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Published online before print March 21, 2002, 10.1148/radiol.2232010501
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(Radiology 2002;223:417-425.)
© RSNA, 2002

Experimental Studies

Gamma Camera Dual Imaging with a Somatostatin Receptor and Thymidine Kinase after Gene Transfer with a Bicistronic Adenovirus in Mice1

Kurt R. Zinn, DVM, PhD, Tandra R. Chaudhuri, PhD, Victor N. Krasnykh, PhD, Donald J. Buchsbaum, PhD, Natalya Belousova, MS, William E. Grizzle, MD, David T. Curiel, MD and Buck E. Rogers, PhD

1 From the Depts of Radiology (K.R.Z., T.R.C.), Radiation Oncology (D.J.B., B.E.R.), Pathology (W.E.G.), Division of Human Gene Therapy, Depts of Medicine, Surgery, and Pathology (V.N.K., D.T.C.), and Gene Therapy Center (K.R.Z., V.N.K., D.J.B., N.B., D.T.C., B.E.R.), University of Alabama at Birmingham, Boshell Bldg, BDB 11, 1530 3rd Ave S, Birmingham, AL 35294-0012. Received Feb 21, 2001; revision requested Mar 29; revision received Aug 2; accepted Sep 17. Supported by the National Cancer Institute grants CA80104 and CO97110. Address correspondence to K.R.Z. (e-mail: kurtzinn@uab.edu).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 APPENDIX
 REFERENCES
 
PURPOSE: To compare two systems for assessing gene transfer to cancer cells and xenograft tumors with noninvasive gamma camera imaging.

MATERIALS AND METHODS: A replication-incompetent adenovirus encoding the human type 2 somatostatin receptor (hSSTr2) and the herpes simplex virus thymidine kinase (TK) enzyme (Ad-hSSTr2-TK) was constructed. A-427 human lung cancer cells were infected in vitro and mixed with uninfected cells at different ratios. A-427 tumors in nude mice (n = 23) were injected with 1 x 106 to 5 x 108 plaque-forming units (pfu) of Ad-hSSTr2-TK. The expressed hSSTr2 and TK proteins were imaged owing to internally bound, or trapped, technetium 99m (99mTc)-labeled hSSTr2-binding peptide (P2045) and radioiodinated 2'-deoxy-2'-fluoro-ß-D-arabinofuranosyl-5-iodouracil (FIAU), respectively. Iodine 125 (125I)-labeled FIAU was used in vitro and iodine 131 (131I)-labeled FIAU, in vivo. The 99mTc-labeled P2045 and 125I- or 131I-labeled FIAU were imaged simultaneously with different window settings with an Anger gamma camera. Treatment effects were tested with analysis of variance.

RESULTS: Infected cells in culture trapped 125I-labeled FIAU and 99mTc-labeled P2045; uptake correlated with the percentage of Ad-hSSTr2-TK-positive cells. For 100% of infected cells, 24% ± 0.4 (mean ± SD) of the added 99mTc-labeled P2045 was trapped, which is significantly lower (P < .05) than the 40% ± 2 of 125I-labeled FIAU that was trapped. For the highest Ad-hSSTr2-TK tumor dose (5 x 108 pfu), the uptake of 99mTc-labeled P2045 was 11.1% ± 2.9 of injected dose per gram of tumor (thereafter, dose per gram), significantly higher (P < .05) than the uptake of 131I-labeled FIAU at 1.6% ± 0.4 dose per gram. 99mTc-labeled P2045 imaging consistently depicted hSSTr2 gene transfer in tumors at all adenovirus doses. Tumor uptake of 99mTc-labeled P2045 positively correlated with Ad-hSSTr2-TK dose; 131I-labeled FIAU tumor uptake did not correlate with vector dose.

CONCLUSION: The hSSTr2 and TK proteins were simultaneously imaged following dual gene transfer with an adenovirus vector.

© RSNA, 2002

Index terms: Animals • Experimental studies • Genes and genetics • Molecular analysis • Radionuclide imaging


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
 APPENDIX
 REFERENCES
 
Gene therapy is a new medical discipline with the potential to improve human health. Translation of preclinical animal studies to human clinical trials will be facilitated with improved in vivo imaging modalities that validate the location and extent of gene transfer. This is particularly true for gene therapy vectors that are injected directly into patients, regardless of the route. Positron emission tomography (PET), single photon emission computed tomography (SPECT), and gamma camera imaging can depict the location of the radiotracer, which emits gamma rays and accumulates in target sites in vivo, owing to localized expression of a reporter gene product (1–3). In this regard, the administered radiotracers must be cleared from the normal tissues so that specific accumulation can be detected above the background radioactivity. This approach has been applied to imaging of the in vivo expression of four reporter genes: the human type 2 somatostatin receptor (hSSTr2) (4–6), the herpes simplex virus thymidine kinase (TK) (7–16), the type 2 dopamine receptor (8,12,16,17), and the thyroid sodium iodide symporter gene (18).

