Effects Of Exosomes Released By Endoplasmic Reticulum Stress On Antitumor Activity Of Dendritic Cells In Hepatocellular Carcinoma

Background Hepatocellular carcinoma(HCC),as the primary histological type of liver cancer,is characterized by high morbidity,fast disease progression and high-grade malignancy.Therefore,most of the HCC patients are usually diagnosed at the advanced stage of the cancer.However,advanced liver cancer can be treated with very limited means.Due to unsatisfactory effects,the patients treated with traditional radiotherapy are susceptible to the recurrence and metastasis of cancer.In this context,it is critical to find new therapies for HCC.The rise of immunotherapy has spawned many new antitumor therapies based on body immunity,including the dendritic cell therapy.Dendritic cells(DCs)are regarded as the most effective antigen-presenting cell in the body,because it can recognize and present tumor-associated antigens and initiate the corresponding antitumor process.Some studies have shown that DCs have antitumor effects,but their clinical application is less than satisfactory.The key to treating tumors with DCs is to find more effective tumor antigens,which can strongly activate DCs and facilitate their maturation to mediate the occurrence of antitumor immunity in the body.During the growth of many malignant tumors,the tumor cells,affected by a variety of factors like lack of nutrients and oxidative stress,will be under endoplasmic reticulum stress(ERS),thus activating the unfolded protein reaction(UPR).To a certain extent,ERS can promote the growth of tumor cells,inhibit antitumor immunity and induce drug resistance in tumor cells.The occurrence of ERS has bidirectional effects on tumor cells,but its impact on antitumor activity of DCs is not yet entirely clear.Exosomes(Exo)are small vesicles with a range of diameters from 40 to 100 nm.As an important instrument for cell communication,it carries a lot of biological information.The exosomes of tumor cells contain a large number of tumor antigens,which may provide some new ideas for the prevention and treatment of tumors.The present study intends to investigate how the exosomes released from tumor cells after ERS affect the function of DCs.Objective1.The effects of exosomes released from HCC cells after ERS on the activation of DCs and lymphocyte proliferation were explored.2.An animal experiment was conducted to investigate how the DCs activated by such exosomes affect the tumorigenesis,lymphocyte subsets,and cytokine levels in mice.Methods1.Hepa1-6 cells with good growth status were co-cultured with different concentrations of TM(1.25,2.5,5umol / L)for 12,24,and 48 hours,and the expression of ERS-related proteins in the cells was detected by western-blot.Stimulation conditions with obvious effects and good cell status were selected as experimental conditions for inducing cell ERS.2.Exo Quick-TC kit was not used to extract exosomes from the supernatant of stimulated hepa1-6 cells,labeled Exo-Con;TM stimulated hepa1-6 cells to generate ERS,and then Exo Quick-TC kit was used to extract exosomes,Marked as Exo-TM.The morphology was observed by cryo-electron microscopy,and the expression of CD63,TSG101,and Calnexin was detected by western-blot.3.Cell experiments were divided into control group and experimental group.The control group was divided into a negative control(Mock group)and a positive control(LPS group).DCs in the Mock group were not treated.DCs in the LPS group were co-cultured with LPS for 48 hours to activate the cells.The experimental components were Con group and TM group.DCs were co-cultured with the same amount ofExo-Con and Exo-TM for 48 h.Flow cytometry was used to detect the surface molecular levels of DCs and observe the activation of DCs.Finally,the treated DCs were co-cultured with mouse lymphocytes,and the lymphocyte proliferation was observed with CFSE staining.4.Animal experiments were divided into control group and experimental group.The experimental components were i DC group,Con group and TM group,with 5 mice in each group.The mice in the experimental group were injected with 100 ul of tail vein containing 1×10~6 untreated DCs,DCs co-cultured with Exo-Con for 48 hours and DCs co-cultured with Exo-TM for 48 hours.100 ul sterile PBS.Inject once every other week for three times.One week after three injections,each mouse was subcutaneously injected with 7×10~5 hepa1-6 cells to observe the effect of DCs on tumor formation in mice.5.Two weeks later,the mice were sacrificed to measure the size of the tumor.The lymphocytes in the spleen and lymph nodes of mice were collected,and the changes of lymphocyte subsets in the experimental and control groups were detected by flow cytometry.Finally,the expression levels of INF-γ in the serum of mice in different groups were detected.Results1.The western-blot results showed that the ERS-related protein in hepatocellular carcinoma cell line hepa1-6 was most significantly upregulated when stimulated with2.5umol TM for 24 hours.Therefore,stimulation with 2.5umol TM for 24 hours was selected as the stimulation condition.2.Small vesicles with a disc-shaped double-layer membrane structure with a diameter of 40-100 nm can be observed under an electron microscope;western-blot detection showed that CD63 and TSG101 were expressed but Calnexin was not expressed,which is consistent with literature reports.3.Flow cytometry results showed that the surface molecules of DCs in the positivecontrol group were significantly up-regulated,indicating that DCs activation was successfully induced.In the experimental group,the surface molecules of DCs in TM group were significantly up-regulated,and the cells were significantly activated;DCs in TM group had the highest degree of activation.CFSE staining results showed that DCs in the Con and TM groups could significantly induce lymphocyte proliferation compared to the control group(p <0.01),but the TM group had the most significant effect.4.Animal experiments showed that all five mice in the Blank group were successfully tumor-bearing and the tumors were larger;three mice in the i DC group were tumor-bearing and the tumors were smaller;two mice in the Con group were tumor-bearing and the tumors were smaller.The tumor size of the i DC group and the Con group was significantly smaller than that of the Blank group(p <0.01);none of the five mice in the TM group were successfully tumor-bearing.The results showed that injection of DCs before tumor loading could inhibit the tumorigenicity of tumor cells,and the TM group had the most significant effect.5.Flow cytometry results showed that the CD8~+ / CD4~+ ratio of lymphocytes in the spleen and lymph nodes of TM group mice increased significantly compared with the control group(p <0.05),indicating that DCs in the TM group could stimulate T lymphocyte proliferation,while the TM group mice The level of INF-γ in serum was significantly higher than in Blank group(p <0.05).Conclusions1.The exosomes released from HCC cells after ERS can stimulate the activation of DCs and induce lymphocyte proliferation.2.The results of animal experiment show that the DCs activated by such exosomes can activate antitumor immunity in vivo.The tumorigenesis of tumor cells was significantly inhibited by injecting DCs,as evidenced by the increased proportion of CD8+/CD4+ lymphocytes in the spleen and lymph nodes of mice and the increased INF-γ level in serum.