Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Cancer Therapy: Preclinical Targeted Radiotherapy for Prostate Cancer with an Oncolytic Adenovirus Coding for Human Sodium Iodide Symporter Tanja Hakkarainen,1,3 Maria Rajecki,1,3 Mirkka Sarparanta,2 Mikko Tenhunen,4 Anu J. Airaksinen,2 Renée A. Desmond,5 Kalevi Kairemo,4 and Akseli Hemminki1,3 Abstract Purpose: Oncolytic adenoviruses are promising tools for cancer therapy. Although several clinical reports have indicated both safety and promising antitumor capabilities for these viruses, there are only a few examples of complete tumor eradication. Thus, the antitumor efficacy of oncolytic adenoviruses needs to be improved. One potentially useful approach is combination with radiotherapy. Experimental Design: To target systemically administered radioiodide to tumors, we created Ad5/3-Δ24-human sodium iodide symporter (hNIS), a Rb-p16 pathway selective infectivity enhanced oncolytic adenovirus encoding hNIS. Results: Ad5/3-Δ24-hNIS replication effectively killed prostate cancer cells in vitro and in vivo. Also, the virus-mediated radioiodide uptake into prostate cancer cells in vitro and into tumors in vivo. Furthermore, Ad5/3-Δ24-hNIS with radioiodide was significantly more effective than virus alone in mice with prostate cancer xenografts. Conclusions: These results suggest that oncolytic adenovirus-mediated targeted radiotherapy might be a potentially useful option for enhancing the efficacy or adenoviral virotherapy. (Clin Cancer Res 2009;15(17):5396–403) Prostate cancer is the most common cancer in the male population and the second leading cause of cancer deaths in western men. Although radiation therapy and surgery can cure many patients, and watchful waiting is an option for some, >30% of treated patients relapse. For disseminated or recurrent disease, androgen ablation therapies are often initially effective, but Authors' Affiliations: 1Cancer Gene Therapy Group, Molecular Cancer Biology Program & Transplantation Laboratory & Haartman Institute & Finnish Institute for Molecular Medicine and 2Laboratory of Radiochemistry, University of Helsinki; 3 HUSLAB, Helsinki University Central Hospital; 4Department of Oncology, Helsinki University Central Hospital, Helsinki, Finland and 5 Comprehensive Cancer Center, Biostatistics and Bioinformatics Unit, University of Alabama at Birmingham, Birmingham, Alabama Received 10/5/08; revised 5/14/09; accepted 6/12/09; published OnlineFirst 8/25/09. Grant support: European Research Council, EU FP6 APOTHERAPY and THERADPOX, Helsinki University Central Hospital Research Funds (EVO), Finnish Cancer Society, Sigrid Juselius Foundation, Academy of Finland, Biocentrum Helsinki, University of Helsinki, and Helsinki Biomedical Graduate School. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). A. Hemminki is K. Albin Johansson Research Professor of the Foundation for the Finnish Cancer Institute. Requests for reprints: Akseli Hemminki, Cancer Gene Therapy Group, Biomedicum, University of Helsinki, P.O. Box 63, 00014 Helsinki, Finland. Phone: 358-9-1912-5464; Fax: 358-9-1912-5465; E-mail: akseli.hemminki@ helsinki.fi. F 2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-08-2571 Clin Cancer Res 2009;15(17) September 1, 2009 emergence of androgen-independent locally recurrent or distant disease is usually fatal. Therefore, there is a need for developing new antitumor approaches for androgen-independent prostate cancer. The main target for prostate cancer metastasis is bone, but dissemination to lung and liver is also common (1). Although prostate cancer metastases are not insensitive to radiation therapy, relatively high radiation doses are needed. In the context of local disease, this can be achieved with external beam radiation therapy or brachytherapy. However, side effects increase with field size; therefore, treatment of disseminated disease with external beam radiation results in only palliation because a lower dose must be used. Thus, it would be useful if radiation could be targeted to tumors only. Adenoviruses are the most commonly used vectors for cancer gene therapy. Oncolytic adenoviruses, which can destroy target cells via viral replication (2), are promising tools for developing novel treatment modalities for cancer. Oncolytic Ad5-based viruses have shown efficacy and safety in preclinical (3–6) and clinical trials (7, 8), including the treatment of prostate cancer (9–11). However, variable expression of the primary Ad5 receptor, the coxsackie-adenovirus receptor, may limit the efficacy of Ad5-based constructs (12). Various approaches can be used to improve adenoviral transduction of cancer cells. For example, switching the Ad5 fiber knob to serotype 3 knob improves the transduction and enhances the cell-killing capacity of the virus in the context of many cancer types including prostate cancer (3, 6, 13, 14). Although several clinical reports have indicated promising antitumor capabilities with oncolytic viruses, there are only a few examples of complete tumor eradication. One possibility to improve therapeutic efficacy is to use the synergy of radiation and oncolytic adenoviruses (15–17). However, the systemic 5396 www.aacrjournals.org Targeted Radioiodide Therapy for Prostate Cancer Translational Relevance Treatment of metastatic solid tumors requires