Reciprocal activity of AgRP and POMC neurons governs coordinated control of feeding and metabolism.

Agouti-related peptide (AgRP)-expressing and proopiomelanocortin (POMC)-expressing neurons reciprocally regulate food intake. Here, we combine non-interacting recombinases to simultaneously express functionally opposing chemogenetic receptors in AgRP and POMC neurons for comparing metabolic responses in male and female mice with simultaneous activation of AgRP and inhibition of POMC neurons with isolated activation of AgRP neurons or isolated inhibition of POMC neurons. We show that food intake is regulated by the additive effect of AgRP neuron activation and POMC neuron inhibition, while systemic insulin sensitivity and gluconeogenesis are differentially modulated by isolated-versus-simultaneous regulation of AgRP and POMC neurons. We identify a neurocircuit engaging Npy1R-expressing neurons in the paraventricular nucleus of the hypothalamus, where activated AgRP neurons and inhibited POMC neurons cooperate to promote food consumption and activate Th+ neurons in the nucleus tractus solitarii. Collectively, these results unveil how food intake is precisely regulated by the simultaneous bidirectional interplay between AgRP and POMC neurocircuits.

Functionally distinct POMC-expressing neuron subpopulations in hypothalamus revealed by intersectional targeting.

Pro-opiomelanocortin (POMC)-expressing neurons in the arcuate nucleus of the hypothalamus represent key regulators of metabolic homeostasis. Electrophysiological and single-cell sequencing experiments have revealed a remarkable degree of heterogeneity of these neurons. However, the exact molecular basis and functional consequences of this heterogeneity have not yet been addressed. Here, we have developed new mouse models in which intersectional Cre/Dre-dependent recombination allowed for successful labeling, translational profiling and functional characterization of distinct POMC neurons expressing the leptin receptor (Lepr) and glucagon like peptide 1 receptor (Glp1r). Our experiments reveal that POMCLepr+ and POMCGlp1r+ neurons represent largely nonoverlapping subpopulations with distinct basic electrophysiological properties. They exhibit a specific anatomical distribution within the arcuate nucleus and differentially express receptors for energy-state communicating hormones and neurotransmitters. Finally, we identify a differential ability of these subpopulations to suppress feeding. Collectively, we reveal a notably distinct functional microarchitecture of critical metabolism-regulatory neurons.

Detecting tissue deterioration after brain injury: regional blood flow level versus capacity to raise blood flow.

Regional cerebral blood flow (rCBF) is spatially and temporally adjusted to local energy needs. This coupling involves dilation of vessels both at the site of metabolite exchange and upstream of the activated region. Deficits in upstream blood supply limit the 'capacity to raise rCBF' in response to functional activation and therefore compromise brain function. We here demonstrate in rats that the 'capacity to raise rCBF' can be determined from real-time measurements of rCBF using laser speckle imaging during an energy challenge induced by cortical spreading depolarizations (CSDs). Cortical spreading depolarizations (CSDs) occur with high incidence in stroke and various other brain injuries and cause large metabolic changes. Various conditions of cerebral perfusion were induced, either by modifying microvascular tone, or by altering upstream blood supply independently. The increase in rCBF per unit of time in response to CSD was linearly correlated to the upstream blood supply. In an experimental model of stroke, we found that this marker of the capacity to raise rCBF which, in pathologic tissue may be additionally limited by impaired vasoactive signaling, was a better indicator of the functional status of cerebral tissue than local rCBF levels.

Imaging microglial activation and glucose consumption in a mouse model of Alzheimer's disease.

In Alzheimer's disease (AD), persistent microglial activation as sign of chronic neuroinflammation contributes to disease progression. Our study aimed to in vivo visualize and quantify microglial activation in 13- to 15-month-old AD mice using [(11)C]-(R)-PK11195 and positron emission tomography (PET). We attempted to modulate neuroinflammation by subjecting the animals to an anti-inflammatory treatment with pioglitazone (5-weeks' treatment, 5-week wash-out period). [(11)C]-(R)-PK11195 distribution volume values in AD mice were significantly higher compared with control mice after the wash-out period at 15 months, which was supported by immunohistochemistry data. However, [(11)C]-(R)-PK11195 µPET could not demonstrate genotype- or treatment-dependent differences in the 13- to 14-month-old animals, suggesting that microglial activation in AD mice at this age and disease stage is too mild to be detected by this imaging method.

