To evaluate the efficiency of gene delivery in gene therapy strategies for malignant brain tumors, it is important to determine the distribution and magnitude of transgene expression in target tumor cells over time. Here, we assess the time- and vector dose-dependent kinetics of recombinant herpes simplex virus (HSV)-1 vector-mediated gene expression and vector replication in culture and in vivo by a recently developed radiotracer method for noninvasive imaging of gene expression (J. G. Tjuvajev et al., Cancer Res., 55: 6126–6132, 1995).

The kinetics of viral infection of rat 9L gliosarcoma cells by the replication-conditional HSV-1 vector, hrR3, was studied by measuring the accumulation rate of 2-[14C]-fluoro-5-iodo-1-β-d-arabinofuranosyl-uracil (FIAU), a selective substrate for viral thymidine kinase (TK). The level of viral TK activity in 9L cells was monitored by the radiotracer assay to assess various vector doses and infection times, allowing vector replication and spread. In parallel, viral yields and levels of Escherichia coli β-galactosidase activity were assessed quantitatively. To study vector replication, spread and HSV-1-tk and lacZ gene coexpression in vivo, first- or second-generation recombinant HSV-1 vectors (hrR3 or MGH-1) were injected into s.c. growing rat 9L or human U87ΔEGFR gliomas in nude rats at various times (8 h to 8 days) and at various vector doses [1 × 106 to 2 × 109 plaque-forming units (PFUs)] prior to imaging. For noninvasive assessment of HSV-1-tk gene expression (124I-labeled FIAU % dose/g), 0.15 mCi of 124I-labeled FIAU was injected i.v. 8 h after the last vector administration, and FIAU positron emission tomography (PET) was performed 48 h later. For the assessment of HSV-1-tk and lacZ gene coexpression, 0.2 mCi of 131I-labeled FIAU was injected i.v. 24 h after the last vector administration. Forty-eight h later, animals were killed, and tumors were dissected for quantitative autoradiographical and histochemical assessment of regional distribution of radioactivity (TK expression measured as 131I-labeled FIAU % dose/g) and coexpressed lacZ gene activity.

The rates of FIAU accumulation (Ki) in hrR3-infected 9L cells in culture, which reflect the levels of HSV-1-tk gene expression, ranged between 0.12 and 3.4 ml/g/min. They increased in a vector dose- and infection time-dependent manner and correlated with the virus yield (PFUs/ml), where the PFUs:Ki ratios remained relatively constant over time. Moreover, a linear relationship was observed between lacZ gene expression and FIAU accumulation 5–40 h after infection of 9L cells with a multiplicity of infection of 1.5. At later times (>52 h postinjection), high vector doses (multiplicity of infection, 1.5) led to a decrease of FIAU accumulation rates, viral yield, and cell pellet weights, indicating vector-mediated cell toxicity. Various levels of HSV-1-tk gene expression could be assessed by FIAU-PET after in vivo infection of s.c. tumors. The levels of FIAU accumulation were comparatively low (∼ ranging from 0.00013 to 0.003% injected dose/g) and were spatially localized; this may reflect viral-induced cytolysis of infected tumor cells and limited lateral spread of the virus. Image coregistration of tumor histology, HSV-1-tk related radioactivity (assessed by autoradiography), and lacZ gene expression (assessed by β-galactosidase staining) demonstrated a characteristic pattern of gene expression around the injection sites. A rim of lacZ gene expression immediately adjacent to necrotic tumor areas was observed, and this zone was surrounded by a narrow band of HSV-1-tk-related radioactivity, primarily in viable-appearing tumor tissue.

These results demonstrate that recombinant HSV-1 vector-mediated HSV-1-tk gene expression can be monitored noninvasively by PET, where the areas of FIAU-derived radioactivity identify the viable portion of infected tumor tissue that retains FIAU accumulation ability, and that the accumulation rate of FIAU in culture, Ki, reflects the number of HSV-1 viral particles in the infected tumor cell population [4.1 ± 0.6 × 106 PFUs/Ki unit (PFUs ÷ ml/min/g)]. Moreover, time-dependent and spatial relationships of HSV-1-tk and lacZ gene coexpression in culture and in vivo indicate the potential for indirect in vivo imaging of therapeutic gene expression in tumor tissue infected with any recombinant HSV-1 vector where a therapeutic gene is substituted for the lacZ gene.

Modern recombinant DNA technology has led to the development of different viral and nonviral vector systems to facilitate the delivery of specific genes of interest into target cell populations. More than 2100 patients are currently enrolled in >270 clinical Phase I and II studies (1, 2) to assess the clinical safety and efficiency of different modes of gene delivery. To date, most patients enrolled carry malignancies (68%), the most commonly used vectors are retroviral in origin (56%), and the transgenes in most protocols are reporter genes such as lacZ and neoR, immune-enhancing genes such as HLA B7 and interleukin 2, and the prodrug activating gene, HSV-1-tk.3 Retrovirus-mediated HSV-1-tk suicide gene therapy was successful in experimental brain tumor models (3). Clinical studies revealed that the HSV-1-tk/ganciclovir approach as an adjuvant to the surgical resection of recurrent high-grade brain tumors can be performed safely, although clinical responses were observed in only a few patients with very small brain tumors (4, 5). The lack of therapeutic efficiency of this replication-deficient retrovirus vector system in clinical settings may be attributable to: (a) inability to distribute vector-producer cells throughout the tumor; (b) vector-producer cell instability; (c) low transduction efficiency of retrovirions; and (d) tumor heterogeneity. Therefore, replication-competent oncolytic vectors in combination with new or improved prodrug-suicide gene systems are being evaluated as a part of a multimodal approach in current research (6, 7, 8, 9, 10).

Replication-defective and -competent HSV-1 mutants have great potential for the delivery and expression of transgenes to normal cells in the central nervous system, as well as for the efficient and selective destruction of cancer cells using suicide genes or lytic infection limited to dividing cells (conditional replication; Refs. 6, 8, and 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). Clinical trials are currently under way using a replication-conditional HSV-1 vector bearing the HSV-1-tk and lacZ genes in patients with brain tumors (21, 22, 23). The HSV-1 genome can be altered in different ways for specific therapeutic strategies (reviewed in Refs. 8 and 24). For virus therapy of brain tumors, different single or multimutated replication-conditional HSV-1 vectors have been used. These vectors allow replication only in dividing cells and reduce neuronal toxicity. They include mutations involving the tk gene [e.g., RH105 (25); dlsptk (17, 26); KOS-SB (27, 28); dl8.36tk (29); G92A (20)] and the RR gene [e.g., hrR3 (12, 30, 35)], together with the γ34.5 gene or mutations of the γ34.5 gene alone [e.g., R3616 (32, 33, 34); 1716 (18, 23, 35); G207 (19); and MGH-1 (16)]. The RH105 and the hrR3 vector bear lacZ insertional mutations within the tk gene (25) or the large subunit, ICP6, of the gene encoding RR (30), respectively, restricting replication of these vectors to proliferating cells (e.g., tumor cells) that express complementary endogenous TK or RR activity. The MGH-1 and G207 vectors bear additional mutations at both γ34.5 genes, reducing further the risk of recombination and neuroinvasion (16, 21). hrR3, G207, and MGH-1 vectors have been used for the selective destruction of tumor cells in experimental brain tumor models with little or no pathological changes observed within normal brain tissue. These vectors retain an intact viral tk gene encoding a key enzyme in the de novo synthesis of nucleotide precursors (36). The TK enzyme catalyzes the phosphorylation of thymidine to thymidylate using ATP as the phosphate donor. Whereas cellular (mammalian) TK specifically phosphorylates thymidine (37), HSV-1-TK has a much broader range of substrates, including pyrimidine nucleosides and guanosine derivatives, such as ganciclovir, as well as other purine and pyrimidine nucleoside analogues (38). Administration of ganciclovir to animals bearing brain tumors treated with hrR3 served to increase the therapeutic capacity of the vector in some experimental models (32).

