The Effects And Mechanisms Of Exosome Produced By Hepatoma Cell Under Endoplasmic Reticulum Stress On The Immune Function Of Macrophage

BackgroundHepatocellular carcinoma(HCC)is one of the most common malignant tumor in china,surgery is the most effective treatment,but more than 80% of the patients have lost the opportunity when diagnosed.Chemotherapy is a main therapeutic strategy for patients with advanced disease,but neither single agents nor combination therapy achieve significant survival benefits.Therefore,how to improve the treatment effect of HCC becomes a hot spot within tumor research field.Tumor microenvironment is composed by tumor cells,mesenchymal cells,microvascular,interstitial fluid and some of the infiltrated immune cells,such as dendritic cells,macrophages and T lymphocytes.It has a close relationship with the development and metastasis of tumors.In the process of occurrence and development,hypoxia,decreased nutrition supply and low extracellular p H induced by rapid proliferation of tumor cells and poorly developed blood vessel,which collectively induce tumor cells endoplasmic reticulum stress(ERS)to maintain homeostasis.Macrophages have very strong plasticity,and play an important role in maintaining homeostasis and regulation of new blood vessels formation.An increasing number of studies confirmed that macrophages are an important composition of the tumor microenvironment,and the phenotype of macrophages in the tumor microenvironment are often changes into a tumor associated macrophage(TAM),which then turn into promoting tumor proliferation and progression.However,by which way tumor cells“educate” macrophages from an antitumor phenotype to a tumor promoting phenotype is not clear.Exosomes are a class of 40-100 nm-sized membrane vesicles that are released into the extracellular environment and play an important role in cell-to-cell communication.Therefore,there is an important clinical significance to investigate by which way tumor cells transmit stress signals to macrophages,and further change its immune functions.In the current study,we aim to discuss the the precise mechanism by which way hepatocellular carcinoma cells under endoplasmic reticulum stress transmit signals to macrophages and influence its functions.ObjectiveTo evaluated the impact of ER stress on the immune response of macrophages,the expression of inflammatory factors and PD-L1 was detected after macrophages were co-incubated with exosomes derived from HCC cells under either ERS or not for 24 hours.Furthermore,the impact of ER stress on HCC cells exosomal-mi RNAs encapasule was assessed by the high-throughput sequencing,and the specific mechanisms of ER stress related exosomes on PD-L1 expression was also investigated.Methods(1)TM at different concentrations for 24 hours or 3?M for different times was administrated to HCC cells,respectively.And GRP78 protein,a marker of endoplasmic reticulum stress,was detected by Western Blot analysis.(2)HCC cells were co-cultured with 3 ?M of TM or not for 24 hours,then the supernatants were collected and exosomes were isolated by using Exo Quick-TC kit.The morphology or size and marker proteins of exosomes(CD63,TSG101 and Calnexin)were evaluated by transmission electron microscopy(TEM)and Western Blot analysis,respectively.(3)Exosomes wer dyed by PKH67,and incubated with macrophages in vitro or injected intravenously into nude mice via the tail vein,then confocal microscopy or flow cytometry(FCM)was used to detect PKH67 labeled exosomes were intake by THP-1macrophages(induced by 100 ng/ml PMA for 48 hours)or peritoneal macrophages.(4)THP-1 macrophage were co-cultured with different exosomes(exo-con and exo-TM)for 24 hours,then cells and supernatants were collected for evaluating the expression of PD-L1 and inflammatory factors by flow cytometry(FCM)and a human inflammatory cytokines kit,respectively.(5)THP-1 macrophages were co-culture with different exosomes(exo-con and exo-TM)for 24 hours,then cells were collected for total RNA extraction.Affymetrix m RNA chip assay was carried out to detect the m RNA levels of PD-L1 and various inflammatory factors.(6)BALB/c nude mouse were injected with 10?g exosomes(exo-con and exo-TM)or PBS through tail intravenously every other day for 10 times.Mice were euthanized 24 hours after the last injection,and macrophages were collected from the peritoneal lavage fluids and allowed to adhere for 2 hours,thereafter the suspension cells were discarded.The remaining cells were cultured overnight,and cells and the supernatant were collected for detecting PD-L1 and inflammatory cytokines expression by flow cytometry(FCM)and a mouse inflammatory cytokines kit,respectively.(7)High throughput sequencing was employed to analyse the impact of ERS on the expression of mi RNAs encapsuled by exosomes derived from Hep G2 cells either under ERS or not.KEGG pathway analysis and GO analysis were used to evaluate the association of differentially expressed exosomal-mi RNAs with the functions of THP-1macrophages.