The expression of hSSTr2 (a membrane receptor) was imaged with radiolabeled peptide ligands, including technetium 99m (99mTc)-labeled P829 (6), rhenium 188 (188Re)-labeled P829 (6), 99mTc-labeled P2045 (4,5), and indium 111 (111In)-labeled octreotide (4). The expression of TK (an enzyme) was imaged with several radiolabeled substrates, including iodine 131 (131I)-labeled 2'-deoxy-2'-fluoro-ß-D-arabinofuranosyl-5-iodouracil (FIAU) (12,14,15), iodine 124 (124I)-labeled FIAU (7,12,13), 8-[fluorine 18]fluoroganciclovir (8–10,12), 8-[18F]fluoropenciclovir (8,11,12,16), 9-[(3-[18F]fluoro-1-hydroxy-2-propoxy)methyl]guanine (19), and others (12). Type 2 dopamine receptor expression was imaged with 3-(2'-[18F]fluoroethyl)spiperone (8,12,16,17) and carbon 11-labeled raclopride (20). The expressed reporter gene products were imaged in xenograft tumors (4–6,9,11–17), liver (8–12), and striatum (20). In the majority of xenograft tumor studies, the reporter gene was transferred to the tumor cells prior to implantation in the animal (7,11–13,15–17), or vector-producer cells were injected into an established tumor (7,14,15). Transfer of the reporter gene by means of an intratumor injection was accomplished with adenovirus encoding hSSTr2 (Ad-hSSTr2) for subcutaneous tumors (4–6), adenovirus encoding TK (Ad-TK) for intrahepatic tumors (14), Ad-TK for subcutaneous tumors (19), and adenovirus encoding the thyroid sodium iodide symporter gene (18). Transfer of the TK gene to the rat striatum was accomplished with direct injection of Ad-TK (20). Most recently, 124I-labeled FIAU PET was applied to monitor TK expression that resulted from replication and spread of a replication-conditional herpes simplex virus (encoding TK) into a subcutaneous tumor (21).

The purpose of this study was to compare hSSTr2 and TK systems for assessing gene transfer to cancer cells and xenograft tumors by using noninvasive gamma camera imaging.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 APPENDIX
 REFERENCES
 
Cell Line and Adenovirus Vectors
Human non–small cell lung cancer cells (A-427; American Type Culture Collection, Rockville, Md) were used; their growth conditions are provided in the Appendix. Adenovirus vectors were replication-incompetent. Studies were performed with three adenovirus vectors. Two adenovirus vectors each encoded one gene under the control of cytomegalovirus promoter, either hSSTr2 or TK, and thus are referred to as Ad-hSSTr2 and Ad-TK, respectively. The third adenovirus vector, Ad-hSSTr2-TK, encoded both hSSTr2 and TK, each under the control of a separate cytomegalovirus promoter. Details about the adenovirus vectors are in the Appendix.

Radiolabeling
Chemicals were obtained from one source (Fisher, Pittsburgh, Pa) unless otherwise noted. P2045, a proprietary peptide (1450 Da; Diatide Research Laboratories, Londonderry, NH), is chemically related to P829 (NeoTect; Amersham Health, Princeton, NJ), a somatostatin-receptor imaging agent approved by the U.S. Food and Drug Administration for solitary pulmonary nodules (22–25). The P2045 was radiolabeled with 99mTcO4- (Central Pharmacy, Birmingham, Ala), and quality control was conducted as previously described (26,27). The 99mTc-labeled P2045 was more than 95% pure for all studies. The specific activity of the 99mTc-labeled P2045 for all experiments averaged 45 MBq/nmol ± 4.

FIAU and the radiolabeling precursor, 2'-deoxy-2'-fluoro-ß-D-arabinofuranosyluracil, or FAU, were obtained (Moravek Biochemicals, Brea, Calif). FAU was radioiodinated with 125I and 131I (DuPont/NEN Research Products, Boston, Mass), purified at high-pressure liquid chromatography, vacuum dried, and further tested for purity by means of high-pressure liquid chromatography analyses (28). Radiolabeled FIAU and unlabeled FIAU were dissolved in 50% ethanol (2 mg/mL) prior to further dilution. The 125I- and 131I-labeled FIAU were more than 95% pure, as determined by means of high-pressure liquid chromatographic analysis, with specific activities calculated to be 81 and 35.2 MBq/nmol, respectively. The specific activity calculations were based on information provided by the manufacturer.

In Vitro Plate Imaging
Figure 1 summarizes the nine steps of the protocol. A-427 cells were infected with Ad-hSSTr2-TK at 10 plaque-forming units (pfu) per cell, a level of adenovirus sufficient to infect 100% of the cells (27). The adenovirus was removed after 2 hours, the cells were washed, and the complete media were added. After 24 hours, the cells infected with Ad-hSSTr2-TK were diluted with uninfected A-427 cells to achieve 1%, 5%, 10%, 25%, 50%, 75%, and 100% of Ad-hSSTr2-TK-positive cells. Each of the dilutions was plated in six individual wells of a 12-well plate (one-half of the plate) for a total of four plates. The Figure shows one plate containing 100% and 75% of Ad-hSSTr2-TK-infected cells. Uninfected A-427 cells were also included in half of one plate.



