Multimodality in Vivo Molecular-Genetic Imaging - Bioconjugate

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Bioconjugate Chem. 2004, 15, 1376−1388

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REVIEWS Multimodality in Vivo Molecular-Genetic Imaging Michael Doubrovin, Inna Serganova, Philipp Mayer-Kuckuk, Vladimir Ponomarev, and Ronald G. Blasberg* Departments of Neurology and Radiology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021. Received June 18, 2004

Multimodality imaging is increasingly being used in molecular-genetic studies in small animals. The coupling of nuclear and optical reporter genes represents the beginning of a far wider application of this technology. Optical imaging and optical reporter systems are cost-effective and time-efficient, they require less resources and space than PET or MRI, and they are particularly well suited for small animal imaging and for in vitro assays to validate different reporter systems. However, optical imaging techniques are limited by depth of light penetration and scatter and do not yet provide optimal quantitative or tomographic information. These issues are not limiting for PET- or MRI-based reporter systems, and PET- and MRI-based animal studies are more easily generalized to human applications. Many of the shortcomings of each modality alone can be overcome by the use of dual- or triple-modality reporter constructs that incorporate the opportunity for PET, fluorescence and bioluminescence imaging. We optimistically expect that some form of tomographic, small animal optical imaging capability will be developed soon, and that this will provide the opportunity for the colocalization of optical signals to anatomical structures provided by tomographic CT and MR imaging.

INTRODUCTION

As this was the first presentation at the Symposium on Chemistry and Biological Applications of Imaging Agents and Molecular Beacons, and is the initial article in this series of reviews, it might be advantageous to provide some background on the origins and development of “molecular imaging”. The past several decades have witnessed an astonishing increase in our knowledge and understanding of the genetics and molecular biology of human disease. Significant progress has been made in our understanding of the molecular-genetic mechanisms of many diseases, and this has been achieved with the advent of the modern molecular-biological assays. In parallel with the developments in genetics and molecular biology, the imaging sciences have also made remarkable advances in technology for visualizing tissue structure and function. This progress includes the development of new instruments (e.g., microPET, microCT, high-field strength magnets, and novel optical and ultrasound technology) for imaging small animals with improved image resolution and sensitivity (1-5). There have been corresponding improvements in clinical imaging instrumentation as well. The molecular/cellular and imaging disciplines rarely interacted until the 1990s. In the mid 1990s, several investigators and the NCI began to promote “cellular and molecular imaging”, and this convergence of the “molecular” and “imaging” sciences was nurtured by Richard Klausner, then director of the NCI. Molecular imaging-cancer imaging was designated as one of six “Extraordinary Scientific Opportunities for * To whom correspondence should be addressed. E-mail: [email protected].

Investment” in 1997, and this initiative was strongly supported by two imaging-based NCI programs (the Small Animal Imaging Resource Program, SAIRP, and the In Vivo, Cellular and Molecular Imaging Centers, ICMIC). More recently, other NIH institutes and the Department of Energy (DOE) have also developed “molecular imaging” program support. The convergence of molecular biology and noninvasive imaging was timely and addresses several experimental objectives. For example, ex vivo molecular assays in animal models of disease and in transgenic animals require invasive sampling procedures. Tissue sampling may not always adequately represent the biochemical or pathological process under investigation due to tissue heterogeneity, which is especially characteristic of cancer. Furthermore, time-course studies require large numbers of animals that are sacrificed at specific time points in order to achieve a statistically significant temporal profile. The development of versatile and sensitive assays to monitor molecular-genetic and cellular processes in vivo would be of considerable value in human subjects, as well as in experimental animal models of different diseases. Noninvasive imaging of molecular-genetic and cellular processes would compliment existing ex vivo molecular-biological assays and can provide a spatial as well as a temporal dimension to our understanding of various diseases. At this point it may be helpful to provide the readership of this series on “molecular imaging” with a definition of this newly emerging and rapidly evolving field. We developed the following definition several years ago: “the visualization of cellular processes in space and time at a molecular or genetic level of function”. Other

10.1021/bc0498572 CCC: $27.50 © 2004 American Chemical Society Published on Web 11/02/2004

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Figure 1. HSV1-tk reporter construct and the indirect reporter imaging paradigm. The basic structure of a reporter gene complex is shown, in this case herpes simplex virus type 1 thymidine kinase (HSV1-tk). The control and regulation of gene expression is performed through promoter and enhancer regions that are located at the 5′ end (“upstream”) of the reporter gene. These promoter/ enhancer elements can be “constitutive” and result in continuous gene expression (“always on”), or they can be “inducible” and sensitive to activation by endogenous transcription factors and promoters. Following the initiation of transcription and translation, the gene product, a protein, accumulates. In this case the reporter gene product is an enzyme (HSV1-TK). HSV1-TK will phosphorylate selected thymidine analogues (e.g., FIAU or FHBG), whereas these probes are not phosphorylated by endogenous mammalian TK1. The phosphorylated probe does not cross the cell membrane readily; it is “trapped” and accumulates within transduced cells. Thus, the magnitude of probe accumulation in the cell (level of radioactivity) reflects the level of HSV1-TK enzyme activity and level of HSV1-tk gene expression.

