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Intratumoral delivery of IL-18 naked DNA induces T-cell activation and Th1 response in a mouse hepatic cancer model

Intratumoral delivery of IL-18 naked DNA induces T-cell activation and Th1 response in a mouse... Background: The novel cytokine, interleukin (IL)-18, is a strong interferon-γ inducer and costimulatory factor in Th1 cell activation. IL-18 triggers IFN-γ production and enhances cytolytic activity in both T and NK cells. However, the exact mechanism of antitumor action of IL-18 remains to be clarified. To determine the effects of IL-18 plasmid DNA on hepatic cancer in mice, CT26 murine colon adenocarcinoma cells were established in mouse liver. Methods: Plasmid vectors encoding IL-18 were transferred directly into the liver 7 days after tumor injection to restrict IL-18 expression within the tumor site. The IL-18 protein level was increased in the liver 4 days after plasmid injection, and a marked antitumoral effect was observed at day 7. Antitumor effects were evaluated by measuring tumor regression, immune cell population, and IFN-γ production. Results: The IL-18 plasmid controlled the growth of hepatic tumors and proliferation of splenic immune cells. Moreover, treatment of CT26 tumors with the IL-18 plasmid significantly enhanced the population of the effector T and NK cells in the spleen and peripheral blood. In spleen, the + Low population of CD4 CD62 cells was augmented in response to IL-18 on day 7. These results are + + consistent with the increase in CD4 T cells secreting IFN-γ, but not CD8 T cells. The marked reduction of tumor growth in tumor-bearing mice was associated with the maintenance of IFN-γ production in spleen in response to IL-18. These antitumoral effects were maintained until 14 days after plasmid injection. Conclusion: Our results suggest that direct plasmid DNA transfer of IL-18 with no accompanying reagents to augment transfection efficiency may be useful in tumor immunotherapy. Page 1 of 7 (page number not for citation purposes) BMC Cancer 2007, 7:87 http://www.biomedcentral.com/1471-2407/7/87 phy method, according to the manufacturer's protocol Background Effective eradication of established tumors and generation (Qiagen, Chatsworth, CA). of a lasting systemic immune response with a simple gene delivery system are important goals for cancer gene immu- Tumor model and therapeutic protocol notherapy [1]. Cytokines are the most extensively studied CT26, an undifferentiated murine adenocarcinoma, was immunostimulatory agents in cancer gene therapy [2]. induced by rectal injection of N-nitroso-N-methyl- Interferon-γ-inducing factor (IL-18) is a recently character- urethane in BALB/c mice. Colorectal cancer is often meta- ized murine and human cytokine. The murine IL-18 gene static, the most common site of metastasis being the liver. encodes a precursor protein of 192 amino acids, which is CT26 cells were suspended for implantation at 1 × 10 processed to a mature protein containing 157 residues [3]. cells/50 μl saline. Colon carcinoma was established in the This cytokine, produced by Kupffer cells, is a potent left lateral lobe of 6- to 8-week-old male BALB/c mice by inducer of IFN-γ production by T cells and a costimulatory an abdominal operation. Treatments began 7 days after a factor for T cell activation [4,5]. Accumulating evidence defined solid tumor growth was identified within the that IL-18 is a multifunctional cytokine that shares several injected lobe. For intratumoral injection, naked DNA (10, biological activities with IL-12 has led to a series of studies 25, and 50 μg) was diluted in 50 μl saline, and injected on its effects on T and NK cells [4]. Analogous to IL-12, IL- into the parenchyma of the lower surface of the left liver 18 stimulates T cell proliferation and NK cell activity [3]. lobe via insulin syringes (31 gauge, 0.8 inch needles; Bec- The finding that IL-18 stimulates the differentiation of ton Dickinson, Franklin Lake, NJ). Mice were sacrificed at Th1 cells that produce cytokines necessary for the devel- 1, 4, 7, and 14 days after DNA treatment, and tumor opment of cell-mediated immune responses suggests a growth monitored by measuring liver weight [13]. prominent role in defense against tumors [6]. Similarly, high IFN-γ levels are induced by IL-18 in splenic-derived Flow cytometric analysis CD4 T cells in the presence of B cells or adherent cells [7]. Total nuclear cells in the peripheral blood were isolated Furthermore, IL-18-transfected tumor cell vaccines and by erythrocyte lysis with ammonium chloride solution local delivery of the IL-18 gene as naked DNA via a gene (PharM Lyse, Becton Dickinson). Single spleen cell sus- gun or viral vectors has been extensively investigated. pensions were obtained by teasing apart spleen tissue and Numerous animal studies show that IL-18 has potent disaggregating cells through a 70 μm mesh. Briefly, splen- antitumor effects, but induces side-effects upon systemic ocytes and PBMCs were incubated with PE-, Cy5, or FITC- administration [8]. Direct intratumoral DNA administra- conjugated Abs, and the corresponding isotypes (pur- tion is