Hydrodynamic Injection Mediated IL-12 Gene Therapy In A Mice Model Of Orthotopic Liver Cancer

Primary liver cancer is a highly progressive and aggressive malignant tumor. Currently, the therapeutic procedures are mainly surgical resection, radiation therapy and chemotherapy. Unfortunately, the recurrence rate remains high after liver cancer resection. The radiation therapy and chemotherapy with severe side effects are not sufficiently effective. Therefore it is very necessary to explore a new approach for the treatment of liver cancer.IL-12 is a potent antitumor cytokine that plays a crucial role in antitumor immunity. The antitumor mechanism involves inducing production of IFN-γand NO, enhancing the cellular immune function and inhibiting tumor angiogenesis.IL-12 gene transfer mediated by viral vectors is highly effective and results in the retardation of tumor growth or eradication of the tumor. Unfortunately, the production, amplification and purification of viral vectors is laborious. Moreover, the antiviral immunity induced by viral vectors not only results in side effects, but also precludes re-administration of the vector. The naked DNA of non-viral vectors is an attractive alternative because of its advantages including safety, simplicity, and absent or low immunogenicity. However, the low transfection efficiency limits its application.In our study, we used a novel and effective non-viral vector gene transfer technique, the hydrodynamic injection for naked DNA to investigate the antitumor effects of plasmid DNA encoding the IL-12 gene and possible mechanism. The aim of this research was to evaluate the antitumor efficiency of IL-12 gene mediated by hydrodynamic injection in a mice model of orthotopic liver cancer. MethodsTo establish the primary liver cancer model, H₂₂ hepatoma cells were inoculated into the liver of mice. Varying doses of pCMV-mIL-12 plasmid were injected into mice by a hydrodynamic injection 3 days after H₂₂ cells inoculation. Thereafter, the blood was collected at indicated time points. The expression of IL-12 and IFN-γin serum was determined by ELISA. NO production was measured using a commercial kit. The parts of mice were sacrificed 14 days after treatment with plasmid. The tumor sizes were measured. HE staining and immunohistochemical staining were applied to evaluate the proliferative activity of tumor cells, angiogenesis in tumor and metastasis to lung. The other mice were kept to monitor the survival.To prepare the malignant ascites model, H₂₂ hepatoma cells were injected into peritoneal cavity of mice. pCMV-mIL-12 plasmid was administered into mice by a hydrodynamic injection 3 days after H22 cells injection. Thereafter, the blood was collected at indicated time points. The expression of IL-12 and IFN-γand NO in serum were determined as above. Three mice were sacrificed 14 days after treatment with plasmid. The volume of ascites was measured. The cells in ascites were stained with hematoxylin and eosin or AO/EB. The other mice were kept to monitor the survival.Results1. To test the potential of plasmid DNA IL-12 gene transfer to delay tumor growth, we administered 2.5μg or 10μg pCMV-mIL-12 to the mice by hydrodynamic injection 3 days after H₂₂ cells inoculation in the liver. The mice were sacrificed at 14 days after plasmid injection. The hydrodynamic injection of pCMVβor saline had no effect on the growth of liver tumor and ascites formation. In contrast, the tumor size and ascites volume in pCMV-mIL-12-treated mice was substantially reduced.2. Immunohistochemical analysis of the tumors indicated extensive microvessel density and strong PCNA proliferative activity of liver cancer tissue grown in mice treated with pCMVβor saline. In contrast, the smaller tumors with extensive necrotic areas from mice treated with pCMV-mIL-12 showed a marked paucity of microvascular structures and weak PCNA proliferative activity.3. Compared with mice treated with pCMVβor saline, the survival of mice treated with pCMV-mIL-12 was obviously prolonged and the mice were rarely accompanied by pulmonary metastases.4. Compared with mice treated with pCMVβor saline, the significant elevation of IL-12 and IFN-γand NO levels in serum were detected in pCMV-mIL-12-treated mice.5. To determine whether hydrodynamic injection of pCMV-mIL-12 can induce lasting immunological memory, we randomly chose two of mice that survived after 2.5μg pCMV-mIL-12 treatment, and rechallenged mice with intraperitoneal administration of H₂₂ cells at 40 days and 70 days respectively after pCMV-mIL-12 treatment. No ascites was detected in both of two mice that previously received pCMV-mIL-12. By contrast, the normal mouse developed ascites and die at 17-19 days after tumor cells intraperitoneal injection.6. In malignant ascites model, compared with mice treated with pCMVβor saline, less alive tumor cells, more apoptotic cells and inflammatory infiltration were observed in mice treated with pCMV-mIL-12. Longer survival was found in these mice.Conclusions1. Hydrodynamic injection-mediated IL-12 gene transfer increased the IL-12 level in serum of mice. The expression of IL-12 delayed the growth of orthotopic liver cancer and suppressed its metastases and prolonged the survival of mice.2. The possible antitumor mechanism involved inducing production of IFN-γand NO, reducing the proliferation activity of tumor cells and inhibiting tumor angiogenesis.3. Gene therapy using pCMV-mIL-12 mediated by hydrodynamic injection can induce long-term protection against tumor recurrence. 4. Hydrodynamic injection-mediated IL-12 gene transfer decreased the production of hemorrhagic ascites and prolonged the survival of mice bearing malignant ascites. The possible mechanism involved inducing apoptosis of tumor cells and decreasing the permeability of peritoneal blood vessel.