definitions of “molecular imaging” abound and include variations on the theme and some emphasize different aspects to explain the advent, development, and evolution of this new discipline. The readers of this chapter are urged to consult other reviews of imaging transgene expression and molecularbiological reporter gene imaging that have been written (6-13), and other perspectives on molecular imaging (14-20). In addition, there are two new journals that are specifically serving the molecular imaging community: Molecular Imaging (MIT Press; the official journal of the Society of Molecular Imaging, www.molecularimaging.com) and Molecular Imaging and Biology (Elsevier, the official journal of the Academy of Molecular Imaging, (http://149.142.143.206/index.html). Molecular Imaging Strategies. Three imaging strategies have been described: “direct”, “indirect”, and “biomarker”. Direct imaging strategies are based on imaging the target directly, usually with a target-specific probe. This strategy has been established using nuclear, optical, and MR imaging technology. The resultant image of probe localization and concentration (signal intensity) is directly related to its interaction with the target. Imaging cell surface specific antigens with radiolabeled antibodies and genetically engineered antibody fragments (e.g., minibodies) are examples of direct molecular imaging that has evolved over the past 30 years. In vivo imaging of receptor density/occupancy using small radiolabeled ligands has also been widely used, particularly in neuroscience research, over the past two decades. These examples represent some of the first “molecular imaging” applications used in clinical nuclear medicine research. More recent research has focused on chemistry and the synthesis of small radiolabeled or fluorescent probes (and paramagnetic nanoparticles) that target specific receptors (e.g, the estrogen or androgen receptors) and florescent probes that are activated by endogenous proteases (21). For example, the alpha(v)beta3 integrin is highly expressed on tumor vasculature and plays an important role in metastasis and tumor-induced angiogenesis; initial studies targeting and imaging of the alpha(v)beta3 integrin with radiolabeled glycosylated

RGD-containing peptides are very encouraging (22). The development of other novel direct-imaging probes and ligands will be discussed by other contributors to this series, including C. Meares, R. Pandrey, D. Bornhop, J. Katzenellenbogen, W. Eckelman, D. Hnatowich, W. Welsh, and others. Indirect molecular imaging strategies are a little more complex. One example of indirect imaging that is now being widely used is “reporter gene imaging”. It requires “pretargeting” (delivery) of the reporter gene to the target tissue (by transfection/transduction), and it usually includes transcriptional control components that function as “molecular-genetic sensors” and initiate reporter gene expression. Reporter gene imaging may also require a specific probe that must interact with the reporter gene product in order to produce an image. In this case, the “target” molecule (or the activity of a specific intracellular signal transduction pathway) is not imaged directly. A simplified description and cartoon of a reporter gene is shown in Figure 1, and a representation of different reporter genes for imaging transduced cells is shown in Figure 2. The resultant image reflects multiple processes that include the transfer of the reporter gene into the target cells, followed by the initiation of reporter gene transcription and subsequent translation of the reporter protein (the reporter gene product). These issues and others related to the intracellular or membrane localization and the degradation or ubiquination of the reporter protein, the biodistribution and clearance of reporter probes, and the optimal conditions for fluorescence and bioluminescence imaging are more fully addressed below. Biomarker or surrogate marker imaging can be used to assess downstream effects of one or more endogenous molecular-genetic processes. This approach is particularly attractive for potential translation into clinical studies in the near-term, because existing radiopharmaceuticals and imaging paradigms may be useful for monitoring downstream effects of changes in specific moleculargenetic pathways in diseases such as cancer. A recent example of clinically useful biomarker imaging for “early” assessment of treatment response is FDG PET imaging of gastrointestinal stromal tumors (GIST) pre- and post-

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Figure 2. Different reporter systems. The reporter gene complex is transfected into target cells by a vector (e.g., a virus). Inside the transfected cell, the reporter gene may or may not be integrated into the host-cell genome; transcription of the reporter gene to mRNA is initiated by “constitutive” or “inducible” promoters, and translation of the mRNA to a protein, occurs on the ribosomes. The reporter gene product can be a cytoplasmic or nuclear enzyme, a transporter in the cell membrane, a receptor at the cell surface or part of cytoplasmic or nuclear complex, an artificial cell surface antigen, or a fluorescent protein. Often, a complimentary reporter probe (e.g, a radiolabeled, magnetic, or bioluminescent molecule) is given, and the probe concentrates (or emits light) at the site of reporter gene expression. The level of probe concentration (or intensity of light) is usually proportional to the level of reporter gene expression and can reflect several processes, including the level of transcription, the modulation and regulation of translation, proteinprotein interactions, and posttranslational regulation of protein conformation and degradation. Table 1. The Most Widely Used Imaging Modalities • optical (fluorescence, bioluminescence, spectroscopy, optical coherence tomography) • magnetic resonance (contrast, diffusion-weighted imaging, spectroscopy) • radionuclide (PET, SPECT, gamma camera, autoradiography)

