| Background Hepatocellular carcinoma(HCC),one of the most malignant cancers in the digestive system,ranking as the fifth cause of cancer-related deaths in the world,continues to have a high prevalence of drug resistance and lack of curative treatment.In the last several decades,many patients have succumbed to liver cancer despite extraordinary advances in the technologies related to diagnosis and therapeutic modalities.Surgery is the most efficient method in tumor treatment,however most people have lost the surgical chance when tumor is diagnosed.Chemotherapy is a main therapeutic strategy for patients with tumor,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.Nowadays,immunotherapy has become an efficient method in tumor treatment,working out to find specific mechanism in immunotherapy and prolong patients' lives is very important and meaningful.Tumor microenvironment is composed by tumor cells,mesenchymal cells,and immune cells,such as dendritic cells,macrophages and T lymphocytes.It has a close relationship with the development and metastasis of tumors.Immune cells can maintain the stability of human body through immune surveillance and immune clearance.In the process of tumor occurrence and development,tumor cells can influence the microenvironment to cause immunosupression status and achieve progression.Macrophages have strong plasticity,and play a very important role in maintaining homeostasis of the body.Besides,macrophages make up the immune system as one of the important components.A large number of studies have confirmed that macrophages are a significant composition of the microenvironment,and the status of macrophages in the tumor microenvironment can often change into a tumor associated macrophage(TAM),which will turn into promoting tumor progression and proliferation.However,by which way tumor cells “educate” macrophages from an antitumor status to tumor promotion and whether melatonin could reverse it are not clear.Exosomes are micro-membrane vesicles sized 30-100 nm which are released into the extracellular environment and mediating cell-to-cell communication.Therefore,it is of an important clinical significance to study how tumor cells transmit signals to macrophages,and further change their immune functions.In the current study,we aim to find whether exosomes delivered from melatonin treated hepatoma carcinoma cells could influence macrophages imuune functions and the possible mechanism it may be involved in.Objective To evaluate the impact of different exosomes on the immune status of macrophages,the expression of inflammatory cytokines and PD-L1 was detected after macrophages were co-cultured with exosomes derived from HCC cells treated with either melatonin or not for 24 hours.Furthermore,the possible pathway STAT3 which might be related to exosomes on PD-L1 as well as cytokines expression was investigated.Methods(1)HCC cells were co-cultured with 0.1m M melatonin(MT)or not for 24 hours,then exosomes were isolated from the collected supernatants by using Exo Quick-TC kit.The morphology size and protein markers of exosomes(CD63 and Calnexin)were detected by transmission electron microscopy(TEM)as well as Western Blot analysis,respectively.(2)Exosomes were labled by PKH67,and then incubated with macrophages in vitro or injected into nude mice from the tail vein,after that a confocal microscopy and flow cytometry(FCM)were used to detect PKH67 marked exosomes could be intake by THP-1 macrophages induced by PMA(100ng/ml,48h)or peritoneal macrophages.(3)THP-1 macrophages were co-cultured with different exosomes(Exo-con and Exo-MT)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.(4)THP-1 macrophages were co-cultured with different exosomes(Exo-con and Exo-MT)for 24 hours,then cells were collected for detection of PD-L1 and various inflammatory factors by flow cytometry.(5)BALB/c nude normal mouse were injected with 100?l exosomes(Exo-con and Exo-MT)or PBS from tail vein every other day for 10 times.Mice were sacrificed 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,cells and the supernatant were collected for detecting inflammatory cytokines and PD-L1 expression by flow cytometry(FCM)and a mouse inflammatory cytokines kit,respectively.(6)THP-1 macrophages were co-cultured with different exosomes(Exo-con and Exo-MT)for 24 hours,cells were collected for total protein extraction.Western Blot analysis was used to detect the p-STAT3 protein level.(7)STAT3 expression in macrophages was interfered by using a specific inhibitor S3I-201.The protein level of STAT3 pathway was detected by Western Blot analysis,while the PD-L1 expression on macrophages was measured by FCM.Results(1)The results showed that the pellet harvested from the supernatants of HCC cells treated with MT or not were round or class round vesicles with membrane like structures sized in 30 to 100 nm under transmission electron microscope(TEM),which were typical characteristics of exosomes.CD63 but not ER molecular chaperones Calnexin were observed in the harvested pellets,which further confirmed that the pellets were real exosomes 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 MT didn't increase the secretion of exosomes in HCC cells.(2)PKH-67 labeled exosomes were observably 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.(3)Exo-con co-incubated with THP-1 macrophages significantly increased PD-L1 expression compared with control group while Exo-MT decreased PD-L1 expression which were demonstrated both by flow cytometry analysis and immunohistochemistry assay.Meanwhile,the expression of inflammatory cytokines,such as IL-1?,IL-6,IL-10 and TNF-?significantly increased in the Exo-con group compared with control group,while Exo-MT group had a lower increasing.Similarly,Exo-con significantly increased the PD-L1 expression and inflammatory cytokines such as IL-6,IL-10 and TNF-?in peritoneal macrophages after mice were injected from the tail vein with exosomes for 10 times.(4)Western Blot analysis showed that Exo-con co-cultured with macrophages for 24 hours could significantly increase the levels of p-STAT3 protein level while it decreased in Exo-MT group,which means it is of high possibility that exosomes regulate PD-L1 and cytokines expression through STAT3 signal pathway.(5)When macrophages were co-cultured with specific STAT3 inhibitor S3I-201and Exo-con,the expression of p-STAT3 level was decreased,while the PD-L1 expression was also decreased.This result further confirmed that exosomes indeed regulate the PD-L1 expression through STAT3 pathway.Conclusions(1)Melatonin has no impact on the secretion amount of exosome in liver cancer cells.(2)Exosome delivered from HCC cells treated either with melatonin or not can be phagocyted by macrophage,and regulate the immune function of macrophage by regulating the PD-L1 expression and inflammatory cytokines such as IL-6,IL-1?,IL-10 and TNF-?.(3)Exosomes induced by melatonin treated HCC cells regulate PD-L1 expression through STAT3 pathway. |
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