Antitumor and antiangiogenic effects of interleukin 12 gene therapy in murine head and neck carcinoma model

Auris Nasus Larynx 31 (2004) 239–245

Antitumor and antiangiogenic effects of interleukin 12 gene therapy in murine head and neck carcinoma model Yukari Imagawa a,∗ , Kenichi Satake a , Yasumasa Kato a , Hideaki Tahara b , Mamoru Tsukuda a a

b

Department of Biology and Function in the Head and Neck, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan Department of Surgery and Bioengineering, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654, Japan Received 5 January 2004; accepted 19 March 2004 Available online 20 June 2004

Abstract Interleukin-12 (IL-12) plays a critical role in producing an immune response, as indicated in many ways, e.g., induction of interferon-␥ (IFN-␥), and augmentation of the cytotoxic activity of resting activated T cells and natural killer (NK) cells. In this study, we examined whether intratumoral injection of a recombinant retrovirus vector expressing IL-12s induce antitumor and antiangiogenic effects in a murine model using a murine head and neck squamous cell carcinoma (NR-S1). In vitro the levels of vascular endothelial growth factor (VEGF) mRNA and protein expression were decreased in IL-12 gene transfected NR-S1 cell. in vivo direct IL-12 gene therapy resulted in significantly remarkable inhibition of tumor growth compared to the control group. The tumor regression by direct IL-12 gene therapy was also associated with decreased vessel density, and apoptosis and increased infiltration of CD8+ T cells and CD56+ NK cells in the tumor increased. Also, the number of IFN-␥ expressed cells of spleen cells was increased in the treatment group compared with the control group. These results suggested that direct IL-12 gene therapy appears to be effective in reducing tumor growth by triggering both antiangiogenic effects and an immunological enhancing mechanism through induction of IFN-␥. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Interleukin 12; Gene therapy; Head and neck squamous cell carcinoma; Direct intratumoral injection; Antitumor; Antiangiogenesis

1. Introduction Interleukin-12 (IL-12) is a key cytokine in numerous immune functions [1]. It is produced primarily by antigen-presenting cells (APCs) and binds to receptors on T cells and NK cells, activates them, and promotes the induction of T helper type 1 phenotype (Th1) response in vitro as well as in vivo [2,3]. More importantly, IL-12 exerts antitumor activity on a variety of cancers through activation of innate and adaptive immunity, as well as through inhibition of angiogenesis [4–8]. These effects are thought to be largely due to the local IFN-␥ production by T and NK Cells [9,10]. Neither IL-12 nor IFN-␥ has been reported to exhibit antiproliferative effects. However, it is not easy to demon∗ Corresponding author. Tel.: +81-45-787-2687; fax: +81-45-783-2580. E-mail address: [email protected] (Y. Imagawa).

strate that the antiangiogenesis effect of IL-12 is responsible for its antitumor effect because the antitumor immunity and the antiangiogenesis are induced simultaneously. Systemic IL-12 therapy was effective in multiple tumor models and clinical studies [11,12], however preclinical studies and early clinical trials have demonstrated that it causes systemic toxicity [13,14]. Based on these results, strategies aimed at localized delivery of cytokine have received increasing attention. Recently, the advantages of intratumoral paracrine delivery of IL-12 using gene therapy have been documented [15,16]. The mechanisms underlying this antitumor activity by local delivery of IL-12 are incompletely understood but may be related to the ability of IL-12 to inhibit angiogenesis [17] and/or stimulate T cells and NK cells [4]. In this study, we examined that intratumoral injection of a recombinant retrovirus vector expressing IL-12 induced antitumor and antiangiogenic effects in a murine model using a

0385-8146/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.anl.2004.03.008

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murine head and neck squamous cell carcinoma. In vitro we analyzed the antiangiogenesis effects of direct IL-12 gene transfection. In addition, we demonstrated for the first time the mechanisms of antiangiogenesis by immunohistochemistry and enzyme-linked immunospot (ELISPOT) assay in vivo. We also investigated that the inhibition of angiogenesis and induction of apoptosis by direct IL-12 gene therapy resulted in the reduction of tumor size.