STI571 (Gleevec) treatment (23, 24). Although glucose uptake and glycolytic enzyme activity is homeostatically regulated, glucose metabolism has been shown to be regulated by extracellular signals mediated by cell surface receptors. Receptor-mediated regulation of glucose uptake is thought to involve activation of PI 3-kinase, Akt, mTOR, and S6 kinase. A likely explanation for the dramatic effect of STI571 (Gleevec) on glucose uptake in GIST is that Kit receptor signaling regulates glucose uptake as well as glucose metabolism. Imaging Modalities. The most widely used imaging modalities are listed in Table 1, although other modalities, such as ultrasound and CT, are seeing increasing application and could be included. The convergence of these different modalities to support current molecular imaging efforts is largely based on the development of multimodality reporter systems for both optical and radionuclide imaging (see below). A number of different strategies are also being developed for magnetic resonance reporter gene imaging, and they are being combined with other reporter systems to broaden the range of biological applications (25-28). MR spectroscopy can detect and measure the concentration of specific molecules such as 5FU. MR spectroscopy was used in combination with 5FC and yeast cytosine deaminase (yCD) in suicide gene therapy protocol and was shown to be effective (29). The interaction of several disciplines, including molecular-cell biology and chemistry with different imaging modalities, is illustrated by the Ven diagram (Figure 3) and illustrates the theme of this presentation. Namely, a mutimodality, mutidisciplinary approach to molecular imaging provides distinct advantages. Several of the imaging modalities (fluorescence,

Figure 3. Mutidisciplinary approach to molecular imaging. Molecular and cell biology, along with chemistry, provide the foundation and resources for developing novel in vivo molecular constructs and imaging paradigms. Three imaging modalities currently dominate the field, but this will soon expand to include other modalities. The Ven diagram emphasizes the interaction between disciplines and different imaging modalities; it is this interaction that provides a broad new approach to the field.

bioluminescence, and nuclear) will be illustrated in the following paragraph, and their combined advantage will be highlighted. Reporter Gene Imaging. This report will focus on noninvasive multimodality nuclear and optical reporter imaging in small animals. It will highlight the application and advantages of a multimodality imaging approach, as well as discuss the potential limits of reporter-gene imaging and its translation into clinical studies. It is important to note at the outset of this discussion that imaging transgene expression requires transfection/ transduction of a reporter gene construct into target cells.

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This is usually accomplished with a vector that is capable of transfecting/transducing the target tissue (cells), and several currently available vector types can be used (e.g., plasmid, retrovirus, adenovirus, adeno-associated virus, lentivirus). The reporter transgene can encode for an enzyme, (e.g., HSV1-tk (30) or luciferase (31)), a receptor (e.g., hD2R (32)) or hSSTR2 (33)), or a transporter (e.g., hNIS) (34)), or it can encode for a fluorescent protein (e.g., eGFP (35)) (Figure 2). The paradigm for quantitative imaging of transgene expression involves several key steps, including the transfer of the reporter construct into target tissue or cells, the initiation of transcription (that can be controlled by specific promoter/enhancer elements), the process of DNA transcription, and subsequent translation of mRNA into the gene product. As outlined below, recent studies have demonstrated that all these key events can be specifically visualized in vivo by utilizing reporter gene imaging. A general paradigm for gene imaging using radiolabeled probes was initially described in 1995 (30) and our first in vivo (autoradiographic) imaging study using herpes simplex virus type 1 thymidine kinase (HSV1-tk) as a reporter gene and 5-iodo-2′-fluoro-2′deoxy1-β-D-arabino-furanosyl-uracil (FIAU) as a reporter probe is shown in Figure 4B. Subsequent imaging was performed with [131I]FIAU (36) and a gamma camera (Figure 4C) and then with [124I]FIAU (37) and PET (Figure 4D). A common feature of all reporter constructs is the cDNA expression cassette containing the reporter gene(s) of interest (e.g., HSV1-tk) which can be placed under the control of specific promoter-enhancer elements. The versatility of reporter gene imaging results in part from the flexibility to tailor the expression cassette to an individual need. In most cases, standard molecular biology techniques are sufficient to design a suitable expression cassette. Using specific expression cassettes, reporter genes can be “always turned on” by constitutive promoters (such as LTR, RSV, CMV, PGK, EF1, etc.) and used to monitor cell trafficking by identifying the location, migration, targeting, and proliferation of stably transduced cells. Alternatively, the promoter/enhancer elements can be constructed to be “inducible” and “sensitive” to activation in selected cell types and regulation by specific endogenous pathways. Promoter activation can be tissue specific, where promoter/enhancer elements are activated by transcription factors that are overexpressed in specific tissue, e.g. the PSMA promoter shows significant activity in prostate cancer cells (38), the albumin promoter is expressed in liver (39), and the CEA promoter is highly expressed in colorectal cancer (40). Inducible reporter genes can also be constructed to be sensitive to specific endogenous molecular processes, including the regulation of endogenous gene expression (41), the activity of specific signal transduction pathways (42), and specific protein-protein interactions (43, 44), and posttranscriptional regulation of protein expression (45). Another example of the versatility of reporter systems is the ability to create multimodality reporter constructs. For example, a fusion gene containing cDNA from two or three different reporter genes can be constructed, where the gene product yields a single fusion protein that retains functionality of each of the composite gene products (46) (Figure 5). A second strategy links two reporter genes by a type II internal ribosomal entry site (IRES) sequence, where the IRES element enables translation initiation within the bicistronic mRNA, thus permitting gene coexpression by cap-dependent translation of the first cistron and cap-independent, IRESmediated translation of the second cistron (47, 48). A

