APRIL 2009 Volume 20, Number 4 Copyright 2009 by the American Chemical Society
REVIEWS Molecular Imaging with Bioconjugates in Mouse Models of Cancer Stephen Mather* Barts and The London Queen Mary’s School of Medicine and Dentistry, Centre for Cancer Imaging Institute of Cancer and the CR-UK Clinical Centre, St. Bartholomew’s Hos, London, EC1A 7BE, United Kingdon. Received September 18, 2008; Revised Manuscript Received November 12, 2008
The definition of molecular imaging provided by the Society of Nuclear Medicine is “the visualization, characterization and measurement of biological processes at the molecular and cellular levels in humans and other living systems”. This review gives an overview of the technologies available for and the potential benefits from molecular imaging at the preclinical stage. It focuses on the use of imaging probes based on bioconjugates and for reasons of brevity confines itself to discussion of applications in the field of oncology, although molecular imaging can be equally useful in many fields including cardiovascular medicine, neurosciences, infection, and others.
WHY PRECLINICAL MOLECULAR IMAGING Researchers may wish to use molecular imaging for a number of reasons. Basic researchers in the in vivo sciences may wish to measure physiological parameters such as receptor or enzyme concentrations, organ function such as cardiac output or renal excretion, or metabolic processes such as bone turnover or glucose metabolism. All of these parameters are accessible by imaging. More common will be a desire to measure how these parameters change during disease. Since a major reason for the study of disease in small animals is to try and model human diseases in such species, it is important to compare the effects of a particular disease in mouse and man. If such a disease results in a change in a biological function, then there will normally be a desire to correct this change by the use of drugs or other interventions, and it will therefore be valuable to screen drugs to compare their effects on this function using imaging in an animal model before proceeding to clinical trials. If this is successful, then the subsequent application of the imaging * E-mail:
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technology in human subjects may prove useful as a biomarker for the assessment of the performance of a drug in such trials. The disposition of drugs in the body may be monitored by imaging in some case directly or alternatively after tagging with an imaging probe, and last (but not least), imaging studies in small animals can be very useful (essential in fact) for the development of clinically useful imaging agents and, to a lesser extent, for the validation of clinically applicable imaging protocols.
ANIMAL MODELS OF CANCER Normal Animals. As the genetic mutations responsible for malignancy are identified, there is a desire to duplicate these changes in animal models in order to determine their phenotypic consequences, and many highly sophisticated systems have now been developed as described below. However, useful information can also often be gained by the use of much simpler animal models including normal rodents. The physiology of such animals bears many similarities to and some differences from that of man. Thus, the basic layout and function of most organs is preserved, and many of the functional molecules expressed
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in healthy tissues are also conserved. In the early stages of the development of a new biomolecular contrast agent, there is a need to assess its pattern of biodistribution, its stability in vivo, and its ability to interact with a molecular target in a biological environment. Such studies can often be performed in normal animals. Neuropeptide receptors, for example, are targets of current interest in radiopharmaceutical development (1), and such receptors are widely expressed in mice and often interact as efficiently as human receptors with radiolabeled complementary peptides. For example, the development of radiolabeled somatostatin analogues has been significantly informed by such studies (2). However, this is not always the case. For example, Maina et al. found significant differences between bombesin analogues in their ability to interact with gastrin-releasing peptide receptors originating from mouse and man (3). While such receptors may be highly conserved throughout mammals, their pattern of distribution may not be; for example, many neuropeptide receptors are highly expressed in the mouse pancreas but may not be expressed in the human organ (4). While the functional binding sites of receptors may be highly conserved across mammalian species, this is not necessarily true of the their nonfunctional framework regions, and this can cause problems in the study of monoclonal antibodies and their related constructs, which represent a very important class of biomolecular conjugates (5). Such molecules only rarely bind to the same sites as the native ligands, and many antibody-based agents that bind well to human-derived targets will not bind to their rodent equivalents. For such targets, it is therefore necessary to express the human homologue in a mouse background. Other differences between rodent and human physiology are quantitative rather than qualitative. It is well-recognized, for example, that the mouse heart beats much faster than the human and that mice breathe much faster. Because of the much smaller size, tissue perfusion times are much shorter, and compounds will show more rapid pharmacokinetics in mouse than man. Gastrointestinal transit times in man are roughly twice those in mice, and there are also differences in relative organ perfusion so that, for example, renal blood flow in the mouse is 75% of the blood volume per minute, while in man, it is only 15% (6). Tumor-Bearing Animals. If the purpose of the preclinical imaging study is to predict the likely behavior of a drug or contrast agent in a patient with cancer, then it is likely that, at some stage, tumor-bearing animals will be utilized. These fall into several