2. Materials and methods 2.1. Cell line and animals NR-S1, a murine squamous cell carcinoma cell line originated from the bucal mucosa, was obtained from the Department of Biochemistry, Research Institute, Kanagawa Cancer Center (kind gift of Dr. Yanoma). This cell line was maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM glutamine, 100 units/ml penicillin and 100 ␮g/ml streptomycin (complete medium) in a humidified 5% CO2 atmosphere at 37 ◦ C. Female 5-week-old C3H/HeNCrj mice were obtained (Charles River Japan Inc., Kanagawa). The animals were housed in a room kept at 24 ± 2 ◦ C temperatures and 40–70% humidity with a 12 h light/dark cycle. 2.2. Recombinant retroviral vectors The genes that code for the subunits of murine IL-12 (p35 and p40) have been subcloned into the mammalian expression vector TFG-mIL-12, as previously described [18]. This vector was obtained from the Department of Surgery, University of Pittsburgh School of Medicine (Pittsburgh, PA; kind gift of Dr. Tahara).TFG-mIL-12 was transfected into the CRIP packaging cell line, and CRIP-TFG-nIL-12 clones were selected with Geneticin (G-418 sulfate: 0.75 mg/ml) (Invitrogen, Carlsbad, CA, USA). Retroviral supernatant was generated from CRIP-TFG-mIL-12 clones at a concentration of 5×104 to 5×105 colony-forming units (CFU)/ml. A second retroviral vector containing only the Neomycin phosphotransferase gene, which served as a negative control in all of our experiments, was obtained from Dr. Tahara (Department of Surgery, University of Pittsburgh School of Medicine). 2.3. Transfection of NR-S1 cell line NR-S1 cell was infected with CRIP-TFG-mIL-12 by incubating 5 × 105 cells in sterile 25 cm2 flasks 24 h before infection. After aspiration of the medium, 2 ml of CRIP-TFG-mIL-12 or Neomycin viral supernatant (5 × 104 to 5 × 105 CFU/ml) was added to each flask with gentle rocking. Two microliters of Polybrene (Invitrogen: 8 mg/ml) was added to each flask followed by incubation at 37 ◦ C for 2 h. Flasks were rocked every 15 min throughout the

2-h incubation. Eight milliliters of RPMI 1640 was added to the flasks, which were then incubated overnight at 37 ◦ C in 5% CO2 . Fresh medium was added to the flasks the following day, and cells were split at a 1:5 dilution in RPMI 1640 with 0.75 mg/ml G-418 when confluent. Infection with the Neo retrovirus for generation of the NR-S1/Neo cell line was carried out in the same manner as described for NR-S1/mIL-12. NR-S1/mIL-12 and NR-S1/Neo transfectants were selected in G-418 sulfate (0.75 mg/ml) medium for more than 2 weeks with replacement of the medium every 3–4 days. 2.4. Analysis of vascular endothelial growth factor (VEGF) by enzyme-linked immunosorbent assay (ELISA) Supernatants from the NR-S1 and NR-S1/mIL-12 cells were analyzed using a mouse VEGF ELISA kit provided by R&D Systems (Minneapolis, MN). Immunoassays were performed according to the instructions of the manufacturers. 2.5. Detection of VEGF mRNA by quantitative RT-PCR using the TaqMan method Total RNA was extracted from NR-S1 and NR-S1/mIL-12 cells with phenol solution (“ISOGEN”; Nippon Gene Inc., Tokyo) according to the manufacturer’s protocol. One microgram of total RNA was converted into cDNA by using TaKaRa RNA PCR Kit (AMV) Ver.2.1 (TAKARA BIO Inc., Tokyo). The quantitation of relative expression levels was carried out using a fluorescence detection method using TaqMan PCR Reagent Kit (Applied Biosystems, Foster City, CA, USA). PCR was conducted using the following cycle parameters: 50 ◦ C for 2 min, 95 ◦ C for 10 min, followed by 40 cycles at 95 ◦ C for 15 s and 60 ◦ C 1 min. Primer and probes were designed using the computer program Primer Express (PE Biosystems), following the instructions of the manufacturer. The primers for sense and anti sense, respectively, were: VEGF cDNA 5 -CTGTGCAGGCTGCTGTAACG-3 and 5 -CGCATGATCTGCATGGTGAT-3 ; ␤-actin cDNA 5 -GGACCTGACGGACTACCTCATG-3 and 5 -GCCATCTCCTGCTCGAAGTCTA-3 . The primers were obtained from Invitrogen. The TaqMan fluorogenic probe consisted of the following sequences: VEGF, 5 -FAM-AGCCCTGGAGTGCGTGCCCAC-TAMRA-3 ; ␤-actin, 5 FAM-CTGACCGAGCGTGGCTACAGCTTCA-TAMRA-3 . Both purchased from Sawady Technology Co., Ltd., Tokyo). 2.6. In vivo model of gene therapy NR-S1 cells (5 × 106 ) were injected s.c. into the left flank in mice (five mice per group). At day 7 after tumor inoculation s.c., animals received treatments of 5 × 105 CFU/ml of retroviral vectors (CRIP-TFG-mIL-12 and Neo) in 100 ␮l of serum free medium, injected directly into the established tumor twice a week for 3 weeks. The local tumor was mea-

