Affiliations of authors: D. R. Siemens, J. C. Austin, S. P. Hedican, Department of Urology, The University of Iowa, Iowa City; J. Tartaglia, Virogenetics Corporation, Troy, NY; T. L. Ratliff, Department of Urology, The University of Iowa Cancer Center and The University of Iowa Prostate Cancer Research Group, Iowa City.
Correspondence to: Timothy L. Ratliff, Ph.D., Department of Urology, The University of Iowa, 200 Hawkins Dr., 3 RCP, Iowa City, IA 52242-1089 (e-mail: tim-ratliff{at}uiowa.edu).
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ABSTRACT |
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INTRODUCTION |
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We have previously reported one approach to cancer immunotherapy involving the transfer
of genes encoding the cytokines interleukin 2 (IL-2) and tumor necrosis factor-
(TNF-
) utilizing the canarypox viral vector ALVAC (1). The
ALVAC virus was shown to efficiently infect murine prostate cancer cells, RM-1, and to produce
high levels of extrinsic gene product. In addition, antitumor immunity was induced when tumor
cells were infected by ALVAC cytokine recombinants and injected subcutaneously in the flanks
of male C57BL/6 mice. The ALVAC virus is particularly well suited for the direct injection of
tumors because the inability of the canarypox virus vectors to replicate in human cells greatly
reduces the risk of systemic adverse events. The absence of replication also means that the virus
will not propagate through the tumor mass; therefore, the expression of the delivered gene
products is restricted to the sites of delivery. Our preliminary studies have shown a restricted
pattern of expression after direct injection of fluid-phase ALVAC into the prostate or a tumor at a
subcutaneous site. Moreover, any clinical gene therapy protocol for prostate cancer involving the
intraprostatic delivery of therapeutic genes will be hampered by the multifocal and often cryptic
nature of neoplastic and preneoplastic lesions in the prostate. Thus, methods to enhance the
distribution and expression of recombinant genes are needed.
Numerous substances have been used as carriers to enhance and sustain the delivery of
soluble products to both neoplastic and non-neoplastic tissue (2,3). In
these studies, a gelatin sponge matrix was determined to be the most efficient in vitro
and was shown to enhance delivery and, hence, reporter gene expression when injected
intratumorally. The previously described tumor suppression in this model by recombinant virus
encoding genes for IL-2, interleukin 12 (IL-12), and TNF- was also markedly improved
when the vector was delivered by the gelatin matrix as compared with fluid-phase injection.
In this study, we tested the ability of different matrices to act as a carrier vehicle for the canarypox virus (ALVAC) to improve the delivery of the vector in a heterotopic murine prostate cancer model.
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MATERIALS AND METHODS |
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The murine prostate cancer cell line RM-1 used for these studies mimics multistep carcinogenesis by activating the ras and myc oncogenes and is used to induce an aggressive prostate carcinoma in vivo. This cell line retains many features of prostate cancer, including androgen responsiveness early in culture, expression of androgen receptor, and progression to androgen independence with time (4). MB-49, a chemically induced mouse bladder tumor, was used in concert with RM-1 throughout the in vitro and in vivo gene expression experiments. Both RM-1 and MB-49 are syngeneic to C57BL/6 mice. The myc- and ras-transformed BALB/c RM-11 prostate cancer cell line was used for complementary in vivo tumor outgrowth studies. Cultured cells were maintained in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS). Mice (6-8 weeks old at the time of study initiation) were obtained through the National Cancer Institute, Bethesda, MD, and were allowed free access to food and water. All animal studies were approved by the Animal Review Board of the University of Iowa and were performed in accordance with institutional guidelines.
Gene Transfer Vectors
ALVAC is a canarypox virus that can infect mammalian cells but is restricted to avian
species for replication (5). It has been shown to be a safe and effective
vector in both humans and animals (6,7). The viral strain from which
ALVAC was obtained was isolated from a pox lesion on an infected canary. Parental ALVAC,
ALVAC vectors encoding murine IL-2, murine IL-12 and murine TNF-, as well as the
reporter gene constructs ß-galactosidase (ALVAC-lacZ), green fluorescent protein
(ALVAC-GFP), and luciferase (ALVAC-luciferase) were developed at Virogenetics Corporation
(Troy, NY).
