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To optimize the sensitivity of imaging HSV1-tk/GFP reporter gene expression, a series of HSV1-tk/GFP mutants was developed with altered nuclear localization and better cellular enzymatic activity, compared to that of the native HSV1-tk/GFP fusion protein (HSV1-tk/GFP). Several modifications of HSV1-tk/GFP reporter gene were performed, including targeted inactivating mutations in the nuclear localization signal (NLS), the addition of a nuclear export signal (NES), a combination of both mutation types, and a truncation of the first 135 bp of the native hsv1-tk coding sequence containing a "cryptic" testicular promoter and the NLS. A recombinant HSV1-tk/GFP protein and a highly sensitive sandwich enzyme-linked immunosorbent assay for HSV1-tk/GFP were developed to quantitate the amount of reporter gene product in different assays to allow normalization of the data. These different mutations resulted in various degrees of nuclear clearance, predominant cytoplasmic distribution, and increased total cellular enzymatic activity of the HSV1-tk/GFP mutants, compared to native HSV1-tk/GFP when expressed at the same levels. This appears to be the result of improved metabolic bioavailability of cytoplasmically retargeted mutant HSV1-tk/GFP enzymes for reaction with the radiolabeled probe (e.g., FIAU). The analysis of enzymatic properties of different HSV1-tk/GFP mutants using FIAU as a substrate revealed no significant differences from that of the native HSV1-tk/GFP. Improved total cellular enzymatic activity of cytoplasmically retargeted HSV1-tk/GFP mutants observed in vitro was confirmed by noninvasive imaging of transduced subcutaneous tumor xenografts bearing these reporters using [(131)I]FIAU and a gamma-camera.

Molecular imaging is a relatively new discipline, which developed over the past decade, initially driven by in situ reporter imaging technology. Noninvasive in vivo molecular-genetic imaging developed more recently and is based on nuclear (positron emission tomography [PET], gamma camera, autoradiography) imaging as well as magnetic resonance (MR) and in vivo optical imaging. Molecular-genetic imaging has its roots in both molecular biology and cell biology, as well as in new imaging technologies. The focus of this presentation will be nuclear-based molecular-genetic imaging, but it will comment on the value and utility of combining different imaging modalities. Nuclear-based molecular imaging can be viewed in terms of three different imaging strategies: (1) "indirect" reporter gene imaging; (2) "direct" imaging of endogenous molecules; or (3) "surrogate" or "bio-marker" imaging. Examples of each imaging strategy will be presented and discussed. The rapid growth of in vivo molecular imaging is due to the established base of in vivo imaging technologies, the established programs in molecular and cell biology, and the convergence of these disciplines. The development of versatile and sensitive assays that do not require tissue samples will be of considerable value for monitoring molecular-genetic and cellular processes in animal models of human disease, as well as for studies in human subjects in the future. Noninvasive imaging of molecular-genetic and cellular processes will complement established ex vivo molecular-biological assays that require tissue sampling, and will provide a spatial as well as a temporal dimension to our understanding of various diseases and disease processes.

Noninvasive in vivo molecular-genetic imaging has developed over the past decade and involves nuclear (PET, gamma camera), magnetic resonance, and in vivo optical imaging systems. Although three different imaging strategies - "direct," "indirect" and "surrogate" - are being used, most current in vivo molecular imaging strategies are "indirect" and involve the coupling of a "reporter gene" with a complimentary "reporter probe." Imaging the level of probe accumulation provides indirect information related to the level of reporter gene expression. Reporter gene constructs are driven by upstream promoter/enhancer elements; reporter gene expression can be "constitutive" leading to continuous transcription and used to identify the site of transduction and to monitor the level and duration of gene (vector) activity. Alternatively, reporter gene expression can be "inducible" leading to controlled gene expression. Controlled gene expression can be tissue-specific and/or responsive to the level of endogenous promoters and transcription factors. Several examples of imaging endogenous biological processes in animals using reporter constructs, radiolabeled probes and PET imaging are reviewed, including: 1) imaging transcriptional regulation (e.g., p53-dependent gene expression), 2) imaging weak promoters (cis- vs. trans-reporter configurations), 3) imaging post-transcriptional regulation of gene expression, 4) imaging protein-protein interactions. The development of versatile and sensitive assays that do not require tissue sampling will be of considerable value for monitoring molecular-genetic and cellular processes in animal models of human disease, as well as for studies in human subjects in the future. Non-invasive imaging of molecular-genetic and cellular processes will compliment established ex vivo molecular-biological assays that require tissue sampling, and will provide a spatial as well as a temporal dimension to our understanding of various diseases.

