Endocrinology Vol. 144, No. 4 1393-1398
Copyright © 2003 by The Endocrine Society
Gene Therapy of a Rat Follicular Thyroid Carcinoma Model with Adenoviral Vectors Transducing Murine Interleukin-12
Rusheng Zhang and
Leslie J. DeGroot
Thyroid Study Unit, Department of Medicine, The University of Chicago, Chicago, Illinois 60637
Address all correspondence and requests for reprints to: Leslie J. DeGroot, M.D., Thyroid Study Unit, Mail Code 3090, The University of Chicago, 5841 South Maryland Avenue, Chicago, Illinois 60637.
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Abstract
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Interleukin-12 (IL12) is a heterodimeric cytokine that plays an important role in the development of cellular immunity. We previously reported the antitumor activity of mouse IL12 (mIL12) transduced by adenovirus in a medullary thyroid carcinoma model. In this study, a rat thyroid follicular cancer cell line (RTC-R2) was employed to develop tumors after sc injection in Wistar rats. In five of five animals, RTC-R2 cells infected with mIL12 transducing adenovirus (AdCMVmIL12) in vitro failed to be tumorigenic in vivo in syngenic rats, whereas four of five animals developed tumors after injection of luciferase transducing adenovirus (AdCMVLuc)-infected cells. After intratumoral treatment with AdCMVmIL12 at 1 x 109 plaque-forming units per rat, 90% (26/29) of animals bearing small (<100 mm3) tumors were apparently cured. Larger tumors treated by injection of AdCMVmIL12 became significantly smaller than AdCMVLuc-treated animals, and growth stabilized. Challenge studies showed that only 3 of 28 animals previously treated and cured with AdCMVmIL12 developed a tumor after sc reinjection of RTC-R2 cells, whereas all animals developed tumors in naïve animals. Thus, AdCMVmIL12-treated animals developed long-term antitumor immunity. We also studied animals with two tumors, injecting virus in one. Tumors regressed at both sites in five of six animals after treatment of one tumor with AdCMVmIL12, and in the sixth animal one site tumor regressed and another tumor continued to grow. In AdCMVLuc-treated animals, both tumors regressed in only one animal, and the reminder continued to grow.
To detect toxicity to liver and other tissues after administration of AdCMVmIL2, the vector was administrated intratumorally or iv at the dose of 2 x 109 plaque-forming units per rat. No change in behavior was observed in any of the treated animals. Rats were killed at different time after virus administration. An overt increase of spleen size was observed 7 d after infection in all animals treated with AdCMVmIL12. All animals given virus IV had some lymphocyte infiltration in the sinusoids and triads of the liver, whereas AdCMVmIL12 injected intratumorally did not cause this effect. Spleens of some virus-treated animals showed decreased white pulp, with apparently increased hematopoiesis. No specific changes were found in lungs and kidneys. Iv administration of AdCMVmIL12 induced a moderate increase of glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase, whereas AdCMVmIL12 injected intratumorally did not.
This study confirms the efficient antitumor activity of an adenovirus expressing mIL12 after in vivo delivery in an animal model and indicates the possibility of application to patients because of the low toxicity.
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Introduction
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INTERLEUKIN-12 (IL12) HAS MULTIPLE effects on both natural killer (NK) and T cells. It is the most potent NK cell stimulator, stimulates the proliferation of NK cells and T cells, and production of interferon-
(IFN-), enhances both ADCC (antibody dependent cell cytotoxicity) and cell mediated cytolytic activity of NK cells, and CD8+ T cells, is a growth factor for these cells, and contributes indirectly to macrophage activation through lymphocyte INF- synthesis. IL12 also stimulates the differentiation of naïve CD4+ T cells into the Th1 subset and thus helps cellular immunity. The activity of IL12 on cellular immunity appears to be the basis of its ability to eradicate tumors in both animal and clinical trials.