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third strategy uses one vector containing two different promoters and two different reporter genes. A fourth strategy uses multiple vectors, where one or more vectors carry a therapeutic gene and other vectors carry the reporter genes (49). All four strategies are based on demonstrating a proportional and constant relationship in the coexpression of two or more transgenes over a wide range of expression levels. Radionuclide-Reporter Gene Imaging. Wild-type HSV1-tk (37) and a mutant HSV1-tk gene, HSV1-sr39tk (50), are the reporter genes most commonly used in current molecular imaging studies using radiolabeled probes and PET imaging. The HSV1-tk and HSV1-sr39tk gene products are enzymes that have less substrate specificity than mammalian thymidine kinase 1 (TK1) and can phosphorylate a wider range of compounds. The viral kinases can phosphorylated acycloguanosines (e.g., acyclovir, ACV; ganciclovir, GCV; 9-[4-fluoro-3-(hydoxymethyl)butyl]guanine, FHBG) and 2′-fluoro-nucleoside analogues of thymidine (e.g., 5-iodo-2′-fluoro-2′deoxy-1β-D-arabino-furanosyl-uracil, FIAU), whereas mammalian TK1 has limited or no ability to phosphorylate these compounds (51). This difference between mammalian and viral TK enzymes permits the development and use of radiolabeled probes that are selectively phosphorylated by HSV1-TK or HSV1-sr39TK. The advantages and disadvantages of different radiolabeled probes for imaging viral TK expression have been compared and extensively discussed in the literature (52, 53). It is most likely that [124I]FIAU (54) and [18F]FHBG (55) will be the radiolabeled probes that will be first introduced into the clinic for imaging HSV1-tk gene expression. As noted above, other radionuclide-based reporter systems have been developed. Several of these reporter systems have distinct benefits with respect to initiating molecular/ reporter imaging in human subjects, since the reporters are derived from human genes. Optical-Reporter Gene Imaging. Optical-based (bioluminescence and fluorescence) reporter systems are receiving increased attention because of their efficiency for sequential imaging, operational simplicity, and substantial cost benefits. Bioluminescence reporter genes are being widely used for whole body imaging in small animals (Figures 5 and 6). The most commonly used bioluminescence reporter systems include the Firefly (FLuc) or Renilla (RLuc) luciferase genes (56). As with nuclear and magnetic resonance reporter systems, bioluminescence imaging depends on the delivery of a specific substrate to the reporter gene expressing cells. Further, the light emitting bioluminescence reaction catalyzed by luciferases depends on the presence of oxygen (57) and for example in the case of FLuc additionally on the cofactor ATP. The Firefly and Renilla luciferase reporter systems, in combination with their corresponding luminescent substrates (luciferin and coelenterazine), have several advantages for imaging small living animals. Autobioluminescence in most cases is essentially nonexistent and results in very low background light emission; this contributes to the very high sensitivity and specificity of this optical imaging technique (58, 59). Semiquantitative accuracy and reproducibility requires that the luciferin, ATP, and oxygen levels are not rate determining, but rather are in excess. Under these conditions, the photon emission flux (light intensity) is directly related to reporter gene expression and the level of reporter gene product, namely, luciferase. Another potential concern is the fact that the substrate for Renilla luciferase, coelenterazine, is a substrate for the MDR1 transporter.