types depending on the source of the tumor, the immunocompetence of the host, and the degree of genetic manipulation employed. Syngeneic Models. Perhaps the simplest model is the use of normal mice or rats bearing tumors derived from their own species. Tumors may arise spontaneously (although this is rare in normal laboratory strains); they may be induced by administration of carcinogens or transplanted by a variety of administration routes. Spontaneous and carcinogen-induced tumors have the advantage that they develop in a “natural” tumor environment. Thus, the blood supply and stromal and cellular interactions can be considered to closely mimic those occurring in human tumors apart from the fact that they involve murine rather than human homologues. Specific genetic mutations can be induced by the selection of appropriate carcinogens (7). The exogenous administration of tumor cells by typically subcutaneous, intravenous, or intraperitoneal routes, on the other hand, often results in the tumors developing in an unnatural locationspancreatic tumors in the lungs, for examplesand this results in changes in the intratumoral signaling that might normally occur. Such approaches are, however, widely used, because it is quicker to produce the large numbers of animals often required for such studies. It is possible to use orthotopic routes of administration, i.e., to transplant the tumor to the same
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site as its origin, but although useful in superficial tissues such as the mammary gland (8), this is technically much more difficult to perform in less accessible organs, and most commonly, the precise anatomical microlocation of origin cannot be reproduced. Xenogeneic Models. Perhaps the main limitation of syngeneic models is that the target molecules expressed within the tumor are of animal rather than human origin. If this represents a problem for the target of interest, then the next level of complexity is to implant cells of human origin in the mouse tissues. These can again be administered by various routes, although subcutaneous administration is by far the most common, since it is technically simple to perform and it is easy to monitor tumor growth, which is clearly visible. Since the administered cells are derived from a different species from the host, the animal’s immune system would normally recognize them as foreign and mount an effective immune response against them, resulting in very low “take” ratessperhaps as low as 1 in 100. To overcome this problem, animals with disabled immune systems are normally used. In the past, this was often done by irradiation or treatment with cytotoxic drugs, but most commonly today, genetically immunodeficient animals are used, the most common being various strains of “nude” mice (or rats), which lack a thymus (9) and therefore cannot generate mature T-cells, or severe combined immunodeficient (SCID) mice (10), which have a mutation that prevents V(D)J recombination of DNA resulting in a complete loss of both humoral and cellular immune systems. “Take”-rates of close to 100% can be achieved following injection of human-derived tumor cells into such strains, making it easy to generate large numbers of relatively homogeneous tumor-bearing mice for imaging studies. Immunodeficient mice are more expensive (typically 5-10 times more) than their normal counterparts and, since they are very prone to infection, must also be housed in cages with a filtered air supply to prevent ingress of microorganisms. However, the nude/SCID mouse-human xenograft probably represents the most widely used animal model for preclinical imaging studies. A large number of well-characterized tumor types are available, which enables the expression of a great number of potential molecular targets in a living environment. Since these targets are of human origin, such models can successfully be used for contrast agents, which do not bind to mouse homologues such as antibody-based agents (5). However, such models still have their limitations: While the tumor cells may be of human origin, the stromal cells and blood vessels around them are derived from the murine host, and in particular, the immune environment in the tumor is very abnormal. In man, target molecule expression is rarely restricted to tumor cells but is also present at lower levels in normal tissues; however, this is not the case in such models where the patterns of expression on the host tissues are quite different from those on the implanted cells. Such problems can, to some extent, be addressed by the use of genetically modified animal models (11). Genetically Modified Models. At the simplest level, genetic modifications can be employed to simply alter the nature of target molecule expression on the transplanted tumor cells. Although many different cell lines derived from spontaneously occurring tumors expressing many different combinations of target molecules exist, the use of transfected cell lines provides the potential for greater control over the nature, pattern, and levels of expression of these molecules. For example, it is possible to restrict expression to a particular receptor subtype, to vary the levels of expression from high to low, and to generate control cell lines which differ only in one specific detail from others. A good example of such an approach is the recent study by Ginj et al. which studied the uptake of somatostatin receptor
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Figure 1. Reporter gene imaging. A gene coding for the expression of a desired reporter construct is introduced into cells. This construct is typically an enzyme such as HSV-TK, a transporter such as the sodium/ iodide symporter, or a receptor such as the dopamine subtype 2 receptor. To image cells in which the transduced gene is present, a contrast agent consisting of a suitably tagged substrate that interacts with the construct is administered. If the reporter gene is present in the cell, it will result in increased uptake of the contrast agent. If the gene is not present, then there will no specific uptake of the agent.