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  		Figure 1. Diagram illustrates the protocol for plate imaging. A-427 cells were infected with Ad-hSSTr2-TK (#1); after 24 hours, they were mixed with different ratios of uninfected cells (#2) and placed in 12-well plates (six wells per dilution). A total of four plates were used for all dilutions (100%, 75%, 50%, 25%, 10%, 5%, 1%, and 0% of infected cells). As an example, one plate with 100% and 75% of infected cells is shown (#3). Excess unlabeled P2045 and FIAU were added to one-half of the wells for each dilution (#4), the two radiotracers were added (#5), and the plates were imaged by using two window settings on the gamma camera (#6). After incubation for 1 hour (#7), the cells were washed (#8), and final images were collected (#9).

 
After an additional 24 hours (48 hours after adenovirus infection), the plate assay protocol was conducted as previously described (26,27). The adherent cells were washed, and 0.3 mL of either incubation buffer or incubation buffer containing a mixture of excess (final concentrations given next) unlabeled P2045 and FIAU was added in triplicate. A constant amount (0.3 mL) of the mixture of 99mTc-labeled P2045 (0.55 MBq) and 125I-labeled FIAU (0.074 MBq) in incubation buffer was added to all wells. The final volume in each well was 0.6 mL, and for wells receiving excess unlabeled P2045 and FIAU (half of the wells), the final concentrations of P2045 and FIAU were 2.1 mol/L and 112 mol/L, respectively.

The four plates (Fig 1) were immediately imaged with an Anger camera (420/550 Mobile Radioisotope Gamma Camera; Technicare, Solon, Ohio) equipped with a low-energy parallel-hole collimator (Model 14S22022). Intrinsic resolution of the detector was 3.0 mm full width at half maximum. Two images were collected with separate window settings (99mTc centerline of 831, window width of 200; 125I centerline of 964, window width of 350), as previously described (26). Next, the plates were incubated at 37°C for 1 hour. The adherent cells were washed again, included was an acid wash (pH 4.0) to remove surface-bound radioactivity. The four plates were imaged a second time to measure radioactivity that was trapped, or internally bound, in the adherent monolayer of cells.

Images were processed with a modified image analysis program (NucMed Image; M. D. Wittry, St Louis University, Mo) by using standard manual region of interest (ROI) analyses. ROIs were drawn around the area representing the individual wells of the cell culture plates. The same ROI was used for the initial and final image analyses. Background regions were drawn outside the plate region. The mean counts per pixel were recorded for all regions. The average counts per pixel for the background region were subtracted from the average counts per pixel in the well ROI. The fraction of internally bound activity for each well was calculated as the ratio between the final (after acid wash) decay- and background-corrected counts per pixel divided by the initial background-corrected counts per pixel in the same well. In addition, cells were lysed, transferred to vials, and counted in a gamma counter to determine the percentage of added radioactivity that was trapped.

Animal Tumor Model, Adenovirus Injections, Radiotracer Doses, and Imaging
Animal experiments were reviewed and approved by the institutional animal care and use committee. Athymic nude mice (Frederick Cancer Research Laboratory, Hartford, Conn) were subcutaneously implanted on the right and left flanks with 1 x 106 pfu of A-427 cells, as previously described (6). After 3 weeks, the adenovirus vectors were injected intratumorally as follows.

Experiment 1.—There were two groups of mice (four mice per group). Group A received Ad-TK injections (5 x 108 pfu in 0.05 mL) in the left tumor, while group B received Ad-hSSTr2 (5 x 108 pfu in 0.05 mL) in the left tumor. All right tumors were injected with Ad-hSSTr2-TK (5 x 108 pfu in 0.05 mL). Mice were given Lugol iodine solution (Humco, Texarkana, Tex) in drinking water (final iodide concentration, 0.003%). At 48 hours after adenovirus injections, mice were intravenously injected with 99mTc-labeled P2045 (17.8 MBq) and 131I-labeled FIAU (5.2 MBq), in separate injections. The mice were imaged at 3 minutes, 5 hours, 8 hours, and 26 hours, then sacrificed.

Experiment 2.—This experiment was conduced with 15 mice (two tumors per mouse) and was blinded to the individuals conducting the imaging and biodistribution analyses. There were five doses of Ad-hSSTr2-TK: 1 x 106, 3 x 106, 1 x 107, 3 x 107, and 3 x 108 pfu. The tumors were randomly assigned to five groups (six tumors per group in six mice), and the adenovirus dose was given intratumorally with the same injection volume of 0.05 mL. After 48 hours, the mice were intravenously injected with 99mTc-labeled P2045 (23 MBq) and 131I-labeled FIAU (2.5 MBq), in separate injections. The mice were imaged at 3 minutes, 5 hours, and 24 hours, then sacrificed.

The mice in both experiments were positioned with the dorsal surface of the mouse adjacent to and beneath the pinhole collimator (Model PHW; Ohio Nuclear, Solon, Ohio), which was attached to an Anger camera (Sigma 410 Radioisotope Gamma Camera; Ohio Nuclear). Two images were collected with separate window settings (99mTc centerline of 607, window width of 200; 131I centerline of 472, window width of 200). The imaging acquisition time for the 99mTc images was 1 minute, 3 minutes, and 20 minutes, which were obtained at 3 minutes, 5 hours, and 24 hours, respectively, after the injection of 99mTc-labeled P2045. The imaging acquisition time for the 131I images was 5 minutes, 7 minutes, and 20 minutes, which were obtained at 3 minutes, 5 hours, and 24 hours, respectively, after the administration of 131I-labeled FIAU.