reliable and reproducible, and may limit the need chased from Pharmingen, San Diego, CA, USA). Anti- for systemic cytokine administration [9-11]. Here, we mouse NK (clone DX5), -CD3, -CD4, -CD8, -Cd62L, - report the effects of direct intratumoral injection of a non- CD69, -CD19, and -CD11b Abs were used to stain the viral plasmid vector encoding murine IL-18 DNA in estab- populations of NK cells, macrophages, T-cells, and B-cells lished CT26 liver tumors [12]. in the spleen. Samples were analyzed for surface pheno- types using FACS Vantage (Becton-Dickinson, San Jose, CA, USA) and CellQuest software (Becton-Dickinson Methods Animals and cell lines Labware). BALB/c (H-2 ) mice (6 to 8 weeks old) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). All Flow cytometric assessment of intracellular cytokine mice were housed in specific pathogen-free conditions, in production accordance with institutional guidelines. CT26 tumor For parallel evaluation of intracellular cytokines, surface cells were maintained in RPMI 1640 cell culture medium staining was initially performed for CT26 lysate-stimu- (Biowhittaker, Walkersville, MD, USA) supplemented lated splenocytes from each group. Cells were fixed with with 10% heat-inactivated fetal bovine serum, 2 mM L- 4% paraformaldehyde solution for 20 min, treated with glutamine, 100 U/ml streptomycin, and 100 μg/ml peni- permeabilization buffer (0.1% saponin, 1% FBS in PBS) cillin. for 5 min at RT in the dark, and washed. Intracellular staining was performed by incubation of mAb to IFN-γ Plasmids with cell pellets for 20 min at RT in the dark. Cells were The 11 kb mIL-18 DNA expression plasmid vector, washed, and measured using a flow cytometer. pCEP4-mIL18, was constructed using a CMV early Assessment of cytokines (IL-18, IFN-γ) enhancer/promoter/EBNA-1-based pCEP4 plasmid vector (Invitrogen, San Diego, CA) with an ampicillin selection We obtained total liver protein by homogenization of fro- gene. Plasmid DNA was purified in the absence of ethid- zen tissue in extraction buffer containing 1% Triton X- ium bromide using a commercial column chromatogra- 100, 10 mM Tris-HCL (pH 7.6), 5 mM EDTA, 50 mM NaCl, 30 mM Na P O , 50 mM NaF, 200 μM Na VO , 2 4 2 7 3 4 Page 2 of 7 (page number not for citation purposes) BMC Cancer 2007, 7:87 http://www.biomedcentral.com/1471-2407/7/87 mM PMSF, 5 μg/ml aprotinin, 1 μg/ml pepstatin A, and 2 gest that a ~ 2.5-fold higher concentration of IL-18 mod- μg of leupeptin. The suspension was centrifuged at 14,000 ulates the immune cell population (T cells and NK cells) r.p.m. for 20 min at 4°, and the supernatant stored at - around the time of tumor regression. 70°. Supernatant fractions were analyzed with the IL-18 ELISA kit (MBL, Nagoya, Japan). IFN-γ concentrations in Regression of established CT26 hepatic tumors after direct the splenocyte culture supernatants were determined with intratumoral mIL-18 gene transfer specific ELISA performed according to the manufacturer's CT26-tumor bearing mice were treated with 50 μg pCEP4 instructions (R&D Systems, Minneapolis, MN, USA). The or mIL-18-pCEP4, and tumor growth measured at the absolute cytokine levels were calculated by comparison to indicated times (Figure 3). Tumor growth was signifi- assay performance in the presence of known quantities of cantly lower in the groups treated with mIL-18 plasmid recombinant cytokine standards. than those administered control vector (pCEP4). Most of the control mice developed multiple large tumors in the Statistical Analysis liver, but pIL-18 gene therapy markedly inhibited tumor growth at 14 days and 21 days after tumor implantation An unpaired t-test was employed to compare tumor (see additional file 1). Differences between the control weight, quantitative cytokine production, and flow and mIL-18-treated groups were statistically significant (p cytometry analyses. All analyses were performed with Sig- < 0.049 on day 14), as confirmed with the Student's t test. maPlot 2000. P < 0.05 was considered significant. Regression phenotypic changes of immune effector cells in spleen and blood samples Results Levels of transgene expression within the treated tumor Changes in cell surface activation markers define specific site phases of T cell activation, and may be used to distinguish To determine the efficiency of gene transfection, mouse between naïve, effector, and memory T cell populations IL-18 protein expression in the tumor site was evaluated [18]. Resting naïve T cells express low levels of CD44 and by ELISA. mIL-18 protein was detected in all samples integrins, LFA-1 and VLA-4, and high levels of CD45RB injected with control vector or IL-18 plasmid (Figure 1). and CD62L (L-selectin). Upon antigen stimulation, naïve However, significant differences were evident in the mIL- T cells transform into large blastoid cells, and the pheno- hi hi 18 protein levels between the control and the 50 μg plas- types of these effector cells become CD44 LFA-1 VLA- mid-injected group. Protein levels in CT26 treated with control or mIL-18 plasmid vector ranged from 500 to 900 mg in the injected lobe, since tumors developed differen- tially after each treatment. The