Fig 2: HMGB1 enhances the sensitivity of TMZ therapy by inducing M1-like polarization of TAMs. A-B Bioluminescent images (A) and quantification of tumors in mice (B) implanted with GL261 cells with treatment of Ctrl, TMZ, rmHMGB1 and TMZ plus rmHMGB1 for 7, 14 and 21 days. C The survival curves of tumor-bearing mice implanted with GL261 cells for indicated treatments. n = 8. D Representative flow cytometric analysis of tumor-infiltrating CD11c+ TAMs gated on live CD45+ CD11b+ TAMs isolated from 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 the statistical analysis (right panel). n = 4. E Representative flow cytometric analysis of tumor-infiltrating CD206+ 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 the statistical analysis (right panel). n = 4. F Representative IF of HMGB1 (purple), pan macrophage marker IBA1 (red) and M1-like TAM marker CD16/32 (green) in GL261 cell-derived xenograft tumors treated with TMZ plus rmHMGB1 (left panel). Quantitation of IBA1+/CD16/32+ TAM population in Ctrl, TMZ, rmHMGB1 and TMZ plus rmHMGB1 treatment groups (right panel). Scale bars = 50 μm. G Representative IF of HMGB1 (purple), IBA1 (red) and M2-like TAM marker CD206 (green) in GL261 cell-derived xenograft tumors treated with TMZ plus rmHMGB1 (left panel). Quantitation of IBA1+/CD206+ TAM population in Ctrl, TMZ, rmHMGB1 and TMZ plus rmHMGB1 treatment groups (right panel). Scale bars = 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ns = no significance

Fig 3: HMGB1 binds to RAGE on TAMs. A Co-IP assays of interaction of HMGB1 with RAGE in THP1 cell line-derived macrophages (left panel) and RAW264.7 macrophages (right panel). Cell lysates were immunoprecipitated with anti-HMGB1 antibody, then immunoblotted with anti-HMGB1 and anti-RAGE antibodies. B Co-IP assays of interaction of RAGE with HMGB1 in THP1 cell line-derived macrophages (left panel) and RAW264.7 macrophages (right panel). Cell lysates were immunoprecipitated with anti-RAGE antibody, then immunoblotted with anti-HMGB1 and anti-RAGE antibodies. C THP1 cell line-derived macrophages (left panel) and RAW264.7 macrophages (right panel) were treated with RFP-labeled rhHMGB1 or FITC-labeled rmHMGB1 for 5 and 30 min, respectively. Scale bars = 10 μm. D Colocalization of HMGB1 (green) and RAGE (red) in human GB samples (GB8060 and GB9080) with TMZ treatment. Scale bars = 10 μm. E The mRNA expression of HMGB1 receptors (TLR2, TLR4, TLR9, RAGE) by THP1 cell line-derived macrophages after stimulation with rhHMGB1 at indicated concentrations for 24 h (left panel). Immunoblot of RAGE in THP1 cell line-derived macrophages with or without rhHMGB1 treatment for 48 h (right panel). F The mRNA expression of HMGB1 receptors (Tlr2, Tlr4, Tlr9, Rage) by RAW264.7 macrophages after stimulation with rmHMGB1 at indicated concentrations for 24 h (left panel). Immunoblot of RAGE in RAW264.7 macrophages with or without rmHMGB1 treatment for 48 h (right panel). *P < 0.05, **P < 0.01, ***P < 0.001, ns = no significance

Fig 4: 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

Fig 5: Release of HMGB1 induced by TMZ in GB is dependent on the autophagic vacuoles. A Immunoblot of HMGB1, SQSTM1 and LC3B-I to LC3B-II conversion in primary GB1 cells (GB1 cells) treated with 1000 μM TMZ (left panel). ELISA determining the HMGB1 in supernatants of primary GB1 cells (GB1 cells) (right panel). n = 3. B Immunoblot of HMGB1, SQSTM1 and LC3B-I to LC3B-II conversion in GB1 cells treated with TMZ for 72 h (left panel). ELISA determining the HMGB1 in supernatants of GB1 cells (right panel). n = 3. C Colocalization of LC3B (red) with HMGB1 (green) in GB1 cells under TMZ (left panel). Colocalization tracer profile along the line (white arrows) is indicated as merged images (middle panel). Pearson’s colocalization coefficient for LC3B and HMGB1 derived from three independent experiments with five fields (right panel). Scale bars = 10 μm. D Immunoblot of HMGB1 and LC3B-I to LC3B-II conversion in GB1 cells with or without 3-MA (5 mM) and LY294002 (100 nM) for 4 h, then stimulated with or without TMZ (1000 μM) for 24 h (left panel). HMGB1 was detected in the supernatants by ELISA (right panel). E Immunoblot of ATG5 and LC3B-I to LC3B-II conversion in GB1 cells transiently transfected with scrambled siRNA (siCtrl) or siRNA ATG5 (siATG5) for 48 h then treated with TMZ (1000 μM) for 24 h (left panel). HMGB1 in the supernatants was detected by ELISA (right panel). F GB1 cells transfected with scrambled siRNA (siCtrl) or siRNA GORASP2 (siGORASP2) for 36 h were transfected with AAV-mCherryGFP-LC3B then treated with TMZ (1000 μM) for 24 h (left panel). The number of autophagosomes was analyzed in ten random fields for each independent experiment (right panel). G Immunoblot of GORASP2 and LC3B-I to LC3B-II conversion in GB1 cells transiently transfected with siCtrl and siGORASP2 for 48 h then with TMZ (1000 μM) treatment for 24 h (left panel). HMGB1 in the supernatants was detected by ELISA (right panel). *P < 0.05, **P < 0.01,***P < 0.001, ns = no significance