HIF-1α Regulates The Transcription Of Tumor Cells And Its Exosomes PD-L1 And Affects The Anti-tumor Immune Response After Radiotherapy

Objectives:This study was designed to explore the mechanism of inhibiting hypoxia-inducible factor（HIF）-1αin mouse Lewis lung carcinoma cell line（LLC）and the influence of their exosomes to radiotherapy,as well as how HIF-1αregulates the transcription level of PD-L1 and affects immunological effect mechanism of DC and T cells.Methods:1.After treating with HIF-1αinhibitor PX-478,LLC cells cultured under hypoxia（100μmol/L Co Cl₂）were radiated to observe the nuclear damage,apoptosis,proliferation,invasion and migration in each group.Flow cytometry was applied to observe apoptosis of LLC cells.DNA damage markerγH2AX was detected by immunofluorescence.Western Blot was used to analyze the expression of apoptosis-related proteins.LLC cell proliferation was detected by clone formation test.Transwell experiments were used to assess the effects of radiation on the invasion and migration of LLC cells.2.Ultracentrifugation was used to extract exosomes from LLC cell culture supernatants.The exosomes were identified by transmission electron microscopy,nanoparticle tracing method and Western Blot.The expression levels of HIF-1α,PD-L1,Galectin-9 in tumor cells and their culture supernatant exosomes of hypoxia group,radiotherapy group and PX-478 combined radiotherapy treatment group were detected by q PCR and Western Blot.3.Chromatin immunoprecipitation（Ch IP）was applied to identify the binding site between the transcription factor HIF-1αand the downstream target gene PD-L1in LLC cells.4.Confocal microscope was used to observe PKH67 immunofluorescence-labeled exosomes derived from LLC cells treated with cobalt dichloride（Co Cl₂）and HIF-1αinhibitor PX-478 that were taken up by DC.5.LLC cells were divided into hypoxia group,PX-478 hypoxia group,PX-478combined with radiotherapy hypoxia group,exosome inhibitor GW4869 treatment group,tumor cell freeze-thaw antigen treatment group;LLC cell freeze-thaw antigens extracted by repeated freeze-thaw method and exosomes extracted by ultracentrifugation from the cell culture supernatant of each group,respectively,shocked the dendritic cells（DC）derived from mouse bone marrow on the 6th day of culture.Flow cytometry was applied to detect the effects of LLC cell lysates and exosomes on the DC mature phenotype（MHCII,CD80 and CD86）and immunosuppressive molecular ligand（PD-L1）expression after 24 hours.6.DC and heterogeneous mouse spleen T lymphocytes were mixed for 72 hours,CCK8 was applied to observe the ability of DC to stimulate the proliferation of T cells.Flow cytometry was used to detect the expression of Treg（CD4⁺CD25⁺Foxp3⁺）and CD8⁺IFN-γ⁺T cells,CD8⁺Granzyme B⁺T cells,CD4⁺IFN-γ⁺T cells,CD4⁺Granzyme B⁺T cells,and T cell exhaustive molecule（PD-1,TIM-3,BTLA）expression.7.Established tumor-bearing mouse models were grouped as follows:normal saline control group（control）,radiotherapy group（RT）,HIF-1αinhibitor group（PX-478）,HIF-1αinhibitor combined with radiotherapy group（PX-478+RT）.The tumor growth was measured.The mice were sacrificed on the 24th day after inoculation.The tumor tissues were taken and weighed.The spleen mononuclear cells of the mice in each experimental group were collected.Splenic mononuclear cells were collected from the mice in each experimental group,and were analyzed by flow cytometry to detect the DC mature phenotype（MHCII,CD80 and CD86）and the expression of immunosuppressive molecular ligands（PD-L1,LAG-3）.Additionally,CD4⁺/CD8⁺IFN-γ⁺T cells,CD4⁺CD25⁺Foxp3⁺Treg and T cell exhaustion phenotype（PD-1,TIM-3,BTLA）were determined by flow cytometry.ELISA was used to detect the level of serum cytokine secretion of IL-2 and IFN-γ.q PCR was used to determine the level of HIF-1α,PD-L1,Galectin-9 in tumor tissue and spleen of each group of mice.Results:1.Confocal microscopy observed that the DNA breakage and damage of HIF-1αinhibition group increased significantly after radiotherapy（p<0.05）;cell apoptosis was significantly increased by flow cytometry（p<0.05）;the expression of PARP,Caspase-3,and BAX proteins were up-regulated,while Bcl-2 protein was decreased in the HIF-1αinhibitor group（p<0.05）;the clone formation experiment showed that the higher