IFNγ-inducible CXCL10/CXCR3 Axis Alters Sensitivity Of Tumor Cells To Ionizing Radiation

Background:Radiotherapies are important strategies to treat cancers.However,the radiation resistance weakens the effect of radiotherapy.To enhance the damaging effects on neoplastic tissues by ionizing radiation and to improve the susceptibility of neoplastic tissues to ionizing radiation have been a research hotspot.In recent studies,the immune system was proven to be involved in efficacious radiotherapies,and deficiency of the immune system can dwindle the quality and effectiveness of radiotherapies.Moreover,a combination of radiotherapy and immunotherapy can produce more robust anti-tumor outcomes due to the synergic effect of the two methods.IFN-γ is one of the most important cytokines in both innate immunity and adaptive immunity,and it plays a critical role in anti-viral function,anti-tumor function,and immunomodulation.Therefore,it hints the potential use of IFN-γ in combined radiotherapy with immunotherapy,which could ameliorate the poor outcome of radiation-resistant radiotherapy.IFN-γ can induce production of CXCL10,and CXCL10 subsequently interacts with CXCR3.The role of CXCL10/CXCR3 axis in the immune system is mainly to regulate the migration,differentiation and activation of immune cells.By regulating the differentiation of Thl cells,this axis stimulates CTL and NK cells to produce IFN-γ and IL-2,and enhances anti-tumor immunity.In addition,the CXCL10/CXCR3 axis also plays an important role in the occurrence and development of tumors.On the one hand,it has a promoting effect on tumors,and on the other hand,it has inhibitory effects on tumors.However,the role of this axis in radiotherapy is still unclear.The CXCL10/CXCR3 axis may also play an important role in radiotherapy to regulate the radiosensitivity of tumor cells.Objective:The aim of the study is to explore the role of IFN-γ and its downstream gene on ionizing radiation.Methods:In the study,we pre-treated radiation-resistant HEp-2(carcinoma of the larynx)cells with 200 ng/mL IFN-γ,which was followed by radiation at a dose of 0 Gy,2 Gy,4 Gy,6 Gy,8 Gy,and 10 Gy.Subsequently,we evaluated the effects of radiation on cell growth via colony formation assay,and used ’single-hit,multiple target’ model to fit cell survival curves and scored radiobiological parameters,including K,N,D0,Dq,SER,and SF2.We also upregulated and downregulated expression level of CXCL10 in HEp-2 cells through construction of overexpression vectors,RNA interference and CRISPR/Cas9.Radiosensitivity of HEp-2 cells was evaluated by applying the same model.Next,we performed in vivo experiments to compare tumor growth and radiosensitivity of CXCL10-KO HEp-2 tumors and parent HEp-2 tumors after a single dose of 20 Gy of radiation.Finally,we validated the change of radiosensitivity of HEp-2 cells by the CXCR3 gene and confirmed that the change is mediated by the interaction of CXCL10 and CXCR3.Results:First,the colony formation assay shows that distinct cancer cells have different levels of radiosensitivity,and the SF2 value of HEp-2 cells was more high among studied cancer cell lines,at 0.68.Namely,68%of cancer cells survived after 2 Gy of radiation.The high SF2 value of cells represents low radiosensitivity or strong resistance.Therefore,we define HEp-2 as radiation-resistant cancer cells.Second,after pre-treating HEp-2 cells with IFN-γ,the survival rate of cells declined,and D0 value and SF2 value decrease,SER>1.These altogether prove the radiosensitizing function of IFN-γ.After knocking out IFN-γ,the survival curve shifts to the right compared with the control group.Also,the survival score,D0 value,Dq value and SF2 value increase,and cells gained higher repair capacity following sublethal damage.Thus,knockout of IFN-γ increases the radio-resistance of HEp-2 cells.Third,the mRNA expression level of CXCL10 in HEp-2 cells were increased after pre-treating cells with 100ng/mL and 200 ng/mL IFN-γ,respectively.Otherwise,the CXCL10 protein level was obviously increased after IFN-γ treatment.It suggests that CXCL10 could be an essential gene in regulating IFN-γ production and the improvement of radiosensitivity.Fourth,CXCL10 may change the radiosensitivity of HEp-2 cells.’single-hit,multiple target’ model is used to fit cell survival curves and radiobiological parameters.The survival curve of HEp-2 cells overexpressing CXCL10 shifts to the left compared with the HEp-2 cells transfected with empty plasmids.Moreover,the surviving score decreases,and the D0 value decline,SER>1.It illustrates that overexpression of CXCL10 can enhance the radiosensitivity of HEp-2 cells.On the other hand,knockdown of CXCL10 significantly increases the survival score,D0 value、Dq value and SF2 value increase,SER<1,and HEp-2 cells are more resistant to radiation.Importantly,we obtained similar results for knockout of CXCL10.Fifth,in vivo experiment 1 and experiment 2,at the end of the experiment at Day14,the control group HEp-2 tumor volume was 0.00±0.00mm3,but the experimental group CXCL10-KO HEp-2 tumor volume was 120.36±48.79 mm3(p<0.05,vs control group)and 20.21 ± 19.29 mm3(p<0.05,vs control group)respectively.The tumor volume of the experimental group was significantly higher than that of the control group.It is suggested that the lack of CXCL10 reduces the radiosensitivity of HEp-2 cells,and inhibits the radiotherapy.Finally,silencing CXCR3 gene resulted in a higher cell survival score with rising D0 value,Dq value,and SF2 value.It relates gene silencing of CXCR3 to enhanced radiation resistance of HEp-2 cells.However,the survival curve of HEp-2 cells overexpressing CXCR3 shifts to the left compared with the HEp-2 cells transfected with empty plasmids.Moreover,the surviving score decreases,and the D0 value,Dq value,and SF2 value decline,SER>1.It illustrates that overexpression of CXCR3 can enhance the radiosensitivity of HEp-2 cells.The above results suggest that the mechanism of CXCL10 enhancing the radiosensitivity to HEp-2 cells may be through its binding to the CXCR3 receptor,thus affecting the killing effect of radiotherapy on HEp-2 cells.Conclusion:IFN-y is a sensitizer of radiotherapy in terms of enhancing the damaging effects to radiation-resistant HEp-2.Overexpression of CXCL10 enhances the radiosensitivity of HEp-2 cells,while silencing CXCL10 reduces the radiosensitivity of HEp-2 cells.The results of animal experiments indicate that the volume of CXCL10-KO HEp-2 tumor is significantly higher than that of HEp-2 tumor,and the tumor tissuelacking CXCL10 is not sensitive to radiotherapy.The CXCR3 also can regulate the sensitivity of HEp-2 cells.And the possible mechanism could be the production of CXCL10 induced by IFN-y binds to the CXCR3,which ultimately facilitates radiotherapies in anti-tumor treatments.