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Figure 4. Autoradiographic imaging HSV1-tk expression. An animal with a stably transduced RG2TK+ xenograft in the left hemisphere and a wild-type (nontransduced) RG2 xenograft in the right hemisphere was studied. The histology and autoradiographic images were generated from the same tissue section (panels A and B). Both xenografts in the brain of this rat are clearly seen in the toluidine blue stained histological section (panel A). Twenty four hours after iv administration of [14C]FIAU, the RG2TK+ tumor is clearly visualized in the autoradiographic image (panel B), whereas the RG2 tumor is barely detectable; the surrounding brain is at background levels. Figure adapted from ref 30. Gamma camera imaging HSV1-tk expression (panel C). Gamma camera imaging was performed at 4, 24, and 36 h after [131I]FIAU injection in an animals bearing bilateral RG2 flank xenografts; the images have been normalized to a reference standard (not shown in the field of view). The site of inoculation of HSV1-tk retroviral vector producer cells (gp-STK-A2) into the left flank xenograft is indicated by the arrow. The sequential images demonstrate washout of radioactivity from the body, with specific retention of activity in the area of gp-STK-A2 cell inoculation and transduction of RG2 tumor cells with the HSV1-tk reporter (see the 24 and 36 h images). The nontransduced contralateral xenograft (right flank) and other tissues did not show any retention of radioactivity. Figure adapted from ref 36. PET imaging HSV1-tk expression (panel D). Three xenografts were produced in rnu Sprague Dawley R-Nu rats. A W256TK+ (positive control) xenograft was produced from stably transduced W256TK+ cells and is located in the left flank, and a wild-type W256 (negative control) xenograft was produced in the dorsum of the neck and in the right flank. The neck tumor (wild-type) was inoculated with 106 gp-STK-A2 vector-producer cells (retroviral titer: 106-107 cfu/mL) to induce HSV1-tk transduction of the tumor in vivo. Fourteen days after gp-STK-A2 cell inoculation, no-carrieradded [124I]FIAU (25 µCi) was injected iv and PET imaging (GE Advance) was performed 30 h later. Localization of radioactivity is clearly seen in left flank tumor (positive control) and in the in vivo transduced neck tumor (test), but only low background levels of radioactivity were observed in the right flank tumor (negative control). Figure adapted from ref 37.

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Figure 5. Multimodality triple-reporter system. Schematic structure of a retroviral vector (TGL) for mammalian expression of a triple-fusion reporter gene (HSV1-thymidine kinase, TK; enhanced green fluorescent protein, eGFP; firefly luciferase, FLuc) driven by an LTR promoter (left panel). Noninvasive, multimodality imaging of mice bearing subcutaneous xenografts produced from HSV1-tk/eGFP-cmvFluc transduced U87 cells (right shoulder) and wild-type (nontransduced) U87 cells (left shoulder). The xenografts were 4-5 mm in diameter. Whole-body fluorescence imaging (A), whole-body bioluminescence imaging (B), and axial and coronal microPET images of [18F]FEAU accumulation are shown for the same mouse. Figure adapted from ref 77.

It has recently been shown that coelenterazine is rapidly exported from cell lines that express MDR1, and this could impact on the photon emission flux from the coelenterazine-Renilla luciferase reporter system in these cells (60). In vivo bioluminescence imaging has been successfully applied to many novel reporter systems and is rapidly expanding in the molecular and cell biology communities. Fluorescent protein-based reporter gene systems started with different spectral shifted variants of Aequorea victoria green fluorescent protein (GFP), including an enhanced GFP (eGFP) (61-67). A number of red fluorescent proteins including Discosoma species (dsRed1 and dsRed2) (68, 69) and Heteractis crispa (HcRed) (70) have also been described. Fluorescence imaging has been shown to be very useful for in vitro applications such as (1) tracking the translocation of proteins within cells, (2) identifying and selecting transduced cells using FACS, and (3) cost-effective in vitro assays to validate the function and sensitivity of specific-inducible reporter systems. The development of whole body transcutaneous fluorescent imaging technology provides for (1) imaging reporter gene expression in small living animals (Figures 5 and 6), and (2) localizing transduced cells and/or the expression of inducible reporter systems (e.g., expression of p53, Figure 7) in tissue sections at the microscopic level by in situ fluorescence imaging (16, 71). Limitations of fluorescence reporter imaging include the requirement of an external source of light and the exponentially decreasing intensity of light with increasing depth of the target. However, a new class of red fluorescent proteins and near-infrared dyes are providing better deep tissue imaging characteristics (68-70) and are a focus of current research development. Endogenous autofluorescence of tissues frequently results in substantial background emissions that limit the sensitivity and specificity of this imaging technique, and this contributes to an important advantage of bioluminescence over fluorescence reporters. However, the use of selective filters or the application of spectral analysis can significantly reduce the contribution of autofluorescence to the acquired images. Nevertheless, in vivo bioluminescence reporter imaging remains more sensitive than in vivo fluorescence reporter imaging.

Luciferase may be well suited to monitor transcription due to its relatively fast induction (72) and to the considerable short biological half-life of luciferin and luciferase (73). This is an advantage compared to the longer-lived eGFP. However, short-lived (rapidly degradable) variants of eGFP have been recently developed, and eGFP can be used for higher resolution imaging in cells in vitro. Combining these reporter genes into a single gene could provide additional tools for the analysis of cancer cells in vivo and ex vivo. Such a dual-function reporter gene was created and the single encoded protein was shown to be fluorescent and bioluminescent (74). Multimodality Nuclear and Optical Reporter Imaging. The coupling of a nuclear reporter gene (e.g. HSV1-tk) with an optical reporter gene (e.g., eGFP) has been reported (75). More recently a series of TKeGFP mutants were developed with altered nuclear localization and better cellular enzymatic activity to optimize the sensitivity for imaging HSV1-tk/eGFP reporter gene expression (46). The TKeGFP reporter gene has been introduced into several different reporter systems to assess different molecular-biological pathways (41, 42). A mutant thymidine kinase (HSV1-sr39tk)-Renilla luciferase (RL) fusion reporter construct (tk20rl) was recently developed for both nuclear and optical imaging (76). This study demonstrated the specificity and sensitivity of bioluminescence imaging and showed a good correlation between the nuclear (microPET) and optical (CCD camera) readouts of the dual reporter system. More recently, a triple-fusion reporter construct, TKeGFPLuc (TGL), has been developed (77, 78), and representative images are shown in Figure 5 and 6. A single reporter construct (vector) with a gene product that can be assayed by three different imaging technologies (fluorescence, bioluminescence, and nuclear or magnetic resonance) provides the benefits of each modality. It will facilitate the development, validation, and testing of new reporter systems in small animals, as well as provide preliminary data that will facilitate the translation of such studies into humans. Although optical imaging does not yet provide optimal quantitative or tomographic information, these issues are not limiting for PET-based reporter systems, and PET