Delivery Systems
Four solid-state delivery matrices were compared with fluid-phase delivery of viral vector to cells in vitro and in vivo: polyglycolic acid, chromic catgut, catgut spacer, and gelatin sponge. These substances were chosen because of their absorbable nature and relatively low tissue reaction. Polyglycolic acid (Davis and Geck, Inc., Wayne, NJ) and chromic catgut (Ethicon, Inc., Somerville, NJ) are both absorbable suture materials. Plain catgut spacer material (MDTech, Gainesville, FL) is a commercially available product used for prostate cancer brachytherapy protocols. An absorbable gelatin sponge (Gelfoam; Pharmacia and Upjohn, Kalamazoo, MI) is prepared from purified pork skin gelatin granules and is used as a hemostatic agent. All delivery systems were prepared in 6-mm lengths and were tested in vitro and in vivo through an 18-gauge B-D spinal needle (Becton Dickinson and Co., Franklin, NJ) to better mimic intraprostatic injection in a clinical trial.
As previously described for the carrier delivery for insulin (2), the virion concentration to be delivered by an individual matrix was determined by weighing the matrices before and after viral absorption. The dry and wet weights of the matrices after 1 minute of viral absorption were recorded, and the subsequent volume delivered was calculated. Subsequently, to ensure that the gelatin sponge matrix was reliably delivering this weight-calculated number of viral particles, comparisons were made with the use of particle determination by measurement of the optical density (OD) at 260 nm after digestion of the gelatin matrix. A calculated particle concentration was delivered into solution by the gelatin sponge matrix and then digested by a combination of collagenase (0.16%), bovine serum albumin (2.5%), and deoxyribonuclease (0.001%) in phosphate-buffered saline (PBS) (pH 7.2); these three reagents were obtained from Sigma Chemical Co., St. Louis, MO.
Infection and Reporter Gene Assays
For in vitro analysis, RM-1 or MB-49 cells were harvested from tissue culture plates and replated with DMEM containing 10% FCS and 10 mM HEPES buffer (pH 7.0) on the day before infection. The medium was changed to DMEM with 2% FCS at the time of viral infection with either ALVAC-luciferase or ALVAC-lacZ delivered directly into the culture or via the delivery matrices. The viral vectors were added to the cells at the multiplicity of infection (MOI)-plaque-forming units (pfu) per cell shown in each experiment. The cells were then incubated for 6 hours at 37 °C in an atmosphere of 5% CO2 when the medium was changed back to DMEM with 10% FCS. Reporter assays were performed 48 hours after virus addition (unless otherwise stated). All in vitro experiments were performed in triplicate and repeated in at least two independent experiments.
For the in vivo gene expression studies, RM-1 or MB-49 cells were harvested from the tissue culture plates by treatment with 10 mM EDTA and were washed with PBS. The cells were then resuspended in DMEM in a concentration of 5 x 106 pfu/mL, and 0.1 mL was injected subcutaneously into the backs of mice. The ALVAC vectors recombinant for luciferase or ß-galactosidase were injected either directly (fluid phase) or via the delivery systems at a concentration of 3 x 106 pfu/mL approximately 10 days after tumor implantation. Tumors at that time were approximately 8 mm by 8 mm (approximately 200 mg wet weight). Tumors were harvested for reporter gene assays at various times after infection as described for individual experiments. Experiments were performed at least twice for the RM-1 and the MB-49 tumors in vivo.