IL-18 protein level was 2– 2.5 fold higher than background when mice were sacri- ficed 4 days after plasmid introduction. The results suggest that mIL-18 DNA is expressed in the tumor site, and releases bioactive IL-18. In vivo kinetic studies of the immune cell population in spleen We performed phenotype analysis of spleen cells from pCEP4, mIL-18 DNA-treated CT26 tumor-bearing mice on days 2, 5, and 7 after gene treatment, using flow cytom- etry. The antitumoral response of tumor-bearing mice treated with 50 μg of DNA was significantly greater than that of mice administered 10 or 25 μg DNA. Intratumoral delivery of 50 μg of plasmid DNA for each of four treat- Detection of mIL-18 si Figure 1 tes plasmid expression in DNA treatment ments resulted in immune cell augmentation in CD4 Detection of mIL-18 plasmid expression in DNA cells (30.6% total lymphocytes versus 15% in control), treatment sites. BALB/c mice were inoculated with murine CD8 cells (13.5% total lymphocytes versus 6% in con- CT26 cells in the left liver lobe, and treated intratumorally trol), and NK cells (5.6% total lymphocytes versus 3.5% with varying doses of control plasmid or that expressing IL- in control) on day 7 (Figure 2). Other groups report that 18 until day 7 after tumor inoculation. Liver tissue was IL-18 upregulates ICAM-1 expression on monocytes and T obtained on day 4 after direct injection of IL-18 plasmid cells, presenting further evidence of IL-18-stimulated T DNA, and ELISA used to evaluate IL-18 protein levels. Rep- resentative data determined from two separate experiments. cell recruitment [14-17]. These findings, together with the above kinetic data on the immune cell populations, sug- Page 3 of 7 (page number not for citation purposes) BMC Cancer 2007, 7:87 http://www.biomedcentral.com/1471-2407/7/87 IL-18 tumors Figure 3 gene transfer results in regression of CT26 liver IL-18 gene transfer results in regression of CT26 liver tumors. CT26 cells (1.0 × 10 ) were injected into a left lobe of the livers of 6-8-week-old BALB/c mice. pCEP4 or mIL-18 plasmid DNA was injected directly into the tumors on day 7. Tumor growth was measured on days 1, 4, 7, and 14 after plasmid implantation. Data are presented as the mean of tumor weights of 8 mice/group. hi lo - 4 CD45RB CD62L [18,19]. Other activation markers, such as CD25 and CD69, are additionally upregulated [20,21]. Accordingly, flow cytometric analysis of spleen and blood cells from pCEP4 or mIL-18 plasmid-treated CT26 tumor-bearing mice at 7 days after gene treatment was performed. As expected (Table 1), a marked increase + Low (p < 0.04) in the CD4 CD62 subset was evident in spleen and blood cells following injection of mIL-18 plas- mid DNA (30.05 ± 1.85% in spleen and 30.58 ± 4.59% in blood), compared with the pCEP4 DNA-treated control group (17.75 ± 0.92% in spleen and 17.62 ± 2.15% in blood). IL-18 induces IFN-γ production in T cells [5,7,22]. To fur- ther characterize the mIL-18- induced immune response, intracellular cytokine staining for IFN-γ production was performed for subsets of CT26-specific T cells. Splenocytes were prepared and pooled from CT26 tumor-bearing mice treated with pCEP4 or mIL-18 plasmid incubated with medium alone or supplemented with CT26 lysates. The frequencies of functional CT26-specific T lymphocytes were estimated by intracellular IFN-γ staining, and quan- Immune r IL-18 plasmid DNA Figure 2 esponse is enhanced by a single injection of naked + + + + titation of activated CD69 , IFN-γ , CD4 , and CD8 cells Immune response is enhanced by a single injection of (Figure 4). While control mice treated with pCEP4 did not naked IL-18 plasmid DNA. Each group (consisting of 4 generate a CT26-specific T cell response, those adminis- mice) was administered an intratumoral injection of pCEP4 tered pmIL-18 contained a significant number of IFN-γ or naked mIL-18 DNA. Spleens of CT26 tumor-bearing mice + + producing CD4 T cells, but not CD8 T cells (data not treated with plasmids were harvested at 7 days after plasmid shown). The data suggest that the IL-18-mediated injection, and cells analyzed by flow cytometry using the cor- responding FITC- or PE- or PE-Cy5-labeled antibodies and immune response predominantly involves IFN-γ-produc- isotype control. ing CD4 T cells. Page 4 of 7 (page number not for citation purposes) BMC Cancer 2007, 7:87 http://www.biomedcentral.com/1471-2407/7/87 + Low + Low + + Table 1: Frequency of CD4 CD62 , CD8 CD62 , DX5 , CD19 cells in spleen and blood: major subsets Treatment Group Spleens Blood + Low + Low + + + Low + Low + + CD4 CD62 (%) CD8 CD62 (%) DX5 (%) CD19 (%) CD4 CD62 (%) CD8 CD62 (%) DX5 (%) CD19 (%) pCEP4 17.75 ± 0.92 0.997 ± 0.05 3.39 ± 1.26 44.51 ± 3.40 17.62 ± 2.15 5.93 ± 0.96 15.45 ± 1.19 28.97 ± 3.52 a a pIL-18 30.05 ± 1.85 1.995 ± 0.34 1.73 ± 0.08 41.57 ± 1.09 30.58 ± 4.59 9.88 ± 0.94 18.4 ± 2.91 36.49 ± 5.95 Spleen and blood samples of CT26 tumor-bearing mice treated with pCEP4 or mIL-18 plasmid DNA were harvested 7 days after gene treatment, and cells analyzed by flow cytometry using the corresponding FITC-, PE-, or PE-Cy5-conjugated antibodies and isotype control. Data are presented