Systemic administration of recombinant IL12 (rIL12) causes dose-dependent toxicity in animals (1), and in human trials (2). To reduce systemic toxicity, local expression of the IL12 gene in tumors has been explored. Fibroblast cells were engineered to produce mouse IL12 (mIL12) using gene gun mediated transfection. Delivered locally to the tumor site, the cells successfully suppressed tumor growth and the animals developed an antitumor immune response (3, 4, 5). However, the expression level of IL12 was relatively low, and it is obviously difficult to generate many different cell lines producing IL12 for clinical cancer therapy. An alternative means for delivering IL12 is through the use of viral expression vectors. Viral vectors may infect many host cell lines and can localize cytokine expression at the site of injection. Most initial studies have used retrovirus transducing IL12 (4, 6, 7, 8) because the virus can integrate the transgenes into genomic DNA, and IL12-secreting cell lines are obtained. However, retroviruses can only infect proliferating cells, have low in vivo transduction efficiency, are sensitive to serum complement, achieve low viral titers, and are difficult for large-scale production (9). Adenovirus vectors are an attractive alternative. These vectors can efficiently transduce both replicating and nonreplicating tumor cells in vivo and have fewer limitations than retroviral vectors (10). One concern is the immune response to adenovirus. Development of antiviral immunity makes it difficult to maintain long-term expression because of the eradication of the virus. However, in immune gene cancer therapy, it is not necessary to express the cytokine gene for a long time. Once antitumor immunity is elicited, the expression of the transgene is less important. The immune response to viral proteins may actually serve as an adjuvant, enhancing the antitumor activity.
Other researchers and our group have demonstrated that adenovirus vectors can be used to efficiently transduce specific cytokine production in vivo and in vitro (11, 12, 13, 14).
The rat tumor cell line, RTC-R2, was cloned from an anaplastic tumor of a Wistar rat, which was induced chemically. Because of the loss of thyroglubulin promoter activity in this tumor cell line, we employed the cytomegalovirus (CMV) promoter to induce transgene expression. Most anaplastic thyroid tumor cells, including human thyroid carcinomas, have very low or no thyroglobulin promoter activity.
In this article, we describe the expression of functional IL12 in transduced RTC-R2 cells, and antitumor efficacy in our RTC-R2 animal model.
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Materials and Methods
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Animals, cells, and reagents
The animal study protocol was approved by Institutional Animal Care and Use Committee at the University of Chicago. Wistar rats were bred in the Carlson Animal Research Center of the University of Chicago (Chicago, IL). Four- to 6-wk-old rats were used in our studies. Plasticwares were purchased from Fisher Scientific (Pittsburgh, PA).
All cell culture media were from Life Technologies, Inc. (Grand Island, NY). A rat follicular thyroid carcinoma cell line (RTC-R2) was cloned by our laboratory from a tumor-bearing Wistar rat. This cell line was grown in DMEM supplemented with 10% horse serum (Life Technologies, Inc.), 100 U/ml penicillin (Life Technologies, Inc.), and 100 µg/ml streptomycin (Life Technologies, Inc.). The low passage 293 cell line, a human embryonic kidney cell line that expresses the early region 1 of the type 5 adenoviral genome, was purchased from the Microbix Biosystems Inc. (MBI, Ontario, Canada; M8Z3A8), and was maintained in MEM supplemented with 10% heat inactivated fetal bovine serum (FBS) (Life Technologies, Inc.), 100 U/ml penicillin, and 100 µg/ml streptomycin. Human recombinant IL12 was kindly provided by Dr. Thomas Gajewski (The University of Chicago).
Replication-defective adenovirus vectors
Construction of the replication-defective adenoviral vectors containing luciferase (Luc) gene, or mIL12 gene, under the transcriptional control of the human CMV immediate early promoter/enhancer system (AdCMVLuc), has been described (15). Viral stocks were prepared by infection of 293 cells. The viruses were harvested about 48 h after infection and purified by double cesium chloride gradient ultracentrifugation (16). Viral titers (plaque forming units per ml, pfu/ml) were determined by plaque assay using 293 cells. Viral stocks were stored in 10% glycerol at -80 C.
Toxicity of AdCMVmIL12 after in vivo administration
AdCMVmIL12 was administrated either iv or sc at doses of 2 x 109 pfu in 100 µl PBS buffer. Treated animals were inspected every day for their behavior, and killed 3, 7, or 14 d after treatment. Blood and tissues were retained at the time the animals were killed. Serum was obtained from clotted blood for liver function test, and organs and tissues were harvested for pathological examination.
Transaminase assay
A commercial kit was used for quantitative colormetric determination of glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) (Sigma, St. Louis, MO). The procedure from the company was followed.
