Figure 2. Microglia in the TIME of GBM is associated with an immunosuppressive milieu.

(A) A schematic model to describe intracranial glioma xenograft establishment. Primary microglia were pretreated by H₂O₂ (500 µM) in the absence or presence of NAC (5 mM) for 12 hours. Glioma cells (GL261-luc, 2×10⁵ cells; CT2A-luc, 2×10⁴ cells) incubated with conditional medium of pretreated primary microglia cells for 12 hours. Treated glioma cells and treated primary microglia (1:1) were inoculated together into the frontal region of cerebral cortex. In vivo bioluminescence-based imaging was conducted on day 10–14 after orthotopic injection.

(B) Representative in vivo bioluminescence-based images and Hematoxylin and Eosin (H&E) staining of GL261-luc glioma-bearing C57BL/6J mice in respective group. Scale bars, 2 mm.

(C) Quantification of tumor volume based on bioluminescence in respective group (n=5 per group).

(D) Survival curves of C57BL/6J mice implanted with GL261-luc in five groups (n=5 per group).

(E, F) Representative in vivo bioluminescence-based images (E) of CT2A-luc glioma-bearing C57BL/6J mice and quantification (F) of tumor volume based on bioluminescence (n≥4 per group).

(G) Survival curves of C57BL/6J mice implanted with CT2A-luc in five groups (n=6 per group).

(H) A schematic model to describe establishment of intracranial tumor xenograft followed by flow cytometry analysis for tumor infiltrating T cells on day 14 after orthotopic injection.

(I-K) Flow cytometry analysis to evaluate the abundance of infiltrated CD8⁺ T cells and CD4⁺ T cells of glioma in respective groups (I). Quantification of CD8⁺ T cells (J) and CD4⁺ T cells (K) in CD45⁺ T cells (n=3 per group).

(L-N) Flow cytometry analysis to evaluate the expression of PD1 in CD8⁺ T cells with respective treatment (L). Quantification of PD1 (M) and IFN-γ (N) levels in CD8⁺ T cells (n=3 per group).

(O-P) Flow cytometry analysis to evaluate the abundance of infiltrated CD8⁺ T cells and CD4⁺ T cells levels (O), PD1 and IFN-γ (P) levels in CD8⁺ T cells of CT2A-luc glioma in respective groups (n=3 per group).

Data are shown as mean ± SEM. In (D) and (G), survival difference was calculated by log-rank test; in (C), (F), (J), (K), (M), (N), (O) and (P), P value was calculated using one-way ANOVA analysis. *p < 0.05, **p < 0.01, ***p < 0.001.