as means ± SD of 8 mice/group. P < 0.04, compared with the pCEP4 control-treated group. DNA vaccination of mice resulted in broad immune (1.026 ± 0.03 ng/10 cells/72 h; P = 0.006). This may responses, characterized by the activation of B cells, helper explain the continued maintenance of tumor suppression + + and cytotoxic CD8 T cells [23]. Based on these CD4 in mIL-18-treated mice, compared to control mice (Figure results, we propose that mIL-18 overexpression in the 5). Interestingly, in our studies with tumor-bearing mice, tumor site induces phenotypic changes in the T cell and B intratumoral injection with an IL-18 plasmid did not pre- cell subsets, and protects against tumor development by vent CT26 tumor development, although augmentation stimulating IFN-γ production by CD4 T cells. of T cells and maintenance of the IFN-γ level during 2 weeks appeared to reject tumors. Thus, while direct Intratumoral injection of mIL-18 plasmid DNA elevates cytokine gene transfer possibly has therapeutic potential, IFN-γ production by splenocytes expression of cytokine genes may be elevated and sus- The IFN-γ levels of cultured splenocyte supernatants were tained significantly during tumor development. Other compared. Enhanced secretion of IFN-γ by cultured splen- reports show that the effects of cotransfection of ICE and ocytes was evident in animals treated with plasmid mIL- pro-IL-18 cDNA are superior to those of pro-IL-18 cDNA 18 DNA (1.278 ± 0.06 ng/10 cells/72 h), compared to alone, and result in enhanced bioactivity of IL-18 [24]. splenocytes from mice treated with pCEP4 control vector Intrace Figure 4 llular IFN-γ production by CT26-specific T cells from pmIL-18-treated mice Intracellular IFN-γ production by CT26-specific T cells from pmIL-18-treated mice. Splenocytes from CT26-tumor bearing mice treated with three rounds of pCEP4 or mIL-18 were pooled and incubated in medium alone or that containing + + + CT26 lysates. Numbers within the gates in the FACS plots depict the percentage of CD69 IFN-γ CD4 cells. Data are pre- sented from experiments that were repeated at least twice. Page 5 of 7 (page number not for citation purposes) BMC Cancer 2007, 7:87 http://www.biomedcentral.com/1471-2407/7/87 Additional material Additional File 1 Representative results of pIL-18 gene therapy in hepatic tumor bearing mice. Visible tumor foci are generally formed in the liver within a week of injection of CT26 cells, and these grew to a few millimeters in diameter by day 21. Treatment was initiated 7 days after tumor cell injection and representative results of gene therapy were showed at 14 days and 21 days after tumor cell injection. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2407-7-87-S1.jpeg] Acknowledgements This research was supported by grants from the IN-SUNG Foundation for Medical Research, Seoul, Korea El m Figure 5 evated I IL-18 plasmid DNA FN-γ levels after intratumoral injection of nonviral References Elevated IFN-γ levels after intratumoral injection of 1. Somiari S, Glasspool-Malone J, Drabick JJ, Gilbert RA, Heller R, Jaroszeski MJ, Malone RW: Theory and in vivo application of nonviral mIL-18 plasmid DNA. Splenocytes from CT26 electroporative gene delivery. Mol Ther 2000, 2(3):178-187. tumor-bearing mice after gene therapy were collected at 4, 2. Hartikka J, Sukhu L, Buchner C, Hazard D, Bozoukova V, Margalith M, 7, and 14 days after gene treatment, and cultured with anti- Nishioka WK, Wheeler CJ, Manthorp M, Sawdey M: Electropora- CD3 and anti-CD28 antibodies for 3 days before performing tion-facilitated delivery of plasmid DNA in skeletal muscle: plasmid dependence of muscle damage and effect of ELISA (* = 0.006). poloxamer 188. Mol Ther 2001, 4(5):407-415. 3. Okamura H, Tsutsi H, Komatsu T, Yutsudo M, Hakura A, Tanimoto T, Torigoe K, Okura T, Nukada Y, Hattori K, et al.: Cloning of a new cytokine that induces IFN-gamma production by T cells. Nature 1995, 378(6552):88-91. Conclusion 4. Dinarello CA, Novick D, Puren AJ, Fantuzzi G, Shapiro L, Muhl H, Recent trials using cytokine genes show that intratumoral Yoon DY, Reznikov LL, Kim SH, Rubinstein M: Overview of inter- leukin-18: more than an interferon-gamma inducing factor. IL-18 gene transfer is a feasible procedure, but exerts only J Leukoc Biol 1998, 63(6):658-664. mild antitumor effects [24]. Gene transfer with increased 5. Kohno K, Kataoka J, Ohtsuki T, Suemoto Y, Okamoto I, Usui M, Ikeda mIL-18 doses may enhance antitumor efficacy. In this M, Kurimoto M: IFN-gamma-inducing factor (IGIF) is a costim- ulatory factor on the activation of Th1 but not Th2 cells and study, we validate the antitumoral efficacy of mIL-18 over- exerts its effect independently of IL-12. J Immunol 1997, expression in CT26 tumor-bearing mice by using plasmid 158(4):1541-1550. 6. Jiang J, Yamato E, Miyazaki J: Intravenous delivery of naked plas- vectors to transfer the mouse IL-18 gene alone. Our data mid DNA for in vivo cytokine expression. Biochem Biophys Res show marked inhibition of tumor growth and significant Commun 2001, 289(5):1088-1092. phenotypic changes of immune cells, suggesting that mIL- 7. 