EGFR-TKI enhances abscopal effect of radiotherapy in mice with lung cancer via cGAS-STING signaling pathway
Objective To investigate the effect of epidermal growth factor receptor-tyrosine kinase inhibitor(EGFR-TKI)on the abscopal effect of radiotherapy in mice with lung cancer via the cyclic GMP-AMP synthase(cGAS)-stimulator of interferon genes(STING)signaling pathway and the underlying mechanism.Methods Lung cancer LLC1 cells in the logarithmic growth phase were randomly divided into the control group and irradiation group.Cells in the control group were cultured normally without intervention,while cells in the irradiation group were irradiated with 4 Gy(2 Gy/min for 2 min).Twenty-four hours later,the mRNA levels of MB21D1,TMEM173,TBK1,IRF3,IRF5,IRF7,IFNAR1,IFNAR2,MX1,MX2,IFIT1,and IFIT related genes in the cells were determined by real-time PCR(rtPCR).In addition,100 μL of LLC1 cell suspension in the logarithmic growth phase was injected subcutaneously into the groin of mice to prepare bilateral subcu-taneous tumor-bearing models.The tumor-bearing mice were randomly divided into the control group,8 Gy group,EGFR-TKI group,and 8 Gy+EGFR-TKI group,with 3 mice in each group.The tumor-bearing mice in the control group were not intervened.The tumor-bearing mice in the 8 Gy group were radiated at 8 Gy for 3 times,the tumor-bearing mice in the EG-FR-TKI group were intragastrically administered 1 mg/g EGFR-TKI for 3 times,and the tumor-bearing mice in the 8 Gy+EGFR-TKI group received both radiation and EGFR-TKI as above.The tumor on the right side of the mice was taken out af-ter 17 days of intervention.The levels of cGAS,STING,p-STING,and type I interferon(IFN-1)proteins in the tumor tis-sues were determined by Western blotting.Tumor cell proliferation antigen(ki67)and tumor microenvironment-related pro-teins CD4,CD8,epidermal growth factor receptor(EGFR),forkhead transcription factor p3(Foxp3),calreticulin(CRT),and major histocompatibility complex(MHC)were detected by immunohistochemistry.Results Compared with the con-trol group,LLC1 cells in the radiation group showed significantly higher mRNA expression levels of MB21D1,TMEM173,TBK1,IRF3,IRF5,IFNAR1,IFNAR2,MX1,MX2,IFIT1,and IFIT(all P<0.05),and lower IRF7 mRNA expression(P<0.05).After 17 days of intervention,compared with the control group,tumor volume,tumor weight and relative ex-pression of ki67 protein decreased in all three intervention groups(all P<0.05),with the largest decrease in the 8 Gy+EGFR-TKI group.Compared with the control group,the EGFR-TKI group showed no significant difference in expression levels of cGAS,STING,p-STING or IFN-1(all P>0.05);the 8 Gy group showed no significant difference in expression levels of STING or IFN-1 proteins(both P>0.05),but significantly elevated cGAS and p-STING protein expression(both P<0.05);the 8 Gy+EGFR-TKI group showed significantly elevated expression of all cGAS,STING,p-STING and IFN-1 proteins(all P<0.05).Compared with the 8 Gy+EGFR-TKI group,the expression levels of cGAS,IFN-1,and STING proteins were significantly lower in both the 8 Gy group and EGRF-TKI group(all P<0.05).The expression levels of CD4,CD8,and MHC proteins in all three intervention groups significantly increased(all P<0.05),and the expression levels of EGFR,Foxp3,and CRT proteins significantly decreased in comparison with those of the control group(all P<0.05).Compared with the radiation group,the 8 Gy+EGFR-TKI group had higher CD4,CD8,and MHC proteins,and lower EGFR and CRT protein levels(all P<0.05).Similarly,CD4,CD8,and MHC were significantly higher,and EG-FR,Foxp3,and CRT were significantly lower in the 8 Gy+EGFR-TKI group than in the EGFR-TKI group(all P<0.05).Conclusion EGFR-TKI improves the immune function of the tumor microenvironment by activating the cGAS-STING pathway,thereby enhancing the abscopal effect of radiotherapy in mice with lung cancer.
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