Mechanism Of Morphine-3-Glucuronide Promote The Immune Escape Of Non-small Cell Lung Cancer Via TLR4 Signaling Pathway

On a global scale,malignancies have become the leading cause of death.Inflammation is one of the factors leading to the occurrence and progression of tumors.About 20% of the cancers are caused by chronic infection.Toll like receptor 4(TLR4)belongs to the pattern recognition receptors(PRRs)and participates in the immune regulation and inflammation of the body.Studies have shown that a variety of tumor tissues and tumor cell lines highly express TLR4 and is closely related to tumor immune escape.Pain treatment in patients with advanced cancer mainly depends on morphine,but studies have shown that the demand for morphine and morphine dosage was negatively correlated with prognosis.Morphine-3-glucuronide(M3G),the main metabolite of morphine,can activate TLR4 in the nervous system and promote the release of cytokines involved in morphine tolerance.However,there is no report about the interaction between M3 G and tumor cells expressing TLR4 and their biological behavior.This study aims to explore the interaction of M3 G and TLR4 expressed in non-small cell lung cancer(NSCLC)on tumor biology,especially immune escape,and trys to explain the mechanisms that morphine application could lead to the poor prognosis of cancer patients.Firstly we perform a retrospective study of clinical cases.126 patients with NSCLC who underwent radical resection of lung cancer and morphine analgesia with complete follow-up data were enrolled.Immunohistochemical staining of cancer and para-cancerous tissues showed that the tumor tissue overexpressed TLR4 rather than classic morphine receptor mu-opioid receptor(MOR),and TLR4 expression is closely related to clinical pathological data such as tumor TNM stage and lymph node metastasis.Survival analysis showed that TLR4 expression in tumor tissue was negatively correlated with overall survival(OS)and disease free survival(DFS),and was an independent risk factor for poor prognosis of NSCLC patients.The expression of programmed death ligand 1(PD-L1)were also analyzed in tumor tissue by immunohistochemistry.Although the expression of PD-L1 was not correlated with postoperative OS and DFS,we found that the expression of TLR4 in tumor tissue was positively correlated with PD-L1 expression,which provided the clinical basis for our follow-up in vivo experiments.Next,we used the CDOCKER module(semi-flexible docking program)in the Discovery Studio 2.1 software to perform docking experiments of ligand molecules and receptor active sites.The results showed that the binding of M3 G and morphine to TLR4-MD2 was superior to that of the original ligand,while the binding ability of Morphine-6-glucuronide(M6G)to MD2 was weak.In cytological experiments,we first used western blot and RT-qPCR to screen high expressing TLR4 but low expressing MOR NSCLC cell lines and find A549 and H1299 cell lines.Incubation experiments showed that different concentrations of M3 G and LPS promoted the expression of PD-L1 in A549 and H1299,and this effect could be antagonized by TLR4 pathway inhibitor TAK-242.Flow cytometry analysis further confirmed M3 G treatment on A549 and H1299 had no significant effect on membrane expression of other immune checkpoint molecules such as PD-L2,Gal-9,CD200.ELISA experiments showed that incubation of M3 G with A549 can promote pro-inflammatory factor IL-6 release.We then explored the signaling mechanisms by which M3 G promotes PD-L1 expression in NSCLC cell lines via TLR4.The phosphorylation level of key proteins such as MAPK,PI3 K and NFκB signaling in M3G-treated A549 cells was detected by western blot.The results showed that M3 G could activate PI3 K and NFκB signaling pathway,and this effect could be antagonized by TAK-242.Further by flow cytometry,we found that PI3 K signaling pathway inhibitors rather than NFκB signaling pathway inhibitors can reverse M3G-induced PD-L1 upregulation.Finally,we performed cytotoxic T-cell(CTL)target killing experiments.Through the HLA matching experiment,donor blood samples matching with HLA-A type of target cell A549 cell line were selected.The synthesis of peptides stimulates dentric cells maturation and eventually together stimulate the maturation of CTLs.Cytotoxicity assay and ELISPOT results showed that M3 G can inhibit the cytotoxicity of CTL to A549 cell line and the release of INF-γ,which can promote the immune escape of NSCLC cell line.In conclusion,we found that the expression of TLR4 in tumor tissue and the expression of PD-L1 in NSCLC patients receiving morphine analgesia were positively correlated.M3 G,a major metabolite of morphine,can specifically activate TLR4 in NSCLC cell lines and upregulate PD-L1 expression through the PI3 K signaling pathway,thereby inhibiting CTL cytotoxicity.In addition,M3 G can also promote the migration and invasion of NSCLC cells,and upregulate the release of IL-6,so that promotes tumor immune escape.