Figure 4.

The polarizations of monocyte and GBM progression in a co-cultured model under hypoxic conditions. (A) THP-1 monocytes were co-cultured with U87-green fluorescent protein (GFP) or U251-GFP under hypoxic conditions for 48 h. The co-cultured cells were plotted on a side scatter versus FITC. The THP-1 monocytes (GFP-negative cells) were analyzed to assess the levels of cluster of differentiation (CD) 206, arginase 1 (Arg1), interleukin (IL)-10, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α by flow cytometry. (B) The median fluorescence intensity (MFI) of CD206, Arg1, IL-10, IFN-γ, and TNF-α in THP-1 monocytes in the co-cultured model under hypoxic conditions for 48 h. * p < 0.05 compared with the THP-1 monocyte group. Quantitative data are presented as the mean ± standard error (representative of n = 3). (C) U87-GFP and U251-GFP were treated with U104 (CAIX inhibitor) and subsequently co-cultured with THP-1 monocytes under hypoxic conditions for 48 h. THP-1 monocytes (GFP-negative cells) were analyzed to assess the levels of CD206, Arg1, and IL-10 by flow cytometry. (D) U87-GFP or U251-GFP was treated with U104 and subsequently co-cultured with THP-1 monocytes under hypoxic conditions for 48 h. The co-cultured cells were plotted on a side scatter versus FITC. The GFP-positive gated GBM was analyzed to assess the levels of programmed death ligand 1 (PD-L1) by flow cytometry. (E) The levels of CAIX, PD-L1, tumor growth factor-β, and colony-stimulating factor mRNA in intracranial GBM of ALTS1C1-bearing mice were examined by quantitative polymerase chain reaction on day 24. * p < 0.05 compared with the naive control (ALTS1C1 GBM) (Student’s t-test). Quantitative data are presented as the mean ± standard error (representative of n = 4).