subtype-specific antagonists in nude mice bearing transfected human embryonic kidney (HEK293) cell lines (12). An increasingly popular application of genetically modified transplanted tumor cells is for “reporter-gene imaging” (13). The aim of such approaches is to put the expression of the target molecule under the control of a particular gene so that the effect of subsequent changes on the activity of this gene can be directly imaged by the binding of a contrast agent with the target molecule. As illustrated in Figure 1, the reporter gene will typically consist of a coding regionstranscription and translation of which results in the cellular expression of the target moleculesand a promotor region, which determines the extent and level of transcription of the coding region. The reporting target molecule is typically an enzyme, a receptor, or a transporter, which interacts with a contrast agent in such a way as to increase its uptake by cells in which the reporter gene is active. An example of reporter gene imaging using the sodium/ iodide symporter (NIS) reporter gene system is shown in Figure 2. Control of the level and site of transcription is provided by the promotor region of the reporter gene. Promotors may be strong or weak and result in constitutive expression in all tissues in which the gene is present or provide expression that is restricted to certain tissues (such as the prostate, for example) through the use of tissue-specific promoters. A list of some popular reporter gene systems is shown in Table 1. Promotors can also be rendered inducible by the incorporation of response elements that are either turned on or off by administration of drugs that interact directly or with transcription factors that bind to these elements (11). The advantages of such systems is that they allow transient expression of a protein that may be directly toxic and therefore compromise the design of the study, or even result in embryonic lethality, so that the function of a particular gene in a mature animal could not otherwise be determined. They also allow the results of different combinations of gene transcription to be studied in the same animal. Some examples of inducible promotor systems are shown in Figure 3. Such genetic modifications are most simply applied by stable transduction of cells in culture with the gene of interest, followed
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Figure 2. SPECT-CT scan showing uptake of 99mTc-pertechnetate mediated by the sodium/iodide symporter. Uptake is seen in the thyroid gland, the stomach, and around the rim of a tumor following viral transduction of the symporter gene (30). Table 1. List of the Most Popular Report Gene Imaging Systems (Adapted from Ref 13) imaging modality optical reporter genes nuclear medicine reporter genes
MRI reporter genes
reporter
ref(s)
fluorescent proteins luciferase herpes simplex virusthymidine kinase-1 dopamine-2 receptor sodium/iodide symporter norepinephrine transporter somatostatin receptor transferrin receptor ferritin
14,15 16,17 18,19 20,21 22,23 24,25 26 27 28,29
by administration of the cells to a (typically immunodeficient) animal. However, such models still suffer from many of the disadvantages described above. A model that more closely matches the phenotype of the ultimate human recipient can be provided by the generation of transgenic mice (11). In such animals, typically an additional human gene will be incorporated into the mouse genome so that on translation it is widely expressed in many mouse tissues and not only by cells that are exogenously administered. So-called “knock-in” mice were originally developed by microinjection of the transduct into the fertilized eggs of mice, followed by implantation and maturation in pseudopregnant foster mothers (31). However, this results in random, transient, and rather inefficient insertion into the genome and has more recently largely been replaced by the “gene targeting” approach shown in Figure 4 which resulted in 2007 in the award of the Nobel Prize to Capecchi, Evans, and Smithies (32). This involves the biosynthesis of a vector that contains human DNA homologous to the target mouse gene together with an antibiotic resistance sequence that enables successfully transduced cells to be selected. This vector is introduced into cultured embryonic stems cells obtained from harvested mouse blastocysts whereupon the vector is incorporated into a precise location in the genome of one or more cells
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Figure 3. Inducible promotor systems (adapted from ref 11). (A) A tetracycline regulated transcriptional transactivator system. Cells are co-transfected with genes coding for a tetracycline-dependent transactivator, the desired gene expression of which is controlled by a promotor containing a concatamer of seven TetO sequences. In the presence of tetracycline, binding of the transactivator to the promotor is inhibited resulting in low or no levels of protein expression. In the absence of tetracycline, the transactivator binds and promotes transcription of the desired gene. (B) Tamoxifen-induced expression system. The desired gene is linked with that encoding for the transciption of a mutated ligand binding site from the estrogen receptor, and this results in the production of a fusion protein containing both sequences. In the absence of tamoxifen (4-OHT), heat shock proteins bind to the ER-domain of the protein and prevent its function. Addition of tamoxifen displaces the heat shock protein, allowing the protein to function correctly.
by a process known as homologous recombination. These cells can be selected by antibiotic treatment and implanted into foster mothers, which give birth to chimeric mice bearing cells derived from both the original and transduced cells. Further mating of the offspring with normal mice results in the birth of mice that carry only either the knock-in human gene or the original mouse gene. The human gene will be present in all the cells in the transgenic mice and, depending on its nature, will result in a pattern of molecular expression similar to that seen in man (33). Recent examples of the use of such models for imaging include a study of proliferation in gliomas using PET imaging (34) and the use of MRI for studying spontaneous lesions following insertion of a gene inducing tuberous sclerosis (35).
