Imaging Progress of Herpes Simplex Virus Type 1 Thymidine Kinase

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Imaging progress of herpes simplex virus type 1 thymidine kinase suicide gene therapy in living subjects with positron emission tomography Shahriar S Yaghoubi,1,2,3 Jorge R Barrio,1,4,5 Mohammad Namavari,1,4,5 Nagichettiar Satyamurthy,1,4,5 Michael E Phelps,1,2,4,5 Harvey R Herschman,1,2,5,6,7 and Sanjiv S Gambhir1,2,3,4,5,7,8 1Department of Molecular & Medical Pharmacology, UCLA School of Medicine, Los Angeles, California 90095-1770, USA; 2The Crump Institute for Molecular Imaging, UCLA School of Medicine, Los Angeles, California 90095-1770, USA; 3Department of Radiology Bio-X program, Stanford University School of Medicine, USA; 4The Division of Nuclear Medicine, UCLA School of Medicine, Los Angeles, California 90095-1770, USA; 5UCLA/DOE Laboratory of Structural Biology & Molecular Medicine, UCLA School of Medicine, Los Angeles, California 90095-1770, USA; 6Molecular Biology Institute, UCLA School of Medicine, Los Angeles, California 90095-1770, USA; 7UCLA-Jonsson Comprehensive Cancer Center, UCLA School of Medicine, Los Angeles, California 90095-1770, USA; and 8Department of Biomathematics, UCLA School of Medicine, Los Angeles, California 90095-1770, USA.

Molecular imaging of a suicide transgene’s expression will aid the development of efficient and precise targeting strategies, and imaging for cancer cell viability may assess therapeutic efficacy. We used the PET reporter probe, 9-(4-[18F]fluoro-3- (hydroxymethyl)butyl)guanine ([18F]FHBG) to monitor the expression of a mutant Herpes Simplex Virus 1 thymidine kinase (HSV1-sr39tk) in C6 glioma tumors implanted subcutaneously in nude mice that were repetitively being treated with the pro-drug Ganciclovir (GCV). [18F]-Fluorodeoxyglucose ([18F]FDG), a metabolic tracer, was used to assess tumor cell viability and therapeutic efficacy. C6 glioma tumors stably expressing the HSV1-sr39tk gene (C6sr39) accumulated [18F]FHBG prior to GCV treatment. Significant declines in C6sr39 tumor volumes and [18F]FHBG and [18F]FDG accumulation were observed following 2 weeks of GCV treatment. However, 3 weeks after halting GCV treatment, the tumors re-grew and [18F]FDG accumulation increased significantly; in contrast, tumor [18F]FHBG concentrations remained at background levels. Therefore, [18F]FHBG can be used to detect tumors expressing HSV1-sr39tk, susceptible to regression in response to GCV exposure, and the effectiveness of GCV therapy in eradicating HSV1-sr39tk-expressing cells can be monitored by [18F]FHBG scanning. [18F]FHBG and [18F]FDG imaging data indicate that exposure of C6sr39 tumors to GCV causes the elimination of [18F]FHBG-accumulating C6sr39 cells and selects for regrowth of tumors unable to accumulate [18F]FHBG. Cancer Gene Therapy (2005) 12, 329–339. doi:10.1038/sj.cgt.7700795 Published online 10 December 2004 Keywords: mutant herpes simplex 1 thymidine kinase gene (HSV1-sr39tk); positron-emission tomography; molecular imaging; suicide gene therapy; C6 glioma tumor xenografts