We determined ß-galactosidase transgene expression in vitro after briefly fixing the cells with 0.5% glutaraldehyde for 10 minutes, washing them with PBS, then incubating them in X-gal (5-bromo-4-chloro-indolyl ß-D-galactopyranoside) at 37 °C for at least 4 hours. ß-Galactosidase cleaves this substrate into an indigo compound, such that cells producing the ß-galactosidase gene product are stained blue. We quantified ß-galactosidase expression by visualizing a representative area of the culture plate under high power (x40) and recording the percentage of blue cells. In vivo, after the tumor was harvested and weighed, sections were incubated in the X-gal solution containing the detergents sodium deoxycholate (0.01%) and Nonidet P-40 (0.02%). Representative tissue sections were then scored for percentage of blue-stained cells. Tumors infected with the parental ALVAC (not recombinant for the lacZ gene) were used as negative controls.
The luciferase assay from cell lysates was performed with the use of a commercial luciferase assay kit (Promega Corp., Madison, WI) following the manufacturer's recommendations. The Monolight 2010 Luminometer (Analytical Luminescence Laboratory, Ann Arbor, MI) was used for the luciferase assay. Internal controls were performed by reading background luminescence for each assay as well as the periodic evaluation of the variation between replicates. Luciferase assays of infected subcutaneous tumors were performed after each tumor was homogenized with the Tissue Tearor (Biospeck Products, Inc., Fisher Scientific, Itasca, IL) in 0.5 mL of cell lysis buffer.
Tumor Inhibition Studies
RM-1 (5 x 105) or RM-11 (1 x 105) cells were
injected subcutaneously in the backs of mice in a volume of 0.1 mL as described above.
Approximately 10 days after tumor implantation, a total of 8.4 x 106 pfu of
recombinant ALVAC vector was injected intratumorally. The ALVAC vectors used were the
IL-2, IL-12, and TNF- constructs in equal concentrations (2.8 x 106
pfu each). The vectors absorbed by the gelatin sponge matrix were injected in an 18-gauge
needle and were compared with three separate 33-µL injections of the fluid-phase product
(8.4 x 106 total pfu). Other controls included parental ALVAC absorbed by
the gelatin sponge matrix, matrix only, and a no treatment group.
Tumor outgrowth, determined by tumor size as a function of time, was measured approximately three times a week. Survival of the tumor-bearing mice was also determined. Mice were killed for humane reasons if a single tumor was greater than 25 mm in any dimension or if the mice appeared to be ill from the tumor burden. Each experimental group contained four to six mice, and experiments were repeated at least twice.
Statistical Evaluation
Differences were analyzed by the Mann-Whitney rank sum test, including the nonparametric data for ß-galactosidase and luciferase reporter assays. The gene expression data are recorded as the means and the 95% confidence intervals (CIs). The rank sum test (Mann-Whitney) was also used to compare average tumor volume between matrix-delivered and fluid-phase-delivered treatment groups at individual time points. These data were also presented in the figures as the means and the 95% CIs. A one-way analysis of variance of log-transformed data was also used to compare all control groups with the gelatin sponge matrix-delivered treatment group at individual time points for the tumor outgrowth studies. Survival data were analyzed for significance with the use of the Cox proportional hazards regression model. For all statistical analyses, we used a computer software program, SAS (SAS Institute, Inc., Cary, NC) (8), or Statistix (Analytical Software, Seattle, WA; Version 1.0, 1996). All reported P values are two-sided, and statistical significance was determined as a P value of less than .05.
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RESULTS |
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The calculated volume delivered by the individual matrices was determined by the wet weight after absorption of the virus in the 18-guage delivery needle. The mean wet weight and dry weight of at least five samples were assessed for each delivery matrix, and the results of three separate determinations are presented in Table 1. The known volumes absorbed by the individual matrices allowed comparisons to fluid-phase delivery both in vitro and in vivo by calculation of the number of viral particles or pfu. Thus, pfu delivery via either matrix or fluid was equivalent, given the absorbable capacity of each matrix. Equivalency was verified in the gelatin sponge matrix system as determined by the particle count read as OD at 260 nm (data not shown).