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The last refinement of genetically modified animal model to be described here is the “knockout” mouse. Instead of resulting in the added expression of a human gene to a mouse background, this results in the disruption of function of a selected gene. The gene-targeting approach described above can also be used here, except that an inactivated mouse gene would be substituted for the human gene (36). However, another route to knockout mice is by site-specific recombination approaches such as the Crelox system (Figure 5). In such an approach, two transgenic mice must first be generated. One is a mouse that expresses the enzyme Cre recombinase, and the other is a mouse in which the gene of interest is flanked by two recognition sitesscalled “LoxP” sites. When these two strains are mated, some mice inherit both genes, and in these, the recombinase enzyme binds to the two LoxP sites, thereby tying together the ends of the target gene, preventing its transcription, and resulting in a mouse in which the function of this gene is lacking. Such models are very useful for determining the function of a particular gene or for studying the interaction of its gene product with contrast agents, an example of which is the study by de Jong et al. who elucidated the role of megalin in the tubular reabsorption of labeled peptides by the use of megalin-knockout mice (37). Potential disadvantages of the use of genetically engineered animals is that they are complex to produce and not generally available. These drawbacks are, to some extent, ameliorated by initiatives such as the NIH Transgenic Mouse consortium (http:// www.nih.gov/science/models/mouse/index.html), which aims to improve the availability of transgenic models as well as provide a useful source of information. Molecular Imaging Modalities. Each of the different imaging technologies that can be employed for molecular imaging has its own inherent strengths and weaknesses. These are summarized in Table 2 and will be discussed, together with the basic principles of each modality, below. Radionuclide Imaging. As the name implies, radionuclide imaging maps the in vivo biodistribution of radiotracers. It comes in two distinct flavors, single photon emission tomography (SPECT) and positron emission tomography (PET). (For a useful overview on the instrumentation used for PET and SPECT imaging, see ref 38). SPECT or gamma camera imaging is based on the detection of gamma photons or X-rays emitted by radionuclides decaying by electron capture or isomeric transition. After the administration of a contrast agent labeled with an appropriate radioisotope, the emitted radiation is picked up by a suitable detector (normally a sodium iodide crystal) and the distribution of γ rays converted into an electrical pulse and subsequently into an image. In order to remove scattered radiation and to ensure that the detected events can be traced back to their point of origin in the tissue, a collimator consisting of a sheet of lead containing many small parallel holes is placed between the subject and the detector system as shown in Figure 6. Radionuclides decaying in this fashion emit γ radiation with a range of energies, and for successful imaging, this energy must be high enough to penetrate the tissues of the body and interact with the detector but not so high that it passes completely through the crystal. For clinical imaging, the preferred energies lie in the range 80-250 keV, but for preclinical imaging, lower energies can be used owing to the lower attenuation provided by the smaller body mass. One of the advantages of single photon imaging is that the signals arising from photons of different energies can be discriminated, and so the images arising from two or three different radionuclides can be acquired simultaneously. Although radionuclide imaging is, in principle, one of the most sensitive imaging modalities, the sensitivity of SPECT imaging is degraded by the presence of the lead collimator, which absorbs the great majority of events issuing
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Figure 4. Gene targeting. Adapted from ref (32). Embyronic stem cells from mouse blastocyst are transfected with the gene of interest, which inserts by homologous recombination into the genome of the recipient cell. After selection, the cells are transferred to a recipient blastocsyt and implanted into the uterus of a foster mother. This results in the birth of mice who are chimeric for both the targeted cells and those of the parent blastocyst. Mating of these chimeric mice with normal mice results in the production of mice transgenic for the targeted gene by a process of normal Mendelian inheritance.
from the subject. The consequence is that only about 0.1% of the emitted γ rays are normally measured, and this results in a requirement for relatively high administered doses of radioactivity and long imaging timesstypically on the order of tens of minutes. PET imaging, in contrast, operates on a somewhat different principle. When a positron (a positively charged electron) is emitted by the nucleus of the radioactive atom, it almost immediately interacts with an electron in the immediate vicinity, and this interaction results in the production of two 511 keV γ rays emitted simultaneously in (almost) opposite directions. These photons are detected by a ring of detectors around the subject as shown in Figure 7, and when two such events are recorded simultaneously (“in coincidence”), they can be traced back to the point at which the annihilation occurs, thus creating a map of the distribution of the radioactivity in the animal. Because no collimation is required for PET imaging, it is much (about 100 times) more sensitive than SPECT imaging, and consequently, lower levels of radioactivity can be administered, larger numbers of radioactive events detected, and shorter imaging times used. The list of suitable positron emitting radionuclides for PET is not as extensive as those available for
SPECT imaging, and because all PET isotopes result in the production of 511 keV photons, it is not possible to perform simultaneous multi-isotope acquisitions. Either radionuclide imaging approach is able to produce images with an intermediate resolution, typically around 1 mm for SPECT and 2 mm for PET in practice. For a detailed description of PET imaging technology, especially in combination with other imaging modalities, please see the reviews by Pichler et al. (39, 40). Both radionuclide imaging techniques are to a large degree quantitativesthus, the measured signal is directly proportional to the concentration of radioactivity in the tissue, which in turn may be proportional to the levels of the target molecule to which it binds. Consideration needs to be given to attenuation of the signal by overlying tissues, but this is less of a problem in small animals than in human-sized subjects, and provided that the system is appropriately calibrated, it is possible to draw quantitative conclusions from the images obtained (41). Because of the large numbers of events detected in PET imaging, it is possible, by the use of compartmental modeling, to calculate the molar concentration of radiotracer in a tissue of interest, provided that a measure of the arterial concentration of radioactivity (input function) can be obtained (42).