Double prodrug activation gene therapy using the Escherichia coli cytosine deaminase (CD)herpes simplex virus type 1 thymidine kinase ( HSV1-tk) fusion gene ( CD/TK) with 5-fluorocytosine (5FC), ganciclovir (GCV), and radiotherapy is currently under evaluation for treatment of different tumors. We assessed the efficacy of noninvasive imaging with [124I]FIAU (2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodo-uracil) and positron emission tomography (PET) for monitoring expression of the CD/TK fusion gene. Walker-256 tumor cells were transduced with a retroviral vector bearing the CD/TK gene (W256CD/TK cells). The activity of HSV1-TK and CD subunits of the CD/TK gene product was assessed in different single cell-derived clones of W256CD/TK cells using the FIAU radiotracer accumulation assay in cells and a CD enzyme assay in cell homogenates, respectively. A linear relationship was observed between the levels of CD and HSV1-tk subunit expression in corresponding clones in vitro over a wide range of CD/TK expression levels. Several clones of W256CD/TK cells with significantly different levels of CD/TK expression were selected and used to produce multiple subcutaneous tumors in rats. PET imaging of HSV1-TK subunit activity with [124I]FIAU was performed on these animals and demonstrated that different levels of CD/TK expression in subcutaneous W256CD/TK tumors can be imaged quantitatively. CD expression in subcutaneous tumor sample homogenates was measured using a CD enzyme assay. A comparison of CD and HSV1-TK subunit enzymatic activity of the CD/TK fusion protein in vivo showed a significant correlation. Knowing this relationship, the parametric images of CD subunit activity were generated. Imaging with [124I]FIAU and PET could provide pre- and posttreatment assessments of CD/TK-based double prodrug activation in clinical gene therapy trials.

A noninvasive method for molecular imaging of T-cell activity in vivo would be of considerable value. It would aid in understanding the role of specific genes and signal transduction pathways in the course of normal and pathologic immune responses, could elucidate temporal dynamics and immune regulation at different stages of disease and following therapy. We developed and assessed a novel method for monitoring the T-cell receptor (TCR) -dependent nuclear factor of activated T cells (NFAT) -mediated activation of T cells by optical fluorescence imaging (OFI) and positron emission tomography (PET). The herpes simplex virus type 1 thymidine kinase/green fluorescent protein [HSV1-tk/GFP (TKGFP) ] dual reporter gene was used to monitor NFAT-mediated transcriptional activation in human Jurkat cells. A recombinant retrovirus bearing the NFAT-TKGFP reporter system was constructed in which the TKGFP reporter gene was placed under control of an artificial cis-acting NFAT-specific enhancer. Transduced Jurkat cells were used to establish subcutaneous infiltrates in nude rats. We demonstrated that noninvasive OR and nuclear imaging of T-cell activation is feasible using the NFAT-TKGFP reporter system. PET imaging with [124]FIAU using the NFAT-TKGFP reporter system is sufficiently sensitive to detect T-cell activation in vivo. PET images were confirmed by independent measurements of T-cell activation (e.g., CD69) and induction of GFP fluorescence. PET imaging of TCR-induced NFAT-dependent transcriptional activity may be useful in the assessment of T cell responses, T-cell-based adoptive therapies, vaccination strategies and immunosuppressive drugs.

Analysis of transgene expression in vivo currently requires destructive and invasive molecular assays of tissue specimens. Noninvasive methodology for assessing the location, magnitude, and duration of transgene expression in vivo will facilitate subject-by-subject correlation of therapeutic outcomes with transgene expression and will be useful in vector development. Cytosine deaminase (CD) is a microbial gene undergoing clinical trials in gene-directed enzyme prodrug gene therapy. We hypothesized that in vivo magnetic resonance spectroscopy could be used to measure CD transgene expression in genetically modified tumors by directly observing the CD-catalyzed conversion of the 5-fluorocytosine (5-FC) prodrug to the chemotherapeutic agent 5-fluorouracil (5-FU). The feasibility of this approach is demonstrated in subcutaneous human colorectal carcinoma xenografts in nude mice by using yeast CD (yCD). A three-compartment model was used to analyze the metabolic fluxes of 5-FC and its metabolites. The rate constants for yCD-catalyzed prodrug conversion ( k 1 app ), 5-FU efflux from the observable tumor volume ( k 2 app ), and formation of cytotoxic fluorinated nucleotides from 5-FU ( k 3 app ) were 0.49 ± 0.27 min −1 , 0.766 ± 0.006 min −1 , and 0.0023 ± 0.0007 min −1 , respectively. The best fits of the 5-FU concentration data assumed first-order kinetics, suggesting that yCD was not saturated in vivo in the presence of measured intratumoral 5-FC concentrations well above the in vitro K m . These results demonstrate the feasibility of using magnetic resonance spectroscopy to noninvasively monitor therapeutic transgene expression in tumors. This capability provides an approach for measuring gene expression that will be useful in clinical gene therapy trials.