Volumetry of [¹¹C]-methionine positron emission tomographic uptake as a prognostic marker before treatment of patients with malignant glioma.

The purpose of this positron emission tomography (PET) study was to compare the prognostic value of pretreatment volume of ¹¹C]-methionine (MET) uptake and semiquantitative MET uptake ratio in patients with malignant glioma. The study population comprised 40 patients with malignant glioma. Pretreatment magnetic resonance imaging (MRI) and MET-PET imaging were performed before the initiation of glioma treatment in all patients. The pretreatment MET uptake ratios and volumes were assessed. To create prognostically homogeneous subgroups, patients' pretreatment prognostic factors were stratified according to the six classes of Radiation Therapy Oncology Group recursive partitioning analysis (RTOG RPA). Univariate and multivariate analyses were performed to determine significant prognostic factors. Survival analyses identified the pretreatment volume of MET uptake and a higher RTOG RPA class as significant predictors. In contrast, pretreatment maximum areas of contrast enhancement on MRI and semiquantitative MET uptake ratios could not be identified as significant prognostic factors. The patients' outcomes and Karnofsky Performance Scale scores were significantly correlated with pretreatment volume of MET uptake but not with semiquantitative MET uptake ratio. The data suggest that pretreatment volumetry of MET uptake but not the semiquantitative MET uptake ratio is a useful biologic prognostic marker in patients with malignant glioma.

11C-Methionine positron emission tomographic imaging of biologic activity of a recurrent glioblastoma treated with stereotaxy-guided laser-induced interstitial thermotherapy.

In patients with recurrent glioblastoma multiforme (GBM), local minimally invasive treatment modalities have gained increasing interest recently because they are associated with fewer side effects than open surgery. For example, local tumor coagulation by laser-induced interstitial thermotherapy (LITT) is such a minimally invasive technique. We monitored the metabolic effects of stereotaxy-guided LITT in a patient with a recurrent GBM using amino acid positron emission tomography (PET). Serial 11C-methyl-L-methionine positron emission tomography (MET-PET) and contrast-enhanced computed tomography (CT) were performed using a hybrid PET/CT system in a patient with recurrent GBM before and after LITT. To monitor the biologic activity of the effects of stereotaxy-guided LITT, a threshold-based volume of interest analysis of the metabolically active tumor volume (MET uptake index of ≥ 1.3) was performed. A continuous decline in metabolically active tumor volume after LITT could be observed. MET-PET seems to be useful for monitoring the short-term therapeutic effects of LITT, especially when patients have been pretreated with a multistep therapeutic regimen. MET-PET seems to be an appropriate tool to monitor and guide experimental LITT regimens and should be studied in a larger patient group to confirm its clinical value.

Imaging of non- or very subtle contrast-enhancing malignant gliomas with [¹¹C]-methionine positron emission tomography.

In patients with World Health Organization (WHO) grade III glioma with a lack of or minimal (< 1 cm3) magnetic resonance imaging (MRI) contrast enhancement, the volume of the metabolically active part of the tumor was assessed by [¹¹C]-methionine positron emission tomography (MET-PET). Eleven patients with WHO grade III gliomas underwent MET-PET and MRI (contrast-enhanced T1- and T2-weighted images). To calculate the volumes in cubic centimeters, threshold-based volume of interest analyses of the metabolically active tumor (MET uptake index ≥ 1.3), contrast enhancement, and the T2 lesion were performed after coregistration of all images. In all patients, the metabolically active tumor volume was larger than the volume of gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) enhancement (20.8 ± 18.8 vs 0.29 ± 0.25 cm3; p < .001). With the exception of one patient, the volumes of contrast enhancement were located within the metabolically active tumor volume. In contrast, in the majority of patients, MET uptake overlapped with the T2 lesion and reached beyond it (in 10 of 12 MRIs/MET-PET scans). The present data suggest that in patients with WHO grade III glioma with minimal or a lack of contrast enhancement, MET-PET delineates metabolically active tumor tissue. These findings support the use of combined PET-MRI with radiolabeled amino acids (eg, MET) for the delineating of the true extent of active tumor in the diagnosis and treatment planning of patients with gliomas.

