Research On The Role And Mechanism Of Exosomes In Natural Killer Cell-based Tumor Immunotherapy

Objective:Cancer is a leading cause of human death worldwide.Although surgery,chemotherapy and radiotherapy are effective in the cancer treatment,patients generally have serious side effects,such as hair loss,nausea and bone marrow suppression,significantly lowering the quality of life.Moreover,the five-year survival rate of tumor patients is generally low.Treatments using cell-free exosomes have a significant improvement on experimental cancer therapy.Natural Killer(NK)cells are natural anti-tumor cells,albeit in clinical settings NK cells encounter several problems,such as low overall activation effect,lack of tumor specificity,upregulation of immune checkpoint expression and low burden of tumor gene mutations.All limit the application of NK cells in clinical tumor treatment.This study aims to explore the role and mechanism of memory-like NK cell derived exosomes in tumor treatment.Simultaneously,engineered bispecific exosomes are constructed to activate NK cells and enhance the cytotoxicity of NK cells in antitumor immunity.Methods:1.Human peripheral blood mononuclear cells(PBMCs)were isolated by Ficoll density gradient centrifugation.IL-12,IL-15 and IL-18 co-activated memory-like NK(m NK)cells were cultured from PBMCs.The supernatant of NK cell culture was collected,and exosomes were extracted and purified.The size and concentration of exosomes were examined by nanoparticle tracking analyzer(NTA)system.Western blot was applied to verify the expression of exosome markers CD9,CD63,Alix and cytochrome C.The morphology of exosomes was visualized by confocal microscope.2.The effect of m NK-exo on the viability of tumor cells MGC803,A549 and K562 was studied using CCK-8 assay.The effect of m NK-exo on tumor cell apoptosis was analyzed by annexin V/7-AAD staining of cells,followed by flow cytometry.Human bone marrow-derived mesenchymal stem cells(BMMSCs)were isolated and cultured to study the effect of m NK-exo on the viability of normal cells.3.After staining m NK-exo with the fluorescent dye(Dio),they were incubated with tumor cells,and the uptake of m NK-exo by tumor cells was observed by confocal microscope.To calculate the uptake rate of m NK-exo by tumor cells,the percentage of Dio-positive tumor cells was detected by flow cytometry at different time points.To examine the effect of macropinocytosis on the uptake of m NK-exo,tumor cells were pretreated with macropinocytosis inhibitor EIPA.The proportion of Dio-positive tumor cells and apoptotic tumor cells were hence analyzed to illustrate the cellular uptake and toxicity of m NK-exo,respectively.4.The expressions of proteins along the apoptotic sigalling pathway in tumor cells treated with m NK-exo were revealed by Western blot.To confirm the role of caspase in triggering apoptosis of tumor cells,tumor cells were pretreated with broad-spectrum caspase inhibitors.Consequently,the apoptosis of tumor cells was analyzed by flow cytometry.5.Exosomes derived from cytokine IL-2 activated NK cells were isolated and compared with m NK-exo in their cytotoxicity of tumor cells.The blocking antibody against granulysin was incubated with exosomes at room temperature,and after blocking granulysin the effects of exosomes on tumor cell apoptosis and the expression of apoptosis-related signaling proteins were investigated.6.A bispecific antibody fragment targeting both HER2 on tumor cells and CD16 antigen on NK cell was constructed by merging into lentivirus expression vector,followed by package into lentivirus.To obtain 293 T cells that can stably express bispecific antibody,293 T cells were infected with lentivirus and selected based on puromycin resistance.The exosomes derived from 293 T cells were isolated and identified.The expression of HA tag on the outer surface of exosomes was detected by flow cytometry.The engineered bispecific exosomes when combined with NK cells were used to treat tumor cells.Results:1.Exosomes derived from memory-like NK cells were found to have a diameter of about 122 nm on average by NTA.Western blotting results showed that m NK-exo expressed CD9,CD63 and Alix,but not cytochrome C.The morphology of m NK-exo was round or oval,as visualized by transmission electron microscope(TEM).2.The m NK-exo inhibited the growth of tumor cells and induced tumor cell apoptosis,and the inhibitory effect is both time-and dose-dependent.m NK-exo inhibited the growth of BMMSCs cells at 12 h incubation,but did not cause apoptosis of BMMSCs.3.Confocal microscopy demonstrated that m NK-exo could be efficiently uptaken by tumor cells.The results of flow cytometry showed that tumor cells could engulf m NKexo within 3h,and the cellular uptake reached the peak value in ～12h.Pretreatment of tumor cells with macropinocytosis inhibitor EIPA lowered the uptake of m NK-exo by tumor cells,so lessening the resultant apoptosis.4.Western blot results revealed that m NK-exo upregulated the expression of cleaved caspase 3,cleaved caspase 8 and cytochrome C in tumor cells.After the addition of broad-spectrum caspase inhibitors,the apoptosis of tumor cells decreased.5.Compared with exosomes derived from IL-2 activated NK cells,m NK-exo exhibited a higher toxicity of tumor cells when the same concentration and incubation time were applied.The expression of cytotoxic molecules in both NK cells and their exosomes was detected by Western blot.The results found out that granulysin in m NK cells and their exosomes was significantly higher than that in IL-2 activated NK cells and their exosomes,respectively.Granulysin blocking antibody can alleviate the apoptosis of tumor cells and reduce the expression of apoptosis related proteins in tumor cells treated by m NK-exo.6.Results using Western blot and flow cytometry confirmed that the engineered bispecific exosomes expressed HA fusion protein,and the fusion protein was located on the outer surface of exosomes.Compared with exosomes without expression of bispecific antibody,bispecific exosomes when combined with NK cells increased the apoptosis of tumor cells,showing more cytotoxicity.Conclusion:Exosomes derived from m NK-exo can enter tumor cells through macropinocytosis,induce tumor cell apoptosis through activation of caspase signaling pathway,and inhibit the tumor cell growth.Compared with NK cell-derived exosomes stimulated by single cytokine IL-2,m NK-exos own higher cytotoxicity,where granulysin is at least partially responsible for its enhanced cytotoxicity.In addition,engineered αC16/αHER2bispecific exosomes combined with NK cells can significantly enhance the tumor dismantling effect compared to NK cells alone.