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Missol E, Sochanik A, Szala S: Introduction of murine Il-4 gene CY carried out the molecular immunology studies, includ- into B16(F10) melanoma tumors by direct gene transfer ing virus work, and drafted the manuscript. J, HJ and EY with DNA-liposome complexes. Cancer Lett 1995, 97(2):189-193. performed the in vivo study, including mouse surgery, and 11. Parker SE, Ducharme S, Norman J, Wheeler CJ: Tissue distribution statistical analyses. CH and JW participated in the design of the cytofectin component of a plasmid-DNA/cationic lipid complex following intravenous administration in mice. Hum of the study. SJ conceived the study, and aided in its Gene Ther 1997, 8(4):393-401. design and coordination. All authors read and approved 12. Furuya D, Yagihashi A, Yajima T, Kobayashi D, Orita K, Kurimoto M, the final manuscript. Watanabe N: An immuno-polymerase chain reaction assay for human interleukin-18. J Immunol Methods 2000, 238(1- 2):173-180. 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Ju DW, Yang Y, Tao Q, Song WG, He L, Chen G, Gu S, Ting CC, Cao X: Interleukin-18 gene transfer increases antitumor effects of suicide gene therapy through efficient induction of antitu- mor immunity. Gene Ther 2000, 7(19):1672-1679. 17. Wang Q, Yu H, Ju DW, He L, Pan JP, Xia DJ, Zhang LH, Cao X: Intra- tumoral IL-18 gene transfer improves therapeutic efficacy of antibody-targeted superantigen in established murine melanoma. Gene Ther 2001, 8(7):542-550. 18. Swain SL, Croft M, Dubey C, Haynes L, Rogers P, Zhang X, Bradley LM: From naive to memory T cells. Immunol Rev 1996, 150:143-167. 19. Doherty PC: Cytotoxic T cell effector and memory function in viral immunity. Curr Top Microbiol Immunol 1996, 206:1-14. 20. Leonard WJ, Gnarra JR, Napolitano M, Sharon M: Structure, func- tion, and regulation of the interleukin-2 receptor and identi- fication of a novel immune activation gene. Philos Trans R Soc Lond B Biol Sci 1990, 327(1239):187-192. 21. 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Intratumoral delivery of IL-18 naked DNA induces T-cell activation and Th1 response in a mouse hepatic cancer model

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Springer Journals
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Copyright © 2007 by Chang et al; licensee BioMed Central Ltd.
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Biomedicine; Cancer Research; Oncology; Surgical Oncology; Health Promotion and Disease Prevention; Biomedicine general; Medicine/Public Health, general
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10.1186/1471-2407-7-87
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17519043
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Abstract

Background: The novel cytokine, interleukin (IL)-18, is a strong interferon-γ inducer and costimulatory factor in Th1 cell activation. IL-18 triggers IFN-γ production and enhances cytolytic activity in both T and NK cells. However, the exact mechanism of antitumor action of IL-18 remains to be clarified. To determine the effects of IL-18 plasmid DNA on hepatic cancer in mice, CT26 murine colon adenocarcinoma cells were established in mouse liver. Methods: Plasmid vectors encoding IL-18 were transferred directly into the liver 7 days after tumor injection to restrict IL-18 expression within the tumor site. The IL-18 protein level was increased in the liver 4 days after plasmid injection, and a marked antitumoral effect was observed at day 7. Antitumor effects were evaluated by measuring tumor regression, immune cell population, and IFN-γ production. Results: The IL-18 plasmid controlled the growth of hepatic tumors and proliferation of splenic immune cells. Moreover, treatment of CT26 tumors with the IL-18 plasmid significantly enhanced the population of the effector T and NK cells in the spleen and peripheral blood. In spleen, the + Low population of CD4 CD62 cells was augmented in response to IL-18 on day 7. These results are + + consistent with the increase in CD4 T cells secreting IFN-γ, but not CD8 T cells. The marked reduction of tumor growth in tumor-bearing mice was associated with the maintenance of IFN-γ production in spleen in response to IL-18. These antitumoral effects were maintained until 14 days after plasmid injection. Conclusion: Our results suggest that direct plasmid DNA transfer of IL-18 with no accompanying reagents to augment transfection efficiency may be useful in tumor immunotherapy. Page 1 of 7 (page number not for citation purposes) BMC Cancer 2007, 7:87 http://www.biomedcentral.com/1471-2407/7/87 phy method, according to the manufacturer's protocol Background Effective eradication of established tumors and generation (Qiagen, Chatsworth, CA). of a lasting systemic immune response with a simple gene delivery system are important goals for cancer gene immu- Tumor model and therapeutic protocol notherapy [1]. Cytokines are the most extensively studied CT26, an undifferentiated murine adenocarcinoma, was immunostimulatory agents in cancer gene therapy [2]. induced by rectal injection of N-nitroso-N-methyl- Interferon-γ-inducing factor (IL-18) is a recently character- urethane in BALB/c