Juri Gelovani Tjuvajev

Imaging transcriptional regulation of p53-dependent genes with positron emission tomography in vivo

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 [124I]FIAU (2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-[124I]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-genetic imaging: current and future perspectives

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.

Tumor Growth Modulation by Sense and Antisense Vascular Endothelial Growth Factor Gene Expression: Effects on Angiogenesis, Vascular Permeability, Blood Volume, Blood Flow, Fluorodeoxyglucose Uptake, and Proliferation of Human Melanoma Intracerebral Xenografts

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, has been investigated as a potent mediator of brain tumor angiogenesis and tumor growth. We evaluated the effect of VEGF expression on the pathophysiology of tumor growth in the brain. Human SK-MEL-2 melanoma cells, with minimal VEGF expression, were stably transfected with either sense or antisense mouse VEGF cDNA and used to produce intracerebral xenografts. Vascular permeability, blood volume, blood flow, and tumor fluorodeoxyglucose metabolism were assessed using tissue sampling and quantitative autoradiography. Tumor proliferation was assessed by measuring bromodeoxyuridine labeling indices. Tumor vascular density and morphological status of the blood-brain barrier were evaluated by immunohistochemistry. SK-MEL-2 cells transfected with sense VEGF (V+) expressed large amounts of mouse and human VEGF protein; V+ cells formed well-vascularized, rapidly growing tumors with minimal tumor necrosis. V+ tumors had substantial and significant increases in blood volume, blood flow, vascular permeability, and fluorodeoxyglucose metabolism compared to wild-type and/or V- (antisense VEGF) tumors. VEGF antisense transfected V- expressed no detectable VEGF protein and formed minimally vascularized tumors. V- tumors had a very low initial growth rate with central necrosis; blood volume, blood flow, vascular permeability, and glucose metabolism levels were low compared to wild-type and V+ tumors. A substantial inhibition of intracerebral tumor growth, as well as a decrease in tumor vascularity, blood flow, and vascular permeability may be achieved by down-regulation of endogenous VEGF expression in tumor tissue. VEGF-targeted antiangiogenic gene therapy could be an effective component of a combined strategy to treat VEGF-producing brain tumors.

“Facilitated” Amino Acid Transport Is Upregulated in Brain Tumors

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 (K1ACPC and 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 K1ACPC and K1Ga-DTPA data, 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 K1ACPC values compared to corresponding K1DTPA values were also measured in C6 and RG2 gliomas. The ∂K1ACPC values for C6 glioma were more than twice that of contralateral brain cortex. K1ACPC and ∂K1ACPC values 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 K1DTPA were 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.

Positron emission tomography imaging for herpes virus infection: Implications for oncolytic viral treatments of cancer

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 [124I]-5-iodo-2′-fluoro-1-β-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 × 107 viral particles injected into a 0.5-cm human colorectal tumor can be detected by [124I]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.

Mutant herpes simplex virus induced regression of tumors growing in immunocompetent rats.

SummaryHerpes 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 efficacyin vivo. Subcutaneous W256 tumors in male Sprague-Dawley rats were injected with TK-HSV or virus 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.

Positron emission tomography (PET) imaging of tumor-localized Salmonella expressing HSV1-TK

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-β-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.

Cells exposed to antifolates show increased cellular levels of proteins fused to dihydrofolate reductase: A method to modulate gene expression

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.

In vivo imaging of molecular-genetic targets for cancer therapy

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.

In vivo multiple-mouse imaging at 1.5 T

A multiple‐mouse solenoidal MR coil was developed for in vivo imaging of up to 13 mice simultaneously to screen for tumors on a 1.5 T clinical scanner. For the coil to be effective as a screening tool, it should permit acquisition of MRIs in which orthotopic tumors with diameters >2 mm are detectable in a reasonable period of time (<1 hr magnet time) and their sizes accurately measured. Using a spin echo sequence, we demonstrated that this coil provides sufficient sensitivity for moderately high resolution images (156–176 μm in plane‐resolution, 1.5 mm slice thickness). This spatial resolution permitted detection of primary brain tumors in transgenic/knockout mice and orthotopic xenografts. Brain tumor size as measured by MRI was correlated with size measured by histopathology (P < 0.001). Metastatic tumors in the mouse lung were also successfully imaged in a screening setting. The multiple mouse coil is simple in construction and may be implemented without any significant modification to the hardware or software on a clinical scanner. Magn Reson Med 49:551–557, 2003.