(8)THP-1 macrophages were co-culture with different exosomes(Exo-con and Exo-TM)for 24 hours,then cells were collected for total protein extraction.Western Blot assay was used to detect the protein levels of PTEN,p-PTEN,AKT and p-AKT.(9)PTEN expression in THP-1 macrophages was interfered by using a specific si RNA.The protein level of PTEN-PI3K-AKT pathway was evalusted by Western Blot analysis,while the expression of PD-L1 on macrophages was measured by flow cytometry analysis.(10)mi RNA-23-3p mimics,inhibitor and their corresponding control was transfected into THP-1 macrophages for 48 hours.Western Blot assay and flow cytometry analysis was used to evaluate the impact of mi RNA-23-3p on the PTEN-PI3K-AKT pathway and PD-L1 expression,respectively.Results(1)Results showed that TM could concentration dependently increase GRP78 expression,while only a slightly increasing was abserved when the concentration of TM above 3?M.The expression level of GRP78 protein increased along with co-incubation time,while no difference was obsevered between 24 hours and 48 hours(P=0.671).(2)The pellet harvested from the supernatants of HCC cells treated with TM or not demonstrated round or class round vesicles with membrane like structures diameter in40 to 100 nm under transmission electron microscope(TEM),a typical characteristics of exosomes.CD63 and TSG101,but not the ER molecular chaperones Calnexin were observed in the harvested pellets,which further confirmed that the pellets were true exosome without cellular components.The total protein of exosome was quantitated by using the BCA protein assay kit,and the results demonstrated that co-incubation of TM significantly increase the secretion of exosomes in HCC cells(P<0.01).(3)PKH-67 labeled exosomes were effectively engulfed by THP-1 macrophages and peritoneal macrophage by using a laser confocal microscope.And flow cytometry analysis further confirmed that peritoneal macrophages could up-take PKH-67 labeled exosomes in vivo.(4)Affymetrix m RNA chip showed that the m RNA level of PD-L1 and various inflammatory factors(such as IL-1 ?,IL-1 ?,IL-6,IL-10 and TNF-?)increased significantly in the Exo-TM groups compared with Exo-con and control groups.(5)Exo-TM co-incubated with THP-1 macrophages significantly increased PD-L1 expression compared with either control or Exo-con group as demonstrated by flow cytometry analysis and immunohistochemistry assay.Meanwhile,the expression of inflammatory factors,such as IL-1?,IL-6,IL-10 and TNF-?increased significantly in the Exo-con and Exo-TM group compared with control group,and Exo-TM group had even higher increasing.Similarly,Exo-TM significantly increased the expression of PD-L1 and inflammatory factors(such as IL-6,IL-10 and TNF-?)in peritoneal macrophages after mice were injected intravenously through the tail vein with exosomes for 10 times.(6)High-throughput sequencing analyzed the expression characteristic of mi RNAs between Exo-con and Exo-TM,and showed that 112 mi RNAs were differentially expressed and 14 of them were significantly different.Relative to Exo-con group,there were 8 mi RNAs and 6 mi RNAs upregulated and down-regulated in the Exo-TM group,respectively.q RT-PCR showed that mi R-23a-3p and mi R-29a-3p increased 2.44 and1.95 folds in the Exo-TM group compared with Exo-con group,respectively.While mi R-486-3p and mi R-486-5p decreased 0.87 and 1.96 folds in Exo-TM group.PTEN was preticted to be one of the target genes of mi R-23a-3p according to bioinformatics analysis,and it may involved in regulating PI3K-AKT pathway.(7)Western Blot analysis showed that Exo-TM co-incubated with THP-1 macrophages for 24 hours could significantly decrease the levels of PTEN and increase the levels of p-AKT,respectively.Transfection THP-1 macrophages with PTEN specific si RNA could obviously increasing the expression of PD-L1 on macrophages.(8)Transfection THP-1 macrophages with mi R-23a-3p mimics lowered the expression of PTEN on THP-1 macrophages while increasing p AKT protein levels.On the contrary,transfection THP-1 macrophages with mi R-23a-3p inhibitor increased PTEN level and decreased p AKT protein levels,respectively.Flow cytometry analysis show that transfection mi R-23a-3p mimics can significantly increased the expression level of on PD-L1 THP-1macrophages.Conclusions(1)ER stress can affect the secretion of exosome in liver cancer cells.(2)HCC cells ER stress related exosome can be phagocyted by macrophage,and affect the immune function of macrophage by up-regulating the expression of PD-L1 and inflammatory factors(such as IL-6,IL-10 and TNF-?).(3)HCC cells ER stress related exosome can transmit mi RNA-23a-3p to macrophages and inhibit PTET expression,which further activate PI3K-AKT pathway and up-regulate PD-L1 expression.