The purpose of this study was to determine whether a recently developed radiotracer method for the assessment of viral TK activity, used previously to monitor retroviral and adenoviral vector-mediated transduction of the HSV-1-tk gene (39, 40, 41, 42, 43, 44, 45, 46, 47), could also be used to monitor the replication and spread of a replication-conditional HSV-1 vector in tumor cells. That is, can the HSV-1-tk gene be used as a marker/reporter gene to monitor the kinetics of hrR3 and MGH-1 replication and spread in infected tumor cells? These studies are the first assessments of feasibility for imaging replication-conditional HSV-1 vectors using a radiotracer assay. Monitoring the distribution of the viral vector and knowledge of the location, magnitude, and duration of transgene expression is highly desirable and could have a critical impact on the clinical application, management, and assessment of patients undergoing gene therapy. Potential clinical applications include the ability to monitor the location, spread, and persistence of the viral vector by noninvasive imaging of HSV-1-tk gene expression.

Cell Culture

The rat 9L gliosarcoma cells and African green monkey kidney cells (Vero) were obtained from American Tissue Culture Collection (Rockville, MD). The human U87ΔEGFR glioma cells were kindly provided by Dr. H-J. Su Huang (Ludwig Institute for Cancer Research, San Diego, CA). This cell line was established by retroviral transfer of a mutant epidermal growth factor receptor (Δ2–7EGFR) into the U-87 MG human glioblastoma cell line, enhancing its tumorigenic capacity in the brains of nude mice (48). Rat RG2 glioma cells were kindly provided by Dr. D. Bigner (Duke University Medical Center, Durham, NC). The gp-STK-A2 retroviral vector producer cells, which produces a replication-deficient retroviral vector STK that carries the neoR and HSV-1-tk genes (49), were kindly provided by Dr. F. Moolten (Memorial Veterans Hospital, Bedford, MA). The 9LTK+ and RG2TK+ glioma cells were produced by transfection of the wild-type 9L or RG2 cells, respectively, with an STK retroviral vector, as described previously (44, 45). All cell cultures were grown as monolayers in DMEM supplemented with 10% FBS (Sigma Chemical Co., St. Louis, MO) and 100 units/ml penicillin and 100 μg/ml streptomycin (1% P/S; Sigma) at 37°C in a 5% CO2/95% air atmosphere. The culture medium for gp-STK-A2 producer cells and transduced 9LTK+ and RG2TK+ cells was supplemented with 250 μg/ml of G418 (Sigma).

Viral Stocks

The recombinant HSV-1 virus vectors [hrR3 (30) and RH105 25)] containing lacZ insertional mutations within the RR gene or tk gene loci, respectively, were kindly provided by Dr. Sandra Weller (University of Connecticut, Framington, CT) and Drs. Dora Ho and Edward Mocarski (Stanford University, Stanford, CA), respectively. The HSV-1 mutant, MGH-1, was derived from HSV-1 mutant R3616 (double γ34.5 deletion mutant) containing an additional lacZ insertional mutation at the ICP6 gene (encoding RR; Ref. 16). To propagate HSV-1 mutants, subconfluent monolayers of Vero cells were infected (MOI, <1 PFU/cell), grown for 36–48 h until pronounced cytopathic effects became apparent, and scraped into the medium. Virus was harvested by three freeze-thaw cycles. Cell debris was spun down at 650 × g (relative centrifugal field) for 6 min at 4°C. The liberated virus was concentrated (25,000 × g for 1 h at 4°C) and titered on Vero and 9L cells using standard plaque assay. In brief, cells were plated into six-well plates at a density of 5 × 105 (Vero) or 1 × 106 (9L) cells/well. The next day, cells were infected with serial dilutions of viral stock in DMEM with 2% FBS. Two h p.i., cells were covered with 0.75% agarose (Life Technologies, Inc., Gaithersburg, MD) mixed 1:1 with complete medium to restrict virus spread, except through cell-to-cell contact. Four to 5 days after infection, virus plaques were stained with X-Gal (Fisher, Pittsburgh, PA) at 37°C for 6–8 h and counted. Aliquots of the viral stock (typically 1 × 1010 PFUs/ml) were stored at −80°C and thawed rapidly just before use.

Infection of 9L Cells and Accumulation of Radiolabeled Nucleosides in Culture

Rat 9L gliosarcoma cells were plated into 14-cm tissue culture dishes at 8 × 106 9L cells/dish. The next day, cell monolayers were ∼50% confluent and rinsed once with Dulbecco’s PBS (pH 7.4; Mediatech, Herndon, VA) and then trypsinized. Trypan blue exclusion was used to count live cells using a hemocytometer. The mean number of cells in three dishes was used for calculation of the MOI.

To study nucleoside accumulation at various MOIs (0, 0.05, 0.15, 0.5, and 1.5), 30 dishes were infected with the hrR3 vector by incubation with serial dilutions of vector stock in DMEM with 2% FBS, rotating dishes every 10–15 min for 2 h. Subsequently, complete medium was added to a total volume of 14 ml/dish, and infected cells were incubated at 37°C. Nineteen h p.i., the cell:medium accumulation ratios of radiolabeled nucleosides in hrR3-infected cells were determined as described previously (44). In brief, the culture medium was replaced with 14 ml of complete medium containing a mixture of 0.01 μCi/ml lsqb]14C]FIAU (54 mCi/mmol; Moravek Biochemicals, Brea, CA) and 0.1 μCi/ml methyl-[3H]thymidine (TdR; 65 Ci/mmol; Moravek Biochemicals). After various incubation periods (30, 60, and 90 min), the cells were scraped into the medium and collected by centrifugation. After weighing cells, the radioactivity (dpm) of the cell pellet was assayed using a Packard B1600 TriCarb beta spectrometer and standard 14C and 3H dual-channel counting technique. The cell-free medium was counted before and after incubation. Data were expressed as a harvested cell:medium concentration ratio [(dpm/g cells)/(dpm/ml medium); ml medium/g cells (ml/g)]. The rate of accumulation (Ki) was determined from the slope of the cell:medium ratio versus incubation time and is expressed as units of tracer clearance from the medium (ml medium/min/g cells or ml/min/g). The FIAU:TdR accumulation ratio is a ratio of Ki of FIAU and Ki of TdR. The Ki of FIAU is dependent upon the level of HSV-1-tk activity in the cells, whereas the Ki of TdR reflects the viability and proliferative activity of the cells (endogenous thymidine kinase activity). In other words, the FIAU:TdR ratio is a normalized measure of HSV-1-tk activity normalized to cell proliferation.

To study nucleoside accumulation at various time points after infection at a low MOI (0.15), 30 culture plates were infected, as described above, and accumulation studies were performed 0, 19, 31, 48, and 68 h p.i. An additional 54 dishes were infected with a high MOI (1.5), and accumulation studies were performed 0, 5, 9, 19, 28, 42, 50, 68, and 75 h p.i. The accumulation of radiolabeled nucleosides was also determined in stably transduced 9LTK+ cells (positive control), as well as in parental 9L cells and RH105-infected 9L cells (negative controls). All accumulation studies were performed in duplicate, and mean values were used for statistical evaluation.

Single-Step Growth Analysis

To determine whether nucleoside accumulation was related to viral replication, various dishes of 50% confluent 9L cells were infected with hrR3, as described above (MOIs, 0.0, 0.05, 0.15, 0.5, and 1.5). To determine PFU yields, infected cells were scraped into the medium, and virus was harvested after three freeze-thaw cycles to release intracellular virus at given intervals after infection: 19 h (all MOIs); 31, 48, and 68 h (MOI, 0.15); and 5, 9, 28, 42, 50, 68, and 75 h (MOI, 1.5). Virus was titered by standard plaque assay on Vero cell monolayers, as described above. Experiments were performed in duplicate (MOI, 1.5) or triplicate (all others).