In-vivo visualization of tumor microvessel density and response to anti-angiogenic treatment by high resolution MRI in mice.

PURPOSE: Inhibition of angiogenesis has shown clinical success in patients with cancer. Thus, imaging approaches that allow for the identification of angiogenic tumors and the detection of response to anti-angiogenic treatment are of high clinical relevance. EXPERIMENTAL DESIGN: We established an in vivo magnetic resonance imaging (MRI) approach that allows us to simultaneously image tumor microvessel density and tumor vessel size in a NSCLC model in mice. RESULTS: Using microvessel density imaging we demonstrated an increase in microvessel density within 8 days after tumor implantation, while tumor vessel size decreased indicating a switch from macro- to microvessels during tumor growth. Moreover, we could monitor in vivo inhibition of angiogenesis induced by the angiogenesis inhibitor PTK787, resulting in a decrease of microvessel density and a slight increase in tumor vessel size. CONCLUSIONS: We present an in vivo imaging approach that allows us to monitor both tumor microvessel density and tumor vessel size in the tumor. Moreover, this approach enables us to assess, early-on, treatment effects on tumor microvessel density as well as on tumor vessel size. Thus, this imaging-based strategy of validating anti-angiogenic treatment effects has high potential in applications to preclinical and clinical trials.

Distinct spatiotemporal patterns of spreading depolarizations during early infarct evolution: evidence from real-time imaging.

Experimental and clinical studies indicate that waves of cortical spreading depolarization (CSD) appearing in the ischemic penumbra contribute to secondary lesion growth. We used an embolic stroke model that enabled us to investigate inverse coupling of blood flow by laser speckle imaging (CBF(LSF)) to CSD as a contributing factor to lesion growth already in the early phase after arterial occlusion. Embolization by macrospheres injected into the left carotid artery of anesthetized rats reduced CBF(LSF) in the territories of the middle cerebral artery (MCA) (8/14 animals), the posterior cerebral artery (PCA) (2/14) or in less clearly defined regions (4/14). Analysis of MCA occlusions (MCAOs) revealed a first CSD wave starting off during ischemic decline at the emerging core region, propagating concentrically over large portions of left cortex. Subsequent recurrent waves of CSD did not propagate concentrically but preferentially circled around the ischemic core. In the vicinity of the core region, CSDs were coupled to waves of predominantly vasoconstrictive CBF(LSF) responses, resulting in further decline of CBF in the entire inner penumbra and in expansion of the ischemic core. We conclude that CSDs and corresponding CBF responses follow a defined spatiotemporal order, and contribute to early evolution of ischemic territories.

[18F]FLT PET for non-invasive monitoring of early response to gene therapy in experimental gliomas.

The purpose of this study was to investigate the potential of 3'-deoxy-3'-[¹8F]fluorothymidine ([¹8F]FLT) positron emission tomography (PET) to detect early treatment responses in gliomas. Human glioma cells were stably transduced with genes yielding therapeutic activity, sorted for different levels of exogenous gene expression, and implanted subcutaneously into nude mice. Multimodality imaging during prodrug therapy included (a) magnetic resonance imaging, (b) PET with 9-(4-[¹8F]fluoro-3-hydroxymethylbutyl)guanine assessing exogenous gene expression, and (c) repeat [¹8F]FLT PET assessing antiproliferative therapeutic response. All stably transduced gliomas responded to therapy with significant reduction in tumor volume and [¹8F]FLT accumulation within 3 days after initiation of therapy. The change in [¹8F]FLT uptake before and after treatment correlated to volumetrically calculated growth rates. Therapeutic efficacy as monitored by [¹8F]FLT PET correlated to levels of therapeutic gene expression measured in vivo. Thus, [¹8F]FLT PET assesses early antiproliferative effects, making it a promising radiotracer for the development of novel treatments for glioma.

