The Function And The Effect Significance Of PD-L1 In Mediating Cancer Cell Metastasis And Immno-escape From NK Cell Immunology In Non-small Cell Lung Carcinoma

Objective:Non-small cell lung cancer(NSCLC)has the highest morbidity and mortality among all mallignent tumors in the world.Platinum drugs,especially cis-diammine-dichloroplatinum(DDP),are widely used in clinics.Unfortunely,drug reisistance formatted after repeated use.Programmed death factor ligand-1 is a star molecular owing multipal biological function.But the function of PD-L1 in cisplatin resistant lung cancer is not fully illuminated.Our research is aiming at investigate the role of PD-L1 in regulating cell metastasis and immuno-escape from NK cell cytotocity in cisplatin resistant lung cancer.Methods:Cisplatin resistant NSCLC cell lines(A549Cis R and H157Cis R)were clutured by continuous stimulation of parental cell lines(A549P and H157P)with low dose of cisplatin.The expressions of ATM,MEK/ERK,JAK_1,2/STAT₃,FASN,TGF-?1 and PD-L1were detected by Western Blot,q PCR,immunofluorescence and cell flow cytometry in m RNA and protein levels.The expressions of ATM,MEK/ERK,JAK_1,2/STAT₃,FASN,TGF-?1 and PD-L1 were significantly enhanced or inhibited by lenti-virus mediated gene overexpression and slicence,neutralizing antibody,small molecular inbibitor and human recombined protein treatment.Cell proliferation,migration and invasion were detected by MTT assay,wound healing assay and transwell assay.NK-92 cell lines and primary NK cells isolated from human peripheral blood mononuclear cell were co-cultured with tumor cells to micmic the tumor micro-enverinment.The immuno killings of NK cell to tumor cells were detected by LDH cytotocity quantification,cell flow cytometry,and single cell clonegenic assay in different conditions.The tumor cell derived xenografts in mouse model were constructed to comfirm the cell metastasis data in vitro.Tumor development and metastasis were monitored once a week and the changes in tumor volume assessed using the In Vivo Imaging System(IVIS).Immunohistochemistry was performed to detect the associated protein expressions in xenografts model.Results:The IC50 of the cisplatin resistant cell lines to cisplatin was 6-8 higher than parental cells comfirming the successful construction of cisplatin resistant cell lines.The expression of E-cadherin was significantly decresed in cisplatin resistant cell lines while the expressions of N-cadherin,Vimentin,Twist,VEGF and Snail were significantly increased.The cisplatin resistant cell lines have significantly improved migration and invasion ability via EMT mechanism.These ability of cell EMT and metastasis were associated with ATM/JAK_1,2/STAT₃ signal pathway positive regulation of PD-L1.In in Vivo experiments,cisplatin resistant cell lines derived xenograft mouse model showed severe tumor metastasis monitered by IVIS system.Futhermore,the cisplatin resistant cell lines can significantly escape from NK cell immuno killings due to higher PD-L1expression.Besides,the cisplatin resistant cell lines can induce the PD-1 expression in NK cells after co-culture since the parental cell lines can not.MEK/ERK/PD-L1signal pathway and FASN-TGF-?1-PD-L1 positive feed back loop were existed in cisplatin resistant cell lines.The increased expressions of these genes promote cisplatin resistant cell lines escape from NK cell immunology.Interestingly,the positive immune check point constituded with NKG2D and its ligands including ULBP1,ULBP2,ULBP3,MICA and MICB were also controlled by MEK/ERK/PD-L1signal pathway and FASN-TGF-?1-PD-L1 positive feed back loop.The application of inhibitors of TGF-?1 or ERK combined with PD-L1neutralizing antibody exhibited signicantly better effect in reversing the tumor cell immno-escape from NK cell killings by comparison of single use of associated drugs.Conclusions:The significantly high expression of PD-L1 in cisplatin resistant NSCLC can either promote tumor metastasis via cell EMT or help tumor immuno-escaping from NK cell cytotoxicity.PD-L1 was positively regulated by ATM,JAK_1,2/STAT₃,MEK/ERK and FASN-TGF-?1 signal pathway.Inhibition of signaling pathway mentioned above can reverse tumor EMT,metastasis and immuno-escape from NK cell cytotoxicity.Furthermore,combined inhibiton of TGF-?1 or ERK with PD-L1 provided better outcome than single molecular inhibition in NSCLC immunology therapy.