Extended Data Fig. 8: IFN-γ signaling imparts more tumorigenic and metastatic potential.
IFN-γ treatment upregulates a, STAT1 and P-STAT1 (Data representative of minimum of three independent experiments) b, mRNA expression of IFN-γ target genes. qPCR values were normalized to Gapdh. Experiments were performed twice in technical duplicate, and data presented as mean ± SD. Two-sided Student’s t test used to calculate p-value. c, FACS analysis show upregulation of PD-L1 but not CTLA4 in IFN-γ-treated EpRas cells. Data representative of two independent experiments. d, e, IFN-γ treated EpRas cells form larger and more invasive tumors, n=5 tumors/group. Inset shows higher magnification images. Data represented as mean ± SD. f, g, Small metastatic nodules (black arrows) in lungs of mice injected with IFN-γ treated EpRas cells. g, GFP labelled IFN-γ treated EpRas cells show micro metastatic foci in lungs of mice (n=4), while control untreated cell injected mice show no micro metastatic foci (n=4). EpRas 250,000 cells/MFP were injected into mice for (g). Cells were labeled with GFP (lentiviral vector pLEX-hFL2iG was used) using standard method. White arrows show GFP+ micro metastatic foci in whole mount lung image. (e-g) Experiment was repeated twice with similar results. (h-l) IHC analysis shows high (h) P-STAT1+, (i) S100A8+ cells in IFN-γ treated EpRas tumors. (j) Decreased CD8+T-cells observed in IFN-γ treated tumors. (k, l) Increase in S100A8+ cells observed in IFN-γ treated EpRas lung sections (metastatic site) with quantification in the right panel. Boxplot data represent median, interquartile range, and spikes to upper and lower adjacent values. (h-l). Two-way ANOVA test was used with Bonferroni post hoc test to calculate p value in (d). Two-tailed Mann-Whitney U test was used to compute p-value for h, j, i, l and data is represented as mean ± SEM. n=10 random FOV were evaluated from 3 individual tumors (h, j) n=3 and 5 random FOV were evaluated (i), n=3 and 7 random FOV/group were used for quantification (l). (e-g) Representative images of minimum two independent experiments are presented. Scale bar, (e) 200µm, (f) 2mm, and (g) 500 μm and (h-k) 40 μm respectively. ***p < 0.001. FOV; Field of view.