Quantitation of lacZ Gene Expression in hrR3-infected 9L Cells in Culture

The level of lacZ gene expression was assessed in parallel in different cultures using a quantitative spectrophotometric assay for β-galactosidase activity by measuring the rate of ONPG hydrolysis in cell homogenates. After centrifugation, the cell pellets were weighed (10–20 mg) and homogenized in 1 ml of lysis buffer containing 10 mm Tris-HCl (pH 7.5), 1 mm DTT, 200 μg/ml of Pefabloc SC, 40 μg/ml of aprotinin SC, 5 μg/ml of leupeptin (Boehringer Mannheim, Indianapolis, IN), and 2 mm 2-mercaptoethanol. The cell lysate was obtained by centrifugation after brief ultrasonic disruption at 4°C. One hundred μl of the lysate were added to the reaction mixture (in the spectrophotometric cuvette) containing 2.6 ml of 100 mmd-galactose in 100 mm sodium phosphate buffer (pH 8), 100 μl of 30 mm MgCl solution, 100 μl of 3.36 m 2-mercaptoethanol solution. After equilibration at 37°C, the baseline absorbance at 410 nm was obtained. Thereafter, 100 μl of 68 mm ONPG solution (Sigma) were added to the reaction mixture, and a linear increase in 410 nm absorbance was recorded over 5–25 min with a Beckman DU-65 spectrophotometer (Beckman Instruments, Columbia, MD). The rate of increase in absorbance was converted into units of β-galactosidase using a standard curve obtained with known concentrations of Escherichia coli β-galactosidase (0.05 to 50 units/ml; Sigma) mixed with the wild-type tumor cell lysates (10 mg cells/ml). The results (units/ml) were normalized to the initial cell pellet weight, and the β-galactosidase activity was expressed as units/g cells.

Animal Experiments

Tumor Model and Vector Application in Vivo.

The experimental protocol involving animals was approved by the Institutional Animal Care and Use Committee of the Memorial Sloan-Kettering Cancer Center. Tumor cells (106 cells in 100 μl of DMEM) were injected s.c. into both flanks of R-Nu rats weighing 250–300 g. Three sets of animals were studied.

In the first group (n = 12), four s.c. tumors were produced in each rat. wt9L (n = 5) or U87ΔEFGR (n = 7) glioma cells were injected into the right upper and left upper and lower flanks and HSV-1-tk transduced RG2TK+ cells [single cell-derived clones as described previously (46)] were injected into the right lower flank as positive control. To study vector and vector dose-dependent relationships, the replication-conditional HSV-1 vectors, hrR3 (n = 8) or MGH-1 (n = 4), were injected as three different vector doses (1 × 106 PFUs, 1 × 107 PFUs, 1 × 108 PFUs in 200 μl of DMEM) directly into the dorsomedial part of the tumor, with the needle tract following the cranio-caudal axis of the animal. The lowest vector dose (1 × 106 PFUs) was injected into the tumor growing at the right upper flank, the medium vector dose (1 × 107 PFU) into the tumor growing at the left upper flank, and the highest vector dose (1 × 108 PFU) into the tumor growing at the left lower flank. To study time-dependent relationships or vector replication/spread, respectively, vector applications were performed at various times prior to [124I]FIAU administration: 8 h (n = 2; hrR3 only) or 4 days (n = 5; 3 × hrR3 and 2 × MGH-1) as single vector injections, or 2, 4, and 7 days (n = 5; 3 × hrR3 and 2 × MGH-1) as repeated vector injections with a third of the total applied vector dose at each time (0.33 × 106, 0.33 × 107, or 0.33 × 108 PFUs, respectively).

In the second group (n = 9), three s.c. tumors were produced in each rat: wt9L tumors were injected at left and right lower flanks and 9LTK+ cells [single cell-derived clones as described previously (46)] in the neck. To study time-dependent relationships, vector application with 2 × 109 PFUs hrR3 (in 200 μl of DMEM) was performed at various times prior to [131I]FIAU administration (1, 4, and 8 days; each n = 3). Intratumoral vector injection into the left wt9L tumor was performed into various tumor sites to achieve optimal vector distribution. The right wt9L tumor was injected with 200 μl of DMEM and served as negative control.

In the third group (n = 4), five s.c. tumors were produced in each rat. wt9L tumors were injected into the upper and lower flanks (three for hrR3 infection and one as negative control), and 9LTK+ cells were injected into the neck and served as positive control. The schedule of hrR3 injection with 2 × 109 PFUs in 200 μl of DMEM was equivalent to the previous animal group (1, 4, and 8 days prior to [131I]FIAU administration). The right upper flank tumor was infected at day 1, the left upper flank tumor at day 4, and the left lower flank tumor at day 8 prior to radiotracer administration. The right lower flank tumor was injected at day 4 with 200 μl of plain DMEM and served as negative control.

No Carrier-added Synthesis of [131I]FIAU.

No carrier-added 131I (t½ 8 days) was obtained from New England Nuclear (N. Billerica, MA). No carrier-added [131I]FIAU was prepared as described previously (50). Briefly, an excess of 2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-uracil (51) was heated in the presence of [131I]NaI and IODO-GEN. After cooling, [131I]FIAU was isolated on a C-18 Sep-Pak cartridge system and then eluted off with methanol. After evaporation of the methanol, the product was formulated in sterile, pyrogen-free, physiological saline and then passed through a sterile nonpyrogenic 0.22 μm Millipore filter. The radiochemical purity was determined by radio-TLC (Sigma-Aldrich silica gel plates; eluant was ethyl acetate:acetone:water, 14:8:1) and was found to be >97% pure.

No Carrier-added Synthesis of [124I]FIAU.

No carrier-added 124I ( 4 days) was produced (by J. B. and R. F.) from an enriched tellurium-124 oxide target, which after irradiation was subjected to dry distillation to release the trapped radioiodide (52). The 124I was isolated in a small volume of phosphate buffer. The full details of the preparation of [124I]FIAU will be presented elsewhere.4 In brief, [124I]FIAU was prepared from the stannylated precursor, 2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-(tri-n-butyltin)-uracil (53), which was allowed to react with [124I]NaI in the presence of a mixture of acetic acid/30% hydrogen peroxide. After quenching with sodium metabisulfite, the [124I]FIAU was isolated on a C-18 Sep-Pak cartridge system and then eluted off with methanol. After evaporation of the methanol, the product was reconstituted in sterile, pyrogen-free, physiological saline (with 5% ethanol added) and passed through a sterile nonpyrogenic 0.22 μm Millipore filter. The radiochemical purity was determined by radio-TLC (Sigma-Aldrich silica gel plates; eluant was ethyl acetate:acetone:water, 14:8:1) and was found to be >90% pure.

PET.