The mice in experiment 2 (3 minutes and 5 hours after radiotracer injection) were imaged (99mTc window setting, centerline of 831, window width of 200) with an Anger 420/550 camera (Technicare) equipped with a pinhole collimator. The final 99mTc images (24 hours after dosing) in experiment 2 were obtained only with this gamma camera. Two authors (K.R.Z., T.R.C.), who did not inject the adenovirus vectors, evaluated the images; a consensus was reached regarding the presence or absence of radiotracer uptake in the tumor regions. ROI analyses were conducted as described previously. The tumor uptake was normalized to the initial image of the whole animal; background corrections were based on regions outside the animal. Color photographs were obtained at the same time as gamma camera images by using a digital camera (Coolpix 950; Nikon, Tokyo, Japan). These photographs were used to identify the tumor location and compare it with radiotracer uptake, as determined from the gamma camera images.

Other Measurements
Stock solutions and syringes were assayed by using a dose calibrator (Atomlab 100; Biodex Medical Systems, Shirley, NY). Lysed cell solutions and tissues were counted in a gamma counter (Minaxi Auto-Gamma 5000 series; Packard, Downers Grove, Ill). The tissues were collected immediately after the mice were sacrificed and were weighed before counting.

Immunohistochemistry
From previous study findings, it was known that the Ad-hSSTr2 and Ad-TK tumor injections resulted in focal expression of hSSTr2 and TK, respectively. In this study, it was therefore hypothesized that hSSTr2 and TK would likely be found in the same focal areas, since both genes were transferred with the same bicistronic Ad-hSSTr2-TK. The tumors (n = 13) were first evaluated (W.E.G. and D.J.B.) for expression of TK. When TK expression was found, the areas were compared with the next section in the same xenografted tumor, which was stained for hSSTr2. A careful comparison was made to determine if TK and SSTr2 were expressed in the same focal area. The proportion of cells expressing TK in the focal area was determined and compared with the proportion of cells expressing hSSTr2. Five control tumors injected with a control adenovirus (encoding thyroid hormone releasing receptor) were also evaluated for both hSSTr2 and TK expression.

The xenograft tissues were initially fixed in 10% neutral buffered formalin prior to paraffin processing and embedding. A more detailed description of the methods for performing and evaluating the sections has been reported previously (6,29). The primary antibody to TK, an immunoglobulin (Ig) G rabbit polyclonal antibody developed against herpes simplex virus TK, was obtained (S. M. Albelda and J. V. Hughes, University of Pennsylvania) and diluted 1:50. The primary antibody to the human somatostatin receptor type 2A was an IgG rabbit polyclonal antibody, and it was used at a final concentration of 2 µg/mL. The antibody was affinity-purified (Research Genetics, Huntsville, Ala) with a serum collected from New Zealand white rabbits that were previously injected subcutaneously (three times over 10 weeks) with 0.1 mg of (KLH)-CSDSKQDKSRLNE peptide (30) mixed with an equal volume of Freund adjuvant.

Statistical Evaluation
Statistical comparisons were performed with a statistical analysis program (SAS version 8.0; SAS Institute, Cary, NC) by using one-way analysis of variance protocols (Proc GLM; SAS Institute). Treatment means were also compared by using the least-squares means protocol, and differences at P < .05 were considered significant. A log transformation was used to stabilize the variance when means were highly different and when the coefficient of variation was constant among the groups.

In vitro data.—The following were tested: (a) a method for determining the percentage dose that was internally bound (ROI analyses from images versus counting lysed cells in a gamma counter), (b) the effect of excess unlabeled P2045 and FIAU on the percentage dose that was internally bound, (c) the effect of the percentage of Ad-hSSTr2-TK-positive cells on the amount of internally bound 99mTc-labeled P2045 and 125I-labeled FIAU, and (d) 99mTc-labeled P2045 versus 125I-labeled FIAU for the respective levels of internally bound radioactivity.

In vivo data.—The following were tested: (a) experiment 1, effect of time and adenovirus tumor injection (Ad-hSSTr2-TK vs Ad-TK vs Ad-hSSTr2) on the tumor uptake of 99mTc-labeled P2045 and 131I-labeled FIAU from ROI analyses; (b) experiment 1, effect of adenovirus tumor injection (Ad-hSSTr2-TK vs Ad-TK vs Ad-hSSTr2) on the tumor uptake of 99mTc-labeled P2045 and 131I-labeled FIAU for biodistribution analyses; (c) experiment 2, effect of Ad-hSSTr2-TK dose (five doses) on tumor uptake of 99mTc-labeled P2045 and 131I-labeled FIAU; and (d) experiment 2, 99mTc-labeled P2045 versus 131I-labeled FIAU for levels of respective radioactivity in tumors.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 APPENDIX
 REFERENCES
 