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Figure 5. Adapted from ref 11. Generation of transgenic mice by Cre-Lox site-specific recombination. Introduction of Cre-recombinase and a “floxed” gene into a cell results in the floxed gene being inactivated and a reporter gene transcribed instead. Table 2. Comparison of Small-Animal Imaging Modalities modality resolution sensitivity quantitation US CT MRI
50 µm 50 µm 25 µm
v. low v. low low
+
PET
2 mm
high
+++
SPECT BLI NIR
1 mm 5 mm 5 mm
high v. high high
++ ++ +
time
cost
minutes minutes secs-tens of mins mins-tens of mins tens of mins secs-mins secs-mins
low-medium medium v. high high high low low-medium
Optical Imaging. Optical imaging also comes in two distinct flavorssbioluminescence and fluorescence imaging. For a useful overview of the fundamentals of optical imaging, see ref 43. Bioluminescence imaging is a form of reporter-gene imaging and relies upon the emission of light generated in vivo by the action of luciferase enzymes produced by transduced cells. Although a very useful, sensitive, inexpensive, simple, and rapid imaging technique, it does not depend on the use of contrast agents and will therefore not be further discussed in this article. Fluorescence imaging depends on the detection of fluorescent light induced by excitement of fluorophores in the animal tissues as illustrated in Figure 8. Fluorophores may be endogenously produced by transduction of tissues to express, for example, green or red fluorescent proteins, or may be exogenously administered in the form of fluorescent contrast agents. Fluorescence imaging is also a relatively inexpensive, sensitive, and rapid imaging technique, but the main disadvantage of both optical imaging modalities is the high degree of scatter and absorption of light by overlying tissues. This severely degrades the resolution of the system and also means that, apart from its use in very superficial tissues, it cannot be considered quantitative. A further disadvantage of fluorescent imaging over bioluminescence is the fluorescence that arises from normal tissues, in particular, from blood (hemoglobin) and foods in the stomach and intestines. This latter problem can be reduced by feeding animals on a special diet of non-chlorophyll-containing foods and the former by the use of fluorophores that emit light in the near-infrared (NIR) window of 650-900 nm. A number of new optical imaging techniques are seeking to overcome the problems of light scatter, such as time-domain (fluorescence
Figure 6. Principle of the gamma camera for SPECT imaging. Photons emitted from the patient after injection of a radiotracer pass through a lead collimator and impinge in a crystal producing a flash of light, which is detected by an array of photomultiplier tubes and converted into an image such as that shown in Figure 2.
lifetime) imaging (44), frequency domain imaging (45), and optical coherence tomography (46); however, such techniques to a large extent lose the advantages of simplicity and high sensitivity associated with planar imaging. Ultrasound Imaging. Ultrasound imaging depends on the reflection of high-frequency sound waves by anatomical struc-
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Figure 7. Principle of PET imaging. An emitted positron annihilates with a circulating electron resulting in the emission of two photons in opposite directions. Simultaneous interaction of these photons with crystals in a ring around the subject results in the detection of an event that can be traced back to the point of annihilation. Many millions of these events are built up into a PET image such as the example shown in Figure 11.
tures in the body, especially the boundaries between different tissue types (47) and, through the use of Doppler techniques, is particularly useful for measuring blood flow. An example is shown in Figure 9. It is a relatively inexpensive technique, which is simple and rapid to perform but is considered to be somewhat operator-dependent in its use. Since it is essentially an anatomical imaging tool, until recently ultrasound imaging would not have been considered part of the panel of molecular imaging techniques, but recent developments have resulted in a very significant increase in its use for small animal imaging. These developments hinge upon a combination of (i) the use of very high frequency probes, which emit sound waves with frequencies up to 100 MHz and result in a very high imaging resolution of the order of 25 µm, and (ii) ultrasound contrast agents based on the use of gas-filled microbubbles, although since such agents are too large to leave the vascular space, their application is limited to intravascular structures such as endothelial target molecules. A tradeoff against the excellent resolution of highfrequency ultrasound is its poor tissue penetration, and the sensitivity of detection of contrast agents is low (48). Modern semiautomated small animal ultrasound systems overcome to some extent the intraoperator variability of this modality, but it should still be considered a generally qualitative rather than quantitative tool. Magnetic Resonance Imaging (MRI). MRI depends upon the measurement of the concentration and rates of relaxation of hydrogen atoms in a high magnetic field. The spinning single proton in a hydrogen atom creates a magnetic field, and each hydrogen atom therefore acts like a tiny magnet. In the absence of an external magnetic field, hydrogen nuclei magnetic moments are randomly oriented, but in the presence of an external magnetic field, a small proportion of the hydrogen protons align themselves in one of two directions, parallel or antiparallel to the net magnetic field producing a net magnetic field. The hydrogen atoms are not still but “precess” like a spinning top in the direction of the external magnetic field at a particular frequency known as the Larmor (or precessional) frequency. If a magnetic field pulse at the Larmor frequency is then applied
