Effects Of Radiated Lewis Lung Carcinoma Cell-derived Exosomes On Tumor Growth And Anti-tumor Immunity

Objective: To investigate the effect of radiated tumor cell-derived exosomes on tumor growth and the biological function of immune cells.Methods:1.The effects of different doses of x-rays on the apoptosis and viability of Lewis Lung Carcinoma(LLC)cells were examined by Western Blot and cell clone formation assay.Phospho-Histone H2 a.The DNA damage was detected by Immunofluorescence Staining.2.Extract exosomes from culture supernatant of X-ray irradiated and unirradiated LLC cells by ultrafiltration.Transmission electron microscopy (TEM),Malvern potential analyzer,Western Blot were using to identify the marker protein of exosomes.3.The exosomes were labeled with PKH67 and incubated with LLC cells and dendritic cell cells(DC)for 12 hours.The uptake of exosomes was observed by fluorescence confocal microscope.4.Transwell invasion and migration test was used to examine the effect of exosomes from irradiated and unirradiated LLC cells on invasion and migration.5.The effects of exosomes on the differentiation and maturation of DC cultured in vitro were examined by Flow cytometry.6.In Vivo Experiment: LLC Cells of Lung Cancer were subcutaneously inoculated to establish tumor-bearing mice model.The exosome was injected through caudal vein and then record growth of the tumor.7.Mice were killed on the 21 st day after tumor inoculation,tumor tissue was stripped and weighed,and spleen tissue was taken to prepare single cell suspension.The expression of mature Spleen DC,depletion of T cells,and specific phenotype of regulatory Treg cells was detected by Flow cytometry.8.Serum IFN-γ content in each group of mice were determined by ELISA.Results:1.The expression of PARP,Caspase-3 and Bax in LLC cells increased with the increase of radiotherapy dose.The colony formation of LLC cells decreased with the increased dose of radiotherapy.Confocal microscopy showed positive phospho-histone H2 A staining of LLC nuclear after radiotherapy.2.The exosomes extracted by ultrafiltration method were mostly round in shape and typical in the shape of a chute,with an average diameter of about 118 nm,and expressed the exosome surface marker proteins CD63,CD9 and TGS101.3.A large number of PKH67-positive exosomes entered the cytoplasm after coculture with LLC and DC for 12 hours.4.Transwell cell invasion and migration in vitro experiments showed that unirradiated LLC cell-derived exosomes(LLC-exo)could promote invasion and migration of LLC cells,while radiated LLC cell-derived exosomes(RT-LLC-exo)could inhibit invasion and migration of LLC cells.5.Flow cytometry showed that LLC-exo inhibited DC differentiation as compared with the control,and the earlier LLC-exo was added,the more DC differentiation was inhibited.In addition,LLC-exo inhibited the maturation of DC cells,while RT-LLCexo promoted the maturation of DC.6.In animal experiments,LLC-exo promoted tumor growth,while RT-LLC-exo inhibited tumor growth.Flow cytometry showed that LLC-exo reduced the expression of MHC II and CD40 on spleen CD11 C + DC,the expression of MHC II and CD40 in spleen of tumor-bearing mice treated with RT-LLC-exo was significantly higher than that in control group(p<0.05),LLC-exo can up-regulate the expression of PD-1,Tim-3,Btla and increase the expression of CD4 + CD25 + Foxp3 + Tregs,while RT-LLCexo can improve the depletion of t cells and decrease the expression of Tregs.Elisa showed that LLC-exo could down-regulate IFN-γ and RT-LLC-exo could enhance IFN-γexpression.Conclusions:Radiated LLC cell-derived exosomes(RT-LLC-exo)can inhibit the invasion and migration of tumor cells,and promote the maturation of DC cells,improve T cell depletion,and enhance T cell activation.The exosomes released by radiated tumor cells can activate anti-tumor response.