Plate Imaging
Gamma camera images of the cell culture plates depicted internally trapped 99mTc-labeled P2045 and 125I-labeled FIAU, as shown in Figure 2a and 2b, respectively. The images showed that the relative intensities of both signals were correlated with the percentage of positive infected cells. When 100% of the cells were infected with Ad-hSSTr2-TK, 24.0% ± 0.4 and 40% ± 2 of the added 99mTc-labeled P2045 and 125I-labeled FIAU, respectively, became trapped. The percentage dose that became trapped, as measured with ROI analyses from the images, was not significantly different (P > .05) than that determined by counting the lysed cells in a gamma counter. All wells with excess unlabeled P2045 and FIAU (Fig 2) showed a significant reduction (P < .05) in the trapped 99mTc-labeled P2045 and 125I-labeled FIAU, respectively. Excess P2045 was always better at reducing the level of trapped 99mTc-labeled P2045 compared with excess FIAU at reducing trapped 125I-labeled FIAU level. For example, when 50% of the cells were infected, the level of the trapped 99mTc-labeled P2045 was 17% ± 1 of the added dose, which was significantly reduced (P < .05) to 0.84% ± 0.01 of the added dose in the presence of excess P2045. For the same cells, the level of the trapped 125I-labeled FIAU was 34% ± 1 of the added dose, which was significantly reduced (P < .05) to 5.7% ± 0.2 of the added dose in the presence of excess FIAU.



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  		Figure 2a. Gamma camera images of the cell culture plates. (a) 99mTc gamma camera window shows trapped 99mTc-labeled P2045. (b) 125I gamma camera window shows trapped 125I-labeled FIAU. Images in a and b were collected without moving the plates. The percentages refer to the level of Ad-hSSTr2-TK-positive infected cells. The - and + refer to absence or presence, respectively, of excess unlabeled P2045 and FIAU. Note reduction in trapped 99mTc-labeled P2045 and 125I-labeled FIAU with the addition of excess unlabeled P2045 and FIAU.

 


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  		Figure 2b. Gamma camera images of the cell culture plates. (a) 99mTc gamma camera window shows trapped 99mTc-labeled P2045. (b) 125I gamma camera window shows trapped 125I-labeled FIAU. Images in a and b were collected without moving the plates. The percentages refer to the level of Ad-hSSTr2-TK-positive infected cells. The - and + refer to absence or presence, respectively, of excess unlabeled P2045 and FIAU. Note reduction in trapped 99mTc-labeled P2045 and 125I-labeled FIAU with the addition of excess unlabeled P2045 and FIAU.

 
As shown in Figure 3a, when the percentage of positive infected cells increased, the percentage of internally bound 99mTc-labeled P2045 and 125I-labeled FIAU was also significantly higher (P < .05), with one exception: the internally bound 125I-labeled FIAU for 100% and 75% of positive cells was not different (P > .05). For each level of positive cells (0%–100%), the level of trapped 125I-labeled FIAU was significantly (P < .05) higher than that of the trapped 99mTc-labeled P2045. The relative difference was approximately twofold for a mixture of cells at 100%, 75%, 50%, and 25% of the positive infected cells and increased to threefold for 10% and 5% of the Ad-hSSTr2-TK-positive infected cells. A plot of trapped 125I-labeled FIAU versus trapped 99mTc-labeled P2045 was linear (Fig 3b) (r2 = 0.98, P < .01).



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  		Figure 3a. Analyses of internally bound, or trapped, radiotracers in the adherent A-427 cell monolayer. (a) Plot of the percentage dose trapped in the cell monolayers versus the percentage of positive A-427 cells. Data points indicate means. Error bars indicate SD. (b) Plot of trapped 125I-labeled FIAU versus 99mTc-labeled P2045. The highest point (100% of infected cells) was not included because the percentage trapped dose for the 125I-labeled FIAU was not increased compared with 75% of infected cells. This finding indicated a potential saturation for these conditions.

 


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  		Figure 3b. Analyses of internally bound, or trapped, radiotracers in the adherent A-427 cell monolayer. (a) Plot of the percentage dose trapped in the cell monolayers versus the percentage of positive A-427 cells. Data points indicate means. Error bars indicate SD. (b) Plot of trapped 125I-labeled FIAU versus 99mTc-labeled P2045. The highest point (100% of infected cells) was not included because the percentage trapped dose for the 125I-labeled FIAU was not increased compared with 75% of infected cells. This finding indicated a potential saturation for these conditions.

 
Gene Expression Imaging
Images in Figure 4 were obtained 5 hours after radiotracer injection (experiment 1). Figure 4a shows a representative mouse from group A. The 99mTc image showed hSSTr2 gene expression only in the right (Ad-hSSTr2-TK-injected) tumor, while the 131I image revealed TK expression in left (Ad-TK-injected) and right (Ad-hSSTr2-TK-injected) tumors. Mice in group B showed a distinctly different pattern, as evidenced by the representative images in Figure 4b. The 99mTc image showed hSSTr2 gene expression in left (Ad-hSSTr2-injected) and right (Ad-hSSTr2-TK-injected) tumors, while the 131I image showed TK expression only in the right (Ad-hSSTr2-TK-injected) tumor. ROI analyses of the images showed that by 5 hours approximately 85% of both radiotracers were eliminated in the urine. At 5 hours, the right tumors injected with Ad-hSSTr2-TK accumulated 2.0% ± 0.3 and 0.5% ± 0.1 of the injected dose for 99mTc-labeled P2045 and 131I-labeled FIAU, respectively. At 26 hours, ROI analyses showed that the levels of uptake in the same tumors decreased to 1.5% ± 0.2 and 0.2% ± 0.03 of the injected dose for 99mTc-labeled P2045 and 131I-labeled FIAU, respectively. These results and statistical comparisons are presented in Table 1 along with ROI analyses for tumors injected with Ad-hSSTr2 or Ad-TK.