Spreading depolarizations cycle around and enlarge focal ischaemic brain lesions.

How does infarction in victims of stroke and other types of acute brain injury expand to its definitive size in subsequent days? Spontaneous depolarizations that repeatedly spread across the cerebral cortex, sometimes at remarkably regular intervals, occur in patients with all types of injury. Here, we show experimentally with in vivo real-time imaging that similar, spontaneous depolarizations cycle repeatedly around ischaemic lesions in the cerebral cortex, and enlarge the lesion in step with each cycle. This behaviour results in regular periodicity of depolarization when monitored at a single point in the lesion periphery. We present evidence from clinical monitoring to suggest that depolarizations may cycle in the ischaemic human brain, perhaps explaining progressive growth of infarction. Despite their apparent detrimental role in infarct growth, we argue that cycling of depolarizations around lesions might also initiate upregulation of the neurobiological responses involved in repair and remodelling.

Evaluation of primary brain tumors using 11C-methionine PET with reference to a normal methionine uptake map.

UNLABELLED: (11)C-Methionine PET is a well-established technique for evaluating tumor extent for diagnosis and treatment planning in neurooncology. Image interpretation is typically performed using the ratio of uptake within the tumor to a reference region. The precise location of this reference region is important as local variations in methionine uptake may significantly alter the result, particularly for lesions at the border of gray and white matter. Selection of a reference region can be highly user dependant, and identifying a representative normal region may be complicated by midline or multifocal tumors. We hypothesized that current coregistration methods would enable interpretation of methionine PET images with reference to an averaged normal uptake map, allowing better standardization of scan analysis and increasing the sensitivity to tumor infiltration, particularly of white matter regions. METHODS: A normal methionine uptake map was prepared from the normal hemispheres of 20 scans performed on patients with benign or low-grade lesions. Affine and nonlinear coregistration algorithms were evaluated for spatial normalization of the images to a previously developed PET template. A standardized method for applying the normal uptake map in brain tumors was developed and evaluated in a sample of 18 scans (6 grade II, 6 grade III, and 6 grade IV gliomas). Tumor extent was compared with that derived from a mirrored contralateral reference region method. Correlation coefficients were calculated between the uptake ratios for tumor to normal uptake map versus tumor to mirrored reference region. RESULTS: "RatioMap" images depicting voxel-by-voxel ratios of a patient scan to the normal uptake map revealed increased methionine uptake in white matter regions that could not be identified using the standard method. Uptake ratios within the tumor varied slightly with the normalization methods used but correlated closely with the ratio to a single reference value. Nonlinear coregistration with median ratio intensity normalization gave the strongest correlation (r = 0.97, P < 0.001, n = 17). CONCLUSION: Evaluation of methionine PET data with reference to normal uptake data may improve sensitivity to white matter infiltration. The tumor uptake ratios obtained correlated closely with a standard reference value technique, whereas the described method allowed for better standardization of the image analysis.

Switching on the lights for gene therapy.

Strategies for non-invasive and quantitative imaging of gene expression in vivo have been developed over the past decade. Non-invasive assessment of the dynamics of gene regulation is of interest for the detection of endogenous disease-specific biological alterations (e.g., signal transduction) and for monitoring the induction and regulation of therapeutic genes (e.g., gene therapy). To demonstrate that non-invasive imaging of regulated expression of any type of gene after in vivo transduction by versatile vectors is feasible, we generated regulatable herpes simplex virus type 1 (HSV-1) amplicon vectors carrying hormone (mifepristone) or antibiotic (tetracycline) regulated promoters driving the proportional co-expression of two marker genes. Regulated gene expression was monitored by fluorescence microscopy in culture and by positron emission tomography (PET) or bioluminescence (BLI) in vivo. The induction levels evaluated in glioma models varied depending on the dose of inductor. With fluorescence microscopy and BLI being the tools for assessing gene expression in culture and animal models, and with PET being the technology for possible application in humans, the generated vectors may serve to non-invasively monitor the dynamics of any gene of interest which is proportionally co-expressed with the respective imaging marker gene in research applications aiming towards translation into clinical application.

