The recent landmark Phase III clinical trial with a VEGF-specific antibody suggests that antiangiogenic therapy must be combined with cytotoxic therapy for the treatment of solid tumors. However, there are no guidelines for optimal scheduling of these therapies. Here we show that VEGFR2 blockade creates a "normalization window"--a period during which combined radiation therapy gives the best outcome. This window is characterized by an increase in tumor oxygenation, which is known to enhance radiation response. During the normalization window, but not before or after it, VEGFR2 blockade increases pericyte coverage of brain tumor vessels via upregulation of Ang1 and degrades their pathologically thick basement membrane via MMP activation.

Not all tumor vessels are equal. Tumor-associated vasculature includes immature vessels, regressing vessels, transport vessels undergoing arteriogenesis and peritumor vessels influenced by tumor growth factors. Current techniques for analyzing tumor blood flow do not discriminate between vessel subtypes and only measure average changes from a population of dissimilar vessels. We have developed methodologies for simultaneously quantifying blood flow (velocity, flux, hematocrit and shear rate) in extended networks at single capillary resolution in vivo. Our approach relies on deconvolution of signals produced by labeled red blood cells as they move relative to the scanning laser of a confocal or multiphoton microscope and provides fully-resolved three-dimensional flow profiles within vessel networks. Using this methodology, we show that blood velocity profiles are asymmetric near intussusceptive tissue structures in tumors in mice. Furthermore, we show that subpopulations of vessels, classified by functional parameters, exist in, around a tumor and in normal brain.

[Keywords: sphingosine 1-phosphate; cadherin; angiogenesis; vascular stabilization; endothelial cells; pericytes] Supplemental material is available at http://www.genesdev.org.