In the first set of animals, no carrier-added [124I]FIAU (150 μCi/animal) was injected i.v. 8 h or 2 or 4 days after the last vector injection. Three days prior to [124I]FIAU administration, animals received 0.9% sterile NaI solution (2 ml i.p./animal) for three subsequent days to block thyroid uptake of radioactive iodide. To allow sufficient washout of nontrapped [124I]FIAU, PET imaging was performed 48 h p.i. of [124I]FIAU administration using the GE Advance Tomograph (General Electric, Milwaukee, WI), with a spatial resolution of 5 mm FWHM at the center of the field of view. Two-dimensional emission scans were performed for 45 min and corrected for randoms, dead time, and scatter. Measured attenuation correction was performed using an 8-min duration transmission scan using two 9-mCi germanium transmission sources. Emission scans were reconstructed using an iterative reconstruction method with measured attenuation correction, smoothed with an 8-mm gaussian filter. The reconstruction parameters were 28 subsets, two iterations in a 256 × 256 matrix using a loop filter of 2.15 mm FWHM and a postfilter of 3.0 mm FWHM. For quantification of images and to account for differences in imaging time and radioactive decay, a set of [124I]FIAU reference standards of different radioactivity concentrations was placed within the field of view of the PET scanner. A ROI analysis of s.c. tumors was performed on 12.3-mm thick coronal resliced image sets. The coronal reslice parameters were chosen to eliminate the effects of partial volume averaging inherent in estimates of activity concentration in small objects. Regional tumor radioactivity concentrations (% ID/g) were determined from ROIs placed around areas of [124I]FIAU-related radioactivity within the tumor. To correct for background activity, ROIs were also drawn on the same image sets within the chest (the location of which was determined by the transmission scan). The net activity in the tumor was therefore calculated according to the following formula:

\[(\mathrm{Activity}ROI_{tumor}{-}\mathrm{activity}ROI_{background})\mathrm{{\mu}Ci/ml}\ {\times}\ (\mathrm{Volume}ROI_{tumor})\]

[measured activity within the ROItumor (μCi/ml) − measured activity within the ROIbackground (μCi/ml)] multiplied by the volume of the tumor that was Area ROItumor multiplied by the slice thickness.

Tissue Sampling, Histology, and QAR.

After the PET measurements (first set of animals) or 48 h after [131I]FIAU injection (second and third set of animals), rats were killed, and s.c. tumors were extracted rapidly. In [131I]FIAU-injected animals, the tumors were processed for histology/lacZ gene expression and QAR. In [124I]FIAU-injected animals, the tumors were cut into two equal parts, along the transaxial plane relative to the tumor’s position in the animal: (a) to confirm the validity of PET determination of tumor radioactivity, one-half of each tumor and selected organs were sampled, weighed and assayed for radioactivity using a Packard 5500 gamma spectrometer (Packard); and (b) to coregister coronal PET and QAR images with histology and X-Gal-stained sections for lacZ gene expression, the second half of each tumor was frozen and processed as described previously (45). In brief, 20 μm of fresh frozen sections were prepared by putting adjacent sections on two sets of slides, the first set for histology/lacZ gene expression and the second set for QAR. To study the transaxial distribution of vector-mediated lacZ gene expression, tissue sections were rinsed (1× PBS; 10 min), fixed (0.5% glutaraldehyde in 1× PBS; 30 min), washed (1 mm MgCl2 in 1× PBS; 3 × 10 min), and incubated with X-Gal (Fisher) at 37°C for 2–4 h. Thereafter, sections were rinsed (40 and 60% ethanol; 30 min each), counterstained (H&E; 15 min), fixed (80 and 100% ethanol; 30 min each), and mounted. The spatial distribution of FIAU-derived radioactivity in the second set of transaxial tissue sections was obtained by exposure of the sections to SB-5 film (Eastman Kodak, Rochester, NY). Knowing tissue radioactivity and the ID, the autoradiographic images were converted to parametric images of % ID/g tissue and color scale coded to a range of values. The X-Gal/H&E-stained tissue sections were digitized and coregistered to the corresponding autoradiographic images for precise localization of accumulated radioactivity, and they were also compared with the transaxial PET images.

Statistical Analysis

The mean and SE values of the tracer accumulation rates, tracer accumulation ratios, and β-galactosidase activity, as well as the mean and SD values of PFU yields, were calculated using Microsoft Excel 97 (Microsoft Corp.) and GraphPad Prism 2.0 (GraphPad Software, Inc.). Multiple regression analysis was performed using KaleidaGraph 3.08 (Synergy Software, Inc.).

Overview of Studies.

HSV-1-tk gene expression in cultured hrR3-infected and noninfected rat 9L gliosarcoma cells was evaluated by measuring the accumulation rate of radiolabeled FIAU. FIAU accumulation was compared with viral yield and levels of coexpressed lacZ gene activity. Then, HSV-1-tk gene expression was assessed by [124I]FIAU PET and/or [131I]FIAU QAR in noninfected and hrR3- or MGH-1-infected rat 9L or human U87ΔEGFR gliomas growing s.c. in nude rats.

Calculation of the FIAU Uptake Rate (Ki) and Comparison with Positive and Negative Controls.

Fig. 1,A shows the relationship between the cell:medium accumulation ratios of [14C]FIAU (substrate primarily for viral TK) and [3H]TdR (substrate for both viral TK and cellular TK) over time in 9L cells infected with the hrR3 virus (beginning 19 h after hrR3 infection at an MOI of 1.5). The rate of accumulation (Ki) was determined from the slope of the plot. Tracer clearance from the medium (Ki) was 1.1 ml/min/g cells for FIAU and 1.4 for TdR. Fig. 1 B shows the relationship between the levels of FIAU and TdR accumulation. The slope of this relationship yields the FIAU:TdR accumulation ratio that normalizes FIAU accumulation to a measure of endogenous cellular TK activity. The FIAU:TdR accumulation ratio was shown previously to reflect the level of viral TK activity in RG2 cells stably transduced with HSV-1-tk gene (RG2TK+ cells; Ref. 44). The mean ± SD values in RG2TK+ cells were: FIAU Ki, 0.14 ± 0.08 and FIAU:TdR, 0.31 ± 0.05, and these results have remained stable over 6 years.

The FIAU:TdR ratio was 0.77 ± 0.05 at an MOI of 1.5, 19 h p.i. (Fig. 1,B). These Ki and FIAU:TdR accumulation ratio values were significantly above the level measured in retrovirally transduced 9LTK+ cells (Ki, 0.048 ± 0.005 ml/g/min; FIAU:TdR, 0.19 ± 0.02; Fig. 1 C). They were also significantly above the Ki measured in RH105 (tk− virus; Ref. 25) infected 9L cells (Ki, <0.01 ml/g/min; FIAU:TdR, <0.005) and in noninfected 9L cells (Ki, <0.01 ml/g/min; FIAU:TdR, <0.005), both of which served as negative controls.

FIAU Accumulation Rate (Ki), MOI, and Viral Progeny (PFUs/ml) in hrR3-infected 9L Cells.

The initial hrR3 viral dose (MOIs ranging from 0.0 to 1.5) is highly correlated with the FIAU accumulation rate (Ki; Fig. 2,A), the FIAU:TdR accumulation ratio (Fig. 2,B), and the viral yield (PFUs; Fig. 2,C) at 19 h after infection of 9L cells. In contrast, the TdR accumulation rate (Fig. 2,A), as well as the cell pellet mass (Fig. 2 D), at 19 h after hrR3 infection of 9L cells was relatively unaffected by MOIs ranging from 0.0 to 1.5.

The duration of hrR3 infection of 9L cells also has a substantial effect on FIAU Ki, the FIAU:TdR accumulation ratio, and viral yield; this effect was observed after infection at both a low MOI (0.15; Fig. 3) and a high MOI (1.5; Fig. 4). During the early phase after hrR3 infection, FIAU Ki (Figs. 3 and 4,A), the FIAU:TdR accumulation ratio (Figs. 3 and 4,B) and viral yield (Figs. 3 and 4,C) increased exponentially with time; this occurred during the 19–52 h p.i. period for an MOI of 0.15 (Fig. 3, A–C) and during the 5–28 h p.i. period for an MOI of 1.5 (Fig. 4, A–C). In all sets of experiments, a significant correlation was observed between total viral yield and the FIAU accumulation rate Ki (Fig. 5,A) and the FIAU:TdR accumulation ratio (Fig. 5,B). The slopes of the relationships in Fig. 5,A and 5,B represent a “sensitivity measure” for the radiotracer assay. For Fig. 5,A, the FIAU sensitivity was 4.1 ± 0.6 × 106 PFUs per Ki unit for the hrR3 vector. Moreover, the PFU:Ki ratio remained relatively constant over time (Fig. 5,C), indicating that the FIAU accumulation rate, Ki, reflects the number of viral particles, PFUs/ml, in the infected tumor cell population. Only at later time points (>50 h p.i.) in the MOI of 1.5 set of experiments (Fig. 4) did the uptake of TdR, the viral yield, and the cell pellet mass decrease markedly because of cytolysis of the 9L cells (Fig. 4, C and D; Fig. 6). Therefore, FIAU:TdR accumulation ratios could not be reliably determined at these late time points (Fig. 4 B).

lacZ and HSV-1-tk Coexpression.

