| Objective Abscopal effect,referring to a localized irradiated treatment of metastatic cancer resulting in the regression of untreated tumors at a distance,had been connected to mechanisms involving the immune system.Activation of Sting signaling pathway appeared to be an essential component in inducing the systematic immune response after radiotherapy.However,the relationship between Sting pathway activation and radiation induced abscopal effect is still unclear.This study demonstrated the effect of different radiation doses on the activation of Sting pathway in syngeneic GL261(mouse glioma cell)mice model.We also partly explain how the activated Sting pathway related to the anti-tumor immune effects in localized or abscopal GL261 tumors.Methods:This study consists of in vitro and in vivo experiments.To investigate the effects of different doses of irradiation on the activation of Sting signaling pathway in GL261 cells and the effect of activation of Sting signaling pathway on the abscopal effect.For in vitro studies,GL261 cell line was irradiated with different irradiation doses(0 Gy,8 Gy,20 Gy).Cell viability and immune-related molecules level were measured.For in vivo studies,animal tumor models were established by inoculating tumor cells subcutaneously into the bilateral back in C57 mouse.mice were received X-ray on their one side tumors of different doses(the irradiated side is the local tumor and the unirradiated side is the abscopal tumor).The expression of Sting,IFNβprotein and mRNA in local and abscopal tumor tissues and the infiltration of DC,CD8a+T cells in tumor microenvironment were measured after different doses of irradiation.For in vitro studies:(1)GL261 cell line was cultured for 5 days after single X-ray irradiation with 0 Gy,8 Gy or 20 Gy.CellTiter Glo kit was used to detect the inhibitory effect of X-ray irradiation with different doses.(2)GL261cell line was cultured for 24 hours after single X-ray irradiation with 0 Gy,8 Gy or 20 Gy.The expression of cytoplasmic dsDNA in GL261 cells was detected by double-stranded DNA Assay Kit.(3)The expression of Sting and IFNβ in GL261 cells after irradiation were detected by RT-PCR(4)The quantitation of Sting protein in GL261 cells after irradiation were detected by Western Blotting.For in vivo studies:(1)animal tumor models were established by inoculating tumor cells subcutaneously into the bilateral back in C57 mouse.When tumor volume achieved 150mm3,mice were randomly divided into three groups and received X-ray on their one side tumors of 0 Gy,8 Gy or 20 Gy separately.The mice were sacrificed on day five after the irradiation and the tumor tissues were maintained for further exploration.(2)Expression of Sting and IFNβ in the local and abscopal tumor tissues was detected by RT-PCR.(3)Expression of Sting in the local and abscopal tumor tissues was detected by Western Blotting.(4)Other than that,ELISA kit detected the protein of IFNβ in the local and abscopal tumor tissues.(5)The infiltration of DC cells and CD8a+T cells in the tumor microenvironment locally and abscopally were also examined by Flow cytometry.(6)The infiltration of CD8a+cells in the tumor microenvironment locally were also examined by Immunofluorescence.Results:we detected the expression of related molecular proteins and mRNA of GL261 cells after irradiation.The mouse model was successfully constructed and the tumor tissue was retained after irradiation.The above experiments were completed.The main results are as follows:In vitro studies:(1)CellTiter Glo kit test showed that the relative cell number of GL261 cells after 0 Gy,8 Gy and 20 Gy irradiation was(100.0±2.62)%,(91.40±0.66)%and(86.69±1.31)%respectively,the cell viability was inhibited in 8 Gy and 20 Gy group compared with the control group(F=15.07,P<0.001).Stronger inhibitory effect were observed in 20 Gy irradiation group.(2)Expression level of dsDNA in cytoplasm were detected by dsDNA Assay kit after irradiation with 0 Gy,8 Gy and 20 Gy which was(21.03±0.58)ng,(114.9±1.92)ng,(124.9±1.84)ng respectively,expression of dsDNA in cytoplasm of 8 Gy and 20 Gy were higher than control group(F=1330,P<0.0001).20 Gy is more effective than 8 Gy(P＜0.05).(3)Sting expression measured by RT-PCR,showing a significant increase trend in GL261 cells after irradiation with 8 Gy,20 Gy of that in the control group(P>0.05).Similarly,the expression of IFNβ determined by RT-PCR was higher in GL261 cells treated with 8 Gy(4.01±0.16)or 20 Gy(4.1±0.4)irradiation compared with the control group(1.01±0.13),(F=39.76,P<0.001).(4)The expression of Sting protein also showing a significant increase by Western Blotting,was 1.53±0.12,2.3±0.03 with 8 Gy and 20 Gy respectively,and was higher than the control group(1.0±0.01),(F=74.83,P<0.01).In vivo studies:(1)In mouse model,whereas different situations occurred in animal models.RT-PCR showed that the expression of Sting and IFNβ in the local and abscopal tissues of mice in the 8 Gy group have an increasing trend than those in the control group and 20 Gy group(P>0.05).(2)Expression of Sting protein was consistently increased in both local and abscopal tumor tissues after one-side 8 Gy irradiation treatment,which is 1.99±0.11(P<0.001)and 1.36±0.05(P=0.002)of that in control groups.However,even lower expression of Sting was found in 20 Gy Group in either local(0.34±0.006)or abscopal(0.17±0.008)tumor tissues(P<0.001).(3)In addition,IFNP showed higher expression only in local tumor tissue(68.84±2.40 pg/mL)in 8 Gy group(111.70±13.96 pg/mL)as detected by ELISA(F=4.34,P<0.05).(4)On the other hand,flow cytometry results suggest that both DC and CD8a+T cells infiltration in the primary and abscopal site under 8 Gy irradiation were enhanced in comparison with the control group(3.48±0.44)/total%,(5.28±0.77)/total%,(F=6.79,P<0.05),while that was not happened in 20 Gy group.(5)Immunofluorescence showed that the infiltration of CD8a+cells in the local tissues of mice in the 8 Gy group have an increasing trend than those in the control group and 20 Gy group.Conclusion:(1)For achieving an enhanced systematic anti-tumor immune response by radiotherapy,Appropriate dose of irradiation(8 Gy of X-Ray in this study)was crucial to promote the activation of Sting signaling pathway in GL261 mice model.(2)Activation of Sting signaling pathway can promote the expression of immune-associated factor IFNβ and increase infiltration of DC and CD8a+T cells in local and abscopal tumor tissues to enhance anti-tumor immunity. |
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