mIL12 expression assay
The proliferative response of mouse concanavalin A (Con A)-activated splenocytes (Con A blasts) to mIL12 was measured in a 48-h assay as reported (17), with modification. Briefly, BALB/c splenocytes (1 x 106 ml) were cultured in TCM [1/1 mixture of RPMI 1640 medium and DMEM, supplemented with 0.1 mM nonessential amino acids, 60 µg/ml arginine hydrochloride, 10 mM HEPES buffer, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.05 mM 2-mercaptoethonol (Sigma, St. Louis, MO), 1 mg/ml dextrose (Fisher Scientific), and 5% heat-inactivated FBS] supplemented with 2% heat inactivated FBS, 20 U/ml human rIL2 (kindly provided by Dr. Jose Quintan, The University of Chicago), and 2 µg/ml Con A (Sigma). After 2–3 d, the Con A activated splenocytes were harvested, washed, and resuspended in TCM with 5% heat-inactivated FBS at 2 x 105 cells/ml. One hundred microliter aliquots of serial dilutions of mIL12 containing culture supernatants were added into the wells of 96-well plates (Costar, Corning, NY). The cultures were incubated for 1 d at 37 C in a humidified atmosphere of 5% CO2 in air. Twenty microliters (1 mCi) of 3H-thymidine (1 mCi/ml, ICN Pharmaceutical, Inc., Costa Mesa, CA) dilution were then added to each well. The cultures were incubated overnight, and then harvested onto glass fiber filters. Incorporation of 3H-thymidine into cellular DNA was measured using a liquid scintillation counter. All samples were assayed in triplicate.
mIL12 ELISA
An mIL12 (p70) OptE1A Set (PharMingen, San Diego, CA) was employed in the serum mIL12 assay.
Tumorigenicity of AdCMVmIL12 transduced RTC-R2 cells
RTC-R2 cells were transduced with 10 multiplicity of infection (moi) of each virus for 1 h. Transduced cells were washed with DMEM serum-free medium. Cells (1 x 106) in 100 µl were injected sc into the flank of Wistar rats. Animals were inspected every 2 d for the development of tumors after the injection. Tumor size was calculated by formula (18): ab2/2, where a is the longest diameter of tumor, and b is the shortest diameter of the tumor.
Development of RTC-R2 tumors in syngenic rats
Healthy 4- to 6-wk-old Wistar rats were injected sc with 1 x 106 RTC-R2 cells in 100 µl serum-free DMEM. The injected animals developed palpable tumors after 8–14 d. Tumor growth was followed as described above.
Direct intratumoral delivery of the recombinant virus
AdCMVLuc or AdCMVmIL12 stock solution was diluted to 1 x 1010 pfu with serum-free medium. One hundred microliters (1 x 109 pfu) of diluted virus solution were directly injected into the preestablished tumors.
Two sites tumor treatment study
Tumors were developed in rats on both flanks by sc injection of RTC-R2 cells, using 1 x 106 cells for each site. One tumor was treated as described above. Tumor growth at both sites was recorded.
Challenge study
Cured rats were challenged by sc injection of wild-type RTC-R2 cells using 1 x 106 cells per rat. The challenged animals were inspected thereafter during at least 4 wk for the development of tumor. Naïve rats served as the controls in the study.
Statistical calculations
The Student’s t and 
2 test were used to analyze the data. P < 0.05 was considered significant.
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Results
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Expression of mIL12 in infected tumor cells
Functional expression of mIL12 was confirmed by mouse splenocyte-derived Con A blast cell assay. Rat MTC cells were infected with AdCMVmIL12, or AdCMVLuc, as described in Materials and Methods. A clear proliferative response was obtained using the supernatants of AdCMVmIL12 infected RTC-R2 cells, whereas no stimulatory effect was found using the supernatants of AdCMVLuc infected RTC-R2 cells or using control medium (Fig. 1
). The expression of mIL12 was also confirmed by a mIL12 ELISA assay on supernatants of AdCMVmIL12-treated RTC-R2 cells (Fig. 2).
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Figure 1. Production of functional mIL12 in infected RTC-R2 cells. Rat MTC cells (1 x 106) were infected with AdCMVmIL12 in vitro at 100 moi for 1 h in 500 µl solution. Then infected cells were incubated in six-well plates after washing three times with serum-free medium. Supernatant was harvested after 24 h incubation. The activity of mIL12 in the supernatant was examined by ConA blast proliferation assay. Recombinant IL12 served as positive control. 50x rIL12 = 2 µg/ml. Supernatant from AdCMVLuc infected RTC-R2 cells served as negative control. Specimens were tested after serial dilution up to 6250-fold. Error bars in each dilution point indicate the SD.
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Figure 2. ELISA assay of mIL12 in infected RTC-R2 cells. RTC-R2 cells were infected at 100 moi for 1 h in 1000 µl solution. Then infected cells were washed, incubated in six-well plates at 5 x 105 cells/4 ml. Supernatants were harvested every 24 h for a consecutive 6 d. Supernatants from cultures of uninfected RTC-R2 cells served as negative control. The mIL12 in supernatants was examined using an ELISA kit (OptE1A Set, PharMingen, San Diego, CA). Serial dilutions of recombinant IL12 were used as the standard reference for calculating the concentration of mIL12 in the supernatants. Error bars in each time point indicate the SD.
