of the gene to the target of interest (32). Apart from transgene imaging, gene expression imaging may mean visualizing endogenous gene expression with a reporter probe and marker gene. Receptor-based imaging with PET or SPECT (eg, imaging somatostatin receptors with Octreoscan [indium-111-pentetreotide] for neuroendocrine tumors) can be used to study the end products of gene expression (receptor protein). It is also possible to image enzymes and second messengers involved in gene expression. Radiolabeled antisense probes have been synthesized to image mRNA molecules directly (33). Imaging upstream events in gene expression, such as mRNA synthesis, is more difficult than studying more downstream effects (enzymes and receptors), because mRNA is present in far fewer copies per cell. The end products of translation of mRNA (proteins) represent an inherent amplification mechanism that can be exploited for imaging. MR, optical, and nuclear imaging modalities have been used to image gene expression. Weissleder et al (34) used MR to image the expression of an engineered transferrin receptor (ETR) introduced to 9L gliosarcoma tumors in the flanks of nude mice. ETR can be imaged with MR by virtue of its iron binding capacity. Iron produces magnetic susceptibility changes that can be imaged with MR. The animals were intravenously administered iron-containing particles (monocrystalline iron oxide nanoparticles) that acted as the contrast agent. To the author’s knowledge, this experiment was the first to involve MR in transgene expression imaging, and it illustrated two important points. First, Weissleder et al studied ETR because that system is a potential general marker for monitoring gene therapy— general in that the DNA construct containing the ETR could be built into a more complicated construct containing a gene of greater interest to which its expression is linked, such as p53. When p53 is transcribed, so is ETR, which can be imaged. The amount of ETR produced will be directly proportional to the amount of p53 transcribed. Second, if MR is to be used for imaging in this sense, an amplification mechanism is necessary. There are many thousands of transferrin receptors per cell and about 2,000 iron particles per MION administered to the animals. Also, because native transferrin receptor is tightly regulated by a negative feedback loop that prevents cells from internalizing too much iron, Weissleder

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et al were required to develop a construct that contained ETR without this negative feedback mechanism. Other MR-based techniques for imaging gene expression include the imaging of tyrosinase, ␤-galactosidase (see above), cytosine deaminase, and arginine kinase (with MR spectroscopy) (35). The use of green fluorescent protein (GFP) represents a bridge between in vitro and in vivo molecular imaging. GFP is used widely by cell biologists as a marker protein to study cellular processes, including gene expression, in vitro. It operates as a fusion protein or can be synthesized concurrently with a protein of interest in a bicistronic construct (36), to track protein function. Using a commercial (Hamamatsu) charge-coupled device camera, Yang et al (37) performed whole-body imaging of fluorescent (GFP) tumors and metastases in living mice. Their study was somewhat surprising, because GFP does not emit in the NIR range. Nevertheless, with this technique they were able to visualize a 1.8-mm tumor at a depth of 2.2 mm. Red-shifted GFP mutants are being developed to improve that depth of penetration (38). Transgenic mice with GFP under the influence of the promoter for the vascular endothelial growth factor (VEGF)—with GFP production “driven” by the concurrent production of VEGF— have been developed to study VEGF expression in wound healing and tumors (39). An excellent example of how molecular imaging in vivo often represents an adaptation of an accepted in vitro imaging technique is the use of protease-activatable probes for optical imaging (40). Molecular beacons are used to monitor the synthesis of specific nucleic acids (mRNAs) in solution (41). The principle involves a fluorescent molecule in proximity to a fluorescence quencher within the same molecule (beacon). Once the mRNA sequence of interest is found by the beacon, the beacon changes conformation (after a portion of it undergoes complementary base pairing), separating the quencher and the fluorescent moiety, which is now free to fluoresce and be detected. Tung et al (42) used this principle to image cathepsin, an enzyme that may be important in metastagenesis. In their case, however, the fluorophor and quencher were in proximity on a peptide but on opposite sides of the cleavage site recognized by cathepsin. With cathepsin present, cleavage separated the fluorophor and quencher, enabling fluorescence and detection. Submillimeter tumors were visualized with this mechanism-based optical imaging technique. Gene expression imaging with nuclear techniques is more mature than for other modalities. These techniques

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Figure 5. Induction of endogenous p53 with carmustine (BCNU), as imaged by I-124 –labeled FIAU PET. Upper left image shows the noninduced situation, in which tumor xenografts are placed in the mouse flanks, the left flank without and the right flank transfected with the construct shown in Figure 4. Image at the bottom left shows control tumor that continuously produces HSV-TK. Image at the top right shows the induction of p53 by means of PET imaging with I-124 –labeled FIAU. The bottom right image is still visualized, due to continuous HSV-TK production, but is less intense, perhaps because of the effects of carmustine on the tumor. i.p. ⫽ intraperitoneal. (Reprinted, with permission, from reference 43.)

