Combination Treatment For Pancreatic Cancer Though NLRP3 Inflammasome Inhibition And CIK Cells

BACKGROUND:Pancreatic cancer is amongst the most aggressive of human malignancies and has poor prognosis. Conventional managements including surgery, chemotherapy and radiotherapy can kill tumor cells, which also simultaneously cause inflammation response and immunosuppression. Epidemiological studies and molecular biology have revealed that the development of pancreatic cancer is associated with chronic inflammation. NLRP3, as an important regulator for inflammation, is consisted of NLRP3 protein, ASC adapter protein and pro-caspase-1. When activated, NLRP3 combines with the adaptor molecule ASC and pro-caspase-1 to form a protein complexcalled the inflammasome, which mediates maturation of IL-1β and IL-18, thus regulating the inflammation response. It was revealed that IL-1β can enhance pancreatic cancer cells invasiveness while elevated serum levels of free IL-18 are correlated with poor survival of pancreatic carcinoma. Therefore, there may be an association between NLRP3 inflammasome and the pancreatic cancer. Immunotherapy has been considered as the fourth management approach for cancer. As one kind of immunotherapy, CIK cells has been used in clinical practice. However, CIK cells can kill tumor cells as well as induce amount of cytokines releasing, causing cytokine release syndrome. Otherwise, the anti-tumor cellular immunity is inhibited by NLRP3 inflammasome. Accordingly, NLRP3 inhibition combing CIK cells may improve anti-tumor efficacy though inhibition of cancer related inflammation and restoring and promotion of anti-tumor immunity.OBJECTIVE:To evaluate the significance of NLRP3 inflammasome expression in pancreatic cancer, construct the preparation and detection platform for CIK cells and explore the feasibility and efficacy of combination treatment for pancreatic cancer though NLRP3 Inflammasome inhibition and CIK cells.METHODS:1. NLRP3 inflammasome expression in pancreatic cancer was evaluated though tissue microarray and immunohistochemistry, whose correlation with clinicopathological characteristic and prognosis of patients with pancreatic cancer were analyzed by chi square test and Kaplan-Meier analysis, respectively. 2. In setting of available preparation and detection platform for CIK cells completing, PBMCs were isolated from peripheral blood samples of healthy volunteers, which then were cultured and amplified to CIK cells. The percentage of activated T cells and central memory T cells in CIK cells and PBMC were determined by flow cytometry and compared though Student’s t-test to evaluated the activity and anti-tumor effect of CIK cells.3. Western Blot was used to evaluate the NLRP3 inflammasome expression in human pancreatic cancer lines SW1990 and PANC-1, and analyze the influence of MNS on the NLRP3 inflammasome expression. The impact of NLRP3 inhibition on migration and invasiveness of pancreatic cancer cells were analyzed though wound healing assay and transwell assay, respectively. The influence of NLRP3 inflammasome inhibition on proliferation of pancreatic cancer cells were evaluated by CCK08 assay.4. A subcutaneous model of human pancreatic cancer though SW1990 cell line injected into the left upper flank of the Blab/c nudes. A total of 24 nudes were randomly assigned into four groups, and MNS group was treated though MNS intraperitoneal injection, CIK group was managed with CIK cells tail intravenous injection, MNS+CIK group was treated though combing MNS intraperitoneal injection and CIK cells tail intravenous injection and the Control group. The difference of tumor volume, tumor weight and inhibition rate among four grouped was calculated to evaluate the influence of four different managements on the tumor growth. After 2 weeks, all nude were euthanized, and the tumors were obtained to histopathology examination.RESULTS:1. NLRP3 inflammasome expression was significantly increased in pancreatic cancer, comparing with adjacent normal pancreatic tissue, which also significantly related to clinical stage and lymph node invasion (P<0.05). Furthermore, the increasing expression of NLRP3 inflammasome was associated with poor prognosis of pancreatic cancer. The overall median survival in patients with low NLRP3 was significantly improved compared to that in the those with high NLRP3 (21 months VS 12 months, P<0.01), the same survival benefit was showed in ASC(low,18months; high,13months, P<0.01), Caspase-1(low,17months; high,13months, P<0.01) and IL-1β(low,18months; high,13months, P<β.01).2. The percentage of CD3+CD8+ T cells and CD3+CD56+T cells in CIK cells were71.31%±18.77% and 34.62%±15.67%, respectively. Compared with PBMC, the percentage of activated T cells and central memory T cells in CIK cells were significantly increased (P<0.05).3. Western Blot analysis showed expression of NLRP3 inflammasome in human pancreatic cancer lines SW1990 and PANC-1, and that MNS did significantly inhibit the expression of NLRP3 inflammasome in human pancreatic cancer lines (P<0.05). Transwell assay and wound healing assay revealed that NLRP3 inhibition can significantly decrease the migration and invasiveness of pancreatic cancer cells (P<0.5). CCK-8 assay showed that NLRP3 inhibition cannot suppress the proliferation of CIK cells but pancreatic cancer cells.4. The subcutaneous model of human pancreatic cancer in Blab/c nudes were successfully completed, and tumor initiation rate reached to 100%. In vivo experiments showed that combination treatment with MNS and CIK cells had greatest anti-tumor effect among four treatment groups including control, MNS and CIK (P<0.05). The HE and IHC of subcutaneous tumor in MNS+CIK group showed spotty and flaky necrosis and significantly downregulation of NLRP3 inflammasome.CONCLUSION:1. There was high expression of NLRP3 inflammasome in pancreatic cancer tissue and cell lines.2. NLRP3 inflammasome inhibition can suppress the proliferation and metastasis of pancreatic cancer, but no influence on CIK cells.3. Combination treatment with NLRP3 inflammasome inhibition and CIK cells show greater anti-tumor efficacy though inhibition of cancer related inflammation and restoring and promotion of anti-tumor immunity.