mice. Colorectal cancer is often meta- ized murine and human cytokine. The murine IL-18 gene static, the most common site of metastasis being the liver. encodes a precursor protein of 192 amino acids, which is CT26 cells were suspended for implantation at 1 × 10 processed to a mature protein containing 157 residues [3]. cells/50 μl saline. Colon carcinoma was established in the This cytokine, produced by Kupffer cells, is a potent left lateral lobe of 6- to 8-week-old male BALB/c mice by inducer of IFN-γ production by T cells and a costimulatory an abdominal operation. Treatments began 7 days after a factor for T cell activation [4,5]. Accumulating evidence defined solid tumor growth was identified within the that IL-18 is a multifunctional cytokine that shares several injected lobe. For intratumoral injection, naked DNA (10, biological activities with IL-12 has led to a series of studies 25, and 50 μg) was diluted in 50 μl saline, and injected on its effects on T and NK cells [4]. Analogous to IL-12, IL- into the parenchyma of the lower surface of the left liver 18 stimulates T cell proliferation and NK cell activity [3]. lobe via insulin syringes (31 gauge, 0.8 inch needles; Bec- The finding that IL-18 stimulates the differentiation of ton Dickinson, Franklin Lake, NJ). Mice were sacrificed at Th1 cells that produce cytokines necessary for the devel- 1, 4, 7, and 14 days after DNA treatment, and tumor opment of cell-mediated immune responses suggests a growth monitored by measuring liver weight [13]. prominent role in defense against tumors [6]. Similarly, high IFN-γ levels are induced by IL-18 in splenic-derived Flow cytometric analysis CD4 T cells in the presence of B cells or adherent cells [7]. Total nuclear cells in the peripheral blood were isolated Furthermore, IL-18-transfected tumor cell vaccines and by erythrocyte lysis with ammonium chloride solution local delivery of the IL-18 gene as naked DNA via a gene (PharM Lyse, Becton Dickinson). Single spleen cell sus- gun or viral vectors has been extensively investigated. pensions were obtained by teasing apart spleen tissue and Numerous animal studies show that IL-18 has potent disaggregating cells through a 70 μm mesh. Briefly, splen- antitumor effects, but induces side-effects upon systemic ocytes and PBMCs were incubated with PE-, Cy5, or FITC- administration [8]. Direct intratumoral DNA administra- conjugated Abs, and the corresponding isotypes (pur- tion is reliable and reproducible, and may limit the need chased from Pharmingen, San Diego, CA, USA). Anti- for systemic cytokine administration [9-11]. Here, we mouse NK (clone DX5), -CD3, -CD4, -CD8, -Cd62L, - report the effects of direct intratumoral injection of a non- CD69, -CD19, and -CD11b Abs were used to stain the viral plasmid vector encoding murine IL-18 DNA in estab- populations of NK cells, macrophages, T-cells, and B-cells lished CT26 liver tumors [12]. in the spleen. Samples were analyzed for surface pheno- types using FACS Vantage (Becton-Dickinson, San Jose, CA, USA) and CellQuest software (Becton-Dickinson Methods Animals and cell lines Labware). BALB/c (H-2 ) mice (6 to 8 weeks old) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). All Flow cytometric assessment of intracellular cytokine mice were housed in specific pathogen-free conditions, in production accordance with institutional guidelines. CT26 tumor For parallel evaluation of intracellular cytokines, surface cells were maintained in RPMI 1640 cell culture medium staining was initially performed for CT26 lysate-stimu- (Biowhittaker, Walkersville, MD, USA) supplemented lated splenocytes from each group. Cells were fixed with with 10% heat-inactivated fetal bovine serum, 2 mM L- 4% paraformaldehyde solution for 20 min, treated with glutamine, 100 U/ml streptomycin, and 100 μg/ml peni- permeabilization buffer (0.1% saponin, 1% FBS in PBS) cillin. for 5 min at RT in the dark, and washed. Intracellular staining was performed by incubation of mAb to IFN-γ Plasmids with cell pellets for 20 min at RT in the dark. Cells were The 11 kb mIL-18 DNA expression plasmid vector, washed, and measured using a flow cytometer. pCEP4-mIL18, was constructed using a CMV early Assessment of cytokines (IL-18, IFN-γ) enhancer/promoter/EBNA-1-based pCEP4 plasmid vector (Invitrogen, San Diego, CA) with an ampicillin selection We obtained total liver protein by homogenization of fro- gene. Plasmid DNA was purified in the absence of ethid- zen tissue in extraction buffer containing 1% Triton X- ium bromide using a commercial column chromatogra- 100, 10 mM Tris-HCL (pH 7.6), 5 mM EDTA, 50 mM NaCl, 30 mM Na P O , 50 mM NaF, 200 μM Na VO , 2 4 2 7 3 4 Page 2 of 7 (page number not for citation purposes) BMC Cancer 2007, 7:87 http://www.biomedcentral.com/1471-2407/7/87 mM PMSF, 5 μg/ml aprotinin, 1 μg/ml pepstatin A, and 2 gest that a ~ 2.5-fold higher concentration of IL-18 mod- μg of leupeptin. The suspension was centrifuged at 14,000 ulates the immune cell population (T cells and NK cells) r.p.m. for 20 min at 4°, and the supernatant stored at - around the time of tumor regression. 