| BackgroundHepatocellular carcinoma(HCC)ranks as the fourth leading cause of cancer-related death globally,with approximately 800,000 deaths per year.Comprehensive treatments for HCC include surgery,ablation,arterially directed therapies,radiotherapy,and systemic therapy.However,the overall prognosis of HCC is poor,with a 5 year overall survival rate of only 18% and recurrence rate up to 70%.Therefore,novel treatments for HCC are urgently needed.With the rapid development of radiotherapy and immunotherapy,radiotherapy combined with immunotherapy may further improve the therapeutic effect of HCC.As one of the classic treatments for tumors,radiotherapy has become more widely used in hepatocellular carcinoma.It is widely used clinically for radical radiotherapy for early hepatocellular carcinoma and palliative radiotherapy for advanced hepatocellular carcinoma.The clinical efficacy of radiotherapy for tumor is mainly attributable to tumor cell death in the radiation field owing to the direct or indirect induction of DNA damage by ionizing radiation.More and more evidences show that unirradiated cells near or far from the radiation field also undergo the same biological response as the irradiated cells.It was discovered that the irradiated tumor cells can release signal molecules and affect the biological effect of the unirradiated cells.Radiation-induced bystander effect(RIBE)is a biological effect of radiation,which spreads from irradiated cells to adjacent unirradiated cells,resulting in biological changes in the proliferation of unirradiated tumor cells and damages in the subcellular structure of DNA.Research of RIBE mostly focus on the genotoxic events of normal unirradiated cells.With the use of new unconventional radiotherapy methods that target partial volume tumors,the radiobiological response of local non-targeted tumor cells has become a form of RIBE.It has attracted more and more attention.Radiation-induced abscopal effect(RIAE)is a whole body biological effect induced by local radiation.Local radiation can drive the retreat of unirradiated tumors in the distance outside the radiation field.RIAE is a systemic effect mediated by immunogenic response to distant metastasis sites outside the radiation field.It is generally accepted that radiotherapy has the effect of immune stimulation.After radiotherapy,how to induce increased immunogenicity of tumor cells and how to induce effective anti-tumor immune response via immune effector cells still need to be further clarified.The dose of radiotherapy and the segmentation schemes are also variable factors affecting the direct interaction between irradiated tumor cells and non-irradiated tumor cells as well as the immune-stimulating effect of irradiated tumor cells,which need to be continuously optimized in the future research.Exosomes are extramembrane vesicles derived from endosomes secreted by various cells.Exosomes carry an array of DNA,RNA(messenger RNAs,micro RNAs,and long noncoding RNA),proteins and lipids.Its role as a medium for cell-to-cell communication is particularly important for tumor cells,and it has been a major research topic currently.Some studies have confirmed that the exosomes derived from irradiated tumor cells can exert anti-tumor effect through various mechanisms of immune stimulation.Radiation affects the secretion and the composition of exosomes,including the transcription,translation,cell division and signal transduction of differentially expressed proteins in exosomes,and the changes of miRNA and lipid metabolism in radiation cell-derived exosomes,which may be the factors of tumor inhibition.Exosomes derived from irradiated tumor cells may directly affect the biological effects of unirradiated tumor cells.Studies have shown that the impaired function of DCs may be an important reason for the immune escape of HCC.The treatment based on DCs aims to improve the adaptive immunity against hepatocellular carcinoma cells.Tumor DCs vaccine is an immunotherapy designed based on increasing cytotoxic T lymphocytes or NK cells and other immune cells to enhance tumor-specific anti-tumor immune response.There are various antigen loading methods for DCs vaccines,which play a key role in inducing effective anti-tumor effects.With the continuous development of basic and clinical research,the design and preparation of DCs vaccines are also further optimized in order to maximize their immune induction and anti-tumor effects.Commonly used DCs vaccines are DCs co-incubating with tumor cell lysates containing intact HCC antigens or exosomes derived from hepatocellular carcinoma cells,and DCs transfected with specific tumor antigen adenoviruses.Exosomes derived from tumor cells activate DCs better than tumor cell lysates and induce effective anti-tumor effects.Radiation affects the secretion and the composition of exosomes.Exosomes derived from irradiated tumor cells may have a better activation effect and induce stronger anti-tumor effects than exosomes derived from unirradiated tumor cells.ObjectiveThe objective of the study is to determine the