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  		Figure 4a. In vivo simultaneous imaging for hSSTr2 and TK expression. (a) Photograph of the mouse shows tumor locations and adenovirus doses for group A. (b) Photograph of the mouse shows tumor locations and adenovirus doses for group B. The expression of hSSTr2 was depicted with imaging tumor accumulation of 99mTc-labeled P2045 (bottom left), while TK expression was depicted with imaging tumor accumulation of 131I-labeled FIAU (bottom right). The images were obtained 5 hours after intravenous injection of the radiotracers.

 


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  		Figure 4b. In vivo simultaneous imaging for hSSTr2 and TK expression. (a) Photograph of the mouse shows tumor locations and adenovirus doses for group A. (b) Photograph of the mouse shows tumor locations and adenovirus doses for group B. The expression of hSSTr2 was depicted with imaging tumor accumulation of 99mTc-labeled P2045 (bottom left), while TK expression was depicted with imaging tumor accumulation of 131I-labeled FIAU (bottom right). The images were obtained 5 hours after intravenous injection of the radiotracers.

 

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  		TABLE 1. Imaging ROI Analyses for Tumor Uptake of 99mTc-labeled P2045 and 131I-labeled FIAU

 
Tumor biodistribution results and biodistribution analyses for experiment 1 are summarized in Table 2. The uptake of 99mTc-labeled P2045 and 131I-labeled FIAU for Ad-hSSTr2-TK-injected tumors (n = 8) was 11.1% ± 2.9 and 1.6% ± 0.4% dose per gram, respectively. Ad-hSSTr2-injected tumors (n = 4) accumulated 10.2% ± 2.4 dose per gram of the 99mTc-labeled P2045 and 0.3% ± 0.1 of the 131I-labeled FIAU. Ad-TK-injected tumors (n = 4) had a 0.2% ± 0.1 dose per gram for the 99mTc-labeled P2045 and 3.7% ± 1.2 for 131I-labeled FIAU. The uptake of 99mTc-labeled P2045 in tumors injected with Ad-hSSTr2 was not significantly different (P > .05) from that obtained in tumors injected with Ad-hSSTr2-TK. Likewise, the uptake of 131I-labeled FIAU in tumors injected with Ad-TK was not significantly different (P > .05) from that obtained in tumors injected with Ad-hSSTr2-TK.


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  		TABLE 2. Biodistribution Analyses for Tumor Uptake of 99mTc-labeled P2045 and 131I-labeled FIAU

 
In experiment 2, the expression of hSSTr2 in tumor was detected for all adenovirus doses, as evidenced by 99mTc-labeled P2045 uptake in the tumor region depicted with imaging at 5 and 26 hours after radiotracer injection. For TK gene transfer, imaging of 131I-labeled FIAU did not depict TK expression in tumors in 50% of the animals. This finding was independent of the adenovirus dose. Figure 5 shows representative 99mTc-labeled P2045 images of mice whose tumors were previously injected with low adenovirus doses. The results are presented (Fig 6) in a plot of percentage dose per gram (linear scale) versus adenovirus dose (log scale). As indicated on the plot, the tumor uptake of 99mTc-labeled P2045 was highly correlated in a log fashion (r2 = 0.98, P < .01) with adenovirus dose. In contrast, no correlation was observed for the 131I-labeled FIAU detected in tumor relative to the adenovirus dose.



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  		Figure 5. Imaging hSSTr2 expression resulting from low Ad-hSSTr2-TK doses. The 99mTc-labeled P2045 images from two representative mice demonstrate the sensitivity for detection of hSSTr2 expression following gene transfer with low doses of Ad-hSSTr2-TK.

 


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  		Figure 6. Plot of tumor uptake for 99mTc-labeled P2045 and 131I-labeled FIAU relative to the injected dose of Ad-hSSTr2-TK (log scale). The tumors were counted individually in a gamma counter at the time mice were killed 24 hours after the injection of radiotracers. Data points indicate means. Error bars indicate SD. There were six tumors per adenovirus dose. There was excellent correlation between the uptake of 99mTc-labeled P2045 (percentage dose per gram) and the adenovirus (Ad) dose (r2 = 0.98). Uptake of 131I-labeled FIAU in tumor was Ad-hSSTr2-TK-dependent but completely unrelated to the dose.

 
All 13 Ad-hSSTr2-TK-injected tumors had areas that were negative for staining for TK and hSSTr2 expression. All tumors had a focal area that stained for TK and hSSTr2, and in focal areas that stained for TK, there always was matched staining for hSSTr2. In the focal areas of staining, more than 90% of cells expressed TK; however, expression of hSSTr2 could be identified in only 40% ± 10 of cells. Five mock-infected control tumors did not stain for TK or hSSTr2. Figure 7 presents a representative A-427 tumor previously injected with Ad-hSSTr2-TK. Figure 7, A, B, are adjacent sections stained for TK and hSSTr2, respectively. The same areas of the tumor were positive and negative for the two expressed genes.



