The addition of AG-013736 to fractionated radiation improves tumor response without functionally normalizing the tumor vasculature

Although antiangiogenic strategies have proven highly promising in preclinical studies and some recent clinical trials, generally only combinations with cytotoxic therapies have shown clinical effectiveness. An ongoing question has been whether conventional therapies are enhanced or compromised by antiangiogenic agents. The present studies were designed to determine the pathophysiologic consequences of both single and combined treatments using fractionated radiotherapy plus AG-013736, a receptor tyrosine kinase inhibitor that preferentially inhibits vascular endothelial growth factor receptors. DU145 human prostate xenograft tumors were treated with (a) vehicle alone, (b) AG-013736, (c) 5x2 Gy/wk radiotherapy fractions, or (d) the combination. Automated image processing of immunohistochemical images was used to determine total and perfused blood vessel spacing, overall hypoxia, pericyte/collagen coverage, proliferation, and Apoptosis. Combination therapy produced an increased tumor response compared with either monotherapy alone. Vascular density progressively declined in concert with slightly increased alpha-smooth muscle actin-positive pericyte coverage and increased overall tumor hypoxia (compared with controls). Although functional vessel endothelial Apoptosis was selectively increased, reductions in total and perfused vessels were generally proportionate, suggesting that functional vasculature was not specifically targeted by combination therapy. These results argue against either an AG-013736- or a combination treatment-induced functional normalization of the tumor vasculature. Vascular ablation was mirrored by the increased appearance of dissociated pericytes and empty type IV collagen sleeves. Despite the progressive decrease in tumor oxygenation over 3 weeks of treatment, combination therapy remained effective and tumor progression was minimal.