oninvasive molecular imaging in oncology, originally Changes in the patterns of uptake of different PET Napplied for the detection of tumors and their tracers in tumors, before and after therapy, may not metastasis, has been extended to monitoring the efficacy only reveal whether targeted tumors have been eradi- of therapeutic approaches.1,2 For example, positron cated but also elucidate some of the characteristics of emission tomography (PET)scanning of [ 18F]-2-fluoro- residual or resistant tumors.4–8 An additional application 18 2-deoxy-D-glucose ([ F]FDG or FDG)accumulation of molecular imaging in cancer gene therapy is moni- within tumors has been used to monitor the efficacy of toring specific targeting of a therapeutic transgene. chemotherapy, radiotherapy, and suicide gene therapy.3–5 Precise and accurate targeting is especially important in suicide gene therapy to increase the efficacy and Received March 27, 2004. reduce nonspecific induction of cell death. Methods used Address correspondence and reprint requests to: Professor Sanjiv S to cause selective expression of a suicide gene include Gambhir, MD, PhD, Stanford University School of Medicine, The using vectors that primarily deliver genes into dividing James H. Clark Center, 318 Campus Dr, E150, Stanford, CA 94305- cancer cells, using modified vectors that specifically bind 5427, USA. E-mail: [email protected] receptors on cancer cells, direct injection of vectors into Imaging suicide gene therapy with PET SS Yaghoubi et al 330 tumors, and the use of cancer cell-specific promoters Materials and methods or enhancer sequences to regulate the expression of the Cell lines and culture conditions suicide gene.9 Noninvasive molecular imaging of therapeutic trans- C6 rat glioma cells and the C6 cells stably transfected with genes in patients is possible using positron-emitting a construct carrying the HSV1-sr39tk PRG (C6sr39)were isotopes coupled to substrates/ligands (tracers), which provided by Dr M Black (Washington State University, 26,31 can detect products of a therapeutic transgene’s expres- Pullman, WA). Cells were grown in D-MEM sion using PET. Furthermore, PET imaging is possible in supplemented with 10% FBS, 1% penicillin–streptomy- small animals using dedicated small animal PET scanners, cin, and 1% glutamine (The C6sr39 cell’s media was such as the microPET.10,11 PET reporter gene/PET supplemented by 0.5 mM histidinol chloride). reporter probe (PRG/PRP)systems have been developed for general detection of gene expression in living Drugs and radiolabeled compounds 12,13 subjects, in order to avoid costly and time-consuming The stock cytovene-IV (ganciclovir sodium for injection, development of specific probes for every therapeutic gene. GCV)powder, manufactured by Parksdale Pharmaceu- Currently, there are three basic groups of PRG/PRP ticals, Inc. (Rochester, MI, USA)for Roche Laboratories systems: (1)enzyme-based approaches such as Herpes (Nutley, NJ, USA), was dissolved in sterile 0.9% NaCl Simplex Virus type 1 thymidine kinase (HSV1-tk)that and stored at 41C. GCV solution (0.1 mg/ml)was phosphorylates fluorine-18 radiolabeled acycloguanosine administered as a daily intraperitoneal injection (approx. 18 18 18 analogs ([ F]GCV, [ F]PCV, [ F]FHBG)and iodine- 20–30 ml to achieve 100 mg/kg). 9-(4-[18F]-fluoro-3-hydro- 124 124 radiolabeled uracil nucleoside derivatives ([ I] xymethylbutyl)-guanine ([18F]FHBG)was synthesized as 14–18 FIAU); (2)receptor-based approaches such as the described previously28 at specific activities of 1000– dopamine 2 receptor, which can bind fluorine-18 labeled 2000 Ci/mmol. [18F]FHBG is a side-chain fluorine-18 18 19 spiperone ([ F]FESP); (3)the symporter-based ap- radiolabeled analog of the antiviral drug penciclovir. 18 18 proach of using the sodium/iodide symporter as the 2[ F]-fluoro-2-deoxy-D-glucose ([ F]FDG)was synthe- 20 PRG and iodine-124 as the PRP. The expression of sized by the method described by Hamacher et al32 at these PRGs can be linked to the expression of a specific activities of approximately 5 Ci/mmol. therapeutic transgene and then, using PET, can be used to track the expression of the therapeutic transgene in PET image acquisition 21–25 living subjects. 10 In order to increase the sensitivity of detecting PRG The dedicated small animal scanner, MicroPET, was used to acquire tomographic whole-body images of the expression, we have used the mutant HSV1-TK enzyme 18 18 (HSV1-sr39TK),26 which has higher affinity for the PRP biodistribution of the [ F]FHBG and [ F]FDG tracers 18 in mice. Mice were tail vein injected with approximately 9-(4-[ F]fluoro-3-hydroxymethylbutyl)guanine 18 18 18 18,27,28 200 mCi of [ F]FHBG or [ F]FDG, 1 hour prior to the ([ F]FHBG or FHBG). We have examined the bio- 18 distribution, bio-safety, and dosimetry of the [18F]FHBG start of microPET data acquisition. [ F]FDG-injected mice were anesthetized with ketamine/xylazine (4:1)prior tracer in normal human volunteers and have found it to 18 be safe and potentially useful.29 HSV1-sr39tk was to the injection of [ F]FDG and were kept anesthetized until microPET data acquisition was completed, whereas originally selected from among several mutant HSV1-tk 18 genes because lower doses of GCV are sufficient to inhibit [ F]FHBG-injected mice were only anesthetized for the the growth of C6 glioma tumor xenografts expressing the microPET scan duration. During scanning, the long axis of the mouse is parallel to the long axis of the scanner. HSV1-sr39TK relative to the ones expressing the wild- 3 3 type HSV1-TK.26 Cell culture and in vivo imaging The scanner has a volumetric resolution of 1.8 mm and sensitivity assays have thus far demonstrated that the its axial field of view is 18 mm. The total duration of HSV1-sr39tk/[18F]FHBG combination are among the image data acquisition was 28 minutes (seven bed posi- most sensitive of these group of PRG/PRP systems.30 tions; 4 minutes per bed). Prior to positioning the mice for Our aim in this study was to analyze the effect of PET scans, their bladders were emptied as much as GCV on stably expressing HSV1-sr39tk C6 glioma possible. No transmission scan was performed. (C6sr39)tumor xenografts using PET imaging with the accumulation of [18F]FHBG and [18F]FDG tracers. Subcutaneous (s.c.) C6 glioma tumor xenografts [18F]FHBG was used to monitor levels of HSV1-sr39tk Two types of s.c. tumor groups were implanted and expression in the C6sr39 tumors, prior to, during, and exposed to GCV treatment. One group of tumors after GCV treatment. [18F]FDG was used to monitor consisted of C6sr39 cells and a second group consisted tumor cell viability during the study periods. The of control C6 cells. For C6sr39 tumors, the range of total imaging results indicate that C6sr39 tumors accumu- cell numbers implanted was 2À4 Â 107. However, for C6 lated [18F]FHBG prior to starting GCV treatment tumors the total number of cells implanted was approxi- and regressed in response to GCV treatment. mately 0.6 Â 107 cells. The reason for implanting few C6 However, these tumors re-grew within 2–3 weeks after cells was that the duration of studies usually exceeded 1 halting GCV treatment, and the re-grown tumors did month, by which time (since C6 tumors are not affected not accumulate [18F]FHBG at greater than back- by GCV treatment)the tumor sizes would not have been ground levels. acceptable due to animal welfare issues. C6 or C6sr39 cells