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  		Figure 7. Immunohistologic images validate hSSTr2 and TK expression in Ad-hSSTr2-TK-injected tumor xenografts. A, Photomicrograph shows strong phenotypic expression of TK (brown color). (Anti-TK polyclonal antibody stain; original magnification, x400.) B, Photomicrograph shows relatively weaker phenotypic expression of hSSTr2 (brown color). (Anti-hSSTr2 polyclonal antibody stain; original magnification, x400.) Matching areas within dotted lines (NT = no tumor) are composed primarily of stroma and inflammatory cells that are negative for TK and hSSTr2 expression. The sections in A and B were anatomically adjacent and showed that expression of both TK and hSSTr2 was limited to the same local areas in the tumor.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
 REFERENCES
 
Gamma camera imaging simultaneously depicted the expression of hSSTr2 and TK in A-427 cells growing as monolayers following Ad-hSSTr2-TK infection. Cells infected with Ad-hSSTr2-TK showed specific trapping of both 99mTc-labeled P2045 and 125I-labeled FIAU, which were linearly correlated (r2 = 0.98). The trapping was specific since it was reduced in the presence of excess unlabeled P2405 and FIAU. This is consistent with a reduction in internal binding of the two radiotracers owing to competition with the higher concentrations of unlabeled P2045 and FIAU.

Results demonstrate that both genes encoded in the adenovirus vector were functional. Further, the studies established that TK was more efficient at trapping 125I-labeled FIAU in the in vitro system, compared with the capability of hSSTr2 at trapping 99mTc-labeled P2045. This is likely related to the fact that TK is an enzyme, while hSSTr2 is a receptor. Each TK enzyme can react with many FIAU substrate molecules, but each hSSTr2 can bind only a single P2045 ligand.

Uptake of 99mTc-labeled P2045 in tumors was correlated with dose of Ad-hSSTr2-TK; imaging consistently helped to detect hSSTr2 gene expression that resulted from adenovirus doses as low as 1 x 106 pfu. The hSSTr2 reporter gene showed excellent sensitivity and linearity, a favorable indication for further testing in human trials. This contrasted with the imaging of TK expression, which was not reliably detected. A further problem was the fact that the 131I-labeled FIAU in tumor did not correlate with the adenovirus dose. Tjuvajev et al (14) also showed no significant difference in tumor uptake of the 131I-labeled FIAU for Ad-TK doses of 1 x 108 pfu versus those at 3 x 107 pfu (1.1% ± 0.3 dose per gram vs 0.8% ± 0.2 dose per gram). In the current study, a wider range (1 x 106 to 5 x 108 pfu) of Ad-hSSTr2-TK doses was used, and TK expression was consistently detected with immunohistochemistry at the protein level. With respect to detection by means of noninvasive PET imaging (with 9-[(3-[18F]fluoro-1-hydroxy-2-propoxy)methyl]-guanine), Hustinx et al (19) could not image TK expression in subcutaneous tumors previously injected with Ad-TK (2 x 108 pfu). PET imaging depicted TK expression with a higher Ad-TK tumor dose (2 x 109 pfu) (19).

The TK system may have inherent problems for in vivo imaging. The concentration of radiolabeled FIAU is reduced by excretion and from breakdown due to metabolism. These factors may reduce delivery of active 131I-labeled FIAU to areas of TK expression in vivo, such that differences in TK enzymatic levels are not detected, especially in areas of low blood flow. The TK substrate (FIAU) can diffuse across cell membranes, unlike P2045. Alternatively, the TK enzyme might be either too efficient or in too high of a concentration, such that all 131I-labeled FIAU that reached the TK-positive cells in vivo was trapped. The shape of the curve presented in Figure 3a for the in vitro studies and the ease of detection with immunohistochemistry support this hypothesis. In fact, the trapped 125I-labeled FIAU for 100%, 75%, and 50% of Ad-hSSTr2-TK-positive cells was not significantly different (P < .05, Fig 3a). Additional experiments will be necessary to confirm the precise levels of TK expression in tumors previously injected with different doses of Ad-hSSTr2-TK.

The approach presented in this article can be applied with other radiotracer combinations. For example, gamma cameras can simultaneously depict 99mTc and 123I, or 99mTc and 18F. Dual or even triple radioisotope detection is a unique feature of gamma cameras and cannot be readily accomplished with PET. Potential applications include the simultaneous in vivo monitoring of several gene products.

Practical application: The technology reported here can be readily applied in the field of gene therapy. Inclusion of the hSSTr2 reporter gene in a vector with a therapeutic gene would allow the location and extent of gene transfer to be imaged. NeoTect (99mTc-labeled P829) and Octreoscan (Mallinckrodt, St Louis, Mo) (111In-labeled octreotide) are commercially available agents for this purpose. These agents can be detected with SPECT, thereby affording imaging at high spatial resolution in humans.