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sured once a week in two perpendicular dimensions with a caliper. Tumor size in mm3 was calculated by using the formula (a × b2 )/2, where a is the larger and b the smaller dimension of the tumor. The mice were sacrificed on day 35. 2.7. Immunohistochemistry We performed immunohistochemistry to determine vessel density and subsets of tumor infiltrating lymphocytes (TIL). The vessels in the tumor tissues were stained using an antibody to CD31, an endothelial marker (1:200; BD Biosciences, Tokyo), and the subsets of TIL were stained with an anti-CD4 antibody (1:100; Santa Cruz Biotechnology Inc.), an anti-CD8 antibody (1:100; Santa Cruz Biotechnology Inc.), and an anti-CD56 antibody (1:100; Santa Cruz Biotechnology Inc.). Tumors harvested for immunohistochemistry were immediately frozen in OTC medium for cryosection. Tumor sections at 6 ␮m were prepared and fixed in cold acetone. The sections were blocked with 2% goat serum and 1% BSA in PBS and stained with antibodies. Slides were developed using 3,3 -diaminobenzidine substrate biotinylated peroxidase reagent (Vector Laboratories, Inc., Burlingame, CA, USA). The number of vessels and the subsets were scored from a minimum of five microscopic fields from five independent tumors treated with direct IL-12 gene therapy or control groups. The average number of vessels per field was determined under a microscope at 20× magnification, and the average number of CD4, CD8, and CD56 positive cells per field was determined under a light microscope at ×10 magnification. 2.8. Terminal deoxynucleotidyl transferase-mediated cUDP nick end labeling (TUNEL) methods TUNEL was performed using a commercial kit, Apoptosis in situ Detection Kit (Wako Chemical, Osaka) for frozen sections. Containing of immunoreactive cells was based on the distribution of apoptotic tumor cells in three different fields within the same section; the apoptotic index was expressed as the percentage of TUNEL-positive cells with respect to the total number of the cells. 2.9. Enzyme-linked immunospot (ELISPOT) assay for IL-4 and IFN-γ production The ELISPOT assay was performed as described previously [19]. Briefly, 96-well plates with PVDF membrane inserts (Millipore, Bedford, MA, USA) were coated with capture mAb anti-mouse IL-4 (ENDOGEN, Woburn, MA; 1 ␮g/ml in PBS, pH 7.4) or mAb anti-mouse IFN-␥ (ENDOGEN; 0.5 ␮g/ml in PBS, pH 7.4) and incubated over night at 4 ◦ C. Then spleens were harvested from C3H/HeNCrj mice under sterile conditions. Each spleen was cut and pressed gently through wire gauze. The spleen cell suspension, in a petri dish containing 10 ml of RPMI 1640 medium, was spun

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(2000 rpm, 5 min) and resuspended in 10 ml of red blood cell lysis solution (0.15 M NH4 Cl). Cells were then spun (2000 rpm, 5 min) and washed twice in this medium. Spleen cells were placed in wells of 96-well ELISPOT plates at 105 cells per well. The assay was usually run in triplicate or quadruplicate. After 24 h incubation of the plates in a humidified atmosphere of CO2 in air at 37 ◦ C, the plates were developed to visualize the spots. The spots were counted by computer-assisted image analysis (KS ELISPOT V4.4.35, Carl Zeiss, Hallbergmoos, Germany). The number of specific spots was calculated by subtracting control values from experimental values. The number of spots in control wells was either 0 or 1 for all specimens in this series. The assay was standardized as described earlier [20,21]. 2.10. Statistical analyses The data were expressed as means ± S.D., The Mann–Whitney’s U test was used to obtain P and to compare different treatment groups. A P of <0.01 was considered statistically significant.

