Fig 2: A schematic model. In glioma microenvironment, endothelial cell-derived IL-6 and microenvironmental CSF-1 synergistically activate downstream Akt1/mTOR pathway and induces transcriptional activation of PPAR? in macrophages (M?), in turn leading to HIF-2a-mediated arginase-1 expression, and inducing macrophage alternative polarization. The activation of mTOR also induces cell proliferation, contributing to cell survival and growth of alternatively activated macrophages, eventually leading to glioma progression

Fig 3: HIF-2a is critical for IL-6-mediated arginase-1 expression and alternative macrophage activation. a Mouse BM-derived macrophages were treated with CSF-1 and IL-6 for 3 days. Nuclei proteins were subjected to multiplex profiling analysis for transcriptional factor activation. Activity was normalized with transcription factor IID, and expressed as the folds of control. b Mouse BM-derived macrophages were transduced with lentivirus that expresses CMV promoter-driven renilla luciferase (CMV-rLuc), hypoxia response element-driven firefly luciferase (HRE-fLuc), and mutated HRE-fLuc (muHRE-fLuc), followed by treatment with CSF-1 and IL-6 for 2 days. Reporter activity radio of fLuc versus rLuc was determined by bioluminescence. Results were expressed as the percentage of muHRE (n = 3, mean ± SEM). P value was determined by Student’s t test. c–e Mouse BM-derived macrophages were transduced with lentivirus that expresses shRNAs targeting control scrambled sequence, HIF-1a (#49 and #52) and HIF-2a (#5 and #7), followed by treatment with CSF-1 and IL-6 for 10 days. c Cells were lysed and subjected to immunoblot analysis. d Arginase-1 mRNA was analyzed by real-time RT-PCR. Shown are quantified data (normalized with GAPDH expression, n = 3, mean ± SEM). e Cells were stained with anti-CD11b, anti-CD86, and anti-CD206 antibodies, followed by flow cytometry analysis (n = 3, mean ± SEM)

Fig 4: 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

Fig 5: The effect of participant-derived serum on ADSC proliferation. (A) Cellular proliferation (bars) and serum IL6 concentrations (pg/mL; dotted line). Statistical analysis: non-parametric Dunn’s multiple comparisons test with Kruskal–Wallis post hoc test. ∗p < 0.05 indicates significant difference from healthy lean (C) group. (B) Correlation between the IL6 concentrations in participant-derived serum and ADSC proliferative responses over a period of 24 h. (C) Correlation between increasing physiologically relevant concentrations of recombinant IL6 and ADSC proliferation (n = 3). Statistical analysis: Spearman’s ranked correlation analysis with two-tailed p-value. Level of significance accepted at p < 0.05.