Evans Blue extraction and colorimetric assay

A simplified Evans Blue extraction technique ⁷ was used to evaluate vascular permeability. At 8 h (only for the U-87 MG tumors), day 1, day 4, and day 7 post-treatment with bevacizumab, immediately after PET acquisition, Evans blue (Sigma-Aldrich) was administered intravenously at a dose of 30 mg/kg (n = 4/group). At 4 h after dye injection, the mice were perfused at an elevated perfusion pressure with physiological saline through the left ventricle immediately before tumor excision to remove Evans blue from blood circulation. The collected tumors were weighed and cut into pieces of approximately 50 mg/piece. Then formamide (10 μL/mg of tumor tissue) was added to extract EB from the tumor tissue for 72 h. After centrifugation, the supernatant of each tube was carefully collected, and the absorbance at 620 nm was measured with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc.). Note that EB extraction efficiency was measured for different tumor samples. The concentration of EB was calculated using a standard absorption curve of known concentrations of EB mixed with albumin.