Intratumoral Delivery of IL-18 Naked DNA Induces T-Cell Activation and Th1 Response in a Mouse Title: Hepatic Cancer Model Authors:

results : 2nd line in dose dependent manner → removed Background : 8st line correct reference [4], [5] : 20st line We report here antitumoral effects of nonviral vector encoding murine IL-18 plasmid DNA in established CT26 liver tumors. → We report here the antitumoral effects by direct intratumoral injection of nonviral plasmid vector encoding murine IL-18 plasmid DNA in established CT26 liver tumors. Methods: Tumor model and therapeutic protocol 3rd line: Colon carcinoma was established in the liver by intrahepatic implantation of 1x10^5 cells in the left lateral lobe of 6to 8week old male BALB/c mice. Seven days after implantation, mice with liver tumors were treated with pCEP4 control vector or mouse IL-18 in pCEP4 at various doses (10, 25, and 50 ug) → CT26 cells were suspended for implantation at 1×105 cells/50 μl saline. Colon carcinoma was established in the left lateral lobe of 6to 8-week-old male BALB/c mice by an abdominal operation. Treatments began 7 days later, when tumor results in a defined solid tumor growth within the injected lobe. For intratumoral injections of naked DNA, DNA (10, 25, and 50 μg) was diluted in 50 μl saline, and then injected into the parenchyma of the lower surface of the left liver lobe via insulin syringes (31 gauge, 0.8-inch needles; Becton Dickinson, Fanklin Lake, NJ). Mice were sacrificed at 1, 4, 7, and 14 days after DNA treatments and then tumor growth was monitored by measuring liver weight [13]. Results and discussion levels of transgene expression in treated tumor site : 4st line: dose dependent manner → remove : 5st line 500-900 ug of liver tissue when mice were sacrified at 4 day after plasmid introduction → 500 to 900 mg of injected lobe because tumor developed differentially after each treatment : 6st line the level of IL-18 protein expression was increased in a dose-dependent manner. → The level of IL-18 protein expression was 2-2.5 fold higher than background when mice sacrificed at 4 day after plasmid introduction. In vivo Kinetic Study of Immune Cell Population in Spleen : 2nd line 2,5,7 → 2, 5, and 7 days : 2nd line: A dose-dependent antitumor response against CT26 was noted in group receiving pIL18. → Antitumoral response of tumor-bearing mice when treated with 50 μg of DNA was significantly greater than that of the tumor-bearing mice treated with 10or 25-μg DNA groups. : 8st line: The result suggest that high level of IL-18 modulates immune cell population of T cells and NK cells around the time of tumor regression. → Other groups have also reported that IL-18 has been shown to up-regulate ICAM-1 expression on monocytes and on T cells, providing further mechanisms whereby IL-18 can promote T cell recruitment [14-17]. These data, together with the above data about kinetic of immune cell population, suggested that about -2.5 fold higher concentration of IL-18 modulates immune cell population of T cells and NK cells around the time of tumor regression. Intratumoral injection of mIL-18 plasmid DNA elevated IFN-γ production by splenocytes 6st line: addition However, in our studies with tumor-bearing mice revealed that IL-18 plasmid intratumoral injection does not prevent development of CT26 tumors although such augmentation of T cells and maintenance during of IFN-γ level during 2 weeks appeared to reject tumors. Thus, direct cytokine gene transfer may have a therapeutic potential but expression of cytokine gene may be elevated and sustained significantly during tumor development. Other reports show that the cotransfeciton of ICE and pro-IL-18 cDNA was superior to pro-IL-18 cDNA alone and resulted in enhanced bioactivity of IL-18. Thus dual transfection of pro-IL-18 and ICE cDNA results in secretion of more bioactive IL-18 protein than that of pro-IL-18 cDNA alone [24]. Acknowledgements : This study is supported by a grant I-A4-003 from Samsung Biological Research Institute. → This research was supported by grants from the IN-SUNG Foundation for Medical Research, Seoul, Korea