70°. Supernatant fractions were analyzed with the IL-18 ELISA kit (MBL, Nagoya, Japan). IFN-γ concentrations in Regression of established CT26 hepatic tumors after direct the splenocyte culture supernatants were determined with intratumoral mIL-18 gene transfer specific ELISA performed according to the manufacturer's CT26-tumor bearing mice were treated with 50 μg pCEP4 instructions (R&D Systems, Minneapolis, MN, USA). The or mIL-18-pCEP4, and tumor growth measured at the absolute cytokine levels were calculated by comparison to indicated times (Figure 3). Tumor growth was signifi- assay performance in the presence of known quantities of cantly lower in the groups treated with mIL-18 plasmid recombinant cytokine standards. than those administered control vector (pCEP4). Most of the control mice developed multiple large tumors in the Statistical Analysis liver, but pIL-18 gene therapy markedly inhibited tumor growth at 14 days and 21 days after tumor implantation An unpaired t-test was employed to compare tumor (see additional file 1). Differences between the control weight, quantitative cytokine production, and flow and mIL-18-treated groups were statistically significant (p cytometry analyses. All analyses were performed with Sig- < 0.049 on day 14), as confirmed with the Student's t test. maPlot 2000. P < 0.05 was considered significant. Regression phenotypic changes of immune effector cells in spleen and blood samples Results Levels of transgene expression within the treated tumor Changes in cell surface activation markers define specific site phases of T cell activation, and may be used to distinguish To determine the efficiency of gene transfection, mouse between naïve, effector, and memory T cell populations IL-18 protein expression in the tumor site was evaluated [18]. Resting naïve T cells express low levels of CD44 and by ELISA. mIL-18 protein was detected in all samples integrins, LFA-1 and VLA-4, and high levels of CD45RB injected with control vector or IL-18 plasmid (Figure 1). and CD62L (L-selectin). Upon antigen stimulation, naïve However, significant differences were evident in the mIL- T cells transform into large blastoid cells, and the pheno- hi hi 18 protein levels between the control and the 50 μg plas- types of these effector cells become CD44 LFA-1 VLA- mid-injected group. Protein levels in CT26 treated with control or mIL-18 plasmid vector ranged from 500 to 900 mg in the injected lobe, since tumors developed differen- tially after each treatment. The IL-18 protein level was 2– 2.5 fold higher than background when mice were sacri- ficed 4 days after plasmid introduction. The results suggest that mIL-18 DNA is expressed in the tumor site, and releases bioactive IL-18. In vivo kinetic studies of the immune cell population in spleen We performed phenotype analysis of spleen cells from pCEP4, mIL-18 DNA-treated CT26 tumor-bearing mice on days 2, 5, and 7 after gene treatment, using flow cytom- etry. The antitumoral response of tumor-bearing mice treated with 50 μg of DNA was significantly greater than that of mice administered 10 or 25 μg DNA. Intratumoral delivery of 50 μg of plasmid DNA for each of four treat- Detection of mIL-18 si Figure 1 tes plasmid expression in DNA treatment ments resulted in immune cell augmentation in CD4 Detection of mIL-18 plasmid expression in DNA cells (30.6% total lymphocytes versus 15% in control), treatment sites. BALB/c mice were inoculated with murine CD8 cells (13.5% total lymphocytes versus 6% in con- CT26 cells in the left liver lobe, and treated intratumorally trol), and NK cells (5.6% total lymphocytes versus 3.5% with varying doses of control plasmid or that expressing IL- in control) on day 7 (Figure 2). Other groups report that 18 until day 7 after tumor inoculation. Liver tissue was IL-18 upregulates ICAM-1 expression on monocytes and T obtained on day 4 after direct injection of IL-18 plasmid cells, presenting further evidence of IL-18-stimulated T DNA, and ELISA used to evaluate IL-18 protein levels. Rep- resentative data determined from two separate experiments. cell recruitment [14-17]. These findings, together with the above kinetic data on the immune cell populations, sug- Page 3 of 7 (page number not for citation purposes) BMC Cancer 2007, 7:87 http://www.biomedcentral.com/1471-2407/7/87 IL-18 tumors Figure 3 gene transfer results in regression of CT26 liver IL-18 gene transfer results in regression of CT26 liver tumors. CT26 cells (1.0 × 10 ) were injected into a left lobe of the livers of 6-8-week-old BALB/c mice. pCEP4 or mIL-18 plasmid DNA was injected directly into the tumors on day 7. Tumor growth was measured on days 1, 4, 7, and 14 after plasmid implantation. Data are presented as the mean of tumor weights of 8 mice/group. hi lo - 4 CD45RB CD62L [18,19]. Other activation markers, such as CD25 and CD69, are additionally upregulated [20,21]. Accordingly, flow cytometric analysis of spleen and blood cells from pCEP4 or mIL-18 plasmid-treated CT26 tumor-bearing mice at 7 days after gene treatment was performed. As expected (Table 1), a marked increase + Low (p < 0.04) in the CD4 CD62 subset was evident in spleen and blood cells following injection of mIL-18 