The Regulatory Role And Mechanism Of CCR2 And CX3CR1 In The Anti-tumor Effect Mediated By Radiotherapy Combined With Anti-CD40

Radiotherapy can kill tumor cells by damaging DNA,at the same time,it may mediate immunosuppression or immune activation,even abscopal effect.The abscopal effect induced by radiotherapy and checkpoint inhibitors has great potential,yer far from satisfactory in clinical settings.The immunological mechanism of the abscopal effect,especially that mediated by mononuclear/myeloid cells remains poorly understood.Monocytes can be divided into phenotypically and functionally different classical inflammatory monocytes and non-classical patrol monocytes according to the expression of CCR2 and CX3CR1.In CCR2-deficient and CX3CR1-deficient mice,radiotherapy combined with a myeloid checkpointαCD40 agonist was used to establish the abscopal effect model,and the role and regulatory mechanism of different monocyte subsets in the abscopal effect were explored.Methods:In this study,radiotherapy combinedαCD40 agonist were used to establish unilateral and bilateral MC38 tumor bearing model in mice,and the tumor volume and survival time of each group were compared after treatment.The expression level of CCR2 and CX3CR1 in murine blood and tumor infiltrating myeloid cells were detected by flow cytometry and immunofluorescence.CCR2 knockout mice and CX3CR1 knockout mice were used to explore the role of CCR2 and CX3CR1 in regulating RT combined withαCD40 induced abscopal effect model,and flow cytometry was used to detect the percentage changes of monocytes,dendritic cell and macrophages in mouse blood and tumor.Moreover,macrophages and dendritic cell of various genotypes of mice were cultured in vitro to observe the effects of polarization of macrophages,phagocytosis function and dendritic cell antigen presentation function.Results:αCD40 was observed to increase the therapeutic effect of radiotherapy on both local and distant tumors in mice.The expression of CCR2 and CX3CR1 were upregulated in blood and tumor infiltrating myeloid cells after combined treatment.In the abscopal effect model established by CCR2 knockout mice and CX3CR1 knockout mice,the distant tumor growth of Ccr2^RFP/+Cx3cr1^GFP/+mice was slower than that of Ccr2^RFP/RFPCx3cr1^+/+mice.However,tumor growth was faster than that of Ccr2^+/+Cx3cr1^GFP/GFPmice.The Ly6C^himonocytes,dendritic cell and macrophages of Ccr2^RFP/RFPCx3cr1^+/+mice were less than those of the other two gene mice.The experiment in vitro confirmed thatαCD40 strengthen dendritic cell antigen presentation function and promote the proliferation of CD8⁺T cells.macrophage phagocytosis of Ccr2^+/+Cx3cr1^GFP/GFPmice were stronger than other genotypes.Conclusion:This study verified thatαCD40 can further enhance tumor control by radiotherapy and mediate abscopal effect in unilateral and bilateral MC38 tumor bearing models.At the same time,abscopal effect were improved in CX3CR1 knockout mice,while dampened in CCR2 knockout mice.The mechanism may be related to the enhancement of macrophage phagocytosis in CX3CR1 knockout mice and the decrease of antigen-presenting dendritic cells in CCR2 knockout mice.This study provides new insights in exploring new targets of radiotherapy combined with immunotherapy mediated abscopal effect.