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To examine the ability of each matrix to deliver the calculated virion concentration in cell culture, we determined ß-galactosidase and the firefly luciferase expression of cells 48 hours after infection with ALVAC-lacZ or ALVAC-luciferase vectors. The virus delivered by the matrices was compared with the addition of a known MOI (pfu) of fluid-phase virus in cell culture. As shown in Fig. 1, A, no significant differences were found with the percentage of RM-1 cells infected by the ß-galactosidase vector (MOI 10 : 1) in the gelatin sponge matrix-delivered group (mean = 48%; 95% CI = 41%-55%) versus the fluid-phase group (mean = 49%; 95% CI = 44%-55%). It is interesting that there was a trend to less ß-galactosidase expression for the other delivery systems, especially for the chromic catgut. These results were consistent over a wide range of MOI (10 : 1 to 200 : 1) for the RM-1 cell line, as well as for experiments using the MB-49 cell line.
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Transgene Expression in a Heterotopic Tumor Model
To determine the ability of these carrier systems to deliver the viral vectors in vivo, we first injected ALVAC-luciferase (3 x 106 pfu) into established subcutaneous RM-1, either in fluid-phase or via the various delivery systems. The chromic system was not tested, given its consistently poor transfer of virus to cells in culture. Forty-eight hours after infection, the tumors were harvested and the luciferase assay was performed. The gelatin sponge matrix delivery consistently resulted in significantly (P = .037, Mann-Whitney rank sum test) enhanced gene expression (mean = 34 226 RLU; 95% CI = 14 673-52 012 RLU) over fluid-phase delivery (mean = 2961 RLU; 95% CI = 29-7707 RLU) (Fig. 2, A). The other delivery systems (polyglycolic acid and catgut spacer) did not consistently enhance gene expression in injected tumor nodules. Similar experiments in the heterotopic MB-49 tumor model confirmed the ability of the gelatin sponge matrix to significantly improve (P = .03, Mann-Whitney rank sum test) the delivery of viral vectors compared with direct injection of the fluid-phase product. Fig. 2, B, shows the improved luciferase expression with gelatin sponge delivery in the RM-1 tumor model at different doses (pfu) of the ALVAC-luciferase vector (note log scale for RLU in Fig. 2, B).
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Tumor Outgrowth Studies
To determine if the enhanced gene expression found with the matrix
delivery of vectors translated into improved biologic effect, we
treated established subcutaneous RM-1 tumors (mean tumor volume = 110
mm3) with ALVAC vector expressing IL-2, IL-12, and TNF-
delivered in the gelatin sponge matrix or via fluid-phase injection.
Control groups included 1) parental ALVAC delivered via the matrix, 2)
a matrix only, and 3) a group with no treatment. Statistically
significant (P values for all comparisons <.05,
Mann-Whitney rank sum test) tumor inhibition, as determined by tumor
volume over time, was seen in the treatment group only when delivered
by the matrix (Fig. 5, A). This tumor inhibition was
greatest within the first 6 or 7 days after infection with the
recombinant virus, although the inhibitory effects remained significant
through 13 days. Further comparison between groups was not possible
because many control mice were killed after day 13. Several tumors
(three of five) in the treatment group delivered by the gelatin matrix
demonstrated substantial inhibition of growth for a period of 10 days
after gene transfer, although all tumors did eventually grow out. Tumor
volumes at days 4-13 in the gelatin sponge matrix-delivered group were
statistically significantly smaller (P values for all
comparisons <.05, Mann-Whitney rank sum test) than tumors in the
fluid-phase injection group, as well as those in the control groups.
Similarly, a statistically significant (P<.005; Cox
proportional hazards regression model) increase in survival was seen
for mice treated with the recombinant virus delivered by gelatin matrix
as compared with those mice treated with the fluid-phase injection
(Fig. 5, B). The differences observed between matrix and fluid-phase
delivery have been confirmed in four separate tumor outgrowth experiments.
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DISCUSSION |
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Several reports investigating prostate cancer immunotherapy have been encouraging. Early
studies by Sanda et al. (12) showed that granulocyte-macrophage
colony-stimulating factor (GM-CSF)-transfected rat prostatic adenocarcinomas grew more
slowly than parental tumors. Subsequently, Vieweg et al. (13) showed
that IL-2-transfected rat R3327-MatLyLu prostate cancer cells also induced antitumor activity.