new approaches, as current therapeutic modalities cannot cure most cases. Expression of the human sodium iodide symporter as a transgene is an attractive strategy for targeting systemically applicable radioisotopes to tumors. Previous work has shown the utility of the approach for imaging. However, our article is the first study that combines a therapeutic radioiodide with an oncolytic adenovirus. This approach allows tumor cells to be killed due to the oncolytic effect of the virus and due to radiation-induced cell death and the approach also takes advantage of synergy between radiation and oncolytic adenovirus replication. These preclinical results facilitate clinical testing of the approach. nature of disseminated prostate cancer calls for body-wide targeting of radiation to tumor cells. This might be achievable by incorporating the human sodium iodide symporter (hNIS) transgene into an oncolytic adenovirus. hNIS is an integral plasma membrane glycoprotein mainly expressed in thyroid follicular cells (18). The biological function of hNIS is to mediate active transport of iodine, which is a crucial component for thyroid hormone biosynthesis. This transporting ability of hNIS has been used for half a century in radioiodide therapy of thyroid carcinoma, where radioactive iodide molecules (131I) are used to internally radiate cancer cells of thyroid origin. By introducing hNIS as a transgene, radioiodide therapy can be used to treat cancers of nonthyroid origin. The approach has been successfully studied, for example, in hepatocarcinoma, breast, and prostate cancers (19–23). An additional useful aspect of hNIS is noninvasive imaging of transgene expression (24), which allows monitoring viral spread and persistence (25, 26). Although hNIS has already been used for clinical and preclinical imaging, no previous studies have assessed the utility of radioiodide for enhancing the therapeutic efficacy of oncolytic adenovirus. In this study, we developed a novel oncolytic adenovirus (Ad5/ 3-Δ24-hNIS) expressing hNIS under the endogenous adenoviral E3 promoter. This tightly couples transgene expression with virus replication (3, 27). Tumor specificity was achieved by engineering a 24-bp deletion in constant region 2 of E1A, which renders the virus selective for cells mutant in the Rb-p16 pathway (6, 28–30). Rb-p16 pathway defects are found in practically all cancers including prostate cancer (31, 32). We show that Ad5/3-Δ24-hNIS expresses functional hNIS resulting in intracellular accumulation of radioactive iodide in prostate cancer cells in vitro and in vivo. In addition, the combination of Ad5/3-Δ24-hNIS with radioiodide significantly attenuates tumor growth in prostate cancer bearing mice in comparison with virus only. Materials and Methods Cell culture. Androgen-independent prostate cancer cell lines 22Rv1, PC-3, and DU-145 and lung adenocarcinoma cell line A549 were purchased from the American Type Culture Collection. PC-3MM2 cells are a metastatic hormone-refractory subline of PC-3 (courtesy of Isaiah J. Fidler, M. D. Anderson Cancer Center). Human www.aacrjournals.org embryonic kidney epithelial 293 cells were purchased from Microbix. Cell line 911 was obtained from Dr. Alex J. van der Eb (University of Leiden). All cell lines were cultured in the recommended conditions. Viral constructs. To create Ad5/3-Δ24-hNIS, EcoRI-digested and blunted hNIS fragment (from pcDNA3-hNIS; courtesy of Steve Russell, Mayo Clinic) was inserted into BsiWI-MfeI–digested and blunted pTHSN (27) to obtain pTHSN-hNIS. To make the control virus Ad5/ 3-Δ24-Δgp19K, BsiWI-MfeI–digested and blunted pTHSN was selfligated resulting in pTHSN-Δgp19K. To obtain pAd5/3-Δ24-hNIS and pAd5/3-Δ24Δgp19K, PmeI-linearized pShuttle-Δ24 (33) and pAd5/3E1-hNIS or pAd5/3-E1-Δgp19K were electroporated into BJ5183 cells. Adenoviral plasmids were linearized and transfected into 911 cells with Superfect (Qiagen) following the manufacturer's instructions. Adenovirus colonies were picked and viruses were propagated in A549 cells and purified using standard techniques. Ad5/3-Δ24 and Ad5/ 3luc1 viruses were constructed previously (6, 12). Viral particles (VP) were determined with spectrophotometry and plaque-forming units with TCID50 assay. Titers were Ad5/3-Δ24-hNIS: 1.1 × 1012 VP/mL; 9.0 × 1010 plaque-forming units/mL, Ad5/3-Δ24-Δgp19K: 1.5 × 1012 VP/mL; 2.8 × 1011 plaque-forming units/mL, Ad5/3-Δ24: 1.7 × 1012 VP/mL, and Ad5/3luc1: 6.9 × 1011 VP/mL. Reverse transcription-PCR. Prostate cancer cells were infected with Ad5/3-Δ24-hNIS and Ad5/3-Δ24-Δgp19K (10 VP/cell) for 2 h at 37°C. Cells were harvested 24 and 48 h after infection. Total RNA was isolated using the RNeasy Kit (Qiagen) and treated with DNase before reverse transcription-PCR. Thus, 350 ng total RNA was used for each reaction. Amplification (35 cycles, annealing at 54°C) was carried out with the OneStep Reverse Transcription-PCR Kit (Qiagen) using hNIS-specific primers (forward 5′-CTTCTGAACTCGGTCCTCAC3′ and reverse 5′-TCCAGAATGTATAGCGGCTC-3′) and β-actin–specific primers (forward 5′-CGAGGCCCAGAGCAAGACA-3′ and reverse 5′-CACAGCTTCTCCTTAATGTCACG-3′). PCR product size for hNIS and β-actin was 453 and 482 bp, respectively. Iodide uptake. Prostate cancer cells were infected with Ad5/3-Δ24hNIS and Ad5/3-Δ24-Δgp19K (10 VP/cell) for 2 h at 37°C. Cells were