The time-dependent morphological changes in 9L cells after hrR3 infection (MOI, 1.5) and the increase in the level of lacZ expression (LacZ units/g) are depicted in Figs. 6 and 7. Most cells in the tissue culture dish were infected, and the blue-staining LacZ positive cells demonstrated preserved morphology during the 5–19-h period after infection (Fig. 6, A–C). Thereafter, a cytopathic effect became apparent, which was manifested by rounding of the cell bodies (28 h p.i.; Fig. 6,D) and by a plateau in cell pellet weight (Fig. 4,D). Over time, the number and percentage of rounded cells increased (42 h p.i.; Fig. 6,E), until the cells begin to lyse, and the total number of cells decreased (40–50 h p.i.). This lytic process is reflected by a decreasing cell pellet weight (Fig. 4,D) and decreasing levels of nucleoside accumulation to nonmeasurable levels. After an initial exponential increase in LacZ enzyme levels (5–19 h p.i.), the level of lacZ expression remains stable over 28–50 h p.i. (Fig. 7,A); beyond 50 h p.i., LacZ levels decreased rapidly to unmeasurable levels (data not shown). Most importantly, the levels of lacZ gene expression were related to the viral yield (Fig. 7,B) and to viral tk expression, as assessed by the FIAU:TdR accumulation ratio (Fig. 7C) and the Ki of FIAU accumulation (Fig. 7 D).

[124I]FIAU PET Imaging of Vector-mediated HSV-1-tk Gene Expression.

After infection of s.c. rat 9L gliosarcomas or human U87ΔEGFR gliomas, various levels of HSV-1-tk gene expression measured as % ID/g tumor tissue were found in all animals ranging from 0.00013 to 0.003% ID/g (0.2–4.2 nCi/g; values corrected for background levels of radioactivity). These values were 16–315-fold lower than in the positive control RG2TK+ tumor (21.9–132.2 nCi/g). The transaxial PET images suggest that [124I]FIAU-derived radioactivity was mostly localized around the site of virus injection, i.e., the needle injection tract (Fig. 8). Comparison of the 8-h (Fig. 8,A) and 4-day (Fig. 8 b) postvirus [124I]FIAU PET images revealed little or no progression in the radius of distribution of [124I]FIAU-derived radioactivity in the tumors. The localized areas of [124I]FIAU-derived radioactivity observed in the transaxial PET images were also compared with corresponding transaxial histological sections stained with X-Gal to identify the tumor areas infected with virus (Fig. 9). This comparison confirms the localization of [124I]FIAU-derived radioactivity in the tumor to the areas of virus injection and suggests limited lateral spread of the virus through the tumor over 4 days. In addition, no correlation or trend was observed between local or overall levels of [124I]FIAU-derived radioactivity (as measured in a ROI analysis on the PET images or directly in tumor samples) and vector dose or time allowed for vector replication and spread within the tumors.

[131I]FIAU QAR and X-Gal-stained Histology.

To further analyze the spatial relationships between HSV-1-tk-related radioactivity and lacZ gene expression, coregistration of X-Gal-stained tumor histology and QAR images was performed (Fig. 10). A characteristic pattern of HSV-1-tk and lacZ gene coexpression was found; a rim of lacZ gene expression immediately adjacent to necrotic tumor areas was surrounded by a narrow band of HSV-1-tk-related radioactivity, primarily in viable-appearing tumor tissue.

This study demonstrates four important findings:

(a) Recombinant HSV-1 vector-mediated HSV-1-tk gene expression can be monitored noninvasively by PET, where the areas of FIAU-derived radioactivity identify the viable portion of infected tumor tissue that retains FIAU accumulation ability. This imaging method may provide for the monitoring of recombinant HSV-1 vector replication in future human trials of herpes virus therapy of tumors. Such an imaging modality would allow one to correlate the location, extent, duration, and kinetics of HSV-1 vector-mediated gene expression with its therapeutic efficiency and also to evaluate the safety of administering these types of vectors.

(b) The levels of FIAU accumulation in the HSV-1 vector-infected tumors were comparatively low, suggesting that the levels of thymidine kinase expression driven by the HSV-1 viral tk promoter are at the lower end of detectability by this method. In future studies, the signal:noise ratio might be improved by using recombinant HSV-1 vectors containing expression cassettes in which the tk gene is driven by a stronger promoter (such as the cytomegalovirus IE promoter; pCMV) or a tissue-specific promoter.

(c) The direct correlation between the levels of HSV-1-tk and lacZ gene coexpression in the infected tumor cell population in culture and their partial coregistration in vivo indicate the potential for correlative in vivo imaging of therapeutic gene expression in tumor tissue infected with a recombinant HSV-1 vector. For example, a therapeutic gene, such as the prodrug activation enzyme cytochrome P450B1 (54), could be substituted for the lacZ gene in the hrR3 or MGH-1 vector. In hrR3 and MGH-1, the lacZ gene is inserted into the open reading frame of the RR gene, thus disrupting its expression and placing the lacZ gene under the control of the RR promoter. In both vectors, the tk and lacZ genes are driven by early gene promoters; thus, both genes are temporally coexpressed in the tightly regulated cascade of HSV-1 replication. This explains, at least in part, the observed correlation between the expression levels of HSV-1-tk and lacZ, as measured by FIAU accumulation and the ONPG assay, respectively.

(d) In cell culture, the accumulation rate of FIAU, Ki, reflects not only the level of tk and lacZ gene expression but also the number of HSV-1 infectious viral particles produced (viral propagation) in the infected tumor cell population. The rate of FIAU accumulation increased over time and correlated with the viral yield. The slopes of the relationships shown in Fig. 5,A and Fig. 5 B represent a “sensitivity measure” for the radiotracer assay to detect the number of HSV-1 viral particles in infected tumor tissue. The PFU:Ki ratio remained relatively constant over time in all experiments, irrespective of the MOI under investigation. This constancy was maintained fairly late in the time course, even after the Ki and PFU values started to decline in the MOI of 1.5 experiments. These results indicate that the FIAU accumulation rate reflects active virus propagation, and that the radiotracer assay may be able to monitor the number and spread of viral particles by noninvasive imaging of FIAU accumulation.

This observation is one of the important findings of the current study. The observed correlation of FIAU Ki with MOI and PFU/ml could be explained as follows: (a) the FIAU Ki measures the HSV-1-tk enzyme concentration/activity in cells that is dependent on the number of viral particles that infect each cell and produce HSV-1-tk; (b) the higher the MOI, the more viral particles infect each cell and produce more HSV-1-tk, resulting in more FIAU accumulation (larger Ki); (c) because MOI is a measure of the relative number of viral particles that is used to infect the cells (the “input”), and PFUs/ml values measure the resultant number of viral particles that are produced in the cells and released into the culture medium (the “output”), HSV-1-tk enzyme is produced during viral replication and, therefore, the amounts (concentration of HSV-1-tk and Ki of FIAU) correlate with the number of viral particles produced.

