Antagonism of Ang-Tie2 and Dll4-Notch signaling has opposing effects on tumor endothelial cell proliferation, evidenced by a new flow cytometry method.

Sustained angiogenesis is essential for tumor growth as it provides the tumor with a network of blood vessels that supply both oxygen and essential nutrients. Limiting tumor-associated angiogenesis is a proven strategy for the treatment of human cancer. To date, the rapid detection and quantitation of tumor-associated endothelial cell (TAEC) proliferation has been challenging, largely due to the low frequency of endothelial cells (ECs) within the tumor microenvironment. In this report, we address this problem using a new multiparametric flow cytometry method capable of rapid and precise quantitation of proliferation by measuring bromodeoxyuridine (BrdUrd) uptake in mouse TAECs from established human tumor xenografts. We determined the basal proliferation labeling index of TAECs in two human tumor xenografts representing two distinct histologies, COLO 205 (colorectal cancer) and U-87 (glioblastoma). We then investigated the effects of two large-molecule antiangiogenic agents targeting different biochemical pathways. Blocking angiopoietin-Tie2 signaling with the peptide-Fc fusion protein, trebananib (AMG 386), inhibited proliferation of TAECs, whereas blocking Dll4-Notch signaling with an anti-Dll4-specific antibody induced hyperproliferation of TAECs. These pharmacodynamic studies highlight the sensitivity and utility of this flow cytometry-based method and demonstrate the value of this assay to rapidly assess the in vivo proliferative effects of angiogenesis-targeted agents on both the tumor and the associated vasculature.