Tumor Derived IL-35 Promotes CCL5 Secretion To Recruit Monocytes

Objective IL-35 is a newly discovered two heterologous dimers belongs to the IL-12 family of cytokines. It contains EB virus induced gene EBI3 and IL-27 P35 two subunit. IL-35 has a direct immunosuppressive potential, it has been confirmed that IL-35 is involved in the process of the formation of a variety of diseases, including inflammatory diseases, coronary artery disease and cancer. In the previous work, We found that IL-35 not only in pancreatic cancer in high expression and had correlation with the tumor local infiltration of monocyte/macrophage number and macrophages is an important regulator in the process of tumor metastasis, progression and angiogenesis. Therefore, this paper is to further clarify the way of IL-35 recruitment of monocytes / macrophages.Content and methods1. The relationship between the expression of IL-35 and local infiltration of monocytes/macrophages in pancreatic ductal adenocarcinoma(PDAC).1) Immunohistochemical staining and statistical analysis of the correlation between IL-35 and local infiltration of monocytes/macrophage numbers.2) The correlation between IL-35 and monocytes cells Markers and the effect of monocyte / macrophage number on the survival of patients were analysed by TCGA database.2. Explore the recruitment of monocytes / macrophages by IL-351) Gene sequencing of IL-35 stable cell lines was analyzed, to observe the changes associated with the recruitment of mononuclear macrophage chemotactic factor gene;2) TCGA database to analyze the correlation between IL-35 and monocyte /macrophage associated chemokine;3) QRT-PCR and Western-blot experiments to verify that at the m RNA and protein level, whether there is a link between IL35 and macrophage chemotaxis factor; 4)Western-blot to verify IL-35 to promote macrophage chemotaxis related molecular mechanism of chemokine secretion;5)To verify whether the IL-35 can combinate the macrophage related chemokine promoter by CHIP.3. In vitro experiments confirmed that IL-35 had the function of recruitment of monocytes/macrophage by related chemokines.1)The separation of peripheral blood mononuclear cells by density gradient centrifugation, and purified macrophages by adherence method;2) Using Transwell to verify the effect of the recruitment of stabletransfection cell lines supernatant of macrophage, and the results were statistically analyzed.4. In vivo animal experiments to verify the effect of IL-35 by recruiting related chemokines on monocyte macrophage.1) Black rat subcutaneous Pan02 expression and reduced expression of IL-35 stably transfected cell lines and selective removal of related chemokine group; 2) Close observation of changes in mice with tumor size and the conditions of mice, the time is ripe, tumor bearing mice were complete peeling off and the ground state of a single cell, flow detection of monocytes/macrophages number of intratumoral infiltration between different groups.Results1.1) Immunohistochemistry showed a statistically significant expression level of IL-35 and local infiltration of monocytes/macrophages number, and monocytes/macrophages infiltration quantity and survival is significantly negative correlation.2)TCGA database analysis showed that IL-35 two subunits and CD11 b,CD68, CCR2 and monocyte related marker has a very high correlation.2. The construction of pancreatic cancer IL-35 overexpression of stabletransfection cell lines(PANC-1, Bx PC-3 and Pan02) and reduced expression of IL-35 stabletransfection cell lines(MIA-paca-2, CFPAC-1 and Pan02), and sent to the sequencing of gene sequencing.1) Over expression of IL-35 sequencing of stabletransfection cell lines showed that compared with the related chemokine genes and cells in the control group after overexpression of IL-35(CCL20, CCL2, CCL5,CX3CX1) expression were significantly improved.2) TCGA data analysis showed that the two subunits of IL-35 have a high correlation of two chemokines of CCL5 and CCL2. 3) QRT-PCR, Western-blot at the cellular level from the two aspects of m RNA and protein between IL-35 and CCL2 and CCL5 confirmed the results confirm the correlation between IL-35 and CCL5 is obvious.4) Western-blot to verify the molecular mechanism of IL-35 promoting secretion of CCL5 in JAK-STAT pathway, is mainly STAT1 and STAT4 activation, phosphorylation of P-STAT1 and P-STAT4 in nuclear regulation.5) CHIP verified that in the stimulation of IL-35 of P-STAT1 and P-STAT4 could enter the nucleus and combinated the promoter sequences of CCL5.3.1)Using the density gradient centrifugation of mononuclear cells from peripheral blood, further purified by adherent method of mononuclear cells, mononuclear cells to obtain relatively pure.2) Confirmed by Transwell IL-35 through promoting the secretion of PDAC CCL5 to recruit macrophages, and the results of statistical analysis, the statistical results have significance.4.1) Black rat subcutaneous implantation pan02 over the group of IL-35 expression and decreased expression of IL-35 group and in the over expression of IL-35 based on join CCL5 blocking agent group, IL-35 group was significantly increased in mice tumor volume were observed, blocked CCL5, mice into tumor volume was significantly reduced.2) Results showed that over expression of IL-35 group, tumor infiltrating mononuclear macrophage number was significantly higher, at will to express IL-35 group, tumor infiltrating mononuclear macrophage number and significantly lower, in based on the over expression of IL-35 blocked CCL5, then tumor infiltrating mononuclear macrophage number was significantly reduced.Discussion IL-35 as an immunosuppressive factor, tumor in such a complex micro environmental conditions plays a immeasurable role, IL-35 expression in pancreatic cancer, and can promote pancreatic cancer secretion of CCL5 to recruit mononuclear macrophage. As is known to all, mononuclear macrophages for tumor growth, invasion and metastasis plays an important role, then found IL-35 and CCL5 regulatory relationships may be important progress in antitumor therapy.