Effect Of IL-6 On The Immune Escape From NK Cells In Castration-resistant Prostate Cancer

Background and PurposeThe survival and progression of tumor depend on the tumor microenvironment,where NK cells with independent killing ability are the first line of immunity defense of tumor,and tumor-associated macrophages?TAM?are also involved in this immune process.Studies have shown that IL-6 is involved in the invasion and progression of prostate cancer through the regulation of various cytokines such as JAK and Stat3.PD-L1 is the downstream regulatory factor of JAK and Stat3,it plays an important role in the regulation of tumor immunity by targeting the regulation of T cell function,whereas the combining effect of three factors on the NK cell immune escape in castration-resistant prostate cancer?CRPC?has not been studied.In our study,we focus on this idea.By observing the recruitment of TAM,IL-6 regulates JAK _1?2/Stat3-PD-L1 signaling pathway and influence the effect of NK cell mediatd immune killing of the CRPC,we observed this process and aim to explore the effective way to improving the NK cell mediated immune killing of the CRPC,and provide new ideas for targeted CRPC immunotherapy.Methods1.CRPC cell lines C4-2,CWR22Rv1 were the subjects of this study,lentivirus transfection was used to establish IL-6 knockout cell line siIL-6 and control cell line sc,the effect of IL-6 on the ability of tumor cells to recruit TAM was observed.The cytokines significantly related to recruitment of TAM were screened,then we added the corresponding antibodies to observe changes of the ability to recruit TAM by CRPC.2.The CRPC were co-cultured with TAM culture medium,then the expression of IL-6,PD-L1 and NKG2D ligand?NK cell activating immunoreceptor NKG2D?were observed.The changes in the ability to recruit TAM by CRPC were observed.IL-6antibody were added to observe the expression of PD-L1 and NKG2D ligand in tumor cells.3.The TAM culture medium were co-cultured with CRPC cells,or NK cells respectively;or TAM culture medium,CRPC cells and NK cells were co-cultured.NK cell-mediated cytotoxicity assay were conducted,the IL-6 antibody were added respectively to observe its effect on the immune cytolytic activity of NK cells.CRPC cells were co-cultured with TAM medium,then co-cultured with NK cells.Single cell cloning experiments were carried out to reflect the changes of NK cell killing ability through the cell survival rate.4.CRPC cells were co-cultured with TAM medium,downstream cytokine of IL-6 that can significantly regulate PD-L1 were screened.The tumor cells and TAM culture medium were co-cultured,NK cell-mediated cytotoxicity experiments were conducted.The appropriate inhibitors were added,or in combination with application of PD-L1 antibody,to observe its effect on the immune cytolytic activity of NK cells.5.The C4-2sc and C4-2siIL-6 cell lines were used to establish the castrated CRPC orthotopic transplanted tumor model.The small animal in vivo imaging system?IVIS?assay were carried out weekly to detect the tumors formation and tumor growth.At the second week of establishing the model,the luciferase labeled TAM were injected through the tail vein,differences of TAM recruitment by tumor cells were observed after 48 hours.Tumor samples were collected for testing the expression of IL-6,CD68,F4/80,CCL2,CCL5.6.The same method was used to establish the castrated CRPC orthotopic transplanted tumor model.On the 4th day and the 7th day,the human primary NK cells were injected through the tail vein.Nude mice were sacrificed on the 48th day.The tumor tissue was weighed and the effect of inhibiting IL-6 on the immune cytolytic activity of NK cells in tumor cells were observed.Results1.The siIL-6 cell line can significantly reduce the ability of CRPC to recruit TAM,and can significantly inhibit the recruitment of related cytokines CCL2,CCL5.The CCL2and CCL5 antibodies were added respectively or in combination,then the ability of the tumor cells to recruit TAM were decreased significantly.2.TAM medium and tumor cells were co-cultured,the expression of IL-6 and PD-L1of tumor cells were increased,while the expression of NKG2D ligand decreased.While the adding of the IL-6 antibody can effectively reverse this effect.3.TAM were co-cultured with tumor cells and NK cells respectively or in combination.NK cell-mediated cytotoxicity assay showed that the killing ability of NK cells to tumor cells decreased,and the decrease was most significant in three-cell co-culture mode.Addition of IL-6 antibody can effectively enhance immune cytolytic activity of NK cells to CRPC cells.4.When TAM co-cultured with tumor cells,the expression of downstream effector JAK₁,JAK₂,Stat3 of IL-6 was significantly increased,and the inhibition of JAK₁,JAK₂and Stat3 can significantly reduce the expression of PD-L1 in tumor cells;suggesting that IL-6-JAK_1?2/Stat3-PD-L1 was in upstream and downstream regulation relationship.Combined inhibition of JAK _1?2/PD-L1 is more effective than single inhibition in immune cytolytic activity of NK cells to tumor cells.5.The results of animal experiments showed that inhibition of IL-6 expression could significantly reduce the ability of CRPC to recruit TAM.The expression of IL-6 and TAM markers CD68,F4/80 and TAM-recruiting markers CCL2 and CCL5 in tumor tissue were significantly decreased.Inhibition of IL-6 expression can effectively enhance the immune cytolytic activity of NK cells on tumor cells,thereby inhibiting the growth of the tumor.Conclusions1.Inhibition of IL-6 expression can effectively inhibit the recruitment of TAM by CRPC cells,which is achieved by downregulating CCL2 and CCL5.Compared with single inhibition of CCL2 or CCL5,inhibiton of both factors were more effective in inhibiting the recruitment of TAM by CRPC cells.2.TAM influence CRPC cells and NK cells through paracrine.By upregulating IL-6and PD-L1,downregulating NKG2D ligand in CRPC cells,thereby inhibiting NK cell immunological killing of CRPC cells,while addition of IL-6 antibody significantly reverse this effect.3.IL-6-JAK _1?2/Stat3-PD-L1 signaling pathway form the upstream and downstream regulation relationship,leading to immune escape of CRPC to NK cells.Compared with single inhibition of the signaling pathway,the combined inhibition of JAK _1?2/PD-L1 can enhance the immune cytolytic activity of NK cells to CRPC cells.4.Animal experiments confirmed that inhibition of IL-6 can reduce the recruitment of TAM by CRPC and improve the immune killing of CRPC by NK cells and inhibit the growth of tumors.