   APPENDIX
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 APPENDIX
REFERENCES
 
The A-427 cells were obtained (American Type Culture Collection, Rockville, Md). Cells were grown in Eagle minimum essential medium containing nonessential amino acids and 1 mmol/L sodium pyruvate supplemented with 10% fetal bovine serum. The cells were cultured at 37°C in a humidified atmosphere with 5% carbon dioxide. The replication-incompetent adenovirus (serotype 5) encoding hSSTr2 complementary DNA (type A) under the control of cytomegalovirus promoter element (Ad-hSSTr2) was produced and purified as previously described (31). The replication-incompetent adenovirus (serotype 5) encoding TK under the control of cytomegalovirus promoter element (Ad-TK) was previously described (32).

Figure A1 depicts the Ad-hSSTr2-TK. To produce Ad-hSSTr2-TK, a double-cassette shuttle vector was constructed and pCA13 (Microbix, Toronto, Canada) was used as a basic construct. First, the immediate-early cytomegalovirus promoter-driven expression cassette within the pCA13 was modified. Two derivatives of pCA13 carrying different polylinker sequences and annealing sites for two pairs of primers were generated. Two pairs of complementary oligonucleotides were designed to form upon the annealing of two duplexes, each of which contained a new polylinker/primer annealing site. Linker I contained an annealing site for the T7 primer; unique cloning sites for SalI, ClaI, BclI, PmeI, and KpnI; as well as an annealing site for the T3 primer. Linker II contained an annealing site for the Sp6 primer; unique cloning sites for EcoRI, EcoRV, XbaI, XhoI, and BamHI; as well as an annealing site for the reverse M13 primer. When annealed, both duplexes had SalI- and BamHI-compatible sticky ends. To substitute the linker sequence in pCA13 with newly designed sequences, pCA13 was digested with SalI and BamHI, purified with gel, and ligated with duplexes Linker I and Linker II, resulting in pCA.L1 and pCA.L2, respectively.



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  		Figure A1. Diagram depicts the construction of Ad-hSSTr2-TK, as described in the Appendix.

 

The pCA.L1 was then used as a cloning vector to incorporate Linker II sequence to generate a double-cassette shuttle vector. The pCA.L1 was partially digested with BglII and ligated with a BglII fragment of pCA.L2 containing the expression cassette with Linker II sequence. The resultant plasmid, pCA.DC, contains the Linker II expression cassette localized upstream from the Linker I expression cassette and a head-to-tail relative orientation of the cassettes.

The pAA(cytomegalovirus).hSSTr2 (31) was cleaved with EcoRI-XbaI, and a 1.3-kb fragment containing hSSTR2 cDNA was cloned into EcoRI-XbaI-digested pCA.DC, which resulted in pDC.SSTR2. HSV-TK cDNA was then cloned into PmeI-digested pDC.SSTR2 as a 1.3-kb BglII-BsaI fragment of pAB109 (R. Garver, University of Alabama at Birmingham); the sticky ends were made blunt with Klenow enzyme fill-in reaction. The plasmid with the correct orientation of the insert was designated pDC.hSSTr2-TK. The double expression cassette contained in pDC.hSSTr2-TK was transferred by homologous recombination in Escherichia coli into the E3-deleted adenovirus genome contained in the rescue vectors pVK454. This resulted in the derivation of plasmid pVK455. Details of the cloning strategy and maps of the plasmids are available upon request (victor.krasnykh@ccc.uab.edu). Transfection of 293 cells with the PacI-digested pVK455 led to the derivation of Ad5-hSSTr2-TK. The virus was amplified on the 293 cells and purified on cesium chloride gradients as described previously (33). Particle titer and the infectivity of the virus on 293 cells were determined with standard methods (34), whereas the integrity of its genome was verified with restriction enzyme analysis and partial DNA sequencing.


   ACKNOWLEDGMENTS
 
The authors greatly appreciate the generous supply of P2045 from Diatide Research Laboratories. The authors thank Dr James Mountz and Dr Robert Stanley for review of the manuscript; Qi Wu, Sheila Bright, Rob Stockard, and Richard Kirkman for excellent technical assistance; and Dr Steven M. Albelda and Dr Joseph V. Hughes for the anti-TK antibody.


   FOOTNOTES
 
Abbreviations: Ad-hSSTr2 = adenovirus encoding hSSTr2, Ad-hSSTr2-TK = adenovirus encoding hSSTr2 and herpes simplex virus TK enzyme, Ad-TK = adenovirus encoding TK, FIAU = 2'-deoxy-2'-fluoro-ß-D-arabinofuranosyl-5-iodouracil, hSSTr2 = human type 2 somatostatin receptor, pfu = plaque-forming units, ROI = region of interest, TK = thymidine kinase

Author contributions: Guarantor of integrity of entire study, K.R.Z.; study concepts, D.T.C., K.R.Z., T.R.C.; study design, D.J.B., B.E.R., V.N.K., T.R.C., K.R.Z.; literature research, K.R.Z.; experimental studies, W.E.G., N.B., K.R.Z., T.R.C., B.E.R., V.N.K.; data acquisition, K.R.Z., T.R.C., N.B., B.E.R., W.E.G.; data analysis/interpretation, D.J.B., N.B., W.E.G., K.R.Z., T.R.C.; statistical analysis, K.R.Z.; manuscript preparation, K.R.Z., T.R.C., V.N.K.; manuscript definition of intellectual content, all authors; manuscript editing, K.R.Z.; manuscript revision/review and final version approval, all authors.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 APPENDIX
 REFERENCES
 

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