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perpendicular to the main magnetic field by a radiofrequency coil, the protons will absorb some energy and alter their alignment away from the direction of the main magnetic field. As well as changing direction, the protons also begin to precess “in phase” resulting in a net magnetic moment transverse to the external field, which induces a current and is detected in the transceiver coil. When the RF pulse is switched off, the protons give up the energy they have absorbed and start to return to their previous direction and to precess out of frequency. This results in a gradual increase in the longitudinal magnetizations called T1 or spin-lattice relaxationsand a gradual decrease in the transverse magnetizationscalled T2 or spin-spin relaxation. The rate at which these processes occur vary from tissue to tissue. These parameters can be converted into images that can be “weighted” toward T1 or T2 in order to increase the contrast between the tissues of interest. For a useful overview of the physics of MRI, see ref 49. Contrast agents in MRI work by influencing the T1 and T2 relaxation times. Most contrast agents are based on paramagnetic metals such as gadolinium, which have unpaired electrons in their outer shells. These interact with the water molecules in the vicinity resulting in a reduction in T1 relaxation times and a corresponding increase in signal intensity on T1-weighted images. Superparamagnetic iron oxide (SPIO) based agents on the other hand cause a significant reduction in T2 relaxation with a resultant decrease in image intensity on T2-weighted images. Although MRI is a very powerful high-resolution anatomical imaging tool, its sensitivity for detection of changes at the molecular level is very lowstypically 106 times lower than that of PET imaging, although this can be increased somewhat by the use of superparamagnetic contrast agents (50). It is probably the most expensive of the molecular imaging modalities, and long image acquisition times are often required, especially for high-resolution images and if low-strength magnetic fields are employed. X-ray Computed Tomography (CT). CT imaging is not a molecular imaging tool per se since it provides only anatomical information as a result of the differential absorption of X-rays by different tissues. However, functional molecular imaging especially PET/SPECT is often combined with CT, either by using dual-modality instrumentation or by software image fusion, in order to provide enhanced anatomical localization of the functional information or, in the case of PET imaging, for attenuation correction.
BIOCONJUGATE CONTRAST AGENTS All of the imaging modalities described above depend to some extent on the use of contrast agents to either provide or enhance the signals required for image generation. Irrespective of the modality employed, all molecular imaging contrast agents consist of (at least) two main components: (i) a targeting component that interacts with the target molecule of interest in vivo and (ii) a signaling component, sometimes called a “molecular beacon”, which is detected by the instrument and provides information on the location and concentration of the signal, and consequently the target molecule. These two components are normally conjugated together to form a contrast agent using a range of chemistries, a rich variety of which are described every month in this journal. The targeting requirements for the contrast agent are essentially shared by each of the modalitiessit should bind with high affinity and selectivity to the molecular target and show low-nonspecific binding to other tissues, should be stable in vivo, and be nontoxic. Stability is of particular importance in avoiding metabolites that might contribute to a signal but have lost their capacity for molecular targeting. The signaling component of the contrast agent, however, will be specific to the imaging method being used:
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Figure 8. Fluorescence imaging. The presence of an endogenous or exogenously introduced fluorophore can be imaged following excitation with a laser. Scattering of the emitted light by tissue overlying the event results in absorption and degradation of the imaging resolution.
Figure 9. Example of ultrasound imaging. A subcutaneous MKN45 tumor on the right flank of a female SCID mouse (M1). (a-e) Sonograms showing the tumor 4, 7, 11, 14, and 17 days after inoculation, respectively. (f) Growth of MKN45 tumors measured by 3D ultrasound volume quantification.
radionuclides for PET/SPECT, fluorochrome for fluorescence imaging, microbubble for ultrasound, and paramagnetic chelate for MRI. The methodology used for attachment should ensure that the conjugation does not impact on the desired properties of the contrast agent (binding affinity, stability, etc.) but will depend on the chemical properties of the particular agent to be used, and when short-lived radionuclides are employed, clearance rates need to be commensurate with isotope lifetimes. The reader is again referred to elsewhere in this journal for many examples.
APPLICATIONS Molecular Targets and Design Considerations. The performance of any molecular imaging approach is determined by the characteristics of the modality of choice, the contrast agent
developed, and the molecular target of interest. Ultimately, the most important performance criterion for any imaging approach is the signal-to-noise ratio (SNR) that is achieved where “signal” represents the signal arising from the contrast agent that is bound to the target and “noise” represents the signal arising from background, which comprises nontargeted contrast agent plus that arising from extraneous “noise” intrinsic to the detection system. The higher the SNR, the better, in general terms, is the imaging approach, and therefore, the higher the signal and the lower the noise, the better. There are many thousands of biochemical pathways that could usefully be explored with molecular imaging, and it is outside the scope of this review to discuss more than a few of these, but some general considerations apply in selecting targets worthy of pursuit (52, 53). In the context of optimizing the SNR, the
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Figure 10. Example of a contrast enhanced MRI study. Reproduced with permission from ref 51. Transverse T1 maps of drug-treated and untreated tumors in animals injected with PS-active (GST-C2A-Gd) and PS-inactive (GST-C2A-Gd) contrast agents. Color scale indicates T1 values for image voxels. Images were acquired immediately before injection of contrast agent (a T1 map acquired from a tumor before injection is shown on the left-hand side) and at 24 h after injection. A reference capillary was placed adjacent to the tumors, the position of which is indicated on the grayscale image. (A) Drug-treated tumor in animal injected with PS-active GST-C2A-Gd (TA). (B) Drug-treated tumor in animal injected with PS-inactive GST-C2A-Gd (TI). (C) Untreated tumor in animal injected with PS-active GST-C2A-Gd (UA). (D) Untreated tumor in animal injected with PS-inactive GST-C2AGd (UI). Drug-treated tumor in animal injected with PS-active contrast agent shows greater accumulation 24 h after injection (A).
