Immunotherapeutic Effect Of Recombinant Interleukin-18on Pancreatic Cancer By Selectively Blocking Its Protumoral Activity

Part Ⅰ Detection of IL-18expression in pancreatic cancer patients’ plasmas and pancreatic cancer tissuesObjective Screen potenial immunotherapy agent for pancreatic cancer and validate the results. Detect IL-18expression in pancreatic cancer patients’plasmas and pancreatic cancer tissues.Methods Human cytokine array analysis of plasma from three pancreatic cancer patients and three healthy control plasmas was performed. ELISA was caught out for IL-18detecting in the plasmas from104pancreatic cancer patients,35pancreas benign tumour patients,27duodenal adenocarcinoma patients,77cholangiocarcinoma patients,8hepatic cancer patients,22pancreatitis patients,38health subscribers. IL-18expression in112pairs specimen of pancreatic cancer tissues and adjecant normal pancreas tissues is investigated by IHC.Results IL-18was up-regulated in pancreatic cancer patients’plasma. Elevated plasma levels of IL-18were found in pancreatic cancer patients versus pancreas benign tumour patients, pancreatitis patients and healthy subjects. In addition, IL-18levels were highest in pancreatic cancer patient plasma compared with plasma from patients with common malignant tumours found in abdominal surgery, including pancreatic cancer, hepatic cancer, cholangiocarcinoma, duodenum carcinoma, gastric cancer and colorectal carcinoma. Significant differences in levels of circulating IL-18were observed between3days before surgery and3days after surgery. Furthermore, IL-18plasma levels were beneficially associated with patient overall survival, whereas patients with high IL-18plasma levels had a slightly longer overall survival. IL-18expression differed significantly between normal pancreas and adenocarcinomas. The expression of IL-18was significantly higher in tumours from patients with lymph node involvement and/or distant metastasis than in those without lymph node involvement and/or distant metastasis. Patients with higher grade differentiation according to American Joint Committee on Cancer staging had higher IL-18expression. Importantly, we found that IL-18tissues level was significantly and inversely associated with patient overall survival:patients with high IL-18tissues levels had significantly shorter overall survival.Conclusion IL-18is up-regulated in pancreatic cancer patient plasma and correlates with a better prognosis. And IL-18is up-regulated in pancreatic cancer tissues and correlates with increased metastasis and a poorer prognosis. Part Ⅱ The research of immunotherapeutic effect of single recombinant Interleukin-18on pancreatic cancerObjective Identify the effects of IL-18on main immune cells and whether IL-18has therapeutic effect on pancreatic cancer.Methods PBMCs isolated from healthy volunteers were co-cultured with rhIL-18for24hours. Flow cytometry was used to investigate changes of immunophenotype and activation status of PBMCs. rmIL-18was continuously injected intraperitoneally into C57BL/6J mice for7days and immune cells from spleen and aortic lymph nodes were isolated for flow cytometry. Main immune cell subpopulation in the spleen and aortic lymph nodes of IL-18C57BL/6J mice and wild type C57BL/6J mice were dected by flow cytometry. Orthotopic transplant model in C57BL/6J mice or Balb/c nude mice were established and IL-18intraperitoneal injected. Long-term follow-up was caught out.Results The proportion of cytotoxic T cells (Tc) and NK cells was significantly up-regulated and the proportion of B and T helper (Th) cells was slightly up-regulated in the IL-18treated group. The proportion of Tc and NK cells was significantly up-regulated and the proportion of B cells, Th cells and dendritic cells (DC) cells were slightly up-regulated in the IL-18treated group. Tc and NK cells were significantly down-regulated in IL-18-/-C57BL/6J mice and the proportion of B cells, NK cells and DC cells were also slightly down-regulated. In the C57BL/6J mouse model, IL-18had no effect on the survival state and overall survival time. In Balb/c nude (Balb/c-nu) mice, we found that IL-18accelerated tumour growth and deteriorated the survival state. In the IL-18treated group, mice were maintained in a poor survival state. Long-term follow-up showed that IL-18shortened the overall survival time of Balb/c nude miceConclusion IL-18up-regulates a proportion of the main anti-tumour