Journal: Nature Communications

Article Title: Vascular niche IL-6 induces alternative macrophage activation in glioblastoma through HIF-2α

doi: 10.1038/s41467-018-03050-0

Figure Lengend Snippet: 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 (Mϕ, 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

Article Snippet: Mouse BM cells were co-cultured with mouse brain microvascular ECs, CCL5 (PeproTech, 250-07), or CXCL5 (PeproTech, 300-22) under normoxia or hypoxia (1% O 2 ).

Techniques: Activation Assay, Control, Derivative Assay, Incubation, Cell Culture, Staining, Flow Cytometry, Expressing, Isolation, Western Blot

Journal: Nature Communications

Article Title: Vascular niche IL-6 induces alternative macrophage activation in glioblastoma through HIF-2α

doi: 10.1038/s41467-018-03050-0

Figure Lengend Snippet: 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

Article Snippet: Mouse BM cells were co-cultured with mouse brain microvascular ECs, CCL5 (PeproTech, 250-07), or CXCL5 (PeproTech, 300-22) under normoxia or hypoxia (1% O 2 ).

Techniques: Multiplex Assay, Isolation, Western Blot, Injection, Staining, Immunofluorescence

Journal: Nature Communications

Article Title: Vascular niche IL-6 induces alternative macrophage activation in glioblastoma through HIF-2α

doi: 10.1038/s41467-018-03050-0

Figure Lengend Snippet: 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

Article Snippet: Mouse BM cells were co-cultured with mouse brain microvascular ECs, CCL5 (PeproTech, 250-07), or CXCL5 (PeproTech, 300-22) under normoxia or hypoxia (1% O 2 ).

Techniques: Activation Assay, Derivative Assay, Cell Culture, Control, Staining, Flow Cytometry, Expressing

Journal: Nature Communications

Article Title: Vascular niche IL-6 induces alternative macrophage activation in glioblastoma through HIF-2α

doi: 10.1038/s41467-018-03050-0

Figure Lengend Snippet: Endothelial IL-6 is critical for macrophage alternative activation and GBM growth and progression. The genetically engineered GBM model was induced in Ntv-a ; Ink4a-Arf −/− ; Pten −/− ; LSL-Luc donor mice, followed by orthotopic tumor implantation into Cdh5 - Cre ERT2 ; Il6 fl/fl mice that were treated with (IL-6-ΔEC) or without (Control) tamoxifen. a Animal survival was monitored for 50 days post-injection ( n = 5–6 mice, one representative result from three independent experiments). P values were determined by log-rank (Mantel–Cox) tests. MS, median survival. b Tumor growth was analyzed by bioluminescence. Left, representative images. Right, quantitative analysis of integrated luminescence in tumors at day 12 (mean ± SEM, n = 5–6, one representative result from three independent experiments). P value was determined by Student’s t test. c Tumor sections were stained with hematoxylin and eosin (H&E). Representative images are shown ( n = 10 mice). P pseudopalisades, MP microvascular proliferation, EN extensive necrosis, LI leukocyte infiltration. Bar represents 100 μm. Zoom-in factor: 3. e , f Tumors were excised. Single-cell suspensions were prepared and subjected to flow cytometry analysis. d , e Single-cell suspensions were probed with anti-F4/80, anti-CD86, and anti-CD206 antibodies. CD206 and CD86 expression were analyzed in sorted F4/80 + cells. d Representative sorting. e Quantified results (mean ± SEM, n = 10–14 mice). f Single-cell suspensions were probed with anti-F4/80, anti-IL-10, and anti-IL-12 antibodies. IL-10 and IL-12 expression was analyzed in sorted F4/80 + cells. Show are quantified results (mean ± SEM, n = 8–13 mice). g Tumor sections were stained and analyzed by immunofluorescence. Tumor sections were probed with anti-iNOS, anti-arginase-1, anti-F4/80 antibodies ( n = 10 mice). Bar represents 100 μm

Article Snippet: Mouse BM cells were co-cultured with mouse brain microvascular ECs, CCL5 (PeproTech, 250-07), or CXCL5 (PeproTech, 300-22) under normoxia or hypoxia (1% O 2 ).

Techniques: Activation Assay, Tumor Implantation, Control, Injection, Staining, Flow Cytometry, Expressing, Immunofluorescence