The radiotracer assay was used to assess the dynamics of HSV-1-tk expression in hrR3-infected, proliferating 9L cells in culture and identified clearly definable relationships between FIAU accumulation and: (a) viral dose (MOI); (b) time allowed for infection, replication, and virus spread; (c) viral yield (PFUs/ml); and (d) level of coexpressed lacZ gene. These findings reflect the kinetics of HSV-1 virus replication, where virus genes are temporally expressed in a cascade of α, β, and γ genes (reviewed in Ref. 24). tk, RR, and other early (β) genes of HSV-1 are coexpressed during the early phase of viral DNA synthesis, which starts 2–4 h and peaks 5–7 h after infection. This is followed by the expression of late genes coding for structural proteins (55). The whole cycle of HSV-1 replication takes approximately 18–20 h (time until virus particles are released; Refs. 36 and 55). At 19 h p.i. of 9L cells (approximate duration of one HSV-1 life cycle), only minimal FIAU accumulation was observed after infection with an MOI of 0.05. This is consistent with the low number of infected cells in the tissue culture population at this time (1–5% of cells were positive on histochemical staining for LacZ). Because the half-life of the HSV-1-tk mRNA transcript is ∼8–11 h (56), the observed level of FIAU accumulation at 19 h p.i. reflects continuing HSV-1-tk gene expression from the initial wave of hrR3 replication. At an MOI of 0.15, the level of HSV-1-tk gene expression (FIAU:TdR accumulation ratio, 0.1) at 19 h p.i. was comparable with that in retrovirally transduced 9LTK+ cells, and this accumulation ratio was even higher (∼0.8) when higher MOIs were used.

The FIAU accumulation (and FIAU:TdR ratio) continues to be high, even at 64 h p.i. (Figs. 3 and 4), when the cells have already begun to lyse, as evidenced by cell weight. This is because the radiotracer method with FIAU and TdR measures the accumulation of these tracers in cells grown in monolayers. When the cells are killed by the virus, they detach from the plastic and then lyse. Before adding the radioactive medium, the old medium is removed (with the lysed cell debris), and the accumulation occurs in the remaining viable cells. After harvesting the cells, the cell pellet was weighed, and the radioactivity concentration was expressed per gram of cells (dpm/g cells). That is why all of the data derived from the values of dpm/g cells reflect only what occurs in viable cells. Thus, even when a small portion of cells in the infected cell population is dead after 64 h after infection at low MOI (0.15), the remaining viable cells continue to accumulate FIAU more rapidly over time (because the infection spreads over time and more HSV-1-tk is produced; Fig. 3,C). After infection at a 10-fold higher MOI (1.5), the number of viral particles produced and the FIAU accumulation rate increase exponentially over time until 30 h p.i. Later, the capacity of viable cells to produce HSV-1-tk enzyme, to proliferate, and to produce more viral particles plateaus (Figs. 3 and 4).

This also explains the differences is the dynamics of viral particle production over time, after infection of a cell population at different MOIs (Figs. 3 and 4). That is, after infection at an MOI of 0.15, there is an exponential increase of viral particles in the infected cell population; this reaches a yield of 105 viral particles at 30 h p.i. (Fig. 3,C). After infection at an MOI of 1.5, a viral particle yield of 105 is observed at 4 h p.i. (Fig. 4,C) and continues to increase exponentially with a similar rate until 5 × 105 pfu/ml. Similar coefficients of 0.05 and 0.08 are observed for the exponential phases of viral proliferation after infection of cells at MOIs of 0.15 and 1.5. Therefore, the data presented in Fig. 3 are complimentary to the data presented in Fig. 4, i.e., the “early phase” of viral replication is exponential.

The replication and spread of a recombinant HSV-1 vector within a solid tumor mass may not proceed with the clearly definable kinetics that we observed in cultured cells, and the intensity of images may depend on factors other than the local vector concentration (PFUs/voxel). A very low regional vector concentration (MOI) may result in tk expression that is below the level of detection by PET imaging. Depending on virus dose and time after infection, replication-conditional HSV-1 mutants are cytotoxic and cause lysis of replicating host cells (Figs. 4 and 6). Host cell cytotoxicity will result in lower levels of tk expression and FIAU accumulation in infected tissue (Fig. 4). Thus, in comparison with replication-defective retro- and adenoviral vectors, replication-conditional HSV-1 vectors are somewhat limited by their propensity to eventually disrupt cellular functions and interfere with the enzymatic radiotracer assay. Nevertheless, the radiotracer imaging assay does provide some measure of vector-mediated tk expression and enzyme activity in target tissue. In addition, the images provide important spatial information that identifies the viable portion of transduced tissue at a given “imaging time window.” This imaging window can be defined as the time frame after recombinant HSV-1 vector infection where the FIAU accumulation processes of infected cells are still intact. Our results indicate that there is viral vector persistence during the time frame that has been studied (8 h to 8 days). However, a significant time-dependent difference in the distribution and magnitude of transgene expression was not observed. Vector-related cytotoxicity probably accounts for the low levels of FIAU accumulation in the infected tumors.

The spatial and time-dependent distribution of hrR3 and MGH-1 vector-mediated gene transduction, as assessed by PET, QAR, and histology/lacZ in this study, are consistent with previous studies of virus therapy for various tumors using first- or second-generation HSV-1 mutants (11, 12, 16, 17, 19, 26, 27, 31, 33, 57, 58, 59, 60, 61). The extent of the primary transduction varies and is dependent on: (a) the time of the assay; (b) the state of immunocompetence of the animals; (c) the species, infectivity, and burst size of cell line used; and (d) the delivery method. Variations of transduction efficiency may also be attributable to methodological difficulties in assessing gene expression throughout the tumor by serial sectioning and histology. For hrR3, the overall transduction efficiency in the first days after direct intratumoral vector application ranged from ∼ 8% (57, 58) to ∼75% (59) and after intracarotid vector infusion from 2.7 to 73.7% (60). The time dependency of vector-mediated gene expression and persistence of viral replication have been reported by Lasner et al.(61) using a first-generation γ34.5 deletion HSV-1 mutant (1716). These authors performed histological analyses and viral recovery from tumor tissue homogenates over 10 time points (up to 280 days) after intratumoral vector application in an intracranial D283 medulloblastoma model in nude mice. They noticed a decrease of vector-mediated gene expression and viral recovery within the first 2 weeks after vector application but persistence of viral replication for up to 280 days. Our data are limited but indicate that the kinetics of recombinant HSV-1 vector replication and spread throughout the tumor is slow. However, the persistence of vector replication should be measurable by sequential PET imaging of the same subject over time.

Better assessments of vector transfection and spread in gene therapy are being developed, and imaging now provides a noninvasive way to assess vector-mediated gene transduction and expression in vivo. These imaging assessments can involve either PET or magnetic resonance imaging, and they rely on the detection of radiolabeled or paramagnetic marker substrates (39, 43, 44, 45, 62), receptor binding compounds (63), or oligonucleotides (64). The necessary conditions for noninvasive transgene imaging with PET have been summarized previously (42, 44).

In conclusion, our results demonstrate, that: (a) recombinant HSV-1 vector-mediated HSV-1-tk expression can be monitored noninvasively by PET, where the areas of radioactivity identify the viable portion of infected tumor tissue expressing TK above a certain threshold; (b) the accumulation rate of FIAU in culture, Ki, reflects the number of HSV-1 viral particles in the infected tumor cell population; and (c) the definable viral dose- and time-dependent relationships between the levels of HSV-1-tk and lacZ gene coexpression in the infected tumor cell population in culture and the partial coregistration of HSV-1-tk and lacZ in vivo indicate the potential for indirect in vivo imaging of therapeutic gene expression in tumor tissue infected with any recombinant HSV-1 vector, where a therapeutic gene is substituted for the lacZ gene.

Fig. 1.

FIAU and TdR uptake in hrR3-infected and in retrovirally transduced rat 9L gliosarcoma cells (9LTK+ as positive control). A, the time-dependent accumulation of FIAU and TdR in 9L cells 19 h after hrR3 infection (MOI, 1.5). The rate of tracer accumulation (Ki) can be determined from the slope of the plots. B, the relationship between FIAU and TdR accumulation measured in A. C, the FIAU-TdR accumulation relationship in retrovirally transduced 9LTK+ cells. The slope of the plots in B and C yield the FIAU:TdR accumulation ratio, which normalizes FIAU accumulation to a measure of endogenous TK activity and which has been demonstrated to be a measure of viral TK activity (44).

