A Study On The Mechanism Of NK Cell Dysfunction Induced By Clear Renal Cell Carcinoma Derived Exosomes

Objective: Clear cell renal cell carcinoma(ccRCC)with a high incidence and low rate of early detection is a common urinary tract tumor in China The development and clinical outcome of ccRCC are affected by the regulation of various cells in the microenvironment and antitumor effect of immune cells inhibited by the regulation of ccRCC is closely related with poor prognosis of the clinical progress.As an important part of the innate immune system,Natural killer(NK)cells play a key role in anti-tumor effects.The purpose of this study is to explore the immune response of NK cells in ccRCC and its regulation and mechanism.Methods:(1)The expression,the frequency and number of NK cells of NK cells in the tissues and blood were detected by flow cytometry.(2)Analyze the capacity of degranulate and secrete cytokines in ccRCC infiliating NK cells and the relationship between the NK cell functions and progression of ccRCC.(3)The primary cells extracted from tumor tissue,adjacent tissues and non tumor tissues were incubated with normal NK cells in transwell cells to determine the communication between tumor cells and NK cells.The extracted exosomes from these primary cells was identified by electron microscopy,WB and were further characterized for size distribution analysis.We coculture exosomes with NK cells to analyzed the relationship between exosome and NK cells.We also analyzed the relationship between exosomes concentration and the progress of ccRCC,and found that exosomes caused NK cells dysfunction.(4)The exosomes extracted from the supernatant of the tumor cell line 786-O or the normal renal epithelial cell line HK-2 were incubated with normal NK cells.The level of exosomal TGF-β1 from the supernatant was determined by ELISA.TGF-β1 knockout 786-O cells were also incubated with normal NK cells to detect NK cell cytotoxicty and TGF-β signaling pathway was then detected by WB to claim the mechanism underlying the NK cell dysfunction induced by ccRCC.Results:(1)We found more NK cells infiltrated in adjacent renal tissues and less NK cells infiltrated in control tissues when compared with those in tumor tissue detected by flow cytometry.There was no significant difference in circulating NK cells between tumor tissues and adjacent renal tissues.Tumor infiltrating NK cells displayed an inhibitory property evidenced as upregulation of inhibitory and downregulation of activating receptors.(2)We further determined the functions of NK cells and found that these cells displayed impaired degranulation and cytokine secretion compared with NK cells infiltrated in adjacent tissue or control tissue.And the tumor infiltrating NK cell functions were negatively correlated with the tumor grade.(3)The primary cells and exosomes from tumor tissue,adjacent tissues and non tumor tissues were extracted successfully.Tumor cells had an negative role on NK cell function through exosome way by transwell culture system.The exosomes derived from tumor were negatively correlated with the tumor grade.(4)We found that ccRCC derived exosomes induced NK cell dysfunction through the TGF-β1/SMAD signal pathway.Conclusions:(1)More NK cells infiltrated in adjacent renal tissues and less NK cells infiltrated in control tissues when compared with those in tumor tissue detected by flow cytometry.There was no significant difference in circulating NK cells between tumor tissues and adjacent renal tissues.Tumor infiltrating NK cells displayed an inhibitory property evidenced as upregulation of inhibitory and downregulation of activating receptors.(2)Tumor infiltrating NK cell displayed impaired degranulation and cytokine secretion compared with NK cells infiltrated in adjacent tissue or control tissue.And the tumor infiltrating NK cell functions were negatively correlated with the tumor grade.(3)The primary cells and exosomes from tumor tissue,adjacent tissues and non tumor tissues were extracted successfully.Tumor cells had an negative role on NK cell function through exosome way by transwell culture system.The exosomes derived from tumor were negatively correlated with the tumor grade.(4)ccRCC derived exosomes induced NK cell dysfunction through the TGF-β1/SMAD signal pathway.