Analysis Of Radiation Therapy Enhancing The Response Of Immune Checkpoint Inhibitor Therapy In A Melanoma Murine Model

Background:Long term durable response to immunotherapy remains low,and approaches to overcome resistance are lacking.Combination radiation with immune checkpoint inhibition(ICI)shows promise in clinical trials;however,the mechanism underlying this potentiation of ICI by radiation remains unclear,thereby limiting efforts to predict therapeutic response and further adapt this treatment paradigm.In the present study,we develop a novel murine model to decouple post-irradiation changes in tumor mutational burden(TMB)and intratumoral heterogeneity(ITH)to investigate their effects on response to CTLA-4 and PD-L1 blockade.Methods:Parental mouse melanoma B16F10 cells were irradiated in vitro(5Gy × 3 fractions),then ana priori determined number of resulting colonies were implanted in C57BL/6J immunocompetent mice to create a syngeneic model of unirradiated and irradiated tumors with low-and high-ITH(irradiated-L and irradiated-H,respectively).Mice were subject to treatment with placebo,α-PD-L1,α-CTLA-4 or dual ICI.Survival and tumor growth were monitored.CD4 and CD8 staining were performed to observe T lymphocyte infiltration at baseline levels in different models.CD4 and CD8 immunohistochemical staining of tumors in the post-radiation model(Irradiated-L)were performed to observe the dynamic change of tumor microenvironment after immune checkpoint inhibitor treatments.The DNA and RNA of paired tumor specimens in the Parental and Irradiated-L were extracted for whole exome sequencing and RNA sequencing to explore effect of radiation therapy on tumor cells.Results:Tumors formed from irradiated-L cells showed an increase in TMB(618 exon mutations)and sustained decrease in ITH.Irradiated-L tumors were predicted to express five neoantigens with high VAF(>0.25)consistent with a clonal/public distribution.Mice with irradiated-L and irradiated-H tumors experienced a survival benefit with dual ICI whereas those with parental B16F10 tumors did not(median survival irradiated-L 38.5d,irradiated-H 28.5d,parental B16F10 21d,p=0.0004).On pairwise comparison,overall survival with dual ICI was higher in mice with irradiated-L versus irradiated-H tumors(p=0.045).Only mice with irradiated-L tumors showed an overall survival benefit with single agent ICI(placebo 22.5d,α-PD-L1 26d,α-CTLA-4 34d,dual ICI 38.5d,p=0.0002).Tumor infiltration of CD4 and CD8 increased after radiation in tumor microenvironment and CD8 was increased more significantly.There was no statistical difference between the two post-irradiation tumor models.In the Irradiated-L model,CD4 was significantly elevated in the α-CTLA-4 group and the dual ICI group,and CD8 was significantly increased in each treatment group.Through whole exome sequencing,RNA sequencing,paired sample analysis and neoantigen prediction in the Parental and Irradiated-L groups,it was shown that the tumors after radiation irradiation had increased by 618 exon mutations and were accompanied by an increase in VAF(Variant Allele Frequency).Neoantigen predictions on pre-and post-irradiation pared specimens suggest that radiation induction of neoantigens with semi-maximum inhibitory concentrations(IC50)below the corresponding wild-type epitopes and the corresponding genes for each neoantigen have a high variable allele frequency(VAF>0.25).Conclusions:In the baseline groups,tumor growth was delayed,and survival of Irradiated-L and Irradiated-H achieved significance compared with Parental model.Compared with the baseline of the parental models,the tumor microenvironment of post-irradiation models have a higher CD4 and CD8 T lymphocytes infiltration and the CD8 T cell is more pronounced.In the Parental tumor model,dual immune checkpoint inhibitors showed a tendency to inhibit tumor growth and prolong survival but did not produced statistical differences.In the Irradiated-L tumor model,α-CTLA-4 and dual ICI showed significant tumor growth inhibition and α-PD-L1,α-CTLA-4 and dual ICI significantly prolonged survival.Immunohistochemistry suggests a significant increase in CD4 in the α-CTLA-4 and dual ICI groups,CD8 increased significantly in all treatment groups.In the Irradiated-H tumor model,survival significance was achieved by dual ICI.In addition,Irradiated-L mice had significant improvements in overall survival at dual ICI treatment compared to Irradiated-H mice,suggesting the efficacy of immune checkpoint inhibitors was associated with decreased clones after irradiation.Whole exome sequencing,RNA sequencing and neoantigen prediction with the pared specimens suggest elevated tumor mutation burden post-radiation and the emergence of radiation-induced clonal neoantigens.Suggests that radiation therapy can enhance the tumor immunogenic recognition by increasing tumor TMB and inducing clonal neoantigen expression,improving efficacy of ICI.