Fig. 3

TNFα secreted by GAMs induces EC activation and associates with OS in human GBM. a CM from Mφ-NHA, Mφ-U87, Mφ-U118, Mφ-U251, or Mφ-A172 was analyzed by a multi-analyte inflammatory ELISA array or b concentrated and immunoblotted with anti-TNFα. Total protein was detected using PageBlue. c TNFα ELISA with CM from Mφ-NSC, Mφ-GSC28 or Mφ-GSC267 was performed to validate the results found in the GBM cell lines. d Human cerebral endothelial cells (hCMEC/D3) were incubated with or without 10 ng/mL hTNFα for 24 h and gene expression was analyzed on an RT-PCR angiogenesis array. Relative quantity was calculated by normalizing to –TNFα. n = 3, mean ± max/min, 95% confidence interval *p < 0.05, **p < 0.01, ***p < 0.001. e Representative image of human GBM tissue section co-stained with TNFα (blue) and CD68 (red), double positive cells (arrow). f Representative image of human GBM tissue section co-stained with TNFα (blue) and VCAM1 (red). Scale bar = 50 µm. g. Correlation between extent of VCAM1 staining (Low, Medium or High) and percentage of TNF-alpha expressing GAMs cells per mm2 tumor area was performed in 39 IDH-wt GBM tissues. Log2 transformation was performed on the values of percentage of TNF-alpha expressing GAMs cells per mm2 tumor area (R = 0.8, p = 2.738e-009). h. Correlation of expression between CD68 and TNFα was analyzed in 137 IDH-wt GBM, R = 0.46, p = 1.93E-08 (lower panel), Kaplan–Meier survival curve of 137 IDH-wt GBM grouped by high or low CD68 and TNFα expression, p = 0.027 (upper panel)