Cancer Gene Therapy Imaging suicide gene therapy with PET SS Yaghoubi et al 331 were harvested and after washing once with 1 Â PBS were confident that the tumor sizes calculated based on ROI re-suspended in less than 150 ml1Â PBS. Nude mice areas and averaged planes correlated with the size of the (NU/NU, 25–35 g, Charles River Labs, Wilmington, MA, tumors measured by calipers. We used the [18F]FDG USA)were anesthetized by i.p. injection of 50 mlofa images, because regardless of whether the tumor cells solution containing 80 mg ketamine and 20 mg xylazine/ expressed HSV1-sr39tk the viable cells would take up ml. After the mice were unconscious, the cells were [18F]FDG and give a better delineation of the tumor injected s.c. on the back of the mice. Tumors were grown borders. for approximately 10 days. HSV1-sr39tk gene detection assay Monitoring tumor volume, [18F]FHBG accumulation, 18 A C6sr39 tumor implanted in a nude mouse that had not and [ F]FDG accumulation during and after GCV been treated with GCV and a C6sr39 tumor re-grown 3 treatment weeks after halting GCV treatment in a mouse that had Approximately 10 days after implanting C6sr39 or C6 been treated with GCV (100 mg/kg daily)for 2 weeks were cells s.c. in nude mice, the tumor volumes were measured isolated. DNA was extracted from these tumor tissues and using a caliper. Since the tumors were of different shapes C6sr39 cells grown in cell culture, using the phenol/ (not uniform spheres or ovals), assuming they were cubic chloroform/isoamyl alcohol DNA extraction method36 yielded the best estimations. The width, length, and depth and or the QIAamp DNA kit (Qiagen, Valencia, CA, of the tumors were measured with a caliper and multiplied USA). Genomic DNA were digested by the BamH1 and to obtain the volume in mm3. Mice were then scanned for Nco1 restriction enzymes. The HSV1-sr39tk cDNA was 32 [18F]FHBG (week 0). Following the first [18F]FHBG scan, labeled with [ P]ATP using the Prime-It II Random a daily i.p. injection of GCV (100 mg/kg)was initiated. Primer Labeling Kit (Stratagene, La Jolla, CA, USA). An [18F]FDG scan was done the day prior to or after the The presence of HSV1-sr39tk gene was then assayed by 36 days of [18F]FHBG scans. A week after starting GCV Southern blotting. treatment (week 1), some of the mice were scanned for [18F]FHBG and [18F]FDG and their tumor volumes were measured. In order to avoid competition between GCV Results and discussion and [18F]FHBG as substrates for HSV1-sr39TK enzyme, GCV treatments were halted 48 hours prior to scanning We calculated two separate measures of the activity of 18 with [ F]FHBG. After 2 weeks, GCV treatment was [18F]FHBG and [18F]FDG tracers in the C6sr39 (in GCV- stopped and all of the mice were again scanned for treated and untreated mice)and C6 (in GCV-treated 18 18 [ F]FHBG and [ F]FDG and their tumor volumes were mice)tumors. The percent injected dose per gram (%ID/ measured (week 2). The same procedure was carried out g)is the percentage of total amount of tracer injected into on some of the mice, 2 and or 3 weeks after GCV mice accumulated in the tumor normalized to the entire treatment had been halted. volume of the tumor. Theoretically, if all cells in the tumor accumulate equivalent amounts of the tracer, and Image reconstruction and data analysis all other parameters affecting tracer accumulation into a MicroPET image data were reconstructed using the three- certain tumor remain unchanged, %ID/g will remain dimensional iterative maximum a posteriori (MAP) constant when the tumor grows or regresses. However, algorithm.33 The MAP algorithm has been reported to heterogeneity of cells in the tumors with regard to tracer yield images of higher resolution and image data of accumulation may result in a change in %ID/g when superior quantitative accuracy than the filtered back tumors grow or when they regress following GCV- projection (FBP)algorithm. 34 For each tumor, all of the induced cell death. We also calculated the percentage of planes containing the tumor, as observed by the the total tracer injected dose that accumulated into the investigator on the PET images, were averaged and scaled entire tumor (%ID). Any tumor growth resulting from by the injected dose. Three different-shaped regions of proliferation of tracer-accumulating cells or tumor interest (ROI)were drawn to include the whole tumor regression resulting from death of tracer-accumulating area and the average counts/pixel/second in each ROI and cells should result in an increase or a decrease in %ID, the area of the ROI in each pixel was recorded. Using a respectively. Since %ID was calculated based on the calibration factor, determined by analyzing the MAP tumor volumes measured from the ROI drawn on the images, we evaluated the correlation between tumor image data of a cylinder containing a known dose of 18 [18F]FDG, the average counts were converted to percent volumes calculated from [ F]FDG images and tumor volumes calculated from caliper measurements injected dose per gram of tissue (%ID/g). The average 2 %ID/g in tumors with dimensions from any side less than (R ¼ 0.94). 6 mm (or less than three times full-width at half- 35 GCV treatment causes reduction of C6sr39 tumor size. maximum)were partial volume corrected. The volume 18 18 of each voxel was 0.000064 cm3. Assuming 1 g of tissue is Accumulation of [ F]FHBG and [ F]FDG declines in equal to 1 cm3, the %ID of the tracers per tumor was the regressed tumors calculated based on the recorded area of each ROI and We expected that C6 glioma cells stably expressing the the # of planes that had been averaged. We wanted to be HSV1-sr39tk gene (C6sr39 cells), which were used to

