Quantitative analysis of IHC staining

The quantification of PD-L1, VEGF, MMP-9 and KI-67 expression levels was determined using a computerized image analysis system (Image-Pro Plus version 6.0 software). Firstly, image acquisition of IHC stained slides. Briefly, a digital image (Tag Image File Format format) with characteristic glioma on each slides was acquired under an Nikon Eclipse 80i microscopy equipped with an Nikon DS-Fi1 camera. All Images were acquired under high magnification (400×) with a constant set of imaging parameters (unified light source, exposure time, and autofocus). Secondly, optical density analysis of the images was performed with the Image-Pro Plus software. Images was calibrated and an appropriate threshold value for DAB-positive hues (brown) was set through histogram (hue; saturation; intensity). Then, an area of interest (AOI) was drawn to include most of tumor components in the image and exclude endothelial cells (blood vessels), necrosis and slice edge. After the AOI was defined, the integrated optical density (IOD) of the AOI was determined by the software. Lastly, the MOD of the selected area were calculated (IOD/area of AOI) and represented as the immunoreactivity of the candidate protein within tumor tissue. Intra-tumor heterogeneity was minimized through the elimination of the two fields with the highest and lowest MODs, and the average MOD of the remaining five fields was used to represent the PD-L1 level for an individual.