washed with 1× PBS 24 and 48 h post-infection and incubated with 7.4 kBq sodium iodide [ 125 I]NaI (MAP Medical Technologies Oy) for 20 min at room temperature. Cells were washed twice with 1× PBS followed by lysis using 300 μL Cell Culture Lysis Reagent for each sample (Promega). Radioactivity was quantified with a well counter connected with a multichannel analyzer Atomlab 950 (Biodex Medical System). Cell-killing assays. Prostate cancer cells were infected with Ad5/ 3-Δ24-hNIS, Ad5/3-Δ24-Δgp19K, Ad5/3-Δ24, and Ad5/3luc1 using 0.01, 0.1, 1, 10, and 100 VP/cell. Growth medium was replaced every other day. Cell viability was determined 6 days post-infection for PC3MM2, 7 days post-infection for DU-145 and PC-3, and 9 days postinfection for 22Rv1 cells with CellTiter 96 AQueous One Solution cell proliferation assay (also called the MTS assay; Promega). TCID50 assay Cell line samples. Prostate cancer cells (5 × 104 per well) were plated in triplicates in 1 mL of 5% growth medium on 24-well plates, incubated overnight, and infected with Ad5/3-Δ24-hNIS (5 VP/cell) for 2 h at 37°C. At 8, 24, 48, and 72 h post-infection, cells and growth medium were collected, frozen at -80°C, and freeze-thawed three times. For TCID50, the cells were spun down and the supernatant was added to 293 cells. Tissue samples. Organs collected from mice treated with Ad5/3Δ24-hNIS and 131I were collected, weighed, manually homogenized, and diluted into 800 μL DMEM. They were freeze-thawed at -80°C three times. For TCID50, the tissue samples were spun down and the supernatant was added to 293 cells. For both experiments, 104 293 cells were plated in 100 μL of 5% DMEM into 96-well plates, incubated overnight, and infected with the supernatants of above experiments. After 10 days, the development of CPE was assessed to estimate the titer. 5397 Clin Cancer Res 2009;15(17) September 1, 2009 Cancer Therapy: Preclinical Fig. 1. A schematic presentation of the viruses used in this study. Oncolytic viruses Ad5/3-Δ24-hNIS, Ad5/3-Δ24-Δgp19K, and Ad5/3-Δ24 have a 24-bp deletion in constant region 2 of the adenoviral E1A gene, which confers replication selectivity to Rb-p16 pathway mutant cells. In Ad5/3-Δ24-hNIS, hNIS replaces gp19K in the E3 region and is driven by the native E3 promoter, thereby coupling hNIS expression to virus replication. Ad5/3-Δ24-Δgp19K lacks hNIS but is otherwise isogenic to Ad5/3-Δ24-hNIS, whereas Ad5/3-Δ24 contains an intact E3. Replication-deficient, E1-deleted Ad5/3luc1 encodes luciferase. All the viruses contain chimeric 5/3 fiber, which targets them to Ad3 receptor, highly expressed in prostate cancer. In vivo studies. Male NMRI/nude mice were purchased from Taconic. Tumors were established in 8-week-old mice by subcutaneous injection of 5 × 106 PC-3MM2 cells under medetomidine-ketamine-0.9% saline (1:2:7) anesthesia. In the imaging experiment, three mice received intratumorally saline (top left tumor), 7 × 108 VP Ad5/3-Δ24Δgp19K (top right tumor), and 7 × 108 VP Ad5/3-Δ24-hNIS (bottom right and left tumors) 11 and 12 days after tumor cell inoculation. A day after the last viral injection, mice received intravenously 1.85 MBq sodium iodide [123I]NaI (MAP Medical Technologies Oy) followed by imaging with γ-camera Toshiba 7200A/UI (Toshiba) 0.5, 1, 1.5, 2, 4, and 13 h after iodide exposure. Animal regulation did not allow analysis of later time points. The γ-energy peak 159 keV was recorded using 20% window, and 150 kilocounts were collected per image. The used matrix size was 256 × 256 × 16, and this made the pixel size 1.08 × 1.08 mm. At 13 h, various organs (heart, lung, liver, spleen, kidney, thyroid, stomach, muscle, blood, and bone) and tumors were collected, weighed, and analyzed with a well counter connected with a multichannel analyzer Atomlab 950 (Biodex Medical System). Results are expressed as percentage from initial iodide dose per gram of tissue. For the therapeutic experiment, tumor-bearing mice were randomized into four groups (6 mice per group, 2 tumors per mouse): mock, 131 I, Ad5/3-Δ24-hNIS, and Ad5/3-Δ24-hNIS + 131I. Six and 7 days after tumor cell inoculation, mice received intratumorally either growth medium or 7 × 108 VP Ad5/3-Δ24-hNIS per tumor. A day after the last viral injection, mice received intraperitoneally 50 MBq carrier-free sodium iodide [131I]NaI (MAP Medical Technologies Oy). Tumor size was measured every other day and the volume was calculated using the formula: length × width2 × 0.5. When mice were killed due to tumor size, organs were collected and weighed and their radioactivity was determined with a γ-counter (Wizard 3; Perkin-Elmer) for 180 s. Results are expressed as percentage from initial iodide dose per gram of tissue. Whole-body emitted radioactivity was measured at days 9, 10, 11, 13, 14, 15, and 17. On days 9 to 11, measurements were done on a dose calibrator (CRC-120; Capintec), and after day 11, measurements were done on a whole-body measurement apparatus consisting of a lead measurement chamber and a UniSpec tube-base multichannel analyzer with sodium iodide scintillation crystal (Canberra Industries). The mice were weighed at day 14, and the same weight was used in calculations throughout the experiment. In all experiments, mice were killed when tumor size reached 15 mm in any diameter as required by animal regulations. The