Cancer Gene Therapy Imaging suicide gene therapy with PET SS Yaghoubi et al 332 develop s.c. tumor xenografts in nude mice, would die after exposure to GCV; thus C6sr39 tumor volumes should decrease in response to GCV treatment. We further hypothesized that the eradication of C6sr39 cells will result in decreased accumulation of [18F]FHBG in the C6sr39 tumors. The decreased number of viable cells after GCV treatment should also result in decreased accumulation of [18F]FDG in C6sr39 tumors. Figure 1 illustrates gradual decline in size of a C6sr39 tumor grown s.c. in a nude mouse, during 2 weeks of daily intraperitoneal GCV treatment. During this period of GCV treatment, the C6sr39 tumor (tumor on the left)size reduction is also accompanied by decreasing accumulation of [18F]FHBG in the C6sr39 tumor. Meanwhile, the control tumor (tumor on the right), consisting of control C6 glioma cells that do not express the HSV1-sr39tk gene, does not respond to GCV treatment and continues to grow during the 2 weeks of GCV therapy. In addition, [18F]FDG accumulation into the C6sr39 tumor declines, but increases in the growing C6 tumor. In week 2, lack of [18F]FDG uptake in a portion of the C6 tumor is due to necrosis of that portion of the tumor. Assuming C6sr39 tumors consist of 100% HSV1- sr39tk-expressing C6 cells, each C6sr39 cell traps the same amount of [18F]FHBG tracer, the cells dying in response to GCV are phagocytosed, and the C6sr39 Figure 1 Response of C6sr39 and control C6 tumor xenografts to tumors lack necrotic regions, theoretically the concentra- GCV treatment and activity of [18F]FHBG and [18F]FDG in these tion of [18F]FHBG (%ID/g)should remain relatively tumors throughout the treatment period. C6sr39 cells were s.c. constant even as the tumor volume decreases. In contrast, injected on the left flank near the shoulder of a nu/nu mouse. C6 cells the percentage of total injected [18F]FHBG (%ID) were injected on the right flank. The mouse was scanned for 18 trapped in the C6sr39 tumors should decline in parallel [ F]FHBG (week 0) accumulation the day prior to initiating daily intraperitoneal (IP) GCV (100 mg/kg) treatment. The mouse was with the decline in tumor volume. However, not only is 18 18 again scanned for [ F]FHBG on weeks 1 and 2 of GCV treatment. there a high correlation between [ F]FHBG (%ID)and 18 18 3 [ F]FDG scans were carried out 1 day prior or after the [ F]FHBG C6sr39 tumor volume (mm )(Fig 2a: R ¼ 0.97)as both scans. [18F]FHBG and [18F]FDG activity in the regressing C6sr39 decline during the 2 weeks of GCV treatment, but also tumor decreased after GCV treatment. [18F]FDG activity increased in during the same period there is a correlated decrease in the growing C6 tumor. both [18F]FHBG (%ID/g)and C6sr39 tumor volume (mm3)(Fig 2b: R2 ¼ 0.87). Furthermore, the 92% average decrease in [18F]FHBG (%ID/g)and the 99% average HSV1-sr39TK enzyme molecules. Perhaps the C6sr39 decrease in [18F]FHBG (%ID), as well as the 81% cells expressing the highest number of HSV1-sr39TK average decrease in C6sr39 tumor volume (mm3), after 2 molecules phosphorylate and trap more [18F]FHBG, and weeks of GCV treatment, are all significant (Table 1). also convert more GCV to the phosphorylated toxic These data indicate that at least one of the assumptions metabolite. These cells would be expected to die at an about C6sr39 tumors during GCV treatment is inaccu- earlier point relative to C6sr39 cells expressing fewer rate. The C6sr39 tumor may not consist of 100% C6sr39 HSV1-sr39TK molecules. Although our data provide no cells. It is also possible that not all C6sr39 cells equally direct evidence to support this theory, if true, this could trap the same amount of [18F]FHBG tracer, because not result in a sharper decline in [18F]FHBG entrapment all of the C6sr39 cells may contain the same number of relative to the decline in the number of viable cells,