Current gene therapy technology is limited by the paucity of methodology for determining the location and magnitude of therapeutic transgene expression in vivo . We describe and validate a paradigm for monitoring therapeutic transgene expression by noninvasive imaging of the herpes simplex virus type 1 thymidine kinase (HSV-1- tk ) marker gene expression. To test proportional coexpression of therapeutic and marker genes, a model fusion gene comprising green fluorescent protein (gfp) and HSV-1- tk genes was generated ( tkgfp gene) and assessed for the functional coexpression of the gene product, TKGFP fusion protein, in rat 9L gliosarcoma, RG2 glioma, and W256 carcinoma cells. Analysis of the TKGFP protein demonstrated that it can serve as a therapeutic gene by rendering tkgfp transduced cells sensitive to ganciclovir or as a screening marker useful for identifying transduced cells by fluorescence microscopy or fluorescence-activated cell sorting (FACS). TK and GFP activities in the TKGFP fusion protein were similar to corresponding wild-type proteins and accumulation of the HSV-1- tk -specific radiolabeled substrate, 2′-fluoro-2′-deoxy-1β-D-arabino-furanosyl-5-iodo-uracil (FIAU), in stability transduced clones correlated with gfp-fluorescence intensity over a wide range of expression levels. The tkgfp fusion gene itself may be useful in developing novel cancer gene therapy approaches. Valuable information about the efficiency of gene transfer and expression could be obtained by non-invasive imaging of tkgfp expression with FIAU and clinical imaging devices (gamma camera, positron-emission tomography [PET], single photon emission computed tomography [SPECT]), and/or direct visualization of gfp expression in situ by fluorescence microscopy or endoscopy.

The goal of this study was to determine the magnitude of “facilitated” amino acid transport across tumor and brain capillaries and to evaluate whether amino acid transporter expression is “upregulated” in tumor vessels compared to capillaries in contralateral brain tissue. Aminocyclopentane carboxylic acid (ACPC), a non-metabolized [14C]-labeled amino acid, and a reference molecule for passive vascular permeability, [67Ga]-gallium-diethylenetriaminepentaacetic acid (Ga-DTPA), were used in these studies. Two experimental rat gliomas were studied (C6 and RG2). Brain tissue was rapidly processed for double label quantitative autoradiography 10 minutes after intravenous injection of ACPC and Ga-DTPA. Parametric images of blood-to-brain transport (K1ACPCand K1Ga-DTPA, μL/min/g) produced from the autoradiograms and the histology were obtained from the same tissue section. These three images were registered in an image array processor; regions of interest in tumor and contralateral brain were defined on morphologic criteria (histology) and were transferred to the autoradiographic images to obtain mean values. The facilitated component of ACPC transport (∂KlACPC) was calculated from the K1ACPCand K1Ga-DTPAdata, and paired comparisons between tumor and contralateral brain were performed. ACPC flux, K1ACPC, across normal brain capillaries (22.6 ± 8.1 μL/g/min) was >200-fold greater than that of Ga-DTPA (0.09 ± 0.04 μL/g/min), and this difference was largely (~90%) due to facilitated ACPC transport. Substantially higher K1ACPCvalues compared to corresponding K1DTPAvalues were also measured in C6 and RG2 gliomas. The ∂K1ACPCvalues for C6 glioma were more than twice that of contralateral brain cortex. K1ACPCand ∂K1ACPCvalues for RG2 gliomas was not significantly higher than that of contralateral cortex, although a ~2-fold difference in facilitated transport is obtained after normalization for differences in capillary surface area between RG2 tumors and contralateral cortex. K1ACPC, ∂K1ACPC, and K1DTPAwere directly related to tumor cell density, were higher in regions of “impending” necrosis, and the tumor/contralateral brain ACPC radio-activity ratios (0 to 10 minutes) were very similar to that obtained with 0 to 60 minutes experiments. These results indicate that facilitated transport of ACPC is upregulated across C6 and RG2 glioma capillaries, and that tumors can induce upregulation of amino acid transporter expression in their supporting vasculature. They also suggest that early imaging (e.g., 0 to 20 minutes) with radiolabeled amino acids in a clinical setting may be optimal for defining brain tumors.

