Fig. 5

Inhibition of TNFα prevents EC activation and prolongs survival of mouse glioma model a HUVECs were incubated with EBM or CM from Mφ-U87 or Mφ-U251 in the presence or absence of 0.4 µg/mL anti-TNFα neutralizing antibody or IgG (upper panel) or thalidomide at concentration of 10 µM (lower panel). Relative quantities of genes were analyzed by RT-PCR and normalized to EBM. n = 3, mean ± max/min, 95% confidence interval. b HUVEC were incubated with EBM media alone (Mφ-Unstim) or CM from RAW264.7 cells stimulated with glioma cell lines (Mφ-U87 or Mφ-U251) in the presence or absence of 0.4 µg/mL anti-TNFα neutralizing antibody or IgG. Relative quantities of F3 were analyzed by RT-PCR and normalized to EBM. n = 3, mean ± max/min, 95% confidence interval. c hCMEC/D3 were incubated with CM from RAW264.7 macrophages stimulated with normal mouse astrocytes (Mφ-NMA) or GL261(Mφ-GL261) cells in the presence or absence of MP6-XT22. Gene expression was analyzed as in (a). d Protein expression of TNFα in tumor (T) versus normal contralateral brain tissue (NB) isolated from GL261 syngeneic mice was analyzed by ELISA, mean ± SD, n = 3 or immunoblotting. e Representative images from GL261 syngeneic mice brain tissue co-stained with CD31 (blue) and VCAM1 (red). GL261 tumor-bearing mice were treated with vehicle control (left) or MP6-XT22 (right) (Scale bar = 50 µm). f Co-expression of CD31 and VCAM1was scored by two independent pathologists with adjacent normal brain area or normal contralateral tissue (NB) scoring as 1 (n = 10). g Capillary-depleted brain rich fractions and capillary fractions from untreated and MP6-XT22 treated mice were isolated from NB and tumor tissue, lysed, and analyzed with a rat IgG ELISA. Concentrations were normalized to total protein, mean ± SD, n = 5. h Kaplan–Meier survival curve of GL261 syngeneic mice randomized into control (n = 10) or MP6-XT22-treated (n = 10) groups. p = 0.0008. *p < 0.05, **p < 0.01, ***p < 0.001