immune cells, especially Tc and NK cells. IL-18has no therapeutic effect in C57BL/6J mice pancreatic cancer orthotopic transplant model but a tumour promotion effect in a Balb/c nude mice orthotopic transplant model. Part Ⅲ The research of the effects of IL-18on proliferation and invasion of pancreatic cancer cells and the underlying mechenismsObjective To identify the effects of IL-18on proliferation and invasion of pancreatic cancerMethods Generating stable mia-paca-2and panc-1cell lines overexpressing or knocking-down IL-18using a lentiviral delivery system. Then we detected IL-18’s effect on proliferation, invasion, migration and metastesis by CCK-8assay,3-dimensional culture system, transwell assay and wound healing assay. Next, we established subcutaneous transplant model and liver metastasis model of pancreatic cancer using transfected cells.Results Overexpressing IL-18promoted cell proliferation and growth while knocking-down IL-18suppressing cell proliferation and growth. Over-expression of IL-18promoted tumour proliferation and growth and knocking-down IL-18suppressed tumour proliferation and growth in vivo. In the IL-18knock-down group, the xenograft had slow growth and the mice were maintained in a healthy state. Over-expressed IL-18promoted pancreatic cancer cell invasion and migration while IL-18knock-down suppressed cancer cell invasion and migration. IL-18promoted tumour cell invasion and metastasis and reduced the overall survival time and survival state in vivo. Haematoxylin and eosin (HE) staining of serial sections of whole liver showed increased micrometastases in the IL-18over-expressed group, and reduced micrometastases in the IL-18knock-down group compared with the controls. Simulating pancreatic cancer cells with IL-18(20ng/mL) for120minutes or longer augmented p-IKKα/β expression and reduced the levels of IκBα, a key factor for NF-κB pathway activation. Furthermore, using a dual-luciferase assay we determined that IL-18directly activated the NF-κB pathway. IL-18promoted the translocation of NF-κB from the plasma into the nucleus. An electrophoretic mobility shift assay also confirmed that IL-18could directly activate NF-κB pathway. Small interfering RNA (siRNA)-NF-κB, an NF-κB pathway inhibitor BAY11-7082and curcumin counteracted the IL-18promoting effect. We detected the main NF-κB pathway target genes by real-time PCR after20ng/ml rhIL-18administration for48hours and found that IL-18mainly affected matrix metalloprotease (MMP)-3, MMP-9, cyclin-D1and c-myc expression. Over-expressed IL-18or rhIL-18up-regulated the expression of MMP-3, MMP-9, cyclin-D1and c-myc could be inhibited by siRNA-NF-KB, BAY11-7082or curcumin. MMP-3, MMP-9, cyclin-D1and c-myc were up-regulated in the IL-18administered group of the subcutaneous transplanted Balb/c nude mouse model. Then we found IL-18could augment NF-κB’s recruitment of MMP3, MMP9, c-myc and cyclin-D1promoter.Conclusion IL-18promotes the proliferation and invasion of pancreatic cancer cells both in vitro and in vivo through the NF-κB pathway. Administration of BAY11-7082, a NF-κB inhibitor, prevents the pro-cancer effects of IL-18. Part Ⅳ IL-18has an immunotherapeutic effect on pancreatic cancer with selectively blocking its protumoral activity. Objective To identify the therapeutic effect of IL-18and an NF-κB pathway inhibitor on pancreatic cancerMethods Establish an orthotopic transplant model in C57BL/6J mice and IL-18-/C57BL/6J mice using LTPA cells and IL-18knocked-down LTPA cells. Four groups were investigated:i) LTPA/C57BL/6J; ⅱ) IL-18KD/C57BL/6J; ⅲ) LTPA/IL-18-/-C57BL/6J; and ⅳ) IL-18KD/IL-18-/-C57BL/6J. All mice were weighed and sacrificed when cachexia developed. Establish an orthotopic transplant model in C57BL/6J mice with LTPA cells. Four groups were treated as follows:ⅰ) PBS; ⅱ) IL-18; ⅲ) BAY11-7082; and ⅳ) IL-18and BAY11-7082. All mice were weighed and sacrificed when cachexia developed.Results The IL-18KD/C57BL/6J group mice had the longest survival time while the LTPA/IL-18-/-C57BL/6J group had the shortest survival time. And the LTPA/IL-18C57BL/6J had more liver and lung micro metastasis. The IL-18and BAY11-7082group had the longest survival time of all three groups. There was no difference between the overall survival time between the control group and IL-18group.Conclusion IL-18only had a therapeutic effect when co-administered with an NF-κB pathway inhibitor in pancreatic cancer cells.