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AdCMVmIL12 infected RTC-R2 cells lost their tumorigenicity
Virus infected RTC-R2 cells were transplanted sc into Wistar rats to examine their tumorigenicity. Cells infected with AdCMVmIL12 completely lost their tumorigenicity. No animal developed a tumor. All control animals injected with AdCMVLuc-infected RTC-R2 cells developed tumors (Fig. 3).
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Figure 3. Tumorigenicity of infected RTC-R2 cells. RTC-R2 cells were infected in vitro for 1 h at 10 moi in 500 µl infection solution. Then the infected cells were injected sc into Wistar rats at 1 x 106 cells per rat. The animals were inspected every 2 d for the development of tumors. An animal was considered tumor free if no tumor developed within 8 wk after tumor cell injection. 2 test was used to examine the difference in results between infected and noninfected groups of animals.
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Antitumor efficacy in vivo
The antitumor effect of AdCMVmIL12 was evaluated by direct injection of vector into RTC-R2 tumors. An efficient antitumor effect was obtained in treated animals. In small size tumor (<100 mm3)-bearing animals, most treated tumors regressed (26/29), compared with only 4 of 12 AdCMVLuc-treated tumors. In larger tumors (>100 mm3), most tumors stabilized after treatment, and a significant difference in tumor size between AdCMVmIL12 and AdCMVLuc-treated groups was found 1 wk after treatment Fig. 4). All of the in vivo studies are summarized in Table 1. Sixty-four percent of tumors disappeared after AdCMVmIL12 treatment, and 36% of tumors were stabilized. Only 30% of tumors regressed in the AdCMVLuc-treated group, and none of them stabilized. Animals observed for 8 wk after tumor disappearance, without regrowth, were considered cured.
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Figure 4. In vivo antitumor activity. Tumor-bearing rats were treated either with AdCMVmIL12, or AdCMVLuc, at a dose of 1 x 109 pfu, or with medium alone, in a volume of 100 µl. The treatment was performed in tumors of different sizes. A, Tumor volume was equal or less than 100 mm3. B, Tumor volume was larger than 100 mm3. The tumor sizes were significantly smaller in AdCMVmIL12-treated groups than in control groups 8 d after the treatment (P < 0.05). Error bars in each time point indicate the SD.
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Two-sites tumor treatment study
After development of two tumors in every rat, one tumor was treated by adenoviruses at the same dose as in the one tumor model. Both the treated and untreated tumor decrease in size 2 wk later in AdCMVmIL12-treated animals compared with the AdCMVLuc-treated animals (Fig. 5), indicating the systemic antitumor immunity in the AdCMVmIl12-treated rats.
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Figure 5. Two-site tumor treatment. One tumor in a two tumor model was treated by adenoviruses at the same dose as in Fig. 4 . Both the treated and untreated tumor significantly decrease in size 2 wk later in AdCMVmIL12-treated animals compared with AdCMVLuc-treated controls. Error bars in each time point indicate the SD.
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Long-term antitumor immunity
Two months after treatment induced tumor regression, cured animals were challenged with wild-type RTC-R2 cells. Twenty-five of 28 challenged rats cured by AdCMVmIL12 developed antitumor immunity because no tumor developed after challenge (Table 2).
Cytotoxicity of adenoviral vectors
To detect possible damage to liver and other tissues after administration of AdCMVmIL12, this vector was administrated iv at doses of 2 x 109 pfu per rat. This provides a viral load greater than that given directly into the tumor, but allowed direct iv access to all tissues. There was no change in behavior in any of the treated animals compared with that of untreated animals. Rats were killed on d 3, 7, or 14 after virus administration. Liver is the major target organ after viral treatment. GPT and GOT levels in animals treated by iv administration were elevated compared with untreated control animals (Fig. 6). There was no difference in transaminase levels from the control animals, in animals treated by intratumor or ip virus administration (data not shown). All animals given virus iv had mild to extensive lymphocyte infiltration in the sinusoids and triads of the liver, and in some liver samples central veins were dilated where lymphocyte infiltration was present (Fig. 7). Spleens of some virus-treated animals showed decreased white pulp, and apparently increased hematopoiesis, No specific changes were found in lungs and kidneys.
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Figure 6. Transaminase assays. AdCMVmIL12 was administrated iv at doses of 2 x 109 pfu per rat. Rats were killed on d 3, 7, or 14 after virus administration, the serum was saved for transaminase assay. Sera from untreated rats with normal liver function served as controls. Error bars in each column indicate the SD.