Existing tumors had a decreased rate of outgrowth, and protection was gained against subsequent
tumor challenge. Using the parental R3327G tumor that exhibits hormone responsiveness,
Yoshimura et al. (14) also observed antitumor activity; however, neither
cytotoxic T-lymphocyte activity nor protection against subsequent tumor challenge was
observed. In a previous study (1), we have shown that the ALVAC virus
can efficiently infect prostate cancer cells, produce high levels of extrinsic gene product, and
induce antitumor immunity. RM-1 tumor cells were infected by ALVAC cytokine recombinants
and injected subcutaneously in the flanks of male C57BL/6 mice. As single agents,
ALVAC-IL-2, ALVAC-IL-12, ALVAC-GM-CSF, and ALVAC-TNF- were effective in
partially inhibiting tumor outgrowth. As a combination therapy of ALVAC-TNF-
with
ALVAC-IL-2, ALVAC-IL-12, or ALVAC-GM-CSF, the tumor outgrowth inhibition was
optimized. Subsequent studies assessing the ability of the ALVAC cytokine vectors to induce
regression of existing tumors showed only limited effects (data not shown). Although this system
seems perfectly suited for intratumoral treatment of established tumors, given the inability of the
ALVAC vector to replicate in mammalian species, we hypothesized that the fluid-phase injection
of the viral vector resulted in limited expression and/or distribution of the gene product, as has
been reported in other models (15).
Although numerous clinical gene therapy trials for prostate cancer have been initiated and many investigators have demonstrated great promise for both tumoricidal and corrective gene therapies, improving delivery and enhancing gene expression are imperative. Most preclinical investigations and ongoing phase I clinical protocols for localized prostate cancer rely on direct intraprostatic injection for the delivery of viral vectors (16,17). However, the relative inaccessibility of the prostate and the often isoechoic nature of cancer foci on transrectal ultrasound make detection and localization difficult. Moreover, examination of autopsy and radical prostatectomy specimens has revealed multiple and separate tumor sites, suggesting the possibility of a field change in the prostate (18,19) and necessitating even more efficient and accurate distribution of any therapeutic vector.
Unfortunately, the efficient delivery of genetic material to tumors remains a formidable task for all solid-tumor oncology. Asgari et al. (20) have found significant inhibition of preestablished subcutaneous tumor nodules of human prostate cancer by a single injection of the recombinant adenovirus p53 vector. It is interesting that they were unable to detect p53 overexpression in these adenovirus wild-type p53-infected tumors 48 hours after intratumoral injection. Moreover, multiple injections (1 week apart) did not improve the antitumor effect. These results most likely reflect the poor efficiency of transfer when direct intratumoral injections are employed. These difficulties with vector delivery are not isolated to the prostate. The effective use of gene therapy in glial tumors of the brain is hampered by the inefficient delivery of viral vectors. High titers of adenovirus are required for gene transfer to gliomas (15), and the resulting inflammation of the high inoculum decreases the efficiency of the gene transfer. To improve delivery, Beer et al. (21) found sustained release of recombinant adenovirus when coupled to biodegradable microspheres, allowing the administration of lower doses of viral vectors to glioma tissue.
Numerous methods have been investigated to better deliver soluble products to both neoplastic and non-neoplastic cells. A polymer-based paste has been found to enhance local delivery of chemotherapeutic agents and decrease recurrence rates at tumor resection sites (3). A fibrin- and gelatin-based drug-delivery system has been shown to more slowly release and to improve the therapeutic effect of antibiotics (22). Poloxamer 407 has been shown to improve the delivery of adenoviral vectors in vascular smooth muscle based on ß-gal reporter gene expression (23). The absorbable gelatin sponge employed in this study is primarily used as an intraoperative hemostatic agent, but it has also been used to deliver a number of different compounds, including insulin (2) and various cytokines and growth factors (24,25), in order to improve and sustain delivery.