3. Results 3.1. In vitro IL-12 production by genetically engineered NR-S1 To determine whether infection of NR-S1 with CRIPTFG-mIL-12 produced biologically relevant secretion of IL-12, NR-S1 was infected with CRIP-TFG-mIL-12 at different MOIs. Using a MOI of 5 × 105 CFU/cell, the cells secreted 186 pg/ml per 24 h, thereby confirming the secretion of murine IL-12 by CRIP-TFG-mIL-12 infected NR-S1 (Fig. 1a). 3.2. Expression of VEGF protein and mRNA on NR-S1, NR-S1/Neo, and NR-S1/mIL-12 When cultured for 48 or 72 h, NR-S1 released VEGF into the culture medium (Fig. 1b). In order to determine whether the antiangiogenic effect of IL-12 was a result of decreased expression of VEGF, we analyzed VEGF expression levels of protein in the supernatants and of mRNA in cells. Both protein and mRNA levels of VEGF expression was decreased in the NR-S1/mIL-12 compared with NR-S1/Neo and NR-S1 significantly (Fig. 1b and c). These results suggest that IL-12 gene regulates the levels of VEGF directly. 3.3. Inhibition of NR-S1 tumor growth by direct IL-12 gene therapy The mice were divided into three treatment groups of five animals each. Each group received inoculations of NR-S1 followed 7 days later by intratumoral injection

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Fig. 1. (a) Expression of biologically active murine interleukin 12 (mIL-12) expression in NR-S1, NR-S1/Neo, and NR-S1/mIL-12. NR-S1 was infected with CRIP-TFG-Neo and -mIL-12 at different MOIs. At 48 h after infection, supernatants were harvested and the production of mIL-12 was confirmed by ELISA. (b) Vascular endothelial growth factor (VEGF) expression in NR-S1, NR-S1/Neo, and NR-S1/mIL-12 when these cells were cultured for 48 and 72 h. Supernatants were analyzed for the presence of VEGF by ELISA. (c) Total RNA was prepared from these cells and the expression of VEGF was determined by real-time PCR. Beta-actin (␤-actin) serves as an internal control (house keeping gene) for the expression level of VEGF. To simplify the results, the expression level showed the ratio of VEGF to ␤-actin. Results are representative of three experiments. Statistical analysis, ∗ P < 0.01.

of CRIP-TFG-mIL-12, CRIP-TFG-Neo, or saline. Tumor growth was statistically significantly reduced by the end of 5 weeks in animals receiving IL-12 intratumoral injection compared with the groups receiving control vector and saline (Fig. 2a). 3.4. Inhibition of angiogenesis and induction of apoptosis by direct IL-12 gene therapy To determine the antiangiogenesis responsible for the increased antitumor efficacy exhibited by the direct IL-12 gene therapy, we analyzed the vessel density of the tumors using antibodies against endothelial marker CD31 respectively

Fig. 2. (a) Inhibition of tumor growth by treatment with direct IL-12 gene therapy. NR-S1 cells (5×106 ) were injected s.c. into the left flank in mice (five mice per group). At day 7 post s.c., animals received treatments of 5×105 CFU/ml of CRIP-TFG-mIL-12, LacZ, and saline as control injected directly into the established tumor twice a week for 3 weeks. (b) Inhibition of the number of microvessels by direct IL-12 gene therapy. Direct IL-12 gene therapy had reduced levels of the number of microvessels compared to control. The number of microvessels was determined under a light microscope at 20× magnification from five fields from each mouse tissue. (c) Induction of Apoptosis by Direct IL-12 Gene Therapy. The apoptotic index was significantly higher in tumors from mice that had been treated by direct IL-12 gene therapy as compared with controls. The data was an average counting result of 1000 cells from five light microscope fields (20×) from each mouse tissue. Statistical analysis, ∗ P < 0.01.

