Impact of cholangiocarcinoma cell derived exosomes on natural killer cell function
Objective To investigate the effect of RBE-Exos on the function of natural killer(NK)cells and their underly-ing mechanisms.Methods Immunohistochemical staining was performed to detect the numbers of NK cells infiltrated into cholangiocarcinoma tissues and paracancerous tissues.Exosomes were isolated from RBE cells by ultracentrifugation.The morphology,size and protein markers of RBE-Exos were identified by transmission electron microscopy(TEM),Nanopar-ticle tracking analysis(NTA)and western blot respectively.Immunofluorescence staining was conducted to show the in-ternalization of RBE-Exos into NK-92 cells.The expression levels of adhesion molecules were analyzed by real-time fluo-rescence quantitative polymerase chain reaction(qRT-PCR)and western blot after NK-92 cells were treated with RBE-Exos.LDH detection was applied to analyze the killing activity of NK-92 cells.Results The results of immunohistochemi-cal staining showed that the numbers of CD56(t=2.977,P=0.008)or NKp46(t=4.530,P＜0.001)positive NK cells in tumor tissues were lower than those in paracancerous tissues.The morphology of isolated RBE-Exos was identified by TEM as typical dimpled and cup-shaped particles with expression of CD63 and HSP70.NTA detection showed that the av-erage diameter of RBE-Exos was(102.90±7.00)nm.After 24 hours of incubation,RBE-Exos was observed to enter NK-92 cells by fluorescent staining.NK-92 cells lost the normal character of clustered growing and presented in a dispersed status after treated with RBE-Exos.The results of qRT-PCR indicated that the expression of adhesion molecules CD11a(t=4.999,P=0.038),CD18(t=9.225,P=0.012),and CD54(t=12.050,P=0.007)at the transcriptional levels were reduced in the RBE-Exos-treated NK-92 cells compared with the PBS group.Western blot results displayed that the pro-tein expression of adhesion molecules CD11a(t=5.140,P=0.007),CD18(t=7.177,P=0.002),and CD54(t=8.713,P＜0.001)were all decreased in the RBE-Exos-treated NK-92 cells compared with the PBS group.LDH detection showed that the killing activity of NK-92 cells against target cells was attenuated by RBE-Exos(20:1,t=3.975,P=0.017;10:1,t=5.776,P=0.005).Conclusions Cholangiocarcinoma cells may inhibit the expression of adhesion molecules in NK cells by releasing exosomes,consequently inhibiting NK cells infiltration into tumor site.
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