therapeutic in vivo experiment had to be stopped on day 17 due to animal husbandry constraints. All the animal experiments were approved by the National Committee for Animal Experimentation in Finland (State Provincial Office of Southern Finland). Clin Cancer Res 2009;15(17) September 1, 2009 Statistical analysis. Analysis of iodide uptake was done by one-way ANOVA with Bonferroni's post hoc test. Analysis of tumor volume over time between experimental groups was done using a repeated-measures model with PROC MIXED (SAS version 9.1). The tumor volume measurements were log-transformed for normality. The a priori planned comparisons of specific differences in predicted treatment means averaged over time and at each time point by t statistics and Tukey-Kramer model were used for adjustments. For all analyses, a two-sided P < 0.05 was deemed statistically significant. Results hNIS-specific mRNA is detected in Ad5/3-Δ24-hNIS–infected prostate cancer cells. A new virus containing the Ad3 knob in the Ad5 fiber shaft, a 24-bp deletion in the E1A region, and hNIS inserted in the E3 was constructed (Fig. 1). To assess the expression of the transgene from the virus, hormone-refractory prostate cancer cell lines PC-3MM2, 22Rv1, PC-3, and DU-145 Fig. 2. Detection of hNIS mRNA in Ad5/3-Δ24-hNIS–infected prostate cancer cells. PC-3MM2, 22Rv1, PC-3, and DU-145 prostate cancer cell lines were infected with 10 VP/cell of Ad5/3-Δ24-hNIS (lanes 2 and 3 ) or Ad5/3-Δ24-Δgp19K (lanes 4 and 5) and analyzed with reverse transcription-PCR 24 h (lanes 2 and 4) and 48 h (lanes 3 and 5) after infection. Noninfected cells were used as a negative control (lane 1) and β-actin was used for the normalization of the samples. 5398 www.aacrjournals.org Targeted Radioiodide Therapy for Prostate Cancer Fig. 3. Ad5/3-Δ24-hNIS–mediated iodide uptake in prostate cancer cells. Cells were infected with 10 VP/cell of Ad5/3-Δ24-hNIS or Ad5/3-Δ24-Δgp19K and exposed to 125I 24 and 48 h post-infection. Cells were analyzed with γ-spectrometer to determine the radioactive iodide content of the cells. Noninfected cells were used as a negative control. Bars, SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001. were infected with Ad5/3-Δ24-hNIS or Ad5/3-Δ24-Δgp19K (a control virus without a transgene) and analyzed with reverse transcription-PCR 24 and 48 h after infection (Fig. 2). All Ad5/3Δ24-hNIS–infected cell lines featured a 453-bp, hNIS-specific amplification product, suggesting that hNIS is expressed from the virus genome. In contrast, Ad5/3-Δ24-Δgp19K–infected cells or cells per se did not yield an amplification product. Ad5/3-Δ24-hNIS can mediate efficient iodide uptake into prostate cancer cells. To assess the functionality of the hNIS expressed from the virus, prostate cancer cells were infected with Ad5/3-Δ24-hNIS or Ad5/3-Δ24-Δgp19K followed by exposure to 125 I and quantification of iodide accumulation (Fig. 3). When compared to noninfected or control virus-treated cells, Ad5/3-Δ24-hNIS resulted in significantly higher iodide uptake in all examined cell lines. In comparison with mock infection, Ad5/3-Δ24-hNIS caused up to 2.5 (P < 0.001) and 3 (P < 0.001) times higher iodide accumulation in PC-3MM2 and 22Rv1 cells, respectively. In PC-3 and DU-145 cells, a more modest but still significantly enhanced accumulation was seen compared with mock-treated cells [up to 1.6 (P < 0.05) and 1.3 (P < 0.001) times higher levels, respectively]. Ad5/3-Δ24Δgp19K treatment did not result in iodide accumulation, suggesting that iodide uptake was mediated by hNIS expressed from Ad5/3-Δ24-hNIS rather than from viral infection or replication per se. Ad5/3-Δ24-hNIS replicates in and causes oncolysis of prostate cancer cells. Prostate cancer cell lines were infected with Ad5/3-Δ24-hNIS, Ad5/3-Δ24-Δgp19K, Ad5/3-Δ24, and Ad5/ Fig. 4. Ad5/3-Δ24-hNIS replication and oncolysis in prostate cancer cells without radioiodide. Cells were infected with various viral doses (0-100 VP/cell) and cell viability was determined 6 to 9 d later by MTS assay. A replication-deficient Ad5/3luc1 and oncolytic Ad5/3-Δ24 were used as controls. Bars, SE. www.aacrjournals.org 5399 Clin Cancer Res 2009;15(17) September 1, 2009 Cancer Therapy: Preclinical 3luc1 followed by determination of cell viability 6 days (PC3MM2), 7 days (PC-3 and DU-145), or 9 days (22Rv1) postinfection (Fig. 4). Ad5/3-Δ24-hNIS showed efficient cell killing in all examined prostate cancer cell lines. The most rapid and efficient oncolysis was seen in PC-3MM2 cells, where almost complete cell killing was achieved 6 days after infection using 1 VP/cell of Ad5/3-Δ24-hNIS. In addition, oncolysis was similar to that seen with Ad5/3-Δ24-Δgp19K. In other prostate cancer cell lines, the oncolytic potency of Ad5/3-Δ24-hNIS was slightly less than Ad5/3-Δ24-Δgp19K. In fact, Ad5/3-Δ24Δgp19K, which has the partial deletion in E3, showed the highest oncolytic potency in all examined cell lines. It is possible that the larger genome size of Ad5/3-Δ24-hNIS may affect the speed of oncolysis compared with Ad5/3-Δ24-Δgp19K. The hNIS transgene is ∼2.2 kbp, which together with the 1 kbp Δgp19K keeps the genome size <105%, which has been suggested important for retaining virus functionality (34, 35). The replicativity of Ad5/3-Δ24-hNIS was assessed in PC-3 and 22Rv1 cells where the TCID50 