Figure 2 (a) Correlation between [18F]FHBG(%ID) accumulation and tumor volume (mm3) for C6sr39 tumors during the 2-week period of GCV treatment (R2 ¼ 0.97; y ¼ 0.0044xÀ0.3658; week 0, n ¼ 20; week 1, n ¼ 17; week 2, n ¼ 18). (b) Correlation between [18F]FHBG (%ID/g) concentration and tumor volume (mm3) for C6sr39 tumors during the 2-week period of GCV treatment (R2 ¼ 0.87; y ¼ 0.0033x þ 0.0793; week 0, n ¼ 23; week 1, n ¼ 20; week 2, n ¼ 21). (c) Correlation between [18F]FDG (%ID/g) concentration and [18F]FHBG (%ID/g) concentration in C6sr39 tumors during the 2-week period of GCV treatment (R2 ¼ 0.78; y ¼ 0.4414x þ 1.8921; week 0, n ¼ 23; week 1, n ¼ 20; week 2, n ¼ 21). (d) Correlation between [18F]FHBG (%ID) accumulation and [18F]FDG (%ID) accumulation in C6sr39 tumors during the 2-week period of GCV treatment (R2 ¼ 0.91; y ¼ 1.307xÀ0.6428; week 0, n ¼ 20; week 1, n ¼ 17; week 2, n ¼ 18). (e) Correlation between [18F]FDG (%ID) accumulation and tumor volume (mm3) for C6sr39 tumors at week 0 (n ¼ 20), week 1 (n ¼ 17), week 2 (n ¼ 18), week 4 (n ¼ 8), and week 5 (n ¼ 13) (R2 ¼ 0.98; y ¼ 0.003x þ 0.3564). (f) Lack of correlation between [18F]FDG (%ID/g) concentration and tumor volume (mm3) for C6sr39 tumors during the 2- week period of GCV treatment (R2 ¼ 0.42; y ¼ 0.0011x þ 2.0231; Week 0, n ¼ 23; Week 1, n ¼ 20; Week 2, n ¼ 21). Error bars are standard error of the mean.

Cancer Gene Therapy Imaging suicide gene therapy with PET SS Yaghoubi et al 333 resulting in a decrease in [18F]FHBG (%ID/g)con- first week of GCV treatment, while during the same centration. It is also interesting to note that the significant period the drop in tumor volume was not significant 62% decrease in [18F]FHBG (%ID)occurs during the (Table 1).

Cancer Gene Therapy Imaging suicide gene therapy with PET SS Yaghoubi et al 334 Table 1 Statistical analysis of changes in average FHBG, FDG, and C6sr39 tumor volume

FHBG (% ID/g) FDG (% ID/g) FHBG (%ID/g)/FDG (%ID/g)

Duration % Change Statistics % Change Statistics % Change Statistics

Weeks 0–1 À42.34114 Po0.02 6.436167 NS 14.2353744 NS Weeks 0–2 À92.24247 Po0.001 À35.70847 Po0.02 À91.2365399 Po0.001 Weeks 1–2 À86.54581 Po0.001 À39.59616 NS À92.3285934 Po0.05 Week 2–4 184.9064 Po0.01 54.76119 NS 58.1202591 NS Week 2–5 53.40384 NS 53.62166 NS 0.0783637 NS Weeks 0 and 4 À77.89829 Po0.001 À0.501663 NS À86.1431942 Po0.005 Weeks 0 and 5 À88.09965 Po0.001 À1.234286 NS À91.2296725 Po0.005