Multitracer positron emission tomographic imaging of exogenous gene expression mediated by a universal herpes simplex virus 1 amplicon vector.

To develop efficient and safe gene therapy approaches, the herpes simplex virus type 1 thymidine kinase gene (HSV-1-tk) has been shown to function as a marker gene for the direct noninvasive in vivo localization of thymidine kinase (TK) expression by positron emission tomography (PET) using radiolabeled nucleoside analogues as specific TK substrates. Moreover, the gene encoding dopamine type 2 receptor (d2r) could be used as a PET marker gene using specific radiolabeled receptor binding compounds. Here we describe the quantitative colocalization of d2r and HSV-1-tk gene expression mediated from a universal HSV-1 amplicon vector in a subcutaneous human Gli36dEGFR glioma model by PET. The HSV-1 amplicon vector was constructed using a bicistronic gene cassette to contain (1) the d2r80A mutant, which is able to bind its ligand racloprid but unable to activate downstream signal transduction pathways, and (2) the tk39 mutant with enhanced enzymatic activity toward guanosine analogues fused to the green fluorescent protein gene (tk39gfp) serving as a marker gene in cell culture. After infection of human Gli36dEGFR glioma cells with the HSV-d2r80AIREStk39gfp (HSV-DITG) amplicon vector in cell culture, D2 receptor expression and its targeting to the cell surface were determined by Western blotting and immunolabeling. Vector application in vivo served for quantitative colocalization of d2r80A- and tk39gfp-derived PET signals employing the specific D2 receptor binding compound [(11)C]racloprid and the specific TK39 substrate 9-(4-[(18)F]fluoro-3-hydroxymethylbutyl)guanine. Our results demonstrate that for the range of gene expression studied in vivo, both enzymatic and receptor binding assays give comparable quantitative information on the level of vector-mediated gene expression in vivo. The d2r80A in combination with a specific binding compound passing the intact blood-brain barrier might be an alternative marker gene for the noninvasive assessment of vector-mediated gene expression in the brain using PET.

Imaging-guided gene therapy of experimental gliomas.

To further develop gene therapy for patients with glioblastomas, an experimental gene therapy protocol was established comprising a series of imaging parameters for (i) noninvasive assessment of viable target tissue followed by (ii) targeted application of herpes simplex virus type 1 (HSV-1) amplicon vectors and (iii) quantification of treatment effects by imaging. We show that viable target tissue amenable for application of gene therapy vectors can be identified by multitracer positron emission tomography (PET) using 2-(18)F-fluoro-2-deoxy-D-glucose, methyl-(11)C-L-methionine, or 3'-deoxy-3'-(18)F-fluoro-L-thymidine ([(18)F]FLT). Targeted application of HSV-1 amplicon vectors containing two therapeutic genes with synergistic antitumor activity (Escherichia coli cytosine deaminase, cd, and mutated HSV-1 thymidine kinase, tk39, fused to green fluorescent protein gene, gfp) leads to an overall response rate of 68%, with 18% complete responses and 50% partial responses. Most importantly, we show that the "tissue dose" of HSV-1 amplicon vector-mediated gene expression can be noninvasively assessed by 9-[4-(18)F-fluoro-3-(hydroxymethyl)butyl]guanine ([(18)F]FHBG) PET. Therapeutic effects could be monitored by PET with significant differences in [(18)F]FLT accumulation in all positive control tumors and 72% in vivo transduced tumors (P = 0.01) as early as 4 days after prodrug therapy. For all stably and in vivo transduced tumors, cdIREStk39gfp gene expression as measured by [(18)F]FHBG-PET correlated with therapeutic efficiency as measured by [(18)F]FLT-PET. These data indicate that imaging-guided vector application with determination of tissue dose of vector-mediated gene expression and correlation to induced therapeutic effect using multimodal imaging is feasible. This strategy will help in the development of safe and efficient gene therapy protocols for clinical application.