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Figure 7. Histology of treated animal livers. AdCMVmIL12 was administrated iv at doses of 2 x 109 pfu per rat. Rats were killed on d 3, 7, or 14 after virus administration, the tissues were saved for pathologic studies. Tissues were examined after hematoxylin and eosin staining. All livers of treated animals showed mild to extensive lymphocyte infiltration. A, Liver from 2 x 109 pfu-treated animal, 3 d after treatment, showing minimal pericentral lymphocyte infiltration with slight increase in sinusoidal lymphocyte infiltration. B, Liver from 2 x 109 pfu-treated animal, 7 d after treatment, showing marked portal triad and sinusoidal infiltration of lymphocytes. C, Liver from 2 x 109 pfu-treated animal, 14 d after treatment. D, Liver from untreated animal.
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Discussion
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Direct administration of rIL12 provides an antitumor effect, but also induces dose dependent side effects (19, 20). We chose to use adenovirus to transduce IL12 cDNAs. Recombination viruses were generated that contain the cDNAs of p35 and p40 subunits of murine IL12 in the E1 region, each under the control of the HCMV immediate early promoter. Bramson et al. (21) have reported construction of a double recombinant adenovirus vector expressing the heterodimeric cytokine, mIL12, in which the p35 subunit cDNA was cloned in the E1 region of adenovirus type 5 and the p40 subunit cDNA in E3. It has been reported that transplicing in E3 region could lead to chimeric transcripts between viral and foreign genes (22, 23). Our construct should be more stable because both subunits were inserted into the E1 region.
Infection of RTC-R2 cells by AdCMVIL12 abrogated their tumorigenicity. This is similar to our previous observations using an AdCMVmIL2 vector (24) in a rat medullary thyroid cancer (rMTC) mode. In the in vivo studies, we found that 100% of small size tumors were destroyed by AdCMVmIL12 treatment. This result is far superior to our result using an AdCMVIL2 vector, which caused 40% of tumors to regress after intratumoral injection of double amount of virus. This result also is consistent with several recent reports. In our rMTC animal model, 86% of tumor-bearing animals were apparently cured, and almost all remaining tumors were stabilized after AdCMVmIL12 treatment (15). Challenge studies showed that most animals cured after the first treatment remained tumor free after reinjection of wild-type rMTC cells, indicating that long-term antitumor immunity developed. In a breast tumor model, a single injection of an adenovirus vector expressing mIL12 produced regression in greater than 75% of the treated tumors. The overall response rate was 92% (21). Intratumor injection of tumors with AdmIL12 (1 x 109 pfu) resulted in complete tumor regression in all mice in a murine bladder carcinoma model (25). Intratumor injection of 4 x 108 pfu of AdCMVmIL12 resulted in a complete regression of established (d 7) sc MC38 murine adenocarcinomas and MCA205 murine fibrosarcomas (26). Eighteen (72%) of 25 tumor-bearing mice treated with AdmIL12 exhibited tumor regression in a murine neuroblastoma (neuro-2a) animal model (27). In some studies, efficacy is reduced when studies are performed in mice depleted of CD4+ and CD8+ cells or in nude mice (25). However, in a hepatic metastases animal tumor model, therapeutic efficiency was not diminished in animals depleted of CD4+ or CD8+ T cells. Similarly, in SCID mice, antitumor effect remained after NK cell ablation. These observations suggest a model of metastatic growth inhibition mediated by nonlymphocyte effector cells including macrophages and neutrophils, or possibly antiangiogenic chemokines (28). In our study, most animals did not develop tumors after challenge in previously cured animals, indicating generation of specific antitumor immunity (Table 2).
Our current results confirm the antitumor activity of IL12 and the feasibility of using this adenovirus as a vector to transduce the expression of antitumor cytokines. Further studies are necessary to explore methods for administration of the vector and to define toxicity from the leaked virus.
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Footnotes
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Abbreviations: ADCC, Antibody-dependent cell cytotoxicity; CMV, cytomegalovrius; Con A, concanavalin A; FBS, fetal bovine serum; GOT, glutamic-oxalacetic transaminase; GPT, glutamic-pyruvic transaminase; IFN-, interferon-; IL12, interleukin-12; luc, luciferase; moi, multiplicity of infection; NK, natural killer; pfu, plaque-forming units; rIL12, recombinant IL12; rMTC, rat medullary thyroid cancer; RTC-R2, rat thyroid follicular cancer cell line.
Received September 30, 2002.
Accepted for publication December 23, 2002.
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Abstract
Introduction
