Fig. 7
IL-6 pathway is activated in tumors treated with ICB therapy upregulating NMD activity. (A) Treatment schedule for (A) and (B). (B)Tumor-free and Panc02.gCtrl tumor-bearing C57/BL6 mice were treated with isotype control (ISO) SO or anti-CTLA-4 + anti-PD-1 combination or untreated on day 1, 4 and 7 and n day 14 post tumor inoculation, mice were bled, to analyze TNF-α, MCP-1, IL-12, IL-10, IL-6 and IFN-γ by Cytometric Bead Array (CBA). (B) Cytokine levels measured by CBA in mouse sera from (A). n = 3-7/group. (C) Representative individual sample from (B) for each cytokine. (D) UMAP depicting expression of SMG1, IL6ST and STAT3 in tumor cells. IL-6 signaling factors perfectly co-localize with SMG1 [21]. Only tumor cells are shown in this figure. (E) IL6ST (gp130) and STAT3 expression at scRNAseq in malignant cells from (D) show a highly significant linear correlation with SMG1. (F) IL-6 signaling upregulates NMD expression in B16.gCtrl cell line. B16.gCtrl were incubated in the presence of hyper-IL-6 for 96 h. Protein levels were analyzed by western blot. (G) IL-6 signaling upregulates NMD. B16, Panc02 and 4T1 mouse tumor cells stably transduced with our luciferase-SIINFEKL NMD reporter plasmid were plated and murine hyper-IL-6 or vehicle was added to the media. 96 h later, luciferase signal was measured. n = 3. (H) 4T1.gCtrl or SMG1KD were treated as in (F). Trp53 mRNA levels were measured by qRT-PCR. (I) 4T1.gCtrl and STAT3KD were treated with hyper-IL-6 as in (F). SMG1 mRNA levels were measured by qRT-PCR. (J) STAT3KD or gCtrl Panc02 were injected in the left and right flanks, respectively, of Rag2/IL2rg-/- mice. Activated OT-I splenocytes were administered intravenously as shown in the schedule (right). n = 9. (K) STAT3KD or gCtrl Panc02 were injected in the left and right flanks, respectively of Rag2/IL2rg-/- mice. Activated Pmel splenocytes were administered intravenously as shown in the schedule (right). n = 7. (L) Antitumor effect of IL-6 blockade and NMD knockdown. Balb/c mice were injected with 4T1.gCtrl or SMG1KD cells. Treatment was carried out as shown in the tumor schedule. n = 6-8/group. 2-way ANOVA corrected with Bonferroni’s test was performed for tumor growth and luciferase evolution over time; 1-way ANOVA corrected by Bonferroni’s test was used in (B); 2-tail t-test employed in (F). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ns = non-significant (p > 0.05)