direct effect of exosomes from hepatoma cells irradiated by specific fractionated dose mode on non-irradiated hepatoma cells.Another goal of this work is to investigate the effect of exosomes from hepatoma cells irradiated by specific fractionated dose mode on DCs and whether the DCs can stimulate the proliferation of T lymphocytes in vitro and activate adaptive anti-tumor immunity in vivo,so as to enhance the anti-tumor effect.MethodsAn ExoQuick kit was used in extracting exosomes.The morphology and sizes of the exosomes were observed through transmission electron microscopy,and biomarkers CD63 and TSG101 on the surfaces of the exosomes were detected through Western blotting for the identification of the exosomes.To explore the direct effects of exosomes from irradiated hepatoma cells on non-irradiated hepatoma cells in a specific fractionated dose mode,we detected the proliferation effect of exosomes from irradiated hepatoma cells on non-irradiated hepatoma cells through CCK8 and real-time cell assay.Immunofluorescence detection was performed on the DNA damage marker P53 binding protein 1(53BP1)in the nuclear of non-irradiated hepatoma cells affected by exosomes derived from irradiated hepatoma cells.To explore antitumor immunity mediated by exosomes from irradiated hepatoma cells in a specific fractionated dose mode,we cocultured exosomes from specific fractional doses irradiated hepatoma cells with DC2.4.The phenotype of DC2.4 was analyzed through flow cytometry.The phenotype of bone marrow-derived dendritic cells(BMDCs)was analyzed after the co-culture of exosomes from irradiated hepatoma cells and BMDCs,then the effect of exosomes from irradiated hepatoma cells on DCs were verified.Activated DC2.4 was co-cultured with mouse spleen T lymphocytes,and the proliferation of T lymphocytes was detected through CFSE staining in vitro.Hepa1-6 cell line,Hepa1-6 tumor mass,and passage cells from Hepa1-6 tumor mass were inoculated to homologous C57BL/6 mice subcutaneously.Tumor growth in the tumor-bearing mice was detected,and the subcutaneously tumor-bearing model of the Hepa-1-6 cell line in C57BL/6 was established.PBS,immature DC(i DC),DC co-cultured with exosomes from non-irradiated Hepa1-6 cells(DCexo-con),and DC co-cultured with exosomes from irradiated Hepa1-6 cells(DCexo-ir)were injected into the tail veins of subcutaneous tumor-bearing C57BL/6 mice in groups once a week,then3 × 106 DCs each time,and equal volumes of PBS.Tumor growth in tumor-bearing mice was detected.From five days to seven days after the last injection,the eyeballs of the mice were removed for blood.The mice were sacrificed.Spleens,lymph nodes,and tumor tissues were obtained,and immune cells from peripheral blood,spleens,lymph nodes,and tumor tissues were separated.T cell subtypes were detected through flow cytometry and used in determining the immune status of mice in each group.ResultsThe proliferation of HEPA1-6 cells decreased significantly when Hepa1-6 cells were cultured 48 hours after irradiated with the hypofractionated doses of 8Gy×3f.Exosomes were identified with typical exosomes morphology of cup-dish-shaped double-layer membrane structure and particle size(40-160 nm),exosomal protein markers.Radiation increases the secretion of exosomes from tumor cells,and the quantification of exosomal protein is related to the radiation dose.Exosomes derived from irradiated hepatocellular carcinoma cells induced the bystander effect of non-irradiated carcinoma cells.The exosomes from HepG2 cells irradiated with the conventional fractionated dose of 2Gy showed no proliferation effect in the comparison with non-irradiated HepG2 cells but induced DNA damage in nonirradiated HepG2 cells.Exosomes derived from irradiated hepatocellular carcinoma cells enhance T lymphocyte immunity and anti-tumor response by promoting the maturation of DCs.Exosomes from Hepa1-6 cells irradiated with the hypofractionated doses of 8Gy×3upregulated costimulatory molecules on the surface of DC2.4 and stimulated T lymphocyte proliferation.The subcutaneous tumor-bearing homologous C57BL/6 mice with Hepa1-6 cell line were established.DC2.4 stimulated by exosomes from irradiated Hepa1-6 was injected into subcutaneous tumor-bearing C57BL/6 mice through the tail vein,which increased CD8+T cell in the blood,spleen,inguinal lymph nodes and the tumor,enhanced the anti-tumor immune response and inhibit tumor growth of mice.ConclusionThe above research shows that the exosomes from irradiated hepatoma cells have biological activity and participate in the transmission of DNA damage from irradiated hepatoma cells to non-irradiated hepatoma cells.Moreover,the exosomes from irradiated hepatoma cells inhibited the growth of non-irradiated hepatocellular carcinoma cells in vivo through antigen-presenting cells and T lymphocytes.Our research provides a possible mechanism for determining the non-target effects of irradiation in hepatocellular carcinoma and helps to find safe and effective treatments for hepatocellular carcinoma. |
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