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. 2021 Mar 25;12:1858. doi: 10.1038/s41467-021-22088-1

Fig. 9. MDA5-TBK1-IRF3 signaling within the TME mediates antitumor efficacy.

Fig. 9

a (top) Treatment strategy combining mRIPO with DMSO, Amlexanox (AMX; TBK1:IKKε inhibitor, 250 nmol), or IKK16 (IKKα/β inhibitor, 140 nmol). a (bottom) Tumor volume at the time of harvest (left) and analysis of intratumor CD4+ (middle) and CD8+ (right) T cell expression of GzmB and T-bet (n = 7 DMSO/AMX, n = 6 IKK16; data bars represent mean −/+ SEM; Supplementary Fig. 13 presents extended data). P value indicates Tukey’s post-hoc two-tailed test. b Mice were treated with mock or mRIPO −/+ AMX (250 nm) as in (a) and mean tumor volume + SEM is shown (n = 8 mock + AMX, n = 7/group all others); (#) two-way ANOVA p < 0.05 vs all groups (two-tailed), mRIPO vs: mock p = 0.0003, mock + AMX p = 0.01, mRIPO + AMX p = 0.01. c Mock or Poly(I:C) (30 μg) was co-injected with 100 μg of isotype control IgG antibody or IFNAR-blocking antibody (all intratumor) into B16-F10-OVA tumor-bearing mice; mean tumor volume + SEM is shown (n = 10/group). (#) two-way ANOVA p < 0.05 vs all groups (two-tailed), Poly (I:C) + IgG vs: mock + IgG p = 0.005, mock + α-IFNAR p < 0.001, Poly (I:C) + α-IFNAR p = 0.02. d, e Mock or LPS was injected with and without 5000 IU of IFNα, mean tumor volume + SEM is shown (d, n = 8/group); (#) two-way ANOVA p < 0.05 vs all groups (two-tailed), LPS + IFNα vs mock and IFNα p < 0.0001, LPS p = 0.002. e Thirteen days after treatment, tumors were harvested from tumor-bearing, surviving mice for flow cytometry analysis (n = 3 mock/LPS + IFNα, n = 5 IFNα, n = 4 LPS only); heatmap (left) represents mean % live cells, data bars (right) represent mean −/+ SEM; see Supplementary Fig. 14a for extended analyses; (*) Tukey’s post-hoc p < 0.05 (two-tailed) vs PBS control. f Mouse PEC were treated with mock, Poly(I:C) (10 μg/ml), Poly(I:C)-tfx (250 ng/ml Poly I:C-lyovec), or LPS (100 ng/ml); supernatant cytokines were measured, %maximum values were calculated, and mock values were subtracted from all other values; mean values are presented (n = 2 experiments). g Mice were treated i.t. with mock, PEI, Poly(I:C) (30 μg), or Poly(I:C) (8 μg) complexed with PEI [Poly(I:C)-PEI]. Tumor volume + SEM is shown (n = 7 PEI and Poly(I:C)-PEI, n = 6/group all others); see Supplementary Fig. 14b for associated flow cytometry analysis at day 16. (#) two-way ANOVA p < 0.05 vs all groups (two-tailed), Poly (I:C)-PEI vs all other treatment groups p < 0.0001. h WT or MDA5−/− mice bearing B16 tumors were treated with PEI or Poly(I:C)-PEI as in (g). Tumor volume + SEM is shown (n = 10/group WT; n = 11/group MDA5−/−); (#) two-way ANOVA p < 0.05 vs all groups (two-tailed), MDA5 −/− Poly (I:C)-PEI vs WT and MDA5−/− PEI p < 0.0001, vs MDA5 −/− PEI p = 0.001. i B16 wt tumors from mice treated as in (h, n = 10/group) were harvested eight days post-treatment and analyzed for T-bet and GzmB expression in tumor-infiltrating T cells, see Supplementary Fig. 14c for extended data; data bars depict mean −/+ SEM, (*) Tukey’s post-hoc p < 0.05 (two-tailed) vs WT PEI; from left to right asterisks: p = 0.004, 0.007, 0.02, 0.0006, 0.03. All experiments were repeated at least twice and representative series are shown.