Histology and immunohistochemistry

Sections (8 μm thick) of tumor-bearing cryopreserved brains were prepared. The mean tumor size was determined by measuring the largest tumor area in the horizontal plane on hematoxylin and eosin (H&E)-stained sections from a low-magnification (2.5x) image and the longest perpendicular diameters using the ellipsoid geometric primitive formula [38]. Brain sections were immunostained with Ki-67 antibody (clone SP6, Epitomics, Burlingame, CA), anti-mouse CD31 antibody (clone MEC 13.3, BD Pharmingen, Franklin Lakes, NJ), anti-CD45 antibody (clone 30-F11; Biolegend), anti-CD3 (clone 17A2; BD Biosciences, San Jose, CA) or anti-CD11b (clone M1/70; BD Biosciences) and histofine simple stain mouse MAX PO anti-rabbit secondary antibody (Nichirei Biosciences, Tokyo, Japan) to determine proliferation. Histofine simple stain mouse MAX PO anti-rat (Nichirei Biosciences) and DAB chromogen were used to stain blood vessels. The percentage of Ki-67 − positive tumor nuclei and the mean number of CD31-positive intratumoral vessels per high power field (× 40 objective) were calculated using three randomly selected different microscopic fields of each section for three 3 mice per group or the DAB-positive signals were quantified using the ImageJ software (https://imagej.nih.gov/) [12].