Supplementary Data from Utility of the RIG-I Agonist Triphosphate RNA for Melanoma Therapy

Supplementary Figure S1 shows that RIG-I, MDA and IRF7 mRNA expression are correlated to the Type I IFN signature in melanoma, while MAVS, TBK1 and IRF3 are not. Supplementary Figure S2 shows a list of gene products correlated with RIG-I expression based on TCGA RNAseq data from 384 melanomas. Ranking is done based on Person correlation coefficients. Supplementary Figure S3 shows a graphical illustration of RIG-I and CXCL-10 expression correlation based on TCGA RNAseq data of melanoma samples. Supplementary Figure S4 shows the results of the analysis of mRNA expression of IFN-β, RIG-I and IP-10 following RNA treatment of C8161 parental cells and CRISPR/Cas generated KO clones. Cells were treated with RIG-I agonist ppp-RNA and respective controls at 100nM. Relative mRNA levels were determined by RT-qPCR 48 hours post treatment by reverse transfection with synthetic ppp-RNA and respective OH-RNA controls as indicated. RIG-I knockout variants are characterized by absence of RIG-I agonist triggered mRNA upregulation secretion. Error bars represent standard deviation. Supplementary Figure S5 shows the flow cytometry analysis of surface antigen expression following ppp-RNA treatment of parental and KO variants of C8161 (a-c) and B16-F10 (d-f). Cells were treated with ppp-RNA and respective controls as indicated at 100nM and analyzed 48 hours post treatment. Supplementary Figure S6 shows IP 10 quantification results of C57BL/6 female mice (n=4 per group), carrying Hepa1-6 liver tumors treated with 50ug RNA complexed with In vivo Jet-PEI (i.v.). Serum was collected for IP-10 quantification 4h post RNA delivery. Error bars indicate standard deviation. Supplementary Figure S7 shows the serum cytokine response profiles 6 hours post first intratumoral treatment with RNA as indicated. Error bars reflect standard deviation. Supplementary Figure S8 shows flow cytometry based analysis of immune cell populations isolated from spleens of B16-F10 tumor bearing mice treated intratumorally with ppp-RNA or respective controls. Spleens were harvested 48h after last treatment. Error bars indicate standard deviation. Supplementary Figure S9 shows the serum cytokine response profile 6 hours post first intratumoral treatment with RNA as indicated. Error bars represent standard deviation. Supplementary Table S1 shows a list of genes comprising the type I IFN signature Supplementary Table S2 shows the results of a Pearson Correlation analysis of OVA-specific CTL frequency versus cytokine expression in the respective animals