Fig. 1.

FIAU and TdR uptake in hrR3-infected and in retrovirally transduced rat 9L gliosarcoma cells (9LTK+ as positive control). A, the time-dependent accumulation of FIAU and TdR in 9L cells 19 h after hrR3 infection (MOI, 1.5). The rate of tracer accumulation (Ki) can be determined from the slope of the plots. B, the relationship between FIAU and TdR accumulation measured in A. C, the FIAU-TdR accumulation relationship in retrovirally transduced 9LTK+ cells. The slope of the plots in B and C yield the FIAU:TdR accumulation ratio, which normalizes FIAU accumulation to a measure of endogenous TK activity and which has been demonstrated to be a measure of viral TK activity (44).

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Fig. 2.

Vector dose (MOI)-dependent relationships 19 h after hrR3 infection of 9L cells. The plots demonstrate an hrR3 dose (MOI)-dependent increase of the accumulation rate (Ki) of FIAU (but not TdR; A), of the FIAU:TdR accumulation ratio (B), and of the viral yield (C). D, the relationship between MOI and cell pellet weight per culture dish. Bars, SE.

Fig. 2.

Vector dose (MOI)-dependent relationships 19 h after hrR3 infection of 9L cells. The plots demonstrate an hrR3 dose (MOI)-dependent increase of the accumulation rate (Ki) of FIAU (but not TdR; A), of the FIAU:TdR accumulation ratio (B), and of the viral yield (C). D, the relationship between MOI and cell pellet weight per culture dish. Bars, SE.

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Fig. 3.

Time-dependent relationships after hrR3 infection of 9L cells with a low MOI (0.15). The plots demonstrate the time-dependent increase of the accumulation rate (Ki) of FIAU (but not TdR; A), of the FIAU:TdR accumulation ratio (B), and of the viral yield (C) in the early phase of viral replication. After infection at an MOI of 0.15, there is an exponential increase of viral particles in the infected cell population; this reaches a yield of 105 viral particles at 30 h p.i. (C). After infection at an MOI of 1.5 (Fig. 4), a viral particle yield of 105 was observed at 4 h p.i. (Fig. 4,C) and continues to increase exponentially with a similar rate until 5 × 105 pfu/ml. Similar coefficients of 0.05 and 0.08 were observed for the exponential phases of viral proliferation after infection of cells at MOIs of 0.15 and 1.5. In that, the data presented in Fig. 4 are complementary to the data presented in Fig. 3. D, the time-dependent changes in cell pellet weight per culture dish after hrR3 infection. At 68 h p.i., the TdR accumulation rate and cell pellet mass decreased as an indication of vector-induced cell toxicity and cytolysis. Bars, SE.

Fig. 3.

Time-dependent relationships after hrR3 infection of 9L cells with a low MOI (0.15). The plots demonstrate the time-dependent increase of the accumulation rate (Ki) of FIAU (but not TdR; A), of the FIAU:TdR accumulation ratio (B), and of the viral yield (C) in the early phase of viral replication. After infection at an MOI of 0.15, there is an exponential increase of viral particles in the infected cell population; this reaches a yield of 105 viral particles at 30 h p.i. (C). After infection at an MOI of 1.5 (Fig. 4), a viral particle yield of 105 was observed at 4 h p.i. (Fig. 4,C) and continues to increase exponentially with a similar rate until 5 × 105 pfu/ml. Similar coefficients of 0.05 and 0.08 were observed for the exponential phases of viral proliferation after infection of cells at MOIs of 0.15 and 1.5. In that, the data presented in Fig. 4 are complementary to the data presented in Fig. 3. D, the time-dependent changes in cell pellet weight per culture dish after hrR3 infection. At 68 h p.i., the TdR accumulation rate and cell pellet mass decreased as an indication of vector-induced cell toxicity and cytolysis. Bars, SE.

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Fig. 4.

Time-dependent relationships after hrR3 infection of 9L cells with a high MOI (1.5). The plots demonstrate the time-dependent changes of the accumulation rates (Ki) of FIAU and TdR (A), of the FIAU:TdR accumulation ratio (B), of the viral yield (C), and of the cell pellet weight (D). After infection with a high MOI, viral toxicity results eventually (>28 h p.i.) in a decrease of TdR accumulation (A) and cell pellet weight (D), and a leveling of FIAU accumulation (A and B) and viral yield (C). Bars, SE.

Fig. 4.

Time-dependent relationships after hrR3 infection of 9L cells with a high MOI (1.5). The plots demonstrate the time-dependent changes of the accumulation rates (Ki) of FIAU and TdR (A), of the FIAU:TdR accumulation ratio (B), of the viral yield (C), and of the cell pellet weight (D). After infection with a high MOI, viral toxicity results eventually (>28 h p.i.) in a decrease of TdR accumulation (A) and cell pellet weight (D), and a leveling of FIAU accumulation (A and B) and viral yield (C). Bars, SE.

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Fig. 5.

Relationship between viral yield after hrR3 infection of 9L cells and FIAU accumulation. The comparison between total viral particles (virus propagation) and the FIAU accumulation rate (Ki; A) and the FIAU:TdR accumulation ratio (B) demonstrate high correlations. In A, plotted data were obtained from Fig. 2,A and 2,C (Expt. 2), Fig. 3,A and 3,C (Expt. 3), and Fig. 4,A and Fig. 4,C (Expt. 4). In B, plotted data were obtained from Fig. 2,B and 2,C (Expt. 2), Fig. 3,B and 3,C (Expt. 3) and Fig. 4,B and 4 C (Expt. 4). Note that these relationships are viral dose (MOI) independent. The slopes of the relationships in A and B represent the “sensitivity” of the radiotracer assay; for A, the FIAU sensitivity is 4.1 ± 0.6 × 106 PFUs per Ki unit (PFUs ÷ ml/min/g). The relationship between the viral yield/HSV-1-tk activity ratios (PFUs:Ki) and the incubation time (C) was relatively constant and independent of the MOI.

Fig. 5.

Relationship between viral yield after hrR3 infection of 9L cells and FIAU accumulation. The comparison between total viral particles (virus propagation) and the FIAU accumulation rate (Ki; A) and the FIAU:TdR accumulation ratio (B) demonstrate high correlations. In A, plotted data were obtained from Fig. 2,A and 2,C (Expt. 2), Fig. 3,A and 3,C (Expt. 3), and Fig. 4,A and Fig. 4,C (Expt. 4). In B, plotted data were obtained from Fig. 2,B and 2,C (Expt. 2), Fig. 3,B and 3,C (Expt. 3) and Fig. 4,B and 4 C (Expt. 4). Note that these relationships are viral dose (MOI) independent. The slopes of the relationships in A and B represent the “sensitivity” of the radiotracer assay; for A, the FIAU sensitivity is 4.1 ± 0.6 × 106 PFUs per Ki unit (PFUs ÷ ml/min/g). The relationship between the viral yield/HSV-1-tk activity ratios (PFUs:Ki) and the incubation time (C) was relatively constant and independent of the MOI.

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Fig. 6.

Time-dependent changes in lacZ gene expression and development of cytopathic changes in 9L cells after hrR3 infection (MOI, 1.5). The photomicrographs in A–E were obtained at 5, 19, 28, 42, and 50 h after hrR3 infection, respectively, and show 9L cells that were histochemically stained to visualize the LacZ activity. These images demonstrate the progression (spread) of hrR3 infection in a monolayer of 9L cells as visualized by the increasing fraction of LacZ-positive cells. D and E demonstrate the development of cytopathic changes and marked cytolysis that were observed during the late phase of hrR3 infection.

Fig. 6.