In preclinical studies, genetically engineered Salmonella have the ability to localize, selectively accumulate, and persist within transplantable murine tumors, spontaneous murine tumors and human tumor xenographs, and can express therapeutic proteins at high levels. These strains of engineered non-virulent Salmonella typhimurium display the capacity to accumulate and grow selectively in a variety of tumor types and to inhibit the growth of primary and metastatic tumors following intravenous injection into tumor-bearing mice. One strain of the bacteria (VNP20009) which has endogenous antitumor activity is currently in Phase I clinical trials. The bacteria are highly attenuated and genetically stable. The combination of the lipid mutation and the purine auxotrophy attenuate the virulence of the bacteria by greater than 10 000-fold and enhance the specificity of the bacteria for tumor tissue. These bacteria have been found to be safe in mice, pigs and monkeys when administered intravenously. Second-generation Salmonella vectors will be developed to include transgenes that will express therapeutic agents and reporter transgenes for non-invasive imaging. We have performed a preliminary study to demonstrate localization of [ 14 C]FIAU in tumored mice pretreated with Salmonella expressing HSV1-TK. The [ 14 C]FIAU radioactivity and bacterial count data strongly support a Salmonella TK -dependent [ 14 C]FIAU accumulation of at least 30-fold higher in tumor tissue compared to muscle tissue. These data warrant further investigation on the use of genetically engineered Salmonella as a systemically administered tumor-specific agents for tumor therapy and delivery of diagnostic imaging markers.

The skeleton is a preferred site for breast cancer metastasis. We have developed a multimodality imaging approach to monitor the transforming growth factor β (TGFβ) signaling pathway in bone metastases, sequentially over time in the same animal. As model systems, two MDA-MB-231 breast cancer cells lines with different metastatic tropisms, SCP2 and SCP3, were transduced with constitutive and TGFβ-inducible reporter genes and were tested in vitro and in living animals. The sites and expansion of metastases were visualized by bioluminescence imaging using a constitutive firefly luciferase reporter, while TGFβ signaling in metastases was monitored by microPET imaging of HSV1-TK/GFP expression with [18F]FEAU and by a more sensitive and cost-effective bioluminescence reporter, based on nonsecreted Gaussia luciferase. Concurrent and sequential imaging of metastases in the same animals provided insight into the location and progression of metastases, and the timing and course of TGFβ signaling. The anticipated and newly observed differences in the imaging of tumors from two related cell lines have demonstrated that TGFβ signal transduction pathway activity can be noninvasively imaged with high sensitivity and reproducibility, thereby providing the opportunity for an assessment of novel treatments that target TGFβ signaling.—Serganova, I., Moroz, E., Vider, J., Gogiberidze, G., Moroz, M., Pillarsetty, N., Doubrovin, M., Minn, A., Thaler, H. T., Massague, J., Gelovani, J., Blasberg, R. Multimodality imaging of TGFβ signaling in breast cancer metastases.

Tumor spheroids more faithfully mimic tumor biology than monolayer cultures and require three-dimensional microscopy. Our goal in this study was to overcome the limitations of signal to noise ratio that have traditionally limited three-dimensional imaging to depths of 100 microm or less. We studied the expression of hypoxia-inducible factor 1alpha (HIF-1alpha), the main regulator of cellular hypoxic response in C6 glioma spheroids. In our spheroids, red fluorescent protein is expressed constitutively and green fluorescent protein is expressed conditionally under control of a HIF-1alpha promoter. In this article, we show a series of optimizations that allowed us to obtain excellent quality confocal microscopy images at imaging depths of up to 320 microm. The combined use of special objectives, glass-bottomed culture dishes, and depth-dependent laser output modulation extended our depth range beyond previously accepted limits. This allowed us to image up to the equator of spheroids of 650 microm diameter, allowing interrogation of HIF-1alpha expression from the spheroid periphery to its hypoxic center.