Figure 4. Structure of the reporter vector for imaging p53 induction with I-124 –labeled FIAU PET. (Reprinted, with permission, from reference 43.)

use marker genes that reside within or on the surface of target cells. One technique images cells that have been successfully transfected with the gene encoding the enzyme herpes simplex virus thymidine kinase (HSV-TK) (25). This is an important system, because it is used in gene therapy trials. For therapy, tumor cells transfected

with HSV-tk (the gene) convert ganciclovir to a toxic metabolite. For imaging, tissues transfected with HSV-tk convert iodine-124 –labeled 2⬘-fluoro-2⬘-deoxy-1beta-Darabinofuranosyl-5-iodouracil (FIAU), or some other suitable substrate, to a phosphorylated adduct that cannot leave the cell. As the phosphorylated adducts accumulate in the cell (amplification), PET imaging of transfected cells becomes possible. Tjuvajev et al (25) have used this technique elegantly to image the induction of p53 by carmustine (BCNU), a chemotherapeutic agent. Figure 4 depicts the fusion plasmid containing the marker protein HSV-TK (fused to GFP). GFP is also expressed for correlative fluorescence microscopy, if needed. The important point is that expression of HSV-tk is driven by p53; that is, as p53 is induced, HSV-tk will be transcribed and its protein product, HSV-TK, will be imageable with I-124 –labeled FIAU. The upper left image in Figure 5 shows the noninduced situation. Another technique employs dopamine D2 receptors (D2Rs) and F-18 –labeled fluoroethylspiperone as the imaging agent (44). Because D2Rs are located only in the striatum of the brain, this agent provides an excellent re-

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Figure 6. Mapping of the tumor microenvironment with MR perfusion imaging. (Images courtesy of Zaver Bhujwalla, PhD, Johns Hopkins University, Baltimore, Md.)

porter probe for extracranial imaging, with little or no background activity. The same principle holds for this system as for those described above, that is, that a construct is produced in which D2R expression is linked to a gene of interest. These D2R constructs have entered their second generation, and the potential for signal transduction invoked by the binding of F-18 –labeled fluoroethylspiperone to D2Rs has been avoided by producing a mutant D2R. This further illustrates the power of molecular biology techniques as they are applied to imaging. Other nuclear imaging techniques for gene expression employ SPECT, including transchelation of technetium99m to model peptides and another technique that uses a somatostatin receptor-based system (45). The SPECT systems have several advantages over PET, including wider availability and possible therapeutic implications if Tc99m is replaced with rhenium-188, a ␤-emitter (46). These systems are also located on the cell surface, as is the D2R system, avoiding one more potential barrier (the cell membrane) to facile imaging. As with the MR reporter probe discussed above, a key feature of these nuclear techniques is the fact that they are general. It would be difficult if not impossible to synthesize a new nuclear imaging probe for every possible application. Suitable precursors for new PET ligands are scarce. They are often in the domain of pharmaceutical companies that, for reasons of intellectual property rights, hesitate to dispense them to academic research centers. General probes provide a partial solution to that problem.

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Imaging the Tissue Microenvironment This concept is particularly germane to tumor imaging but equally applicable to other metabolic disorders that affect tissue perfusion and pH, among other parameters. MR techniques are being employed liberally to probe the tumor microenvironment (47). By using coregistration algorithms, researchers can overlay imaging and histologic data to study, in a multiparametric fashion, which conditions of pH, oxygen concentration, or perfusion are required to promote invasion and metastasis. Areas of high vascular permeability, as measured with VEGF immunohistochemistry, do not correlate with areas of high vascular volume, as indicated by areas of high perfusion on MR images (Fig 6) (48). This finding suggests that viable tumor (tumor with high perfusion) tends to have higher vascular integrity than nonviable tumor. Similar techniques will also prove valuable for therapeutic monitoring and drug development, particularly for agents that affect tumor angiogenesis. Development of New Technology The technology of molecular imaging is constantly evolving. Micro CT and small-animal PET scanners are continually being improved. New probes for MR, optical, nuclear, and US applications are being produced more quickly than they can be validated. New modalities are also being developed, such as imaging with electron spin resonance spectroscopy (49).

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imaging promises to facilitate interpretation of relatively low resolution gamma images (Fig 8). The same investigators are also studying combined CT/SPECT. RELEVANCE TO CLINICAL RADIOLOGY

Figure 7. The NIH depth-of-interaction scanner. UFOV ⫽ useful field of view. (Images courtesy of Michael Green, MS, NIH Clinical Center, Bethesda, Md.)

