Fig 1: Blocking IL6 signaling by Tocilizumab attenuates the enhanced proliferation, migration, and invasion of glioma cells, and their crosstalk with macrophages induced by TIM-3 in vitro(A) The elevated proliferation of indicated glioma cells induced by TIM-3 was efficiently decreased by Tocilizumab (500 ng/mL) and anti-TIM-3 antibody (10 µg/mL) (n = 3, means ± SEM, one-way ANOVA; scale bar, 50 µm).(B) Representative bioluminescence (at day 7, 14, 21) images, H&E staining images, and survival plot of mouse brains transplanted with indicated GL261 cells and treated with control, anti-Tim3 antibody or anti-Il6r antibody (n = 6).(C) In vitro proliferation assay showing the influence of indicated Tocilizumab treatment on the growth of GSC40 (n = 3, one-way ANOVA).(D and E) The increased proliferation (D) and invasion (E) of indicated glioma cells induced by TIM-3 was efficiently decreased by Tocilizumab (500 ng/mL) (n = 3, means ± SEM, one-way ANOVA; scale bar, 50 µm).(F) EdU assay showing the increased proliferation of PGC1228 cells induced by TIM-3 was efficiently decreased by Tocilizumab (500 ng/mL) (n = 3, means ± SEM, one-way ANOVA).(G and H) The increased migration (G), and invasion (H) of indicated PGC1228 cells induced by TIM-3 was efficiently decreased by Tocilizumab (500 ng/mL) (n = 3, means ± SEM, one-way ANOVA; scale bar, 50 µm).(I) FACS analyses showed that Tocilizumab (500 ng/mL) inhibited the phenotype transition of PBMC to anti-inflammatory/pro-tumorigenic TAMs induced by IL6 (40 ng/mL) (n = 3, means ± SEM, one-way ANOVA).(J) Representative western blot images of indicated proteins in THP-1-derived macrophages incubated with indicated glioma cell CM (Tocilizumab, 500 ng/mL).(K) Western blot analyses of indicated proteins in THP-1-derived M0 and pro-inflammatory/anti-tumorigenic (M1) macrophages transfected with lentiviral TIM-3 overexpression or control vector, followed with or without Tocilizumab treatment (500 ng/mL) (n = 3, means ± SEM, one-way ANOVA). (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).