assay showed the increase in infectious particles in function of time (Supplementary Fig. S1). Ad5/3-Δ24-hNIS can mediate radioactive iodide uptake into prostate cancer tumors. Subcutaneous PC-3MM2 tumors were established in NMRI/nude male mice. The bottom tumors were infected intratumorally on 2 consecutive days with 7 × 108 VP/ tumor of Ad5/3-Δ24-hNIS and the top right tumor with the same dose of Ad5/3-Δ24-Δgp19k (Fig. 5A and B). The top left tumor was mock treated. A day after the last viral injection, 1.85 MBq 123I/mouse was administered intravenously and mice were imaged with a γ-camera from 0.5 to 13 h after 123I injection. Some iodide accumulation into Ad5/3-Δ24-hNIS–treated tumors was seen already 0.5 h after 123I injection (data not shown). Two hours after iodide exposure, strong iodide accumulation was seen throughout Ad5/3-Δ24-hNIS–treated tumors, whereas tumors injected with control virus or saline did not uptake iodide (Fig. 5A). After 2 h, iodide accumulation reached a plateau and remained relatively constant up to the last imaging time point of the experiment 13 h after iodide injection (Fig. 5B). In addition, iodide was heavily accumulated into thyroid and stomach due to endogenous NIS expression of these organs. High stomach uptake is typically seen in mice but not in humans (36). In patients, it would be possible to protect the normal thyroid from radioiodide, but this was not done in this mouse study. Because clearance of the radioactive iodide occurs through the urine, iodide accumulation in the bladder was seen. To quantify 123I biodistribution in vivo, organs and tumors were collected after imaging and their radioactive content was determined (Fig. 5C). Because of endogenous hNIS expression (36), the thyroid and stomach captured ∼30% of the injected dose. Nevertheless, Ad5/3-Δ24-hNIS–treated tumors accumulated >6% of the total iodide dose. This correlated with large amounts of infectious virus present in tumors, whereas only trace amounts were seen in the thyroid and stomach (Supplementary Fig. S2). Because tumors were collected >10 days after virus injection and TCID 50 measures infectious virus, high TCID50 values probably reflect virus replication. Adenoviruses lose their capsid when they enter cells; thus, input virus cannot be detected with TCID50; it requires production of new virions. Iodide uptake remained low in other organs including mock or Ad5/3-Δ24-Δgp19K–treated tumors (<1% of the initial dose). Ad5/3-Δ24-hNIS with 131I inhibits tumor growth in vivo. To assess the efficacy of Ad5/3-Δ24-hNIS in vivo, prostate tumor- Clin Cancer Res 2009;15(17) September 1, 2009 Fig. 5. Ad5/3-Δ24-hNIS–mediated iodide uptake in vivo. Mice bearing subcutaneous prostate tumors received Ad5/3-Δ24-hNIS intratumorally (bottom flank tumors), Ad5/3-Δ24-Δgp19K (top right tumor), or saline (top left tumor) followed by intravenous 123I injection (1.85 MBq/mouse). Iodide uptake was imaged (A) 2 h and (B) 13 h after intravenous 123I injection with γ-camera. Pixel size was 1.08 × 1.08 mm. C, biodistribution of 123 I 13 h after 123I injection. Bars, SE (n = 3, except n = 6 in hNIS tumors). bearing mice were treated with 131I or Ad5/3-Δ24-hNIS alone or with their combination (Fig. 6). When tumors were treated with Ad5/3-Δ24-hNIS without radioiodide, tumor growth was significantly slower than in the mock or 131I alone groups (P < 0.05 for both). When mice received both Ad5/3-Δ24-hNIS and 131 I, tumor sizes were significantly smaller than in any other group (all P < 0.001). Adjusted pairwise analysis indicated a significant difference between Ad5/3-Δ24-hNIS + 131I versus virus alone, 131I alone, or mock already on day 2 (all P < 0.05). The adjusted pairwise P values between Ad5/3-Δ24-hNIS + 131I versus virus alone, 131I alone, or mock on days 4, 6, and 8 were also all <0.001. Due to animal husbandry constraints, it was predefined that the experiment would be ended on day 17, although some of the mice were still in good condition. It is possible that even more benefit in the combination group might have been evident with a longer follow-up. Whole-body emitted radioactivity was measured starting from 1 day after iodide injection. Radioactivity declined rapidly during the next 3 days as expected by clearance through kidneys (data not shown). When tumor size reached 15 mm in any diameter, mice were killed and organs were collected for radioactivity measurements (Supplementary Fig. S3). Radioactivity declined over time and 5400 www.aacrjournals.org Targeted Radioiodide Therapy for Prostate Cancer most radioactivity was seen in the thyroid gland and stomach and briefly in the heart probably due to propagation of 131I by circulation. In other organs, the radioactivity remained low. Importantly, virus injection did not seem to affect iodide biodistribution; therefore, the safety profile of the approach might resemble that of radioiodide therapy for thyroid cancer. Discussion Radioactive isotopes of iodine were first used for studying thyroid function and later for treatment of hyperthyroidism and other nonmalignant thyroid diseases. As hNIS is present on malignant thyroid cell membranes to variable degree (37), the use of radioiodine was extended to the treatment of thyroid cancer (38). Recently, hNIS has been used as a transgene for concentrating radioiodide to cancers