FHBG (% ID) FDG (%ID) Tumor volume

Duration % Change Statistics % Change Statistics % Change Statistics

Weeks 0–1 À62.37393 Po0.005 À58.32911 Po0.05 À61.6067991 NS Weeks 0–2 À98.78853 Po0.001 À70.89975 Po0.02 À81.1884613 Po0.01 Weeks 1–2 À96.78022 Po0.001 À30.16647 NS À51.0029425 NS Weeks 2–4 3108.597 Po0.002 427.8421 Po0.02 1149.53849 Po0.01 Weeks 2–5 644.9552 Po0.01 551.7077 Po0.005 985.542561 Po0.02 Weeks 0 and 4 À61.12865 Po0.005 53.6034 NS 135.057416 Po0.05 Weeks 0 and 5 À90.97506 Po0.001 89.64858 NS 104.207259 NS Percentage change in average concentration (%ID/g) and total tumor accumulation (% ID) of the FHBG and FDG tracers between weeks during GCV treatment and tumor regrowth. Percentage change in average C6sr39 tumor volume between weeks during GCV treatment and tumor regrowth. P40.05 was considered not significant (NS).

The tumor may also not consist of all viable cells mation can also cause a significant rise in [18F]FDG at the time of imaging and volume measurement, uptake.40 because not all of the dead cells may have been cleared These observations of C6sr39 tumor volume reduction and because the center of tumors (due to hypoxia and decline in [18F]FHBG accumulation support our or malnourishment)may be necrotic. This may perhaps expectations that GCV treatment is effective at eliminat- be another explanation for the significant drop in ing s.c. implanted C6 glioma cells stably expressing the both [18F]FHBG (%ID/g)and [ 18F]FDG (%ID/g)con- HSV1-sr39tk gene. Effectiveness of GCV in killing HSV1- centrations during the 2 weeks of GCV treatment (Table 1 tk and various mutant HSV1-tk expressing C6-glioma and Fig 2c). Our observation that FDG (%ID) always tumors have been previously reported.31,41 As shown in correlates with C6sr39 tumor volume (mm3)(Fig 2e: Figures 1 and 3, C6 glioma tumors (that do not express R2 ¼ 0.98)is consistent with the fact that [ 18F]FDG HSV1-sr39tk)did not respond to GCV treatment by monitors viable cells. Since only viable C6sr39 cells can volume reduction and only background activity of express HSV1-sr39tk, the correlated decline in [18F]FHBG (% ID/g)is observed in C6 tumors. In fact, [18F]FHBG (%ID)and [ 18F]FDG (%ID)is also expected the volume of C6 glioma tumors increased significantly (Fig 2d: R2 ¼ 0.91). (Po0.05)during the 2-week period of GCV treatment. [18F]FDG entrapment into cells is due to enzyme- There was also no significant change in [18F]FDG (%ID/ catalyzed phosphorylation by hexokinase and its uptake g), while [18F]FDG (%ID)increased significantly is dependent on glucose transporters. Therefore, if GCV (Po0.001)due to the increased number of viable C6 exposure causes changes in the activity of hexokinase or gliomas. Figure 1 shows some accumulation of glucose transporters,37 [18F]FDG accumulation in tumors [18F]FHBG into the large C6 glioma tumor at week 2. will be altered. Besides, if any of the tumors have been An above background activity of [18F]FHBG is observed eradicated after 2 weeks, the tracer concentrations will be in very large control tumors when they are scanned an near background levels. However, evidence presented hour after tracer injection, but the tracer is cleared if the below demonstrates that the C6sr39 tumors were not tumors are scanned 2–3 hours after tracer injection (data completely eradicated. Therefore, it was expected that not shown). However, on average, there was no sig- [18F]FDG (%ID/g)only declines by an average of 36% nificant change in [18F]FHBG (%ID/g)and [ 18F]FHBG (Table 1)and [ 18F]FDG (%ID/g)does not decline in (%ID)in the control C6 tumors throughout the 2 weeks correlation with C6sr39 tumor volume (mm3)(Fig 2f: GCV treatment period. R2 ¼ 0.42). We should note that [18F]FDG accumulation These imaging studies demonstrate that [18F]FHBG is a trends might vary when the subject carrying the tumor is suitable tracer for detecting cells susceptible to GCV- immunocompetent, because HSV1-sr39tk/GCV therapy induced cell death, as only the C6sr39 tumors which may induce inflammation at the tumor site.38,39 Inflam- accumulate [18F]FHBG decrease in size in response to

Cancer Gene Therapy Imaging suicide gene therapy with PET SS Yaghoubi et al 335

Figure 3 [18F]FHBG (%ID/g), [18F]FDG (%ID/g), and tumor volume (mm3) for C6 tumors, during GCV treatment (week 0, n ¼ 7; week 1, n ¼ 4; week 2, n ¼ 7). [18F]FHBG (%ID/g) concentration remains relatively constant at background levels because C6 tumor cells did not carry the HSV1-sr39tk gene. [18F]FDG (%ID/g) concentration is also relatively constant at above background levels in proliferating C6 tumors. Error bars are standard error of the mean.