(Fig. 2b). The immunohistochemistry results demonstrated that the vessel density was reduced statistically significantly by direct IL-12 gene therapy compared that with injection of control vector or saline. We then examined whether the direct IL-12 gene therapy was characterized by the induction of apoptosis. Direct IL-12 gene therapy resulted in an increased number of TUNEL-positive cells (Fig. 2c).

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3.5. Increased infiltration of effector cells into tumors To examine the cellular mechanisms responsible for the increased antitumor efficacy exhibited by the direct IL-12 gene therapy, we analyzed the infiltration of effector cells using antibodies against T cells, and NK cells, respectively. The CD8+ T cells and CD56+ NK cells infiltration in the treatment group with direct IL-12 gene therapy significantly increased compared the control group (Fig. 3a). There was no statistically significant increase in the infiltration of CD4+ T cells. These results suggest that the increased antitumor effect of direct IL-12 gene therapy is possibly caused by a CD8+ T cell- and NK cell-mediated immune response. 3.6. Increased the number of IFN-γ secreting spleen cells In order to evaluate a prolonged antitumor activity, we measured the number of IFN-␥ and IL-4 secreting spleen cells by ELISPOT assay. Mice receiving direct IL-12 gene therapy for 3weeks were sacrificed, and the spleen sells were tested for the presence of IFN-␥ and IL-4 secreting spleen cells in an ELISPOT assay. The number of IFN-␥ secreting spleen cells obtained from mice receiving direct IL-12 gene therapy was 5-fold higher than spleen cells obtained from na¨ıve animals (Fig. 3b). In contrast, no significant changes were detected in the number of IL-4 secreting spleen cells

Fig. 3. (a) Quantification of infiltrating cells. Positively stained CD8+ and CD56+ cells were counted from five fields from each mouse tissue under a light microscope at 10× magnifications. Immune cell infiltration was significantly higher in direct IL-12 gene therapy compared to controls. (b) IFN-␥ and IL-4 ELISPOT assay results. The ability of spleen cells to secrete IFN-␥ and IL-4 were evaluated in an ELISPOT assay. The number of IFN-␥ secreting spleen cells was significantly higher in direct IL-12 gene therapy compared to controls. There was no statistically significant increase in the IL-4 secreting spleen cells. Statistical analysis, ∗ P < 0.01.

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among the groups. These results indicated that the mechanism of prolonged antitumor activity was caused by an increase in the IFN-␥ secreting cells after direct IL-12 gene therapy.

4. Discussion IL-12 has received considerable attention as a powerful immunostimulatory cytokine. The role of IL-12 in experimental cancer therapy has been extensively investigated [4–8]. The systemic administration of recombinant IL-12 in a variety of tumor models including carcinomas, sarcomas, melanomas, and lymphomas has significantly inhibited tumor growth and enhanced survival. Although the systemic administration remains strikingly effective in tumor models, often demonstrating complete tumor eradication, it shows severe toxicities including ascites, dry mucus membrane, lack of grooming, hypophagia, and occasional death [15]. Fatal toxicity in a phase II clinical trial utilizing the systemic administration of recombinant IL-12 has also been reported [13]. For these reasons, systems focusing on local delivery of the cytokine to tumor tissue, e.g., gene therapy, have gained popularity. The mechanism by which IL-12 gene therapy elicits an antitumoral activity is complex. The results determined in other tumor models suggest that the mechanism of IL-12 gene action is dependent on the specific models examined. For a melanoma model, the antitumor effect elicited is mainly considered to be caused by its antiangiogenic effect [22], whereas for other tumor models, augmentation of CD8+ T cell cytotoxic activity is thought to be the major mechanism in antitumor effects [4,23,24]. The role of CD4+ T cells in antitumor effect by IL-12 is not clearly understood and may depend on the tumor model and on the amount and timing of IL-12 production [25]. Recently, it has also been reported that IL-12 has an antiangiogenic effect in several experimental settings [9,10]. This effect is exerted in an indirect manner by triggering the high secretion of IFN-␥, which presumably induces the antiangiogenic effect. NK cell involvement in antiangiogenic effect of IL-12 has also been documented [26]. Neither IL-12 nor a small quantity of IFN-␥ was reported to exhibit antitumor effects in vitro. However, it is not easy to demonstrate that the antiangiogenic effect of IL-12 is responsible for its antitumor effect because the antitumor immunity and the antiangiogenesis are induced simultaneously. IFN-␥ is produced by T cells, NK cells, as well as by macrophages and dendritic cells in response to IL-12 [1,27,28]. On the other hand, IFN-␥ also has pleiotropic effects on many cells because of the ubiquitous expression of its receptor on nearly all of the cells [29]. Because of the multiple sources of IFN-␥ production and its wide variety of cellular targets, a critical step in determining the role of IFN-␥ in the IL-12-mediated antitumor response is to identify its source of production and its target cell dur-