of nonthyroid origin in preclinical studies (19–22, 39–43). We sought to use the established synergy between oncolytic adenoviruses and radiation (17) in the context of radioiodide given systemically but targeted to tumors by hNIS. In this approach, tumor cells are killed due to replication of the virus and by the radiation emitted by the radioiodide, and additional benefit may result from the synergy of the approaches (10, 17) and the direct bystander effect of degrading 131I β-particles that can kill untransduced neighboring tumor cells (44). Also, side effects of the treatments are nonoverlapping, which might facilitate increasing efficacy without increasing toxicity (45). Recurrent or metastatic androgen-insensitive prostate cancer requires new treatment approaches. One developmental concept is combination of radiation with oncolytic viruses (45, 46). In the context of local disease, this can be achieved with local virus injections in combination with external beam radiation or brachytherapy. Brachytherapy alone is a powerful treatment option for conformal dose delivery when the tumor size and location allow for injection of radioactive seeds. Low-energy brachytherapy ( 125 I or 103 Pd seeds) delivers a large therapeutic dose to the prostate with little dose to other organs. However, brachytherapy is usually only practical when the tumor is restricted to the prostate. Therefore, additional approaches for targeting radiotherapy and realizing the combination of oncolytic virotherapy with radiation would be useful. When combining direct adenovirus injection with systemic administration of 131I, there is a risk for initial radiation exposure of organs not affected by the disease. However, our data suggest that the tumor specificity of the oncolytic virus results in radiation accumulating in the tumor, whereas excretion of the iodide reduces the burden of nontarget tissue. The systemic application 131I should not be a problem because the administration of radioactive iodide is already widely used in the treatment of thyroid cancer. Moreover, our animal model showed <1% uptake of the initial dose in most organs, except for thyroid and stomach, which would be the critical organs for monitoring toxicity. Oncolytic adenoviruses are not the only type of oncolytic viruses that have been used to express hNIS. Measles virus (47, 48) was used against ovarian cancer and myeloma in preclinical studies that preceded an ongoing phase I trial where virally expressed hNIS is used for imaging the viral propagation. Vesicular stomatitis virus has been coupled to 131I therapy with promising results (40). In contrast, oncolytic adenoviruses have not been previously used for targeting radioiodides with a therapeutic purpose. However, preclinical and clinical studies have featured hNIS armed oncolytic adenoviruses for imaging purposes (24–26). Although other viruses have their merits, oncolytic adenoviruses are appealing because they have been promising in prostate cancer (9, 11, 46) and other tumor types (7, 50, 51) in clinical trials. The safety of oncolytic adenoviruses in cancer patients has been excellent and there is also some evidence of activity, although, in general, efficacy needs improvement (8). Nevertheless, the large body of clinical data available on adenovirus in cancer treatment facilitates clinical translation of the approach described here. We showed that oncolytic adenoviruses can effectively express hNIS as a transgene in prostate cancer cells. Also, we found that it was possible to use Ad5/3-Δ24-hNIS for concentrating sufficient amounts of radioiodide into nonthyroid cells, which has been of concern previously. Finally, viral oncolysis Fig. 6. Ad5/3-Δ24-hNIS antitumor efficacy in vivo. Subcutaneous prostate tumor-bearing mice (6 mice per group, 12 tumors per group) received Ad5/3-Δ24-hNIS or growth medium intratumorally on 2 consecutive days followed by a 50 MBq intraperitoneal injection of 131I or saline. Tumor size was measured every other day. Tumor growth rate was significantly slower in the combination-treated group (Ad5/3-Δ24-hNIS + 131I) when compared with all the other groups. ***, P < 0.001. The experiment had to be ended at a predetermined time point of 17 d after iodide injection due to animal husbandry constraints. www.aacrjournals.org 5401 Clin Cancer Res 2009;15(17) September 1, 2009 Cancer Therapy: Preclinical together with radioiodide accumulation resulted in better antitumor activity in vivo than either treatment alone. Importantly, the biodistribution of radioiodide was not changed by virus injection. This suggests that the transgene was expressed selectively in tumor cells, as expected when placed under the internal adenoviral E3 promoter, which is strongly activated during virus replication. Because Ad5/3-Δ24-hNIS is a tumor-selective virus, replication and subsequent hNIS expression is only expected in tumor cells (6, 28–30). The natural existence of hNIS on most thyroid cancer cells has permitted the use of radioactive iodide for systemically administered local radiotherapy, a good example of which is oral radioiodide treatment in humans. Together with data from other groups, our results suggest that, using virus-mediated hNIS expression, the approach can be extended to treatment of cancers of nonthyroid origin. Further, our results suggest useful combination effects between radioiodide targeting and adenoviral oncolysis. However, further short-term and long-term studies are needed to evaluate the safety and efficacy of the approach. Ad5/3-Δ24-hNIS may be usable in many tumor types, as its selectivity is determined by p16-Rb pathway defects, which are found ubiquitously in solid tumors. In summary, infectivity enhanced oncolytic adenoviruses armed with hNIS seem an attractive platform for improving the treatment of cancers refractory to available treatments. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. References 1. Bubendorf L, Schopfer A, Wagner U, et al. Metastatic patterns of prostate cancer: an autopsy study of 1,589 patients. Hum Pathol 2000;31: 578–83. 2. Alemany R, Balague C, Curiel DT. Replicative adenoviruses for cancer therapy. Nat Biotechnol 2000;18:723–7. 3. Rajecki M, Kanerva A, Stenman UH, et al. Treatment of prostate cancer with Ad5/3Δ24hCG allows non-invasive detection of the magnitude and persistence of virus replication in vivo. Mol Cancer Ther 2007;6:742–51. 4. Bauerschmitz GJ, Kanerva A, Wang M, et al. Evaluation of a selectively oncolytic adenovirus for local and systemic treatment of cervical cancer. Int J Cancer 2004;111:303–9. 5. Ranki T, Sarkioja M, Hakkarainen T, von Smitten K, Kanerva A, Hemminki A. Systemic efficacy of oncolytic adenoviruses in imagable orthotopic models of hormone refractory metastatic breast cancer. Int J Cancer 2007;121:165–74. 6. Kanerva A, Zinn KR, Chaudhuri TR, et al. Enhanced therapeutic efficacy for ovarian cancer with a serotype 3 receptor-targeted oncolytic adenovirus. Mol Ther 2003;8:449–58. 7. Khuri FR, Nemunaitis J, Ganly I, et al. A controlled trial of intratumoral ONYX-015, a selectively-replicating adenovirus, in combination with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer. Nat Med 2000;6: 879–85. 8. Yu W, Fang H. Clinical trials with oncolytic adenovirus in China. Curr Cancer Drug Targets 2007; 7:141–8. 9. Small EJ, Carducci MA, Burke JM, et al. A phase I trial of intravenous CG7870, a replication-selective, prostate-specific antigen-targeted oncolytic adenovirus, for the treatment of hormone-refractory, metastatic prostate cancer. Mol Ther 2006; 14:107–17. 10. Chen Y, DeWeese T, Dilley J, et al. CV706, a prostate cancer-specific adenovirus variant, in combination with radiotherapy produces synergistic antitumor efficacy without increasing toxicity. Cancer Res 2001;61:5453–60. 11. DeWeese TL, van der Poel H, Li S, et al. A phase I trial of CV706, a replication-competent, PSA selective oncolytic adenovirus, for the treatment of locally recurrent prostate cancer following radiation therapy. Cancer Res 2001;61: 7464–72. 12. Kanerva A, Mikheeva GV, Krasnykh V, et al. Targeting adenovirus to the serotype 3 receptor increases gene transfer efficiency to ovarian cancer cells. Clin Cancer Res 2002;8:275–80. 13. Kangasniemi L, Kiviluoto T, Kanerva A, et al. Infectivity-enhanced adenoviruses deliver efficacy in clinical samples and orthotopic models of disseminated gastric cancer. Clin Cancer Res 2006;12:3137–44. 14. Guse K, Ranki T, Ala-Opas M, et al. Treatment of metastatic renal cancer with capsid-modified oncolytic adenoviruses. Mol Cancer Ther 2007;6: 2728–36. 15. Lamfers ML, Idema S, Bosscher L, et al. Differential effects of combined Ad5-Δ24RGD and radiation therapy in in vitro versus in vivo models of malignant glioma. Clin Cancer Res 2007;13: 7451–8. 16. Lamfers ML, Grill J, Dirven CM, et al. Potential of the conditionally replicative adenovirus Ad524RGD in the treatment of malignant gliomas and its enhanced effect with radiotherapy. Cancer Res 2002;62:5736–42. 17. Rajecki M, Kiviharju-af Hällström TM, Hakkarainen T, et al. The combination of oncolytic adenoviru‐ ses and ionizing radiation inhibits DNA double strand break detection which results in autophagocytotic prostate cancer cell death. ASGT; Boston: Mol Ther. 2008, p. S18. 18. Dohan O, De la Vieja A, Paroder V, et al. The sodium/iodide symporter (NIS): characterization, regulation, and medical significance. Endocr Rev 2003;24:48–77. 19. Haberkorn U. Gene therapy with sodium/iodide symporter in hepatocarcinoma. Exp Clin Endocrinol Diabetes 2001;109:60–2. 20. Montiel-Equihua CA, Martin-Duque P, de la Vieja A, et al. Targeting sodium/iodide symporter gene expression for estrogen-regulated imaging and therapy in breast cancer. Cancer Gene Ther 2008;465–73. 21. Spitzweg C, Dietz AB, O'Connor MK, et al. In vivo sodium iodide symporter gene therapy of prostate cancer. Gene Ther 2001;8:1524–31. 22. Kim S, Chung HK, Kang JH, et al. Tumor targeted radionuclide imaging and therapy using human sodium iodide symporter gene driven by a modified telomerase reverse transcriptase promoter. Hum Gene Ther 2008;951–7. 23. Hingorani M, White CL, Zaidi S, et al. Radiation-mediated up-regulation of gene expression from replication-defective adenoviral vectors: implications for sodium iodide symporter gene therapy. Clin Cancer Res 2008;14:4915–24. 24. Niu G, Anderson RD, Madsen MT, Graham MM, Oberley LW, Domann FE. Dual-expressing adenoviral vectors encoding the sodium iodide symporter for use in noninvasive radiological imaging of therapeutic gene transfer. Nucl Med Biol 2006;33:391–8. 25. Barton KN, Stricker H, Brown SL, et al. Phase I study of noninvasive imaging of adenovirusmediated gene expression in the human prostate. Mol Ther 2008;16:1761–9. 