daily i.p. injections of GCV. In addition, imaging with [18F]FHBG, following treatment with GCV, reveals whether C6 glioma cells expressing the HSV1-sr39tk gene remain and may be useful in predicting how long GCV treatment must continue. In our model, it appears that 2 weeks of daily i.p. injection of GCV (100 mg/kg)is Figure 4 (a, b) Comparison between [18F]FHBG (%ID/g and %ID), sufficient for the ablation of the HSV1-sr39tk-expressing [18F]FDG (%ID/g and %ID) and C6sr39 tumor volume (mm3) when cells in the tumors, since we observe an average 99% drop the mouse carrying four C6sr39 tumors was not treated with GCV (a, in total [18F]FHBG (%ID)accumulation and 92% drop b: all weeks, n ¼ 4). [18F]FHBG (%ID/g) and [18F]FDG (%ID/g) in [18F]FHBG (%ID/g)concentration to background concentration remain relatively constant in the tumors as they grow, uptake levels at the sites of C6sr39 tumors. The majority but total [18F]FHBG (%ID) and [18F]FDG (%ID) accumulation of C6sr39 tumors were visually eradicated after 2 weeks of increases as the tumors grow. Error bars are standard error of the GCV treatment, but even in tumors that were still mean. palpable after 2 weeks [18F]FHBG (%ID/g and %ID) was reduced to background levels. To confirm that the decline in C6sr39 tumor volumes (mm3),[18F]FHBG (%ID/g and %ID), and [18F]FDG (%ID/g and %ID) Overall, decrease in [18F]FHBG accumulation can be were mainly due to exposure to GCV, mice carrying s.c. attributed to the following reasons: (1)decrease in the C6sr39 tumor xenografts were monitored for 2 weeks level of HSV1-sr39TK enzyme due to the elimination of without GCV treatment (Fig 4a and b). There were no C6sr39 cells by GCV-induced cell death; (2)decrease in significant changes in [18F]FHBG (%ID/g)and [ 18F]FDG the level of HSV1-sr39TK enzyme due to attenuated (%ID/g). On average, there was a significant (Po0.005) expression of the HSV1-sr39tk gene; (3)decrease in 155% increase in [18F]FHBG (%ID)and a significant HSV1-sr39TK enzymatic activity in vivo; and (4)and (Po0.005)397% increase in [ 18F]FDG (%ID). There was decreased uptake of the tracer. The fact that the decrease also a 377% increase in the volume of C6sr39 tumors that in the C6sr39 tumor volume, in response to GCV had not been exposed to GCV in the 2-week period. treatment, highly correlates with the decrease in

