Fig. 3

HMGB1 contributes to M1-like polarization of macrophages. A The mRNA expression of HMGB1 receptors (TLR2, TLR4, TLR9 and RAGE) in human GB specimens from GEO database (Bone marrow derived macrophage-tumor associated macrophages, BMDM-TAMs; Macroglia-TAMs; Neoplastic cells; Oligodendrocyte progenitor cells, OPCs; Neural progenitor cells, NPCs; Endothelial cells, ECs; Pericytes, PCs; Peripheral blood lymphocytes, PBLs; Neurons.) was analyzed by single cell sequencing. B Immunofluorescence (IF) of HMGB1 receptors TLR2, TLR4, TLR9 and RAGE (green) in TAMs marked by IBA1 (red) in human GB samples (GB6429) without TMZ. Scale bars = 10 μm. C The mRNA level of M1-like phenotype genes in THP1 cell line-derived and RAW264.7 macrophages stimulated with 1 μg/ml recombinant human HMGB1 (rhHMGB1) or recombinant mouse HMGB1 (rmHMGB1) for 24 h. D The mRNA level of M2-like phenotype genes in THP1 cell line-derived and RAW264.7 macrophages stimulated with 1 μg/ml rhHMGB1 or rmHMGB1 for 24 h. E A scheme for in vitro co-culture system (left panel). THP1 cell line-derived macrophages (upper chamber) and shHMGB1 or shCtrl-LN229 cells (low chamber) were co-cultured for 24 h then with TMZ treatment for 24 h. The expression of M1-like phenotype genes, CCL2, IL-1β, IL-6 and TNF-α, was detected by qPCR (right panel). F Representative flow cytometric analysis of CD86⁺ TAMs gated on live CD45⁺ CD11b⁺ TAMs in GL261 cell-derived xenograft tumors with treatment of Ctrl (C), TMZ (T), rmHMGB1 (H) and TMZ plus rmHMGB1 (T + H) for 21 days (left panel). The histogram showed statistical analysis (right panel). n = 4. *P < 0.05, **P < 0.01, ***P < 0.001, ns = no significance