Bone marrow-derived leukocytes aggregate around tumor blood vessels after chemotherapy treatment alone. GL261 tumors from WT host mice were harvested 16 – 20 days after implantation, and: A, at 3, 5, or 7 days after a single low-dose temozolomide injection (25 mg/kg, i.p.) or B, from untreated mice. Tumors were frozen for immunohistochemical analysis of bone marrow-derived CD45-expressing leukocytes (red) and endothelial cells (CD31, green). Nuclei were counterstained with Hoechst (blue). Representative photomicrographs of tumors (A and B) and contralateral brain (A, lower panels) are shown. Original magnification, ×400; Scale bars, 25 μm. C, quantitative analysis was performed to determine the frequency of perivascular leukocyte cuffs per 100 vessels within tumors (white bars) and in contralateral brain (black bars) as a control. Photomicrographs were obtained in a grid pattern at magnification ×200 and analyzed for cuff frequency. Cuffs were counted if they were circumferential and at least 3 cells thick. For each mouse, at least 90 CD31-positive vessels from at least six 200X fields were analyzed for perivascular cuffs. Data are presented as mean ± SEM of 3 mice per time point, from 3 independent experiments. *, P < 0.0003, by ANOVA with Kruskal-Wallis test.