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ing tumor rejection. In an earlier study, the serum level of IL-12-induced IFN-␥ production was not reduced in T-cell deficient nude mice; however, the antitumor effect of IL-12 in these mice was reduced significantly. This observation suggests that T-cell existence and T-cell production of IFN-␥, possibly at the tumor site, may be critical for tumor rejection [30,31]. In the previous study, we evaluated cytotoxic activity of peripheral blood mononuclear cells (PBMC) and TIL immediately after separation, and lymphokine activated killer cells (LAK), IL-2 stimulated TIL (LA-TIL) and IL-2 activated effector cells after mixed lymphocyte tumor cell culture (LA-MLTC) against allogenic and autologous tumor targets in patients with head and neck carcinomas [32]. This study showed that, as compared with LAK cells, LA-TIL cells and LA-MLTC cells were more efficacious in terms of autologous tumor cell killing activity. However, the success rate of IL-2 administration alone in patients with head and neck carcinomas was only about 10%. It was important to concentrate activated effector cells efficiently at the tumor site. However, immunosuppressive factors existing at the tumor site might spoil the cytotoxic function of transferred effector cells. With respect to this problem, it is also necessary to control the production of immunosuppressive substances derived from tumor cells and to regulate such a biological activity of tumor cells in direct IL-12 gene therapy. In the present study, we investigated the mechanism of the antitumor effect of IL-12 in vitro and in vivo. Our data also suggest that two mechanisms for IL-12 antitumor action may exist: a direct antiangiogenesis and an antitumor immune response. In a direct antiangiogenesis, VEGF expression was decreased by IL-12 transfection into tumor cells and decreased vessel density in the tumor receiving direct IL-12 gene therapy without increase in effector cell infiltration (Fig. 1b and c and Fig. 2b). In the antitumor response in vivo, there was a decreased vessel density (Fig. 2b), an increased infiltration of CD8+ T and CD56+ NK cells (Fig. 2c), and increased IFN-␥ secreting cells (Fig. 3b). The therapeutic effect observed in our tumor model was manifested in two phases at the cellular level. The dual effects of therapy, i.e., inhibition of angiogenesis and the immune response, may have been a result of the decreased VEGF expression and the increased secretion of IFN-␥ from the immune cells. We demonstrated the mechanism of antitumor effect by direct IL-12 gene therapy in head and neck squamous cell carcinoma model for the first time. In particular, it is the first time that we measured the number of IFN-␥ secreting cells by ELISPOT assay in the model of direct IL-12 gene therapy. Our data showed that the number of IFN-␥ secreting spleen cells increased by direct IL-12 gene therapy.

5. Conclusion In this study, direct IL-12 gene therapy by intratumoral injection is effective method of therapy against head and neck

squamous cell carcinoma in a murine model. The mechanism of the antitumor effect was likely caused by IL-12-induced expression of IFN-␥ which triggers both the immune response and the antiangiogenic response. These results can serve as the basis for a clinical trial, particularly for the treatment of head and neck squamous cell carcinoma. Now, we are trying to determine the antitumor effect in the combination therapy of direct IL-12 gene therapy and, IL-2 gene therapy, or chemotherapy to eradicate tumor cells completely.