In our study, the ability of a known quantity of recombinant ALVAC absorbed by the gelatin sponge matrix to infect tumor cells in vitro was similar to that seen with infusion of fluid-phase virus into the culture medium. The retained viral particles were able to freely efflux from the gelatin sponge matrix in this liquid environment over a 4- to 6-hour incubation time. The other matrices showed markedly less consistency as a delivery vehicle, most likely explained by some loss of viral particles in the transfer of the matrix to the culture dish or the inability of the retained virus to reenter the media. It is interesting that the gelatin matrix mediated enhanced gene expression and also enhanced biodistribution when compared with the fluid-phase injection of the viral vector in preestablished subcutaneous tumor nodules. The expression of the respective products of ALVAC-GFP or ALVAC-lacZ delivered in fluid phase was limited, i.e., restricted to the needle tract (Fig. 4) and around the tumor margin. Moriuchi et al. (26) have described a similar distribution of viral vector infection after intratumoral injection of an intracerebral tumor, resulting in poor gene expression and biologic response.
In contrast to delivery in the fluid phase, the vectors delivered by the gelatin matrix showed a
broad biodistribution, which often would extend the full breadth of small tumors (approximately
equal to 0.5 cm in diameter). This enhanced gene transfer was shown to translate into an
improved biologic effect in the heterotopic RM-1 murine prostate cancer model with the use of a
cytokine-based immunotherapy protocol. RM-1 tumors with a mean volume of 100-500 mm3 (depending on the experiment) were significantly inhibited when the gelatin
matrix was used to deliver ALVAC-IL-2, ALVAC-IL-12, and ALVAC-TNF-. The
inhibitory effects in these treated mice also resulted in a significant survival advantage with a
single injection. Despite this dramatic increase in gene expression observed in these heterotopic
tumors when viral vectors were delivered with the gelatin sponge matrix, systemic distribution of
vectors was not found to be increased over fluid-phase delivery. Selected organs (spleen, liver,
and kidneys) harvested at the time of these experiments demonstrated no increased luciferase
gene expression when compared with fluid-phase delivery. Similarly, in experiments involving
orthotopic delivery of viral vectors in the ventral prostate of mice, gelatin sponge matrix delivery
resulted in only minimal gene expression in surrounding organs, including the testes, bladder,
and seminal vesicles, that was no greater and often less than that of fluid-phase delivery (data not
shown).
Although gene therapy approaches to immunotherapy have enhanced immune activation and provided enhanced therapy results, the control of preestablished tumors has remained problematic (27-29). Control of the immunogenic RENCA kidney tumors was limited to tumors established for a maximum of 7 days [nonpalpable tumors; (29)]. Likewise, the control of tumor growth by herpes simplex virus thymidine kinase/gancyclovir has been limited to a reduction in tumor growth rate but not tumor regression (30). Our studies show the induction of substantial tumor growth inhibition with a single injection of 8.4 x 106 pfu of ALVAC cytokine vaccine (Fig. 5, A). While the control of tumor growth does not result in a cure, it does provide a significant extension of survival for treated mice. This result is accomplished in spite of the use of the poorly differentiated, highly aggressive RM-1 tumor model (approximate in vitro doubling time of 12 hours) in which treatment was initiated when tumors were large (approximately 215 mm3). The differences between the two means of vector delivery were even more dramatic in the RM-11 tumor model (Fig. 6) and have resulted in the long-term tumor-free survival in approximately 20% of mice over three separate experiments.
Improving the delivery of viral vectors is paramount in any clinical gene therapy trial, and this is especially true for prostate cancer. Delivery in a solid-state matrix, such as the one that we have described, may allow for more efficient and widespread gene transfer for all nonreplicative vectors. The increased efficiency of delivery may also result in circumventing antiviral host immune responses by decreasing the viral concentration needed to attain a desired effect.
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NOTES |
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We thank Jan Rodgers (Department of Pathology, University of Iowa) for her help with histopathology and Dr. Charles Davis (Department of Biostatistics, University of Iowa) for his assistance with statistical analysis.
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Manuscript received May 6, 1999; revised November 29, 1999; accepted December 8, 1999.
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