In order to noninvasively detect Salmonella delivery vectors within tumors, we used a genetically modified Salmonella, VNP20009, that expresses the herpes simplex thymidine kinase (HSV1-tk) reporter gene. VNP20009-TK were able to selectively localize within murine tumor models and to effectively sequester a radiolabeled nucleoside analogue, 2'-fluoro-1-beta-D-arabino-furanosyl-5-iodo-uracil (FIAU). A quantitative relationship between the level of radioactivity accumulated and the number of bacteria in tumor and different tissues was demonstrated. The in vivo accumulation of [14C]FIAU measured in tissue sample homogenates and sections were related to Salmonella number and to immunohistochemical bacterial staining, respectively. Quantitative autoradiography (QAR) revealed the relative intensity of [14C]FIAU accumulation in a tumor cross-section, demonstrating that the peripheral region of the tumor was significantly less active than internal regions. [124I]FIAU positron emission tomography (PET) and subsequent tissue radioactivity and bacterial concentration measurements were compared. A log-log relationship was found, and the PET images could identify multiple tumor sites. The ability to noninvasively detect Salmonella vectors by PET imaging has the potential to be conducted in a clinical setting, and could aid in development of these vectors by demonstrating the efficiency and duration of targeting as well as indicating the locations of tumors.

Medical imaging has undergone a revolution in the past decade. This is largely due to improved technology involving all the major imaging modalities: MRI, computed tomography (CT), positron emission tomography (PET), ultrasound, and optical imaging. These advances and improvements in technology are being rapidly translated into the clinic and have established new standards of medical practice. Cancer imaging was identified as one of six “extraordinary scientific opportunities” by the National Cancer Institute in 1997–1998, and the institute’s subsequent funding initiatives have provided a major stimulus for further developments. A major target of these initiatives has been development of and support for molecular imaging. Molecular-genetic imaging provides visualization in space and time of normal as well as abnormal cellular processes at a molecular or genetic level. Needless to say, current gamma camera, PET, MRI, and optical technologies do not visualize individual cells, much less molecules. Perhaps the most exciting aspect of this emerging new field are the novel imaging paradigms being developed — paradigms that image molecular-genetic processes rather than anatomy. These paradigms can be successful within the inherent spatial-resolution limits of existing imaging systems, provided that the volume element, or voxel, of the tissue (cells) is relatively homogenous. This review will focus primarily on radionuclide imaging, although many of the principles described are directly applicable to optical and MRI technology as well. A more extensive discussion of these issues was recently published (1). Molecular imaging has its roots in molecular biology and cell biology as well as in imaging technology. Three different noninvasive, in vivo imaging technologies have developed more or less in parallel: (a) MRI (2–6); (b) nuclear imaging (quantitative autoradiography, gamma camera, and PET) (7–11); and (c) optical imaging of small animals (12–14). The convergence of these disciplines is at the heart of the molecular imaging success story and constitutes the wellspring for further advances in the field. The development of versatile and sensitive assays that do not require tissue samples will be of considerable value for monitoring of molecular-genetic and cellular processes in animal models of human disease, as well as for studies in human subjects. Noninvasive imaging of molecular-genetic and cellular processes will complement established ex vivo molecular-biological assays that require tissue sampling; it also provides a spatial as well as a temporal dimension to our understanding of various diseases.

We report the development of a novel dual-modality fusion reporter gene system consisting of Escherichia coli xanthine phosphoribosyltransferase (XPRT) for nuclear imaging with radiolabeled xanthine and Discosoma red fluorescent protein for optical fluorescent imaging applications. The dsRed/XPRT fusion gene was successfully created and stably transduced into RG2 glioma cells, and both reporters were shown to be functional. The level of dsRed fluorescence directly correlated with XPRT enzymatic activity as measured by ribophosphorylation of [14C]-xanthine was in vitro ( Ki= 0.124 ± 0.008 vs. 0.00031 ± 0.00005 mL/min/g in parental cell line), and [*]-xanthine octanol/water partition coefficient was 0.20 at pH = 7.4 (log P = 0.69), meeting requirements for the blood-brain barrier (BBB) penetrating tracer. In the in vivo experiment, the concentration of [* C]-xanthine in the normal brain varied from 0.20 to 0.16 + 0.05% dose/g under 0.87 + 0.24% dose/g plasma radiotracer concentration. The accumulation in vivo in the transfected flank tumor was to 2.4 ± 0.3% dose/g, compared to 0.78 ± 0.02% dose/g and 0.64 ± 0.05% dose/g in the control flank tumors and intact muscle, respectively. [14C]-Xanthine appeared to be capable of specific accumulation in the transfected infiltrative brain tumor (RG2-dsRed/XPRT), which corresponded to the 585 nm fluorescent signal obtained from the adjacent cryosections. The images of endogenous gene expression with the “sensory system” have to be normalized for the transfection efficiency based on the “beacon system” image data. Such an approach requires two different “reporter genes” and two different “reporter substrates.” Therefore, the novel dsRed/XPRT fusion gene can be used as a multimodality reporter system in the biological applications requiring two independent reporter genes, including the cells located behind the BBB.