Acknowledgements We thank Dr. Shunsuke Yanoma, the Department of Biochemistry, Research Institute, Kanagawa Cancer Center, for providing murine squamous carcinoma cell line NR-S1. The authors would also like to thank Paul Langman, Ph.D. for his valuable assistance with the English version of this manuscript. This work was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

References [1] Gately MK, Wolitzky AG, Quinn PM, Chizzonite R. Regulation of human cytolytic lymphocyte responses by interleukin-12. Cell Immunol 1992;143:127–42. [2] McKnight AJ, Zimmer GJ, Fogelman I, Wolf SF, Abbas AK. Effects of IL-12 on helper T cell-dependent immune responses in vivo. J Immunol 1994;152:2172–9. [3] Trinchieri G. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity. Annu Rev Immunol 1995;13:251–76. [4] Brunda MJ, Luistro L, Warrier RR, Wright RB, Hubbard BR, Murphy M, et al. Antitumor and antimetastatic activity of interleukin 12 against murine tumors. J Exp Med 1993;178:1223–30. [5] Nastala CL, Edington HD, McKinney TG, Tahara H, Nalesnik MA, Brunda MJ, et al. Recombinant IL-12 administration induces tumor regression in association with IFN-␥ production. J Immunol 1994;153:1697–706. [6] Verbik DJ, Stinson WW, Brunda MJ, Kessinger A, Joshi SS. In vivo therapeutic effects of interleukin-12 against highly metastatic residual lymphoma. Clin Ex Metastasis 1996;14:219–29. [7] Zou JP, Yamamoto N, Fujii T, Takenaka H, Kobayashi M, Herrmann SH, et al. Systemic administration of rIL-12 induces complete tumor regression and protective immunity: response is correlated with a striking reversal of suppressed IFN-␥ production by anti-tumor T cells. Int Immunol 1995;7:1135–45. [8] Strasly M, Cavallo F, Geuna M, Mitola S, Colombo MP, Forni G. IL-12 inhibition of endothelial cell functions and angiogenesis depends on lymphocyte-endothelial cell cross-talk. J Immunol 2001;166:3890–9. [9] Dias S, Thomas H, Balkwill F. Multiple molecular and cellular changes associated with tumour stasis and regression during IL-12 therapy of a murine breast cancer model. Int J Cancer 1998;75:151–7. [10] Voest EE, Kenyon BM, O’Reilly MS, Truitt G, D’Amato RJ, Folkman J. Inhibition of angiogenesis in vivo by interleukin 12. J Natl Cancer Inst 1995;87:581–6.

Y. Imagawa et al. / Auris Nasus Larynx 31 (2004) 239–245 [11] Tannenbaum CS, Wicker N, Armstrong D, Tubbs R, Finke J, Bukowski RM, et al. Cytokine and chemokine expression in tumors of mice receiving systemic therapy with IL-12. J Immunol 1996;156:693–9. [12] Rakhmilevich AL, Turner J, Ford MJ, McCabe D, Sun WH, Sondel PM, et al. Gene gun-mediated skin transfection with interleukin 12 gene results in regression of established primary and metastatic murine tumors. Proc Natl Acad Sci USA 1996;93:6291– 6. [13] Leonard JP, Sherman ML, Fisher GL, Buchanan LJ, Larsen G, Atkins MB, et al. Effects of single-dose interleukin-12 exposure on interleukin-12-associated toxicity and interferon-gamma production. Blood 1997;90:2541–8. [14] Atkins MB, Robertson MJ, Gordon M, Lotze MT, DeCoste M, DuBois JS, et al. Phase I evaluation of intravenous recombinant human interleukin 12 in patients with advanced malignancies. Clin Cancer Res 1997;3:409–17. [15] Chen L, Chen D, Block E, O’Donnell M, Kufe DW, Clinton SK. Eradication of murine bladder carcinoma by intratumor injection of a bicistronic adenoviral vector carrying cDNAs for the IL-12 heterodimer and its inhibition by the IL-12 p40 subunit homodimer. J Immunol 1997;159:351–9. [16] Myers JN, Mank-Seymour A, Zitvogel L, Storkus W, Clarke M, Johnson CS, et al. Interleukin-12 gene therapy prevents establishment of SCC VII squamous cell carcinomas, inhibits tumor growth, and elicits long-term antitumor immunity in syngeneic C3H mice. Laryngoscope 1998;108:261–8. [17] Coughlin CM, Salhany KE, Wysocka M, Aruga E, Kurzawa H, Chang AE, et al. Interleukin-12 and interleukin-18 synergistically induce murine tumor regression which involves inhibition of angiogenesis. J Clin Invest 1998;101:1441–52. [18] Tahara H, Zeh III HJ, Storkus WJ, Pappo I, Watkins SC, Gubler U, et al. Fibroblasts genetically engineered to secrete interleukin 12 can suppress tumor growth and induce antitumor immunity to a murine melanoma in vivo. Cancer Res 1994;54:182–9. [19] Herr W, Schneider J, Lohse AW, Meyer zum Buschenfelde KH, Wolfel T. Detection and quantification of blood-derived CD8+ T lymphocytes secreting tumor necrosis factor alpha in response to HLA-A2.1-binding melanoma and viral peptide antigens. J Immunol Methods 1996;191:131–42. [20] Asai T, Storkus WJ, Whiteside TL. Evaluation of the modified ELISPOT assay for gamma interferon production in cancer patients receiving antitumor vaccines. Clin Diagn Lab Immunol 2000;7:145– 54.

