99mTc-(CO)3 His-Annexin A5 Micro-SPECT Demonstrates Increased Cell Death by Irinotecan During the Vascular Normalization Window Caused by Bevacizumab

Abstract

Colorectal tumors are dependent on angiogenesis for growth, and vascular endothelial growth factor (VEGF) is a key mediator of tumor angiogenesis. Antiangiogenic drugs can induce a transient normalization of the tumor vasculature with improved delivery of coadministered chemotherapy. The efficacy of antihuman VEGF antibody (bevacizumab) with or without irinotecan was evaluated in a colorectal cancer xenograft using (99m)Tc-(CO)(3) His-annexin A5. Colo205-bearing mice were treated with a single dose of bevacizumab (5 mg/kg) during 2, 4, or 6 d. Microvessel density, pericyte coverage (α-smooth-muscle actin immunostaining), collagen-covered tumor vessels (Masson trichrome staining), and tumor hypoxic fraction (pimonidazole staining) were determined at the 3 different time points after treatment with bevacizumab. To investigate the possible synergistic effects of combination therapy with bevacizumab and irinotecan, Colo205-bearing mice were treated with a single dose of bevacizumab 2, 4, or 6 d before administration of a single dose of irinotecan (100 mg/kg) or 0.9% NaCl. The apoptosis-detecting radiotracer (99m)Tc-(CO)(3) His-annexin A5 was injected (18.5 MBq) in mice 12, 24, and 48 h after the start of the irinotecan or NaCl treatment, and micro-SPECT was subsequently performed 3.5 h after injection of the radiotracer. Results were correlated to histologic analysis for apoptosis (caspase-3 activation). Four days after bevacizumab administration, microvessel density decreased significantly, and α-smooth-muscle actin and collagen-covered vessels, compared with control tumors, were increased, suggesting normalization of the tumor vasculature. Hypoxic fraction was slightly reduced 4 d after treatment with bevacizumab. SPECT analyses demonstrated a significant increase in tumoral (99m)Tc-(CO)(3) His-annexin A5 uptake 4 d after bevacizumab treatment and 24 h after irinotecan administration (232.78 ± 24.82 percentage injected dose/tumor weight [g]/body weight [kg], P < 0.05), compared with each monotherapy, indicating a synergistic effect of both therapies. (99m)Tc-(CO)(3) His-annexin A5 micro-SPECT demonstrates increased antitumor activity of irinotecan during the transient vascular normalization period caused by bevacizumab. Our data outline the importance of timing of combined anti-VEGF treatment with chemotherapy.