The past decade has witnessed a remarkable increase in knowledge and understanding of the genetics and molecular biology of human disease, particularly in human cancer (Culver 1994). Significant progress in the understanding of the molecular-genetic mechanisms of many diseases have been achieved with the advent of the modern molecular-biological assays (e.g., Western and Northern blots, PCR, RT-PCR, ELI-SA, etc.). The development of transgenic animal models of human disease, where the molecular basis of that disease can be studied in a living organism, have provided new insights into disease development, progression and treatment. In parallel with these new developments in genetics and molecular biology, the imaging sciences have also made remarkable advances in technology for visualizing tissue structure and function. They include new instruments (e.g., microPET, microCT, high-field strength magnets and novel optical and ultrasound technology) for imaging small animals. There have been corresponding improvements in clinical imaging instrumentation as described in other chapters of this book. The disciplines of molecular/cellular biology and non-invasive imaging rarely interacted until the 1990s. In the mid-1990s, several investigators and the NCI began to promote “cellular and molecular imaging” as a hybrid or convergence of the developments cited above. This convergence and interaction between the “molecular” and “imaging” sciences has been strongly supported by the NCI and was designated as one of six “Extraordinary Scientific Opportunities for Investment”. More recently, other NIH institutes and the Department of Energy (DOE) in the US have also developed “molecular imaging” program support.

New technologies are needed to characterize the migration, survival, and function of antigen-specific T cells in vivo. Here, we demonstrate that Epstein-Barr virus (EBV)--specific T cells transduced with vectors encoding herpes simplex virus-1 thymidine kinase (HSV-TK) selectively accumulate radiolabeled 2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil (FIAU). After adoptive transfer, HSV-TK+ T cells labeled in vitro or in vivo with [131I]FIAU or [124I]FIAU can be noninvasively tracked in SCID mice bearing human tumor xenografts by serial images obtained by scintigraphy or positron emission tomography (PET), respectively. These T cells selectively accumulate in EBV+ tumors expressing the T cells' restricting HLA allele but not in EBV- or HLA-mismatched tumors. The concentrations of transduced T cells detected in tumors and tissues are closely correlated with the concentrations of label retained at each site. Radiolabeled transduced T cells retain their capacity to eliminate targeted tumors selectively. This technique for imaging the migration of ex vivo-transduced antigen-specific T cells in vivo is informative, nontoxic, and potentially applicable to humans.

Human cells exposed to antifolates show a rapid increase in the levels of the enzyme dihydrofolate reductase (DHFR). We hypothesized that this adaptive response mechanism can be used to elevate cellular levels of proteins fused to DHFR. In this study, mouse cells transfected to express a green fluorescent protein-DHFR fusion protein and subsequently exposed to the antifolate trimetrexate (TMTX) showed a specific and time-dependent increase in cellular levels of the fusion protein. Next, human HCT-8 and HCT-116 colon cancer cells retrovirally transduced to express a DHFR-herpes simplex virus 1 thymidine kinase (HSV1 TK) fusion protein and treated with the DHFR inhibitor TMTX exhibited increased levels of the DHFR-HSV1 TK fusion protein and an increase in ganciclovir sensitivity by 250-fold. The level of fusion protein in antifolate-treated human tumor cells was increased in response to a 24-h exposure of methotrexate, trimetrexate, as well as dihydrofolate. This effect depended on the antifolate concentration and was independent of the fusion-protein mRNA levels, consistent with this increase occurring at a translational level. In a xenograft model, nude rats bearing DHFR-HSV1 TK-transduced HCT-8 tumors and treated with TMTX showed, after 24 h, a 2- to 4-fold increase of fusion-protein levels in tumor tissue from treated animals compared with controls, as determined by Western blotting. The fusion-protein increase was imaged with positron-emission tomography, where a substantially enhanced signal of the transduced tumor was detected in animals after antifolate administration. Drug-mediated elevation of cellular DHFR-fused proteins is a very useful method to modulate gene expression in vivo for imaging as well as therapeutic purposes.