Cancer Gene Therapy Imaging suicide gene therapy with PET SS Yaghoubi et al 336 [18F]FHBG accumulation (Fig 2a and b)in addition to period, on average, the C6sr39 tumors grew significantly the fact that [18F]FHBG (%ID/g and %ID)does not (Po0.02)by 986% (Table 1).Although there was no decline when the tumors are not treated with GCV (Fig 4a significant change in [18F]FDG (%ID/g), total [18F]FDG and b)indicates that the decrease in the level of HSV1- (%ID)increased significantly ( Po0.005)by 552% sr39TK enzyme due to the elimination of C6sr39 cells is from week 2 to 5, indicating increased numbers of viable the major reason for decrease in [18F]FHBG (%ID). C6 cells (Table 1). Figures 6a and b illustrate average [18F]FHBG (%ID/g and %ID), [18F]FDG (%ID/g 3 Regressed C6sr39 tumors re-grow after halting GCV and %ID), and C6sr39 tumor volumes (mm )during treatment without recovering [18]FHBG accumulation the 2 weeks of GCV treatment, and 2 and 3 weeks (weeks 4 and 5)after halting GCV treatment. At week 2, After treating the C6sr39 tumors with GCV for 2 weeks, [18F]FHBG (%ID/g and %ID)are at background we had observed that in most cases the tumors were 18 18 levels. Even though total [ F]FHBG (%ID)increases visually eradicated and [ F]FHBG accumulation de- significantly (Po0.01)from week 2 to 5, the average clined to background levels. However, we also observed 18 18 increase in [ F]FHBG (%ID/g)is not significant. that [ F]FDG (%ID/g)concentrations did not always Beside, even though the size of the re-grown tumors at decline to background levels at the tumor sites. Therefore, week 5 was 104% greater than at week 0 (although we monitored the C6sr39 tumors for up to 3 weeks after not a significant change), [18F]FHBG (%ID)was halting GCV treatment, to observe whether they would 18 18 significantly (Po0.001)lower by 91% on week 5 than it re-grow. Figure 5 illustrates [ F]FHBG and [ F]FDG was on week 0. Finally, there is a significant (Po0.001) tracer accumulation in four separate s.c. C6sr39 tumors decline in [18F]FHBG/[18F]FDG (%ID/g)after 2 weeks of during the 2 weeks of GCV treatment and 3 weeks after GCV treatment, whereas the rise in [18F]FHBG/ halting GCV treatment (week 5). During the 3-week [18F]FDG during tumor re-growth is not significant (Table 1). Since [18F]FDG is used to obtain a measure of the number of viable cells, the [18F]FHBG/[18F]FDG ratio may correct for the noise caused by the extracellular material and tumor blood pool nonspecifically taking up the tracer in large tumors. These data demonstrate that a major portion of the C6sr39 cells in the C6sr39 tumors was eradicated. However, at the end of the treatment period, relatively few tumor cells remain alive. These remaining cells are capable of rapid proliferation, resulting in recurrence of tumors after GCV treatment is halted. However, the majority of these proliferating cells are unable to trap [18F]FHBG. There are five possible reasons for the lack of [18F]FHBG accumulation: (1)lack of the HSV1-sr39tk gene; (2)lack of HSV1-sr39TK enzyme expression; (3) lack of HSV1-sr39TK enzyme activity; (4)lack of ability to transport [18F]FHBG into cells; (5)expression of drug- resistant efflux pumps making cells resistant to [18F]FHBG accumulation. Either due to one or a combination of these reasons, GCV treatment of C6sr39 tumors leads to the selection of highly proliferating tumor cells, unable to accumulate [18F]FHBG. This selection process also explains the major decline in [18F]FHBG (%ID/g)concentration during the treatment period. Possibly, while [18F]FHBG-accumulating C6sr39 cells are dying in response to GCV exposure, the C6 cells inside C6sr39 tumors incapable of trapping [18F]FHBG are also incapable of accumulating GCV; thus they survive and proliferate. In order to determine whether Figure 5 Four C6sr39 tumor xenografts were implanted s.c. on four the re-grown tumors carry the HSV1-sr39tk gene, we sites of the nude mouse shown. All four tumors highly accumulated analyzed the genomic DNA extracted from a re-grown 18 18 [ F]FHBG and [ F]FDG prior to starting GCV treatment (week 0). tumor by Southern blot. The HSV1-sr39tk gene was The mouse was administered daily IP injections of GCV (100 mg/kg) detected in both the genomic DNA from the re-grown for 2 weeks, during which time period the tumors regressed (three of them visually eradicated) and [18F]FHBG and [18F]FDG accumula- tumor and the genomic DNA from a C6sr39 tumor, tion declined to background levels. The mouse was monitored up to grown in a mouse that had not been treated with GCV 3 weeks after halting GCV treatment. The tumors re-grew, but only (data not shown). It is possible that a fraction of the cells accumulated [18F]FHBG at background levels, despite robust ability in the re-grown tumors still carry the HSV1-sr39tk gene, to accumulate [18F]FDG. but on average the fraction of these cells in the re-grown

Cancer Gene Therapy Imaging suicide gene therapy with PET SS Yaghoubi et al 337 lower accumulation of [18F]FHBG in the recurrent tumors.

Conclusion

The observations that only the tumors accumulating [18F]FHBG regress after GCV treatment supports using [18F]FHBG imaging to detect cancer cells expressing the HSV1-sr39tk suicide transgene and susceptible to GCV-induced cell death. Furthermore, since [18F]FHBG (%ID)declines as GCV exposed tumors regress, it can be used to monitor the effectiveness of GCV treatment in killing HSV1-sr39tk-expressing cancer cells. However, our imaging investigation also demonstrated that GCV treatment could result in a selection process such that the majority of C6sr39 cells capable of accumulating [18F]FHBG are killed by GCV, while a few cells that are unable to trap [18F]FHBG survive and proliferate. While average C6sr39 tumor volume on week 5 was greater than on week 0, [18F]FHBG (%ID)was 91% lower on week 5 than it was on week 0. Further evidence supporting this conclusion includes a very significant decline in average [18F]FHBG (%ID/g), while average [18F]FDG (%ID)does not decrease to background levels. Therefore, the use of [18F]FHBG and [18F]FDG together may yield clinically significant information on the progress of HSV1-sr39tk/GCV suicide gene therapy of cancer patients.

Acknowledgments

We thank Ron Sumida, David Stout, Waldemar Ladno, and Judy Edwards for assistance with MicroPET imaging and the UCLA cyclotron crew for outstanding support in the synthesis of imaging tracers. This work was partially supported by funding from NIH Grants P50 CA86306, RO1 CA82214-01, and SAIRP R24 CA92865 DOE contract DE-FC03-87ER60615. This work was partially supported by funding from NIH Grants P50 CA86306, RO1 CA82214-01, and SAIRP R24 CA92865 DOE contract DE-FC03-87ER60615.

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