expression of HIF-1αand more cells colonies formed in LLC cells（p<0.01）;at the same time,the cell invasion and migration ability of the HIF-1αinhibition group was weaker than that of the hypoxia group（p<0.01）.2.The results of transmission electron microscopy,nano-particle size tracking technology and Western Blot showed that the exosomes extracted by ultracentrifugation showed a cup-like double-layer membrane structure with an average diameter of about 110 nm.The specific proteins CD63,CD9,TSG101 were expressed in exosomes.In the exosomes isolated from tumor cells treated with HIF-1αinhibitor in combination with radiotherapy,the expression levels of HIF-1α,PD-L1,Galectin-9 m RNA and protein were significantly reduced（p<0.05）.3.Ch IP assay showed that HIF-1αdirectly regulated the expression of PD-L1（p<0.01）as evidenced by binding to the PD-L1 hypoxic promoter region（HER-1,HRE-2/3,HRE-4）.4.Confocal fluorescence microscopy observed that exosomes were absorbed by DC after 12 hours of co-culture.5.Flow cytometry showed that compared with the exosomes derived from hypoxic control group,exosomes derived from the LLC cells treated with PX-478combined with radiotherapy could stimulate DC’s mature phenotype with high expression of MHCII,CD80 and CD86,while the less expression of immunonegative regulatory molecule PD-L1（p<0.01）.Compared with the hypoxic control group,the expression of DC mature phenotype molecules cultured with the radiotherapy group and the inhibitor group exosomes was higher,while the expression of immunosuppressive molecules was lower.There was no difference in the expression of MHCII,CD80,CD86 and PD-L1 between the radiotherapy alone group and the inhibitor group.6.CCK-8 results showed that exosome-shocked DCs derived from the LLC cells treated with PX-478 combined with radiotherapy could activate the proliferation of T lymphocytes more than hypoxia control group（p<0.01）;flow cytometry results show that exosome-treated DC and T cells mixed culture in PX-478 combined with radiotherapy group,T cell exhaustion markers（PD-1,TIM-3,BTLA）were significantly reduced（p<0.05）,Treg cells were significantly reduced（p<0.01）,CD8⁺IFN-γ⁺/Granzyme B⁺T,CD4⁺IFN-γ⁺/Granzyme B⁺T cells increased significantly（p<0.01）.7.Animal experiments showed that using radiotherapy and HIF-1αinhibitor treatment alone can inhibit tumor growth,whereas the combination of the two could further inhibit tumor growth（p<0.001）.The flow cytometry results showed that the combined treatment Up-regulated the expression of MHCII,CD40,CD80,and CD86molecules on the surface of CD11c⁺DC,and down-regulate the expression of immunonegative regulatory molecules such as PD-L1 and LAG-3 on the surface of CD11c⁺DC（p<0.05）In addition,the combination therapy down-regulated the expression of PD-1,TIM-3,BTLA in the spleen T cells of tumor-bearing mice,and reduce the expression of CD4⁺CD25⁺Foxp3⁺Treg（p<0.05）;ELISA showed that the combination therapy up-regulated the cytokines IFN-γand IL-2 expression（p<0.05）.The results of q PCR showed that the expression of HIF-1α,PD-L1,and Galectin-9 in the spleen and tumor tissues of the combined treatment group was significantly reduced（p<0.05）.Conclusion:1.HIF-1αinhibitor PX-478 could enhance the nuclear damage and apoptosis of LLC cells induced by radiotherapy,and could inhibit the invasion and migration of tumor cells.2.HIF-1αcould combine with PD-L1 hypoxia promoter region to regulate its expression,and inhibiting HIF-1αcould down-regulate the expression of PD-L1 in tumor cells.3.Tumor-derived exosomes extracted by ultracentrifugation could be taken up by DC.Exosomes derived from LLC cell after treatment of PX-478 and combined with radiotherapy could promote the maturation of DC cells,reduce T cell exhaustion and Treg cells.This combination treatment also could promote T cell proliferation and activation,suggesting that HIF-1αregulates PD-L1 expression,activated DC antigen presentation and the anti-tumor response ability of T lymphocytes,and reduced the radiotherapy immune tolerance of tumor cells.4.The HIF-1αinhibitor PX-478 combined with radiotherapy could inhibit tumor growth,activate DC antigen presentation and the anti-tumor response ability of T lymphocytes,and reduce the radiotherapy immune tolerance of tumor cells.