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Figure 6. Multimodality imaging system. Multimodality imaging of a mouse bearing two xenografts: a small left shoulder xenograft (dashed oval outline) produced from 2 × 106 U87 cells transduced with the TK-eGFP-FLuc triple-modality reporter, and a large nontransduced U87MG xenograft located in the right shoulder and extending beneath the animal. A white light photograph is initially obtained (A), followed by whole-body fluorescence imaging - without correction for autofluorescence (B), followed by whole-body bioluminescence imaging (C), followed by whole-body scintigraphic imaging of [131I]radioactivity, 24 h after iv [131I]FIAU administration (D). All images were obtained from the same mouse at the same imaging session, and the mouse remained stationary between each imaging session. Note that panels C and D include optical fusion with the white-light photograph shown in panel A. Images were obtained on a Kodak R2000MM multimodal imaging system.

animal studies are more easily generalized to human applications. Using dual or triple modality reporter constructs (PET, fluorescence, and bioluminescence) overcomes many of the shortcomings of each modality alone. Multimodality reporters have been shown to facilitate the development, validation, and testing of new reporter systems in small animals (41, 42), as well as provide preliminary data that will facilitate the translation of such studies into humans. Imaging Biological Processes. Reporter-gene imaging is being used to address biological issues at the cellular and molecular level, even though our imaging resolution is at macroscopic levels. Within the past 5 years, proof-of-principle experiments in small animals have shown that it is possible to noninvasively image tissue-specific gene expression and the activity of several signal transduction pathways, and many of these studies involved multimodality imaging techniques. In addition, feasibility studies have shown that it is possible to image the endogenous regulation of transcription (41, 42), posttranscriptional modulation of translation (45), specific protein-protein interactions (43, 44, 79), protein degradation and the activity of the proteosomal pathway (80), apoptosis (81), etc. Also, noninvasive imaging of viral (82, 83), bacterial (84), and cell trafficking (85) studies have been published. Reporter gene labeling provides the opportunity for repetitive imaging and sequential monitoring of the tumor growth rate and response to treatment (86), as well as imaging metastases (71). Imaging Transcriptional Regulation of Endogenous Genes. Noninvasive imaging of transcriptional regulation in living animals (and potentially in human subjects) will provide a clearer understanding of normal and cancer-related biological processes. A recent paper from our group (42) was the first to show that p53dependent gene expression can be imaged in vivo with

PET and by in situ fluorescence using a retroviral vector containing a Cis-p53/TKeGFP dual-reporter gene under control of a p53-specific response element (Figure 7). DNA damage-induced upregulation of p53 transcriptional activity was demonstrated and correlated with the expression of p53-dependent downstream genes (including p21). These findings were observed in U87 (p53 +/+) cells and xenografts, but not in SaOS (p53 -/-) cells. This was the first demonstration that a Cis-activated dual reporter system (Cis-p53/TKGFP) was sufficiently sensitive to image endogenous gene expression using noninvasive nuclear (PET) imaging. The PET images corresponded with up-regulation of genes in the p53 signal transduction pathway in response to DNA damage induced by BCNU chemotherapy. PET imaging of p53 transcriptional activity in tumors using the Cis-p53TKGFP reporter system could be used to assess the effects of new drugs or other novel therapeutic paradigms that are mediated through p53-dependent pathways. For example, specific p53 gene therapy strategies that are based on p53 overexpression could be monitored by noninvasive imaging. The dual reporter construct (TKeGFP-fusion gene) provides the opportunity for multimodality (both nuclear and optical imaging) imaging of endogenous p53-dependent gene expression, in vivo. The TKeGFP reporter gene could be introduced into other reporter assay systems to assess other molecular-biological pathways. It should also be possible to use the TKeGFP reporter gene in transgenic animals; this will facilitate the monitoring and assessment of newly cloned genes or novel signal transduction pathways. Another advantage of the dual reporter system is the ability to compare the images of reporter gene expression obtained with PET, gamma camera, or autoradiography with corresponding in situ GFP fluorescence images. The comparison between GFP