Clinical applications seem a long way off for the molecular imaging research presented herein. Functional and metabolic imaging, however, are increasingly prevalent in clinical practice. Table 2 lists physiologic processes that are currently measurable in humans. Although only fluorodeoxyglucose PET and MR spectroscopy are used extensively, several other techniques (eg, perfusion MR imaging of the central nervous system) are gaining increasing use. As already mentioned, NIR mammography can be performed on prototype instruments developed by Phillips (30). The first molecular imaging probes to find their way into clinical use will be those that are most clearly aligned with therapy, specifically, those involved in gene therapy trials. The HSV-tk system discussed above is one such example. New nuclear imaging probes will continue to be developed and will be implemented clinically for specific, functional imaging. They will likely gain access to the clinic more readily than those for MR applications because of the aforementioned tracer principle (ie, only minute quantities are necessary for imaging). FUTURE DIRECTIONS

Figure 8. HSV-tk imaging in a mouse xenograft. (Image courtesy of Mark Williams, PhD, University of Virginia, Charlottesville.)

In the realm of PET, increased sensitivity, the use of iterative resolution recovery algorithms, and multimodality imaging are the current goals. A prototype high-resolution/high-sensitivity PET device that uses depth-of-interaction photon detection is under construction in the NIH imaging physics laboratory (Fig 7). Multimodality imaging, for image coregistration of anatomic and physiologic data, is being undertaken at the University of Virginia. Exquisite combined x-ray/gamma

Molecular imaging is a high-growth field within imaging science. The most active areas will initially be oncologic applications, in part because the NCI is the primary funding agency for ICMICs and SAIRPs. In oncology, the primary imaging targets will involve angiogenesis, apoptosis, cytokines, cell cycle, kinases, growth factors and their receptors, and metastasis/invasion. Cell tracking (viral vectors, lymphocytes) is also a high priority in oncology and has already been demonstrated within the central nervous system with MR imaging (51). As we enter the second phase of the genome era, characterizing the functions of genes uncovered during the first phase, many new targets will arise. In terms of technology development, several important hurdles need to be overcome. First, there is a need for a widely available tomographic optical imaging device if optical techniques are to prove viable for translational research in molecular imaging. Second, multimodality devices are needed to enable multiparametric analysis of

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POMPER

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Table 2 Physiologic Processes in Humans Accessible by Means of Functional and Metabolic Imaging Physiologic Processes Glucose metabolism Blood flow or volume Tissue oxygenation Tissue pH Protein synthesis Cell proliferation (mitotic rate) Receptor concentration or occupancy Enzyme kinetics Endogenous metabolite concentration Water diffusion Tissue anisotropy Drug pharmacokinetics/dynamics Vascular permeability

Methods F-18 fluorodeoxyglucose PET* [15O]H2O PET, perfusion MR imaging Nitroimidazole PET, functional MR imaging P-31 MR spectroscopy Amino acid PET Thymidine PET PET PET MR spectroscopy/spectroscopic imaging* Diffusion-weighted MR imaging Diffusion tensor imaging PET, MR spectroscopy Dynamic MR imaging

Source.—Reprinted, with permission, from reference 50. *These techniques are used routinely in clinical practice.

tissues in vivo, or at least to facilitate accurate anatomicphysiologic correlation for longitudinal studies. Third, despite the high resolution obtainable with commercially available small-animal PET systems, even better resolution (ie, submillimeter) will be necessary to enable quantitative studies in mice comparable to those available in larger animals and humans. A new generation of imaging scientists with multidisciplinary training will be required to move molecular imaging forward. This need is reflected in the strong educational component required within ICMICs and SAIRPs. As the scientific community begins to see the utility of molecular imaging by answering their questions more efficiently and in a more relevant—that is, in vivo— context, it will become easier to recruit scientists and engineers to molecular imaging centers. Their efforts, combined with those of enlightened clinicians, will translate molecular imaging into a system that extends beyond diagnosis and therapy to the prediction and prevention of human disease. REFERENCES 1. Cherry SR, Shao Y, Silverman RW, et al. MicroPET: a high resolution PET scanner for imaging small animals. IEEE Trans Nucl Sci 1997; 44: 1161–1166. 2. Foster FS, Burns PN, Simpson DH, et al. Ultrasound for the visualization and quantification of tumor microcirculation. Cancer Metastasis Rev 2000; 19:131–138. 3. Freeman WM, Robertson DJ, Vrana KE. Fundamentals of DNA hybridization arrays for gene expression analysis. Biotechniques 2000; 29: 1042–1046,1048 –1055.

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