26. Merron A, Peerlinck I, Martin-Duque P, et al. Clin Cancer Res 2009;15(17) September 1, 2009 5402 SPECT/CT imaging of oncolytic adenovirus propagation in tumours in vivo using the Na/I symporter as a reporter gene. Gene Ther 2007; 14:1731–8. 27. Kanerva A, Zinn KR, Peng KW, et al. Noninvasive dual modality in vivo monitoring of the persistence and potency of a tumor targeted conditionally replicating adenovirus. Gene Ther 2005;12:87–94. 28. Fueyo J, Gomez-Manzano C, Alemany R, et al. A mutant oncolytic adenovirus targeting the Rb pathway produces anti-glioma effect in vivo. Oncogene 2000;19:2–12. 29. Heise C, Hermiston T, Johnson L, et al. An adenovirus E1A mutant that demonstrates potent and selective systemic anti-tumoral efficacy. Nat Med 2000;6:1134–9. 30. Bauerschmitz GJ, Lam JT, Kanerva A, et al. Treatment of ovarian cancer with a tropism modified oncolytic adenovirus. Cancer Res 2002;62:1266–70. 31. Sherr CJ. Cancer cell cycles. Science 1996;274: 1672–7. 32. Phillips SM, Morton DG, Lee SJ, Wallace DM, Neoptolemos JP. Loss of heterozygosity of the retinoblastoma and adenomatous polyposis susceptibility gene loci and in chromosomes 10p, 10q and 16q in human prostate cancer. Br J Urol 1994;73:390–5. 33. Suzuki K, Alemany R, Yamamoto M, Curiel DT. The presence of the adenovirus E3 region improves the oncolytic potency of conditionally replicative adenoviruses. Clin Cancer Res 2002; 8:3348–59. 34. Raki M, Hakkarainen T, Bauerschmitz GJ, et al. Utility of TK/GCV in the context of highly effective oncolysis mediated by a serotype 3 receptor targeted oncolytic adenovirus. Gene Ther 2007; 14:1380–8. 35. Hawkins LK, Johnson L, Bauzon M, et al. Gene delivery from the E3 region of replicating human adenovirus: evaluation of the 6.7 K/gp19 K region. Gene Ther 2001;8:1123–31. 36. Zuckier LS, Dohan O, Li Y, Chang CJ, Carrasco N, Dadachova E. Kinetics of perrhenate uptake and comparative biodistribution of perrhenate, pertechnetate, and iodide by NaI symporterexpressing tissues in vivo. J Nucl Med 2004;45: 500–7. 37. Smanik PA, Ryu KY, Theil KS, Mazzaferri EL, Jhiang SM. Expression, exon-intron organization, and chromosome mapping of the human sodium iodide symporter. Endocrinology 1997; 138:3555–8. 38. Mazzaferri EL, Kloos RT. Clinical review 128: current approaches to primary therapy for papillary and follicular thyroid cancer. J Clin Endocrinol Metab 2001;86:1447–63. www.aacrjournals.org Targeted Radioiodide Therapy for Prostate Cancer 39. Boland A, Ricard M, Opolon P, et al. Adenovirus-mediated transfer of the thyroid sodium/ iodide symporter gene into tumors for a targeted radiotherapy. Cancer Res 2000;60:3484–92. 40. Goel A, Carlson SK, Classic KL, et al. Radioiodide imaging and radiovirotherapy of multiple myeloma using VSV(Δ51)-NIS, an attenuated vesicular stomatitis virus encoding the sodium iodide symporter gene. Blood 2007;110:2342–50. 41. Park SY, Kwak W, Tapha N, et al. Combination therapy and noninvasive imaging with a dual therapeutic vector expressing MDR1 short hairpin RNA and a sodium iodide symporter. J Nucl Med 2008;1480–8. 42. Kim HJ, Jeon YH, Kang JH, et al. In vivo longterm imaging and radioiodine therapy by sodiumiodide symporter gene expression using a lentiviral system containing ubiquitin C promoter. Cancer Biol Ther 2007;6:1130–5. 43. Dingli D, Diaz RM, Bergert ER, O'Connor MK, Morris JC, Russell SJ. Genetically targeted www.aacrjournals.org radiotherapy for multiple myeloma. Blood 2003;102:489–96. 44. Kassis AI, Adelstein SJ. Radiobiologic principles in radionuclide therapy. J Nucl Med 2005; 46 Suppl 1:4–12S. 45. Freytag SO, Movsas B, Aref I, et al. Phase I trial of replication-competent adenovirus-mediated suicide gene therapy combined with IMRT for prostate cancer. Mol Ther 2007;15:1016–23. 46. Freytag SO, Stricker H, Pegg J, et al. Phase I study of replication-competent adenovirusmediated double-suicide gene therapy in combination with conventional-dose three-dimensional conformal radiation therapy for the treatment of newly diagnosed, intermediate- to high-risk prostate cancer. Cancer Res 2003;63:7497–506. 47. Myers RM, Greiner SM, Harvey ME, et al. Preclinical pharmacology and toxicology of intravenous MV-NIS, an oncolytic measles virus administered with or without cyclophosphamide. Clin Pharmacol Ther 2007;82:700–10. 5403 48. Hasegawa K, Pham L, O'Connor MK, Federspiel MJ, Russell SJ, Peng KW. Dual therapy of ovarian cancer using measles viruses expressing carcinoembryonic antigen and sodium iodide symporter. Clin Cancer Res 2006;12:1868–75. 49. Barton KN, Stricker H, Brown SL, et al. Phase I study of noninvasive imaging of adenovirusmediated gene expression in the human prostate. Mol Ther 2008;16:1761–9. 50. Xia ZJ, Chang JH, Zhang L, et al. Phase III randomized clinical trial of intratumoral injection of E1B gene-deleted adenovirus (H101) combined with cisplatin-based chemotherapy in treating squamous cell cancer of head and neck or esophagus. Ai Zheng 2004;23:1666–70. 51. Lu W, Zheng S, Li XF, Huang JJ, Zheng X, Li Z. Intra-tumor injection of H101, a recombinant adenovirus, in combination with chemotherapy in patients with advanced cancers: a pilot phase II clinical trial. World J Gastroenterol 2004;10:3634–8. Clin Cancer Res 2009;15(17) September 1, 2009