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Figure 7. The p53-sensitive, dual-modality reporter vector (top panel) contains an artificial p53 specific enhancer element that activates expression of the TKeGFP reporter gene. A constitutively expressed neomycin selection gene is also included in the retroviral vector construct. Transaxial PET images (GE Advance tomograph) through the shoulder (A, C) and pelvis (B, D) of two rats are shown (second panel); the images are color-coded to the same radioactivity scale (%dose/g). An untreated animal is shown on the left (A, B), and a BCNU-treated animal, which is known to activate the p53 pathway, is shown on the right (C, D). Both animals have three sc xenografts: a U87p53TKGFP (test) in the right shoulder, a U87 wild-type (negative control) in the left shoulder, and a RG2TKGFP (positive control) in the left thigh. The nontreated animal on the left shows localization of radioactivity only in the positive control tumor (RG2TKGFP); the test (U87p53TKGFP) and negative control (U87wt) xenografts are at background levels. The BCNU-treated animal on the right shows significant radioactivity localization in the test tumor (right shoulder) and in the positive control (left thigh), but no radioactivity above background in the negative control (left shoulder). Fluorescence microscopy and FACS analysis (third panel) of a transduced U87p53/TKGFP cell population in the noninduced (control) state (E, G), and 24 h after a 2 h treatment with BCNU 40 mg/mL (F, H) are shown. Fluorescence microscopic images of post-motem U87p53/TKGFP sc tumor samples obtained from nontreated rats (I) and rats treated with 40 mg/kg BCNU ip (J) are also shown. These results (F-J) demonstrate a corresponding activation of the reporter system (increased fluorescence) due to p53 induction by BCNU treatment. RT-PCR blots from in vitro (K) and in vivo (L) experiments (lower panel) show very low HSV1-tk expression in nontreated U87p53TKGFP transduced cells and xenografts-bearing animals, respectively, and no HSV1-tk expression in wild-type U87 cells and tumor tissue, respectively. When U87p53TKGFP transduced cells and xenografts-bearing animals are treated with BCNU, there is a marked increase in HSV1-tk expression comparable to that in constitutively HSV1-tk expressing RG2TK+ cells and xenografts. Figure adapted from ref 42.

fluorescence and autoradiographic images, coupled with histology of corresponding tissue sections provides for

spatial and quantitative assessments of reporter gene expression at the microscopic as well as macroscopic level.

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Figure 8. Multimodality imaging of autologous bone marrow-derived cells targeting bone. Multimodality imaging: bioluminescence (A); microPET (B); microCT (C); microPET-CT overlay (D); microPET-CT registration, segmentation, and fusion (E). Bone marrowderived cells were transduced with a constitutively expressing triple-modality reporter (Figure 5) The images show targeting of bone 6 days after iv administration. Note that the tomographic display (E) confirms the targeting of transduced cells to bone; a future objective is to perform microPET-MR acquisitions and a similar tomographic microPET-MR registration and fusion to identify soft tissue structures that are targeted or are involved in trafficking of transduced reporter cells. It is also expected that some time in the near-future that tomographic bioluminescence and fluorescence images can be obtained, and that these images will be registered with currently available CT, MR, and PET tomographic images. The image fusion and segmentation was performed by Dr. Luc Bidault, MSKCC.

Imaging the Trafficking of T-Lymphocytes. T cells are an important component of the immune response and participate in the elimination of abnormal cells and infectious agents. Imaging the trafficking of T-lymphocytes has been performed using optical, MR, and PETbased imaging studies. Passive (ex vivo) labeling of T-cells with radioactive isotopes can be unstable and does not account for proliferation of activated T-cells in the body. An objective of ongoing studies in our laboratory is to image the in vivo targeting and the accumulation of EBVlymphoma specific cytotoxic T-cells in allogeneic HLAmatched EBV B-lymphomas using PET and optical imaging modalities. Cytotoxic T-cells (CTL) specific for autologous EBV-transformed B-lymphocytes were obtained and stably transduced with a constitutively expressing dual reporter gene (HSV1tk/egfp fusion gene). Specific accumulation and localization of radioactivity was observed only in the autologous EBV(+) lymphoma, in the allogeneic HLA-matched EBV(+) lymphoma, and in the spleen; no localization was seen in the allogeneic HLA-matched non-EBV lymphoma and the HLA-mismatched EBV(+) lymphoma (85). Sequential imaging over 72 h in another set of animals showed trafficking as well as targeting of the transduced and radiolabeled CTLs. These preliminary studies indicate that it may be feasible to isolate and transduce CTLs (and other immune-specific cells) with reporter constructs and then monitor their targeting and proliferation in the donor or HLA-matched recipient using noninvasive reporter gene PET imaging. Imaging the Trafficking of Bone Marrow-Derived Cells. Multimodality optical-, MR- and PET-based imaging studies can be performed. The use of PET for monitoring bone marrow and progenitor (stem) cell transplantation has lagged behind optical and MR techniques (87, 88). In most cases, PET imaging has been applied to monitoring bone marrow transplantation (BMT), to assess for residual disease (89), or BMT conditioning regime-related toxicities (90). Monitoring the fate of bone marrow stem cells with PET following direct labeling with [18F]fluorobenzoate and transplantation