Time-dependent changes in lacZ gene expression and development of cytopathic changes in 9L cells after hrR3 infection (MOI, 1.5). The photomicrographs in A–E were obtained at 5, 19, 28, 42, and 50 h after hrR3 infection, respectively, and show 9L cells that were histochemically stained to visualize the LacZ activity. These images demonstrate the progression (spread) of hrR3 infection in a monolayer of 9L cells as visualized by the increasing fraction of LacZ-positive cells. D and E demonstrate the development of cytopathic changes and marked cytolysis that were observed during the late phase of hrR3 infection.

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Fig. 7.

Relationships between LacZ enzyme activity and other measures in hrR3-infected 9L cells (MOI, 1.5). A shows the time-dependent increase of LacZ enzyme activity after hrR3 infection; a plateau is approached beyond 19 h p.i. The relationship between viral yield and LacZ enzyme activity in 9L cells after hrR3 infection (measures obtained at different times after infection; see A) is plotted in B. C and D demonstrate the positive correlation between LacZ enzyme activity and FIAU:TdR accumulation ratio and FIAU accumulation rate (Ki), respectively. Bars, SE.

Fig. 7.

Relationships between LacZ enzyme activity and other measures in hrR3-infected 9L cells (MOI, 1.5). A shows the time-dependent increase of LacZ enzyme activity after hrR3 infection; a plateau is approached beyond 19 h p.i. The relationship between viral yield and LacZ enzyme activity in 9L cells after hrR3 infection (measures obtained at different times after infection; see A) is plotted in B. C and D demonstrate the positive correlation between LacZ enzyme activity and FIAU:TdR accumulation ratio and FIAU accumulation rate (Ki), respectively. Bars, SE.

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Fig. 8.

[124I]FIAU PET imaging of hrR3 vector-mediated HSV-1-tk gene expression in a nude rat bearing s.c. U87ΔEGFR tumors. Each animal had three s.c. U87ΔEGFR tumors and one RG2TK+ tumor. Each of the U87ΔEGFR tumors were injected with a different dose (1 × 106, 1 × 107, and 1 × 108 PFUs) of hrR3; the injection track was along a cranio-caudal axis of the animal in the dorsomedial part of each tumor. [124I]FIAU was administered either 8 h (A) or 4 days (B) after virus injection, and PET imaging was performed 48 h later. Transaxial PET images demonstrate regions of HSV-1-tk related radioactivity primarily around injection tracts. A positive control tumor (RG2TK+) growing at the right lower flank demonstrates a 55–98-fold (A) or a 30–46-fold (B) higher level of HSV-1-tk gene expression, respectively. Radioactivity concentration in tumors was assessed from the PET images and background activity (non-tumor tissues) subtracted. The background activity was determined from a comparatively large region drawn over the lungs and heart (vascular pool) and was uniform compared with the infected tumors. The background activity was usually 2–3 nCi/ml. Measurements (% ID/g) performed on the images for tumors are approximations because of the marked heterogeneity of radioactivity within the tumors and because of partial volume effects. Background was not negligible at 48 h because the clearance of FIAU-derived radioactivity from noninfected, control tissues is exponential and has a half-life of ∼6 h (46).

Fig. 8.

[124I]FIAU PET imaging of hrR3 vector-mediated HSV-1-tk gene expression in a nude rat bearing s.c. U87ΔEGFR tumors. Each animal had three s.c. U87ΔEGFR tumors and one RG2TK+ tumor. Each of the U87ΔEGFR tumors were injected with a different dose (1 × 106, 1 × 107, and 1 × 108 PFUs) of hrR3; the injection track was along a cranio-caudal axis of the animal in the dorsomedial part of each tumor. [124I]FIAU was administered either 8 h (A) or 4 days (B) after virus injection, and PET imaging was performed 48 h later. Transaxial PET images demonstrate regions of HSV-1-tk related radioactivity primarily around injection tracts. A positive control tumor (RG2TK+) growing at the right lower flank demonstrates a 55–98-fold (A) or a 30–46-fold (B) higher level of HSV-1-tk gene expression, respectively. Radioactivity concentration in tumors was assessed from the PET images and background activity (non-tumor tissues) subtracted. The background activity was determined from a comparatively large region drawn over the lungs and heart (vascular pool) and was uniform compared with the infected tumors. The background activity was usually 2–3 nCi/ml. Measurements (% ID/g) performed on the images for tumors are approximations because of the marked heterogeneity of radioactivity within the tumors and because of partial volume effects. Background was not negligible at 48 h because the clearance of FIAU-derived radioactivity from noninfected, control tissues is exponential and has a half-life of ∼6 h (46).

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Fig. 9.

Corresponding PET images and X-Gal-stained tissue sections. These comparisons demonstrate the spatial relationship between HSV-1-tk-related radioactivity (shown in the transaxial PET images of Fig. 8) and lacZ gene expression (of corresponding X-Gal-stained transaxial tissue sections) in the same tumor regions. The first two panels correspond to panels 4 and 7 of Fig. 8,A; the third panel corresponds to panel 8 in Fig. 8 B.

Fig. 9.

Corresponding PET images and X-Gal-stained tissue sections. These comparisons demonstrate the spatial relationship between HSV-1-tk-related radioactivity (shown in the transaxial PET images of Fig. 8) and lacZ gene expression (of corresponding X-Gal-stained transaxial tissue sections) in the same tumor regions. The first two panels correspond to panels 4 and 7 of Fig. 8,A; the third panel corresponds to panel 8 in Fig. 8 B.

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Fig. 10.

Coregistration of [131I]FIAU autoradiography with β-galactosidase-stained histology of hrR3-injected tumors. Rat 9L gliosarcomas growing s.c. in nude rats were injected with 2 × 109 PFUs hrR3 2, 4, and 8 days before [131I]FIAU administration. Forty-eight h later, rats were killed, and tumors were processed for tissue sectioning, autoradiography, and β-galactosidase-stained histology. A rim of lacZ gene expression immediately adjacent to necrotic tumor areas was surrounded by HSV-1-tk-related radioactivity, primarily in viable-appearing tumor tissue.

Fig. 10.

Coregistration of [131I]FIAU autoradiography with β-galactosidase-stained histology of hrR3-injected tumors. Rat 9L gliosarcomas growing s.c. in nude rats were injected with 2 × 109 PFUs hrR3 2, 4, and 8 days before [131I]FIAU administration. Forty-eight h later, rats were killed, and tumors were processed for tissue sectioning, autoradiography, and β-galactosidase-stained histology. A rim of lacZ gene expression immediately adjacent to necrotic tumor areas was surrounded by HSV-1-tk-related radioactivity, primarily in viable-appearing tumor tissue.

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Supported in part by the Max-Planck-Society, the Gertrud Reemtsma Foundation (S 104) and MSWWF Grant 516-40000299, Germany (to A. J.); Grant He2636/1-1 (to U. H.) from the Deutsche Forschungsgemeinschaft; National Cancer Institute Grant CA69246 and a grant from the Brain Tumor Society (to X. O. B.); and NIH Grants RO1CA76117, RO1CA60706, and DOE86ER60407 (to J. G. T. and R. G. B.).

3

The abbreviations used are: HSV-1-tk, herpes simplex virus type 1 thymidine kinase; TK, thymidine kinase; RR, ribonucleotide reductase; TdR, thymidine; FBS, fetal bovine serum; MOI, multiplicity of infection; PFU, plaque-forming unit; p.i., postinfection; X-Gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; FIAU, 2′-fluoro-5-iodo-1β-d-arabinofuranosyl-uracil; ONPG, O-nitrophenyl-β-galactoside; wt, wild type; PET, positron emission tomography; FWHM, full width half maximum; ROI, region of interest; % ID/g, percentage of injected dose per gram; QAR, quantitative autoradiography.

4

J. Balatoni et al., manuscript in preparation.

We thank Deborah E. Schuback and Kristen Suilling for excellent technical assistance.

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