18F-fluoro-L-thymidine and 11C-methylmethionine as markers of increased transport and proliferation in brain tumors.

UNLABELLED: Because of the high glucose metabolism in normal brain tissue 18F-FDG is not the ideal tracer for the detection of gliomas. Methyl-11C-l-methionine (11C-MET) is better suited for imaging the extent of gliomas, because it is transported specifically into tumors but only insignificantly into normal brain. 3'-Deoxy-3'-18F-fluorothymidine (18F-FLT) has been introduced as a proliferation marker in a variety of neoplasias and has promising potential for the detection of brain tumors, because its uptake in normal brain is low. Additionally, the longer half-life might permit differentiation between transport and intracellular phosphorylation. METHODS: PET of 18F-FLT and 11C-MET was performed on 23 patients (age range, 20-70 y) with histologically verified gliomas of different grades. On all patients, conventional MRI was performed, and 16 patients additionally underwent contrast-enhanced imaging. Images were coregistered, and the volumes of abnormality were defined for PET and MRI. Uptake ratios and standardized uptake values (SUVs) of various tumors and regions were assessed by region-of-interest analysis. Kinetic modeling was performed on 14 patients for regional time-activity curves of 18F-FLT from tumorous and normal brain tissue. RESULTS: Sensitivity for the detection of tumors was lower for 18F-FLT than for 11C-MET (78.3% vs. 91.3%), especially for low-grade astrocytomas. Tumor volumes detected by 18F-FLT and 11C-MET were larger than tumor regions displaying gadolinium enhancement (P<0.01). Uptake ratios of 18F-FLT were higher than uptake ratios of 11C-MET (P<0.01). Uptake ratios of 18F-FLT were higher in glioblastomas than in astrocytomas (P<0.01). Absolute radiotracer uptake of 18F-FLT was low and significantly lower than that of 11C-MET (SUV, 1.3+/-0.7 vs. 3.1+/-1.0; P<0.01). Some tumor regions were detected only by either 18F-FLT (7 patients) or 11C-MET (13 patients). Kinetic modeling revealed that 18F-FLT uptake in tumor tissue seems to be predominantly due to elevated transport and net influx. However, a moderate correlation was found between uptake ratio and phosphorylation rate k3 (r=0.65 and P=0.01 for grade II-IV gliomas; r=0.76 and P<0.01 for grade III-IV tumors). CONCLUSION: 18F-FLT is a promising tracer for the detection and characterization of primary central nervous system tumors and might help to differentiate between low- and high-grade gliomas. 18F-FLT uptake is mainly due to increased transport, but irreversible incorporation by phosphorylation might also contribute. In some tumors and tumor areas, 18F-FLT uptake is not related to 11C-MET uptake. In view of the high sensitivity and specificity of 11C-MET PET for imaging of gliomas, it cannot be excluded that 18F-FLT PET was false positive in these areas. However, the discrepancies observed for the various imaging modalities (18F-FLT and 11C-MET PET as well as gadolinium-enhanced MRI) yield complementary information on the activity and the extent of gliomas and might improve early evaluation of treatment effects, especially in patients with high-grade gliomas. Further studies are needed, including coregistered histology and kinetic analysis in patients undergoing chemotherapy.

Fast and robust registration of PET and MR images of human brain.