Figure 11. Example of PET-CT image of a mouse bearing a AR42J pancreatic tumor imaged with 18FDG. Uptake is seen in the tumor (in the left thigh), muscle, brown fatty tissue, bladder, and intestines.
two most important considerations are accessibility and abundance. Since an interaction between the contrast agent and its target is the basis of the imaging approach, the tracer must be able to reach its destination. Targets located within the vascular lumen of the blood supply represent the most accessible site and those buried deep within the cell the least, while those expressed on the cell surface of tissues represent the middle ground. Consideration must be given to the way in which the ligand will reach its target. Extravascular delivery demands that the agent is able to leave the bloodstream, and in cancer,
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this is facilitated by the highly fenestrated capillaries present in many tumors. Intracellular delivery requires that the contrast agent pass through the cell membrane and into the appropriate intracellular compartment, and this places severe restrictions on its design. While great emphasis is naturally placed on optimizing the interaction between the target and the contrast agent, in practice a greater impact on SNR can be made by reducing the background signal. Typically, only 1-2% of the administered dose will accumulate at the target at best. This leaves 98-99% of the dose free to contribute to the background. Consideration should therefore be given to reducing this contribution by accelerating blood clearance times, lowering nonspecific protein binding, and reducing accumulation in organs of excretion such as the liver, kidneys, and gastrointestinal tract. A significant impact on such parameters can be achieved by reducing the size and enhancing the hydrophilicity of the contrast agent by judicious choice of conjugation chemistry or by the introduction of pharmacokinetic modifiers such as poly(ethylene glycol) (54). If desired, metabolically labile chemical bonds can also be introduced to accelerate clearance from nontarget tisssues (55). Drug Development. As indicated above, one of the main areas of application of preclinical imaging is in the field of pharmaceutical development. It is hoped that preclinical imaging has the potential to reduce the high attrition rates in the development of new drugs and the long time scales and great expense involved in this process. Applications can be found at all stages of the drug development process from evaluation of target expression through screening of candidate compounds, assessment of activity in animal models of disease to measurement of pharmacokinetics, comparisons of different formulations, and determination of appropriate doses for clinical trials. (For detailed reviews of the role of molecular imaging in drug development, please see refs 56, 57). Biomarkers. In the field of oncology, there is particular interest in the development of imaging biomarkers as a result of the development of molecularly targeted therapies that aim to interact with a defined molecular target rather than induce relatively nonspecific toxicity and the desire the reduce unnecessary drug-related expenditure in the face of the increasing costs of novel drug treatments. Biomarkers provide a means of measuring a particular pathological process that is associated with the progression of a disease or the effectiveness of a drug used for its treatment. Most useful among the range of biomarkers is a “surrogate end point”, which can be considered to provide a direct measure of the severity of a disease such as, for example, the measurement of blood glucose levels in diabetes. However, in the absence of such a direct correlative measure, any biomarker that provides an early indication of drug effectiveness or safety can be useful. Biomarkers can originate from many different types of measurement ranging in complexity (and cost) from the simple measurement of a chemical or cell type in a blood sample, e.g., CD-4 counts in HIV infection, through immunohistochemical analysis of biopsy samples, e.g., the Herceptest used to measure HER-2 expression in breast cancer, through to the use of imaging biomarkers. The most widely used imaging biomarker in oncology is the anatomical imaging techniques to measure the size of tumor masses before and after treatment. To be most valuable, a biomarker should be universally applicable and generalizable to a range of situations and localities, and in order to standardize the use of MRI and CT in this regard, a set of criteria known as the RECIST criteria have been internationally agreed upon (58). However, although a reduction in the size of a tumor can be considered a useful indicator of cytotoxic treatments for cancer, which result directly in cell death, it may not be an appropriate measure of success of a
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molecularly targeted treatment that may result only initially in tumoristasis. In such a situation, molecular imaging biomarkers may prove to be useful in a number of ways: the first requirement is to identify those patients who are most likely to benefit from a particular molecularly targeted treatment. An example is the use of antiangiogenic drugs targeted against the Rvβ3 integrin. Rvβ3 is one of the most widely explored targets for molecular imaging with preclinical imaging applications developed for all of the modalities described above including both gadolinium (59-61) and SPIO-based (62, 63) agents for MRI, targeted microbubbles for ultrasound (64), and NIR probes (65) or quantum dots (66) for optical imaging. However, radionuclide imaging applications for both SPECT and PET have been most widely explored (67). Importantly, developments initially explored in the preclinical imaging arena have since been translated into clinical practice. For example, Beer at al. have shown that PET imaging with [(18)F]Galacto-RGD is able to measure the levels of Rvβ3 integrin in patients with cancer (68) and explored its applicability in the context of breast (69) and head and neck (70) cancers. Having identified those patients most likely to benefit from a