plas- mid DNA (30.05 ± 1.85% in spleen and 30.58 ± 4.59% in blood), compared with the pCEP4 DNA-treated control group (17.75 ± 0.92% in spleen and 17.62 ± 2.15% in blood). IL-18 induces IFN-γ production in T cells [5,7,22]. To fur- ther characterize the mIL-18- induced immune response, intracellular cytokine staining for IFN-γ production was performed for subsets of CT26-specific T cells. Splenocytes were prepared and pooled from CT26 tumor-bearing mice treated with pCEP4 or mIL-18 plasmid incubated with medium alone or supplemented with CT26 lysates. The frequencies of functional CT26-specific T lymphocytes were estimated by intracellular IFN-γ staining, and quan- Immune r IL-18 plasmid DNA Figure 2 esponse is enhanced by a single injection of naked + + + + titation of activated CD69 , IFN-γ , CD4 , and CD8 cells Immune response is enhanced by a single injection of (Figure 4). While control mice treated with pCEP4 did not naked IL-18 plasmid DNA. Each group (consisting of 4 generate a CT26-specific T cell response, those adminis- mice) was administered an intratumoral injection of pCEP4 tered pmIL-18 contained a significant number of IFN-γ or naked mIL-18 DNA. Spleens of CT26 tumor-bearing mice + + producing CD4 T cells, but not CD8 T cells (data not treated with plasmids were harvested at 7 days after plasmid shown). The data suggest that the IL-18-mediated injection, and cells analyzed by flow cytometry using the cor- responding FITC- or PE- or PE-Cy5-labeled antibodies and immune response predominantly involves IFN-γ-produc- isotype control. ing CD4 T cells. Page 4 of 7 (page number not for citation purposes) BMC Cancer 2007, 7:87 http://www.biomedcentral.com/1471-2407/7/87 + Low + Low + + Table 1: Frequency of CD4 CD62 , CD8 CD62 , DX5 , CD19 cells in spleen and blood: major subsets Treatment Group Spleens Blood + Low + Low + + + Low + Low + + CD4 CD62 (%) CD8 CD62 (%) DX5 (%) CD19 (%) CD4 CD62 (%) CD8 CD62 (%) DX5 (%) CD19 (%) pCEP4 17.75 ± 0.92 0.997 ± 0.05 3.39 ± 1.26 44.51 ± 3.40 17.62 ± 2.15 5.93 ± 0.96 15.45 ± 1.19 28.97 ± 3.52 a a pIL-18 30.05 ± 1.85 1.995 ± 0.34 1.73 ± 0.08 41.57 ± 1.09 30.58 ± 4.59 9.88 ± 0.94 18.4 ± 2.91 36.49 ± 5.95 Spleen and blood samples of CT26 tumor-bearing mice treated with pCEP4 or mIL-18 plasmid DNA were harvested 7 days after gene treatment, and cells analyzed by flow cytometry using the corresponding FITC-, PE-, or PE-Cy5-conjugated antibodies and isotype control. Data are presented as means ± SD of 8 mice/group. P < 0.04, compared with the pCEP4 control-treated group. DNA vaccination of mice resulted in broad immune (1.026 ± 0.03 ng/10 cells/72 h; P = 0.006). This may responses, characterized by the activation of B cells, helper explain the continued maintenance of tumor suppression + + and cytotoxic CD8 T cells [23]. Based on these CD4 in mIL-18-treated mice, compared to control mice (Figure results, we propose that mIL-18 overexpression in the 5). Interestingly, in our studies with tumor-bearing mice, tumor site induces phenotypic changes in the T cell and B intratumoral injection with an IL-18 plasmid did not pre- cell subsets, and protects against tumor development by vent CT26 tumor development, although augmentation stimulating IFN-γ production by CD4 T cells. of T cells and maintenance of the IFN-γ level during 2 weeks appeared to reject tumors. Thus, while direct Intratumoral injection of mIL-18 plasmid DNA elevates cytokine gene transfer possibly has therapeutic potential, IFN-γ production by splenocytes expression of cytokine genes may be elevated and sus- The IFN-γ levels of cultured splenocyte supernatants were tained significantly during tumor development. Other compared. Enhanced secretion of IFN-γ by cultured splen- reports show that the effects of cotransfection of ICE and ocytes was evident in animals treated with plasmid mIL- pro-IL-18 cDNA are superior to those of pro-IL-18 cDNA 18 DNA (1.278 ± 0.06 ng/10 cells/72 h), compared to alone, and result in enhanced bioactivity of IL-18 [24]. splenocytes from mice treated with pCEP4 control vector Intrace Figure 4 llular IFN-γ production by CT26-specific T cells from pmIL-18-treated mice Intracellular IFN-γ production by CT26-specific T cells from pmIL-18-treated mice. Splenocytes from CT26-tumor bearing mice treated with three rounds of pCEP4 or mIL-18 were pooled and incubated in medium alone or that containing + + + CT26 lysates. Numbers within the gates in the FACS plots depict the percentage of CD69 IFN-γ CD4 cells. Data are pre- sented from experiments that were repeated at least twice. Page 5 of 7 (page number not for citation purposes) BMC Cancer 2007, 7:87 http://www.biomedcentral.com/1471-2407/7/87 Additional material Additional File 1 Representative results of pIL-18 gene therapy in hepatic tumor bearing mice. Visible tumor foci are generally formed in the liver within a week of injection of CT26 cells, and these grew to a few millimeters in diameter by day 21. Treatment was initiated 7 days after tumor cell injection and representative results of gene therapy were showed at 14 days and 21 days after tumor cell injection. 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