Fig 2: PPAR? induces HIF-2a and arginase-1 expression in macrophages. a, b Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6 for 5 days. a Cell lysates were immunoblotted. b mRNA was extracted and subjected to quantitative RT-PCR analysis. Results were normalized with GAPDH level and expressed as folds of control (n = 3, mean ± SEM). P value was determined by Student’s t test. c, d Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6 for 3 days. c Nuclei protein was incubated with biotin-labeled synthetic DNAs that encode control scrambled or HIF-2a promoter sequence, followed by immunoprecipitation with streptavidin-conjugated beads. Precipitants and nuclei protein were immunoblotted. d Nuclei extracts were subjected to chromatin immunoprecipitation (ChIP) analysis. Immunoprecipitants with control IgG or anti-PPAR? antibody were analyzed by PCR and electrophoresis (upper) or by quantitative PCR (bottom, n = 3, mean ± SD). e Mouse BM-derived macrophages were transduced with lentivirus that expresses shRNAs targeting control scrambled sequence and PPAR? (#1657, #1660, and #25967), followed by treatment with CSF-1 and IL-6 for 10 days. Cells were lysed and subjected to immunoblot analysis. f Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6. At different time points post-treatment, cells were lyzed and subjected to immunoblot analysis. Band density was quantified. g Mouse BM cells were pretreated with or without rapamycin, followed by incubation with CSF-1 and IL-6. Cell viability was determined (n = 3 mice, mean ± SEM)

Fig 3: GBM ECs induce alternative activation of macrophages. a, b Mouse brain microvascular ECs were pretreated with the glioma-conditioned medium (glioma-CM, harvested from medium supernatant of mouse GL26 glioma cells under 1% hypoxia) or control medium for 24 h. Mouse bone marrow (BM)-derived macrophages were incubated with CSF-1 or co-cultured with pretreated ECs for 5 days, stained with anti-CD11b, anti-CD86, anti-CD206 antibodies, and analyzed by flow cytometry. a Representative results of CD206 and CD86 expression in CD11b+ cells. b Quantified data in sorted CD11b+ macrophages (M?, n = 3–4 mice, mean ± SEM). c Human brain microvascular ECs were pretreated with the glioma-CM (harvested from medium supernatant of human U251 glioma cells under 1% hypoxia) or control medium for 24 h. Human peripheral blood mononuclear cell (PBMC)-derived monocytes were incubated with CSF-1 or co-cultured with pretreated ECs for 5 days, stained with anti-CD11b, anti-CD86, anti-CD206 antibodies, and subjected to flow cytometry analysis. Quantified data in sorted CD11b+ cells (n = 3, mean ± SEM). d Human PBMC-derived monocytes were incubated for 5 days with CSF-1, or co-cultured with tumor-associated ECs isolated from different GBM patients or human normal brain microvascular ECs in upper and lower chambers of transwells, respectively. Monocytes were harvested and subjected to immunoblot analysis with anti-arginase-1 and anti-GAPDH antibodies. e Human PBMC-derived monocytes were incubated for 5 days with CSF-1, or co-cultured with tumor-associated ECs isolated from different GBM tumors (n = 4 patients) or human normal brain microvascular ECs. Cells were harvested, stained with anti-CD206, and anti-CD86 antibodies, and subjected to flow cytometry analysis. Representative images are shown. f PBMC-derived monocytes were incubated for 5 days with CSF-1, or co-cultured with tumor-associated ECs isolated from GBM patient #5377 or human normal brain microvascular ECs. Cells were harvested, stained with anti-IL-10 and anti-CD11b antibodies, and subjected to flow cytometry analysis (n = 5, mean ± SEM). P values were determined by Student’s t test

Fig 4: IL-6 is critical for EC-induced macrophage alternative activation. a, b Mouse microvascular brain ECs were pretreated with the glioma-CM for 24 h. Mouse BM-derived macrophages were co-cultured with pretreated ECs for 5 days in the presence of control IgG, anti-CSF-1 antibody, or anti-IL-6 antibody or both antibodies. The cells were stained with anti-CD11b, anti-CD86, anti-CD206 antibodies, and analyzed by flow cytometry. a Representative sorting for CD206 expression in CD11b+ cells. b Quantified data in sorted CD11b+ cells (n = 3–5, mean ± SEM). c–e Mouse BM-derived macrophages were treated with IL-6 and CSF-1 for 5 days. c, d The cells were stained with anti-CD11b, anti-CD86, and anti-CD206 antibodies, and analyzed by flow cytometry. c Representative sorting for CD206 and CD86 expression in CD11b+ cells. d quantified data in sorted CD11b+ cells (n = 3, mean ± SEM). e Cell lysates were immunoblotted

Fig 5: GBM ECs express IL-6. a Human brain ECs were treated with glioma-CM for 24 h, and cell lysates were subjected to multiplex cytokine array analysis. Left, a representative blot. Right, quantified dot intensity of most significantly changed cytokines. b Human microvascular brain ECs were treated with glioma-CM that were harvested from different human glioma cells. Cell lysates were immunoblotted. c Human microvascular brain ECs and tumor-associated ECs isolated from different GBM patients were subjected to immunoblot analysis. d Mouse GBM was induced by orthotopic injection of GL26 glioma cells into wild-type mouse. The brain sections that include normal brains and tumors were stained with anti-CD31, anti-IL-6, and anti-CSF-1 antibodies. Representative immunofluorescence images are shown. Right, enlarged area in normal and tumor tissues. Bar represents 50 µm. Zoom-in factor: 4