In recent years, mutual information has proved to be an excellent criterion for registration of intra-individual images from different modalities. Multi-resolution coarse-to-fine optimization was proposed for speeding-up of the registration process. The aim of our work was to further improve registration speed without compromising robustness or accuracy. We present and evaluate two procedures for co-registration of positron emission tomography (PET) and magnetic resonance (MR) images of human brain that combine a multi-resolution approach with an automatic segmentation of input image volumes into areas of interest and background. We show that an acceleration factor of 10 can be achieved for clinical data and that a suitable preprocessing can improve robustness of registration. Emphasis was laid on creation of an automatic registration system that could be used routinely in a clinical environment. For this purpose, an easy-to-use graphical user interface has been developed. It allows physicians with no special knowledge of the registration algorithm to perform a fast and reliable alignment of images. Registration progress is presented on the fly on a fusion of images and enables visual checking during a registration.

Improved herpes simplex virus type 1 amplicon vectors for proportional coexpression of positron emission tomography marker and therapeutic genes.

For the development of efficient and safe gene therapy protocols for clinical application it is desirable to determine the tissue dose of vector-mediated therapeutic gene expression noninvasively in vivo. The herpes simplex virus type 1 thymidine kinase gene (HSV-1-tk) has been shown to function as a marker gene for the direct noninvasive in vivo localization of thymidine kinase (TK) expression by positron emission tomography (PET). Using bicistronic or multicistronic gene-expressing cassettes with tk as the PET marker gene, the quantitative analysis of tk gene expression may indirectly indicate the distribution and the level of expression of linked and proportionally coexpressed genes. Here, we describe the construction and functional evaluation of HSV-1 amplicon vectors mediating proportional coexpression of HSV-1-tk as PET marker gene and the enhanced green fluorescent protein gene (gfp) as proof of principle and cell culture marker gene and the Escherichia coli cytosine deaminase (cd) as therapeutic gene. Several double-/triple-gene constructs expressing HSV-1-tk, gfp, and E. coli cd were engineered based on gene fusion or the use of an internal ribosome entry site (IRES). Functional analysis in cell culture (green fluorescent protein [GFP] fluorescence and sensitivity to the prodrugs ganciclovir [GCV] and 5-fluorocytosine [5-FC]) and Western blots were carried out after infection of proliferating rat 9L gliosarcoma and human Gli36 glioma cells with helper virus-free packaged HSV-1 amplicon vectors. To study the ability of PET to differentiate various levels of tk expression noninvasively in vivo, retrovirally transduced and selected populations of rat F98 and human Gli36dEGFR glioma cells with defined levels of proportionally coexpressed tk and gfp genes were grown as subcutaneous tumors in nude rats and nude mice, and tk imaging by PET was performed. To study HSV-1 amplicon vector-mediated gene coexpression in vivo, HSV-1 amplicon vectors bearing coexpression constructs were injected (4 x 10(7) to 1 x 10(8) transducing units) into subcutaneously growing Gli36dEGFR gliomas in nude animals, and tk imaging was performed 24 hr later. All vector constructs mediated GFP expression and sensitized 9L and Gli36 cells toward GCV- and 5-FC-mediated cell killing in a drug dose-dependent manner, respectively. The levels of gene expression varied depending on the location of the genes within the constructs indicating the influence of the IRES on the level of expression of the second gene. Moreover, functional proportional coexpression of the PET marker gene HSV-1-tk and the linked therapeutic E. coli cd gene was observed. In selected tumor cell populations, subtle IRES-dependent differences of tk gene expression could be noninvasively distinguished by PET with good correlation between quantitative assays for IRES-dependent attenuated GFP and TK expression in culture and in vivo. After infection of subcutaneously growing gliomas with HSV-1 amplicon vectors, various levels of TK expression were found ranging from 0.011-0.062 percentage injected dose per gram (%ID/g). These values were 4.0- to 5.7-fold lower than positive control tumor cells. TK expression could be imaged by PET in vivo even with the tk gene located at the weak position downstream from the IRES. In conclusion, these HSV-1 amplicon vectors carrying HSV-1-tk as PET marker gene and any linked therapeutic gene will serve an indirect noninvasive assessment of the distribution of therapeutic gene expression by PET. Monitoring the correlation between primary transduction and therapeutic efficiency of a given vector is highly desirable for the development of safe and efficient gene therapy and vector application protocols in clinical applications.