A noninvasive method for molecular imaging of the activity of different signal transduction pathways and the expression of different genes in vivo would be of considerable value. It would aid in understanding the role specific genes and signal transduction pathways have in various diseases, and could elucidate temporal dynamics and regulation at different stages of disease and during various therapeutic interventions. We developed and assessed a method for monitoring the transcriptional activation of endogenous genes by positron-emission tomography (PET) imaging. The HSV1-tk / GFP ( TKGFP ) dual reporter gene was used to monitor transcriptional activation of p53-dependent genes. A retrovirus bearing the Cis -p53/TKGFP reporter system was constructed in which the TKGFP reporter gene was placed under control of an artificial cis-acting p53-specific enhancer. U87 glioma and SaOS-2 osteosarcoma cells were transduced with this retrovirus and used to establish xenografts in rats. We demonstrated that DNA damage-induced up-regulation of p53 transcriptional activity correlated with the expression of p53-dependent downstream genes, such as p21, in U87 (wild-type p53), but not in SaOS-2 osteosarcoma (p53 −/−) cells. We showed that PET, with [ 124 I]FIAU (2′-fluoro-2′-deoxy-1-β- d -arabinofuranosyl-5-[ 124 I]iodouracil) and the Cis -p53TKGFP reporter system, is sufficiently sensitive to image the transcriptional regulation of genes in the p53 signal transduction pathway. These imaging results were confirmed by independent measurements of p53 activity and the expression levels of downstream genes (e.g., p21) by using conventional molecular-biological assays. PET imaging of p53 transcriptional activity in tumor xenografts by using the Cis -p53TKGFP reporter system may be useful in assessing novel therapeutic approaches.

Molecular therapy using viruses would benefit greatly from a non-invasive modality for assessing dissemination of viruses. Here we investigated whether positron emission tomography (PET) scanning using [(124)I]-5-iodo-2'-fluoro-1-beta-d-arabinofuranosyl-uracil (FIAU) could image cells infected with herpes simplex viruses (HSV). Using replication-competent HSV-1 oncolytic viruses with thymidine kinase (TK) under control of different promoters, we demonstrate that viral infection, proliferation and promoter characteristics all interact to influence FIAU accumulation and imaging. In vivo, as few as 1 x 107 viral particles injected into a 0.5-cm human colorectal tumor can be detected by [(124)I]FIAU PET imaging. PET signal intensity is significantly greater at 48 hours compared with that at 8 hours after viral injection, demonstrating that PET scanning can detect changes in TK activity resulting from local viral proliferation. We also show the ability of FIAU-PET scanning to detect differences in viral infectivity at 0.5 log increments. Non-invasive imaging might be useful in assessing biologically relevant distribution of virus in therapies using replication-competent HSV.

A variety of imaging technologies are being investigated as tools for studying gene expression in living subjects. Noninvasive, repetitive and quantitative imaging of gene expression will help both to facilitate human gene therapy trials and to allow for the study of animal models of molecular and cellular therapy. Radionuclide approaches using single photon emission computed tomography (SPECT) and positron emission tomography (PET) are the most mature of the current imaging technologies and offer many advantages for imaging gene expression compared to optical and magnetic resonance imaging (MRI)-based approaches. These advantages include relatively high sensitivity, full quantitative capability (for PET), and the ability to extend small animal assays directly into clinical human applications. We describe a PET scanner (microPET) designed specifically for studies of small animals. We review “marker/reporter gene” imaging approaches using the herpes simplex type 1 virus thymidine kinase (HSV1-tk) and the dopamine type 2 receptor (MR) genes. We describe and contrast several radiolabeled probes that can be used with the HSV1-tk reporter gene both for SPECT and for PET imaging. We also describe the advantages/disadvantages of each of the assays developed and discuss future animal and human applications.

Objective. To develop a non-immunogenic human derived dual reporter gene system for simultaneous imaging of different molecular-genetic processes in humans using nuclear imaging techniques (PET) with potential suicide capabilities.