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[21] Meidenbauer N, Harris DT, Spitler LE, Whiteside TL. Generation of PSA-reactive effector cells after vaccination with a PSA-based vaccine in patients with prostate cancer. Prostate 2000;43:88–100. [22] Asselin-Paturel C, Lassau N, Guinebretiere JM, Zhang J, Gay F, Bex F, et al. Transfer of the murine interleukin-12 gene in vivo by a Semliki Forest virus vector induces B16 tumor regression through inhibition of tumor blood vessel formation monitored by Doppler ultrasonography. Gene Ther 1999;6:606–15. [23] Mendiratta SK, Quezada A, Matar M, Wang J, Hebel HL, Long S, et al. Intratumoral delivery of IL-12 gene by polyvinyl polymeric vector system to murine renal and colon carcinoma results in potent antitumor immunity. Gene Ther 1999;6:833–9. [24] Dow SW, Elmslie RE, Fradkin LG, Liggitt DH, Heath TD, Willson AP, et al. Intravenous cytokine gene delivery by lipid-DNA complexes controls the growth of established lung metastases. Hum Gene Ther 1999;10:2961–72. [25] Puisieux I, Odin L, Poujol D, Moingeon P, Tartaglia J, Cox W, et al. Canarypox virus-mediated interleukin 12 gene transfer into murine mammary adenocarcinoma induces tumor suppression and long-term antitumoral immunity. Hum Gene Ther 1998;9:2481–92. [26] Yao L, Sgadari C, Furuke K, Bloom ET, Teruya-Feldstein J, Tosato G. Contribution of natural killer cells to inhibition of angiogenesis by interleukin-12. Blood 1999;93:1612–21. [27] Chan SH, Perussia B, Gupta JW, Kobayashi M, Pospisil M, Young HA, et al. Induction of interferon gamma production by natural killer cell stimulatory factor: characterization of the responder cells and synergy with other inducers. J Exp Med 1991;173:869–79. [28] Frucht DM, Fukao T, Bogdan C, Schindler H, O’Shea JJ, Koyasu S. IFN-gamma production by antigen-presenting cells: mechanisms emerge. Trends Immunol 2001;22:556–60. [29] Schreiber RD, Farrar MA, Hershey GK, Fernandez-Luna J. The structure and function of interferon-gamma receptors. Int J Immunopharmacol 1992;14:413–9. [30] Tsung K, Meko JB, Peplinski GR, Tsung YL, Norton JA. IL-12 induces T helper 1-directed antitumor response. J Immunol 1997;158:3359–65. [31] Yu WG, Ogawa M, Mu J, Umehara K, Tsujimura T, Fujiwara H, et al. IL-12-induced tumor regression correlates with in situ activity of IFN-gamma produced by tumor-infiltrating cells and its secondary induction of anti-tumor pathways. J Leukoc Biol 1997;62:450–7. [32] Tsukuda M, Mochimatsu I, Sakumoto M, Furukawa S, Yuyama S, Yanoma S, et al. Autologous tumor cell killing activity of tumor-associated lymphocytes in patients with head and neck carcinomas. Biotherapy 1993;6:155–61.