particular treatment, the next aim is to identify a pharmacodynamic response (or lack of it) as soon as possible. This would be valuable for several reasons. Prediction of treatment failure early in the course of therapy would indicate that the treatment should be withdrawn to avoid unnecessary toxicity and expense and would provide an early indication that an individual should change to an alternative therapy before drug resistance mechanisms have been induced. Early detection of tumor sensitivity, on the other hand, indicates that treatment should be continued and also raises the possibility of treatment intensification, since any resulting side effects or increased costs could be justified by the likelihood of improved response. By far, the most widely used imaging biomarker of treatment response is [(18)F] fluorodeoxyglucose (FDG) (see Figure 11). Glucose metabolism is intricately linked to many of the metabolic processes involved in the development of malignancy (for reviews, see refs 71, 72), and as a consequence, glucose transport and utilization are elevated in most types of cancer. Importantly, in the context of assessment of therapy response FDG uptake is often reduced following treatment with anticancer drugs, and its use is beginning to be clinically accepted as a standard biomarker of response (73). However, a number of questions arise relating to the use of FDG imaging in this regard. One issue is the actual cause of reduction in signal. In some cases, this reduction does appear to be due to a reduction in the number of metabolically active cells as a direct consequence of cell death and in such instances could therefore be considered a marker of treatment response; however, in other cases the reduction appears to be due to a translocation of the glucose transporter from the cell membrane to the cytosol (74). In other examples, the reduction in glucose metabolism seems to be a direct consequence of signaling downstream from the molecular target of the drug. Thus, treatment with rapamycin and its analogues results in a direct inhibition of glucose metabolism via the Akt and Hif-1R pathways (75), while in endocrine-sensitive breast cancer, transient stimulation of tumors with estradiol results in a consequent increase in FDG uptake, thereby predicting a likely response to endocrine therapies, because it indicates that the estrogen receptor system is functional (76). It could be argued therefore that although FDG imaging does provide a readout of drug activity, it cannot be considered to provide a surrogate end point in this regard. The other significant drawback of FDG imaging is that it is taken up not only by cancer cells but
Mather
also by some metabolically active normal cells such as macrophages, as well as by microrganisms present in sites of infection. As a result of this perceived lack of specificity, efforts are being made to develop more specific imaging biomarkers of response. One such approach is to try and measure the rate of cell proliferation. Perhaps the most widely explored imaging approach is the use of [(18)F] 3′-deoxy-3′-fluorothymidine (FLT) for PET (77). Retention of FLT by cells is primarily mediated by the action of the enzyme thymidine kinase-1 (TK1), which phosphorylates the tracer resulting in intracellular trapping. If cancer treatment results in a reduction in the rate of cell division within the tumor, then it would result in a down-regulation in the concentration of TK1 and a consequent reduction in FLT uptake. However, a reduction in TK1 can also be caused by drugs such as actinomycin D or cisplatin that have a direct effect on thymidine and DNA metabolism (78). On the other hand, the use of some drugs such as Fluorouracil that deplete the thymidine triphosphate pool can actually cause a transient increase in FLT uptake (79). Interpretation of the use of such agents therefore requires an understanding of the mechanism of action of the drug in use and its effects on DNA and thymidine metabolism. An alternative means of assessing treatment response is to try and image cell death directly. One of the earliest events in the induction of this process in cancer therapy is apoptosissalso known as programmed cell death (80). The molecular events that occur in apoptosis are now well-understood, and a number of attempts have been made to develop imaging biomarkers that interact with these events (81). The most widely exploited of these is the translocation of phosphatidylserine (PS) from the inner to the outer surface of the cell membrane. Molecules such as Annexin V (82) or synaptotagmin (51, 83) that bind to PS can be tagged with Molecular Beacons and potentially used to measure the induction of apoptosis after drug treatment (see Figure 10, for example). Because of the relatively large size of these targeting agents and their extracellular location, a variety of imaging modalities have been studied. PET and SPECT imaging probes again are the most widely explored (81), but contrast agents for optical imaging (84), gadolinium (85), and SPIO (86)-based MRI agents, as well as multimodality contrast agents (87), have been described.
CONCLUSIONS A number of different imaging modalities in combination with a wide range of contrast agents can be employed to provide different types of information from small animal models of cancer and other diseases. Each technology has its own strengths and weaknesses, and each is able to provide unique insights into the status of biological pathways and processes of interest. Great care should be exercised, however, in directly extrapolating the information obtained in animal model systems to the human situation, but particularly in view of the increasing regulatory difficulties in performing experimental studies in human subjects, preclinical imaging can be very helpful in optimizing contrast agents and developing imaging protocols in order to reduce the number and complexity of studies in humans required to answer a particular medical or scientific question.
ACKNOWLEDGMENT Thanks go to Kevin Brindle, Julie Foster, and Andrew Merron for examples of small animal images and to Tim Bacon for the illustrations.
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