Objective. The studies of NK cell based therapies often have to address the question of the NK cells trafficking, homing, tumor targeting and therapeutic effect. We tested feasibility of applying a multi-modality imaging approach for assessment of therapeutic NK-92 cells trafficking and tumor targeting in mice model.

Herpes simplex virus (HSV) mutants kill dividing tumor cells but spare non-proliferating, healthy brain tissue and may be useful in developing new treatment strategies for malignant brain tumors. Two HSV mutants, a thymidine kinase deficient virus (TK-) and a ribonucleotide reductase mutant (RR-), killed 7/7 human tumor cell lines in tissue culture. The TK-HSV killed Rat RG2 glioma and W256 carcinoma lines but not the rat C6 glioma in culture. TK-HSV replication (12 pfu/cell) was similar to wild-type HSV (10 pfu/cell) in rapidly dividing W256 cells in tissue culture, but was minimal (< 1 pfu/cell) in serum-starved cells, suggesting that the proliferative activity of tumor cells at the site and time of TK-HSV injection may influence efficacy in vivo. Subcutaneous W256 tumors in male Sprague-Dawley rats were injected with TK-HSV or free inoculum. A significant effect of TK-HSV therapy on W256 tumor growth was demonstrated compared to controls (p = 0.002). Complete regression was observed in 4/9 experimental tumors, with no recurrence over 6 months. Tumor growth in the remaining 5/9 animals was attenuated during the first 3 to 5 days after treatment, but not beyond 5 days compared to 9 matched control animals; no tumor regression was observed in any of the control animals. These results suggest that HSV mutants are potentially useful as novel therapeutic agents in the treatment of tumors in immunocompetent subjects.

The major factor limiting translation of reporter gene imaging studies to patients is the transduction requirement; target tissue must be transduced with the reporter constructs for reporter gene imaging studies. At least two different reporter constructs will be required in most cases; one will be a “constitutive” reporter that will be used to identify the site, extent and duration of vector delivery, and tissue transduction (the normalizing or denominator term), and one will be an “inducible” reporter that is sensitive to endogenous transcription factors, signaling pathways, or protein-protein interactions, as described above. The initial application of such double-reporter systems in patients will most likely be performed as part of a gene therapy protocol (Jacobs et al., 2001xPositron-emission tomography of vector-mediated gene expression in gene therapy for gliomas. Jacobs, A, Voges, J, Reszka, R, Lercher, M, Gossmann, A, Kracht, L, Kaestle, C, Wagner, R, Wienhard, K, and Heiss, W.D. Lancet. 2001; 358: 727–729Abstract | Full Text | Full Text PDF | PubMed | Scopus (283)See all ReferencesJacobs et al., 2001) or an adoptive therapy protocol where the patients own cells are harvested (e.g., lymphocytes, T cells, or blood-derived progenitor cells) and can be transduced and expanded ex vivo, and then adoptively readministered to the patient. This scenario couples reporter gene imaging with existing adoptive therapies and allows for ex vivo transduction and expansion of harvested cells. For example, adoptive T cell therapy could provide a venue for imaging T cell trafficking, targeting, activation, proliferation, and persistence (Ponomarev et al., 2001xImaging TCR-Dependent NFAT-Mediated T-Cell Activation with Positron Emission Tomography In Vivo. Ponomarev, V, Doubrovin, M, Lyddane, C, Beresten, T, Balatoni, J, Bornman, W, Finn, R, Akhurst, T, Larson, S, Blasberg, R et al. Neoplasia. 2001; 3: 480–488CrossRef | PubMed | Scopus (115)See all References, Koehne et al., 2003xShort and long-term in vivo imaging of targeted migration of HSV-TK transduced human antigen-specific lymphocyets. Koehne, G, Doubrovin, M, Doubrovina, E, Zanzonico, P, Gallardo, H, Ivanova, A, Balatoni, J, Teruya-Feldstein, J, Heller, G, May, C et al. Nat. Biotechnol. 2003; 21: 405–413CrossRef | PubMed | Scopus (158)See all References). This imaging paradigm could address several important questions related to adoptive T cell therapies. For example, is there substantial proliferation of adoptively transferred T cells at the target site or does activation and proliferation occur at other sites (e.g., specific lymphoid organ sites), followed by migration and localization to the target site? This question could potentially be addressed in a quantitative manner by repetitive PET imaging of the double-reporter system described above in the same animal or subject over time.