Download Targeted Radiotherapy for Prostate Cancer with an Oncolytic

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
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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
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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.
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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.
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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.
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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-
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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
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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.
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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.
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