was first reported by Olasz et al. (91). Direct labeling of bone marrow-derived cells is limited by the half-life and quantity of isotope used in the labeling. Reporter gene technology precludes this limitation and allows for extended monitoring of stem cell engraftment. Recently, Cao et al. reported luciferase bioluminescence imaging of hematopoietic stem cells following transplantation into irradiated recipient mice (92). Donor stem cells were derived either from a luciferase or luciferase/GFP transgenic mouse and purified through cell sorting. After systemic administration, repeated optical imaging was used to detect the sites and kinetics of hematopoietic stem cell engraftment. The data suggests that the stem cells initially home to the bone marrow or spleen, while little specificity for a particular bone marrow compartment exists. Interestingly, different subsets of progenitor cells, such as short or long term repopulating cells, showed comparable homing profiles but differences in their proliferative potential. The potential of bioluminescence imaging to monitor engraftment of hematopoietic progenitor cells was previously shown in a mouse model of xenotransplantation of human hematopoietic stem cell populations (93). We have applied reporter gene technology to image the trafficking and distribution of bone marrow cells using a multiple-modality reporter gene approach (94). Coregistration of microPET and microCT images facilitated interpretation of the PET signal and allowed localization radioactive foci to specific anatomical structures (Figure 8). Others have studied effects of the bone marrow transplanted cells on the reconstruction of the ischemic myocardium (95, 96). COMMENTS

Multimodality imaging is increasingly being used in molecular-genetic studies in small animals. Fluorescence and bioluminescence imaging provides an efficient, rapid and low cost imaging alternative to more expensive and time-consuming microPET and MR imaging modalities. Optical reporter genes, including luciferase and eGFP, have unique properties that make each useful for particular experimental applications as described above.

Reviews

Combining these reporter genes into a single fusion or bicistronic gene provides additional advantages. Nevertheless, optical imaging techniques are limited by depth of light penetration and scatter of emitted light photons, and optical imaging does not yet provide optimal quantitative or tomographic information. These issues are not limiting for PET- or MR-based reporter systems. In addition, PET and MR animal studies are more easily generalized to human applications. The use of dualmodality reporters has proven very successful in our laboratory, and this utility has been expanded by the development of a triple reporter system. Advantages of each separate imaging modality include the following: (1) fluorescence reporters provide for shorter development and validation times of new reporter systems by rapid and cost-effective real-time readout of cell-based in vitro assays and by in situ imaging of transduced cells in tissue samples; (2) bioluminescence reporters also provide for rapid, cost-effective real-time reporter imaging in small animals that is particularly useful for generating sequential time-dependent image profiles; (3) nuclear (and magnetic resonance) reporters provide the opportunity to obtain tomographic as well as quantitative measures of reporter gene expression. Since PET and MR are now well developed for imaging in human subjects as well as small animals, they provide a translational link from animal to human studies. Thus, a single reporter construct (vector) with a gene product that can be assayed by three different imaging technologies (fluorescence, bioluminescence, and nuclear or magnetic resonance) would provide the benefits of each modality. It will facilitate the development, validation, and testing of new reporter systems in small animals, as well as provide preliminary data that will facilitate the translation of such studies into humans. In this respect, the triple modality reporter will be substantially greater than the sum of its individual parts. ACKNOWLEDGMENT

A special acknowledgment is given to our friend and colleague, Juri Gelovani-Tjuvajev, who joined our group in 1991. He has recently accepted a well-deserved appointment as Chairman, Department of Experimental Diagnostic Imaging at M.D. Anderson Cancer Center in Houston. He was a leading investigator in our group and initiated many of the projects in our laboratory, including the development of noninvasive multimodality reporter transgene imaging. LITERATURE CITED (1) Jacobs, R. E., and Cherry, S. R. (2001) Complementary emerging techniques: high-resolution PET and MRI. Curr. Opin. Neurobiol. 5, 621-9. (2) Weissleder, R. (2002) Scaling down imaging: molecular mapping of cancer in mice. Nat. Rev. Cancer 1, 11-8. (3) Cherry, S. R. (2004) In vivo molecular and genomic imaging: new challenges for imaging physics. Phys. Med. Biol. 3, R13-48. (4) Jones, T. The imaging science of positron emission tomography. (1996) Eur. J. Nucl. Med. 7, 807-13. (5) Phelps, M. E. (2000) PET: the merging of biology and imaging into molecular imaging. J. Nucl. Med. 4, 661-81. (6) Blasberg, R., and Tjuvajev, J. (1997) In Vivo Monitoring of Gene Therapy by Radiotracer Imaging, in Ernst Shering Research Foundation Workshop 22: Impact of Molecular Biology and New Technical Developments on Diagnostic Imaging, pp 161-189, Berlin-Heidelberg, Springer-Verlag, (7) Gambhir, S. S., Herschman, H. R., Cherry, S. R., Barrio, J. R., Satyamurthy, N., Toyokuni, T., Phelps, M. E., Larson. S. M., Balatoni, J., Finn, R., Sadelain, M., Tjuvajev, J., and

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