DLL4-Notch signaling mediates tumor resistance to anti-VEGF therapy in vivo.

Resistance to VEGF inhibitors is emerging as a major clinical problem. Notch signaling has been implicated in tumor angiogenesis. Therefore, to investigate mechanisms of resistance to angiogenesis inhibitors, we transduced human glioblastoma cells with retroviruses encoding Notch delta-like ligand 4 (DLL4), grew them as tumor xenografts and then treated the murine hosts with the VEGF-A inhibitor bevacizumab. We found that DLL4-mediated tumor resistance to bevacizumab in vivo. The large vessels induced by DLL4-Notch signaling increased tumor blood supply and were insensitive to bevacizumab. However, blockade of Notch signaling by dibenzazepine, a γ-secretase inhibitor, disrupted the large vessels and abolished the tumor resistance. Multiple molecular mechanisms of resistance were shown, including decreased levels of hypoxia-induced VEGF and increased levels of the VEGF receptor VEGFR1 in the tumor stroma, decreased levels of VEGFR2 in large blood vessels, and reduced levels of VEGFR3 overall. DLL4-expressing tumors were also resistant to a VEGFR targeting multikinase inhibitor. We also observed activation of other pathways of tumor resistance driven by DLL4-Notch signaling, including the FGF2-FGFR and EphB4-EprinB2 pathways, the inhibition of which reversed tumor resistance partially. Taken together, our findings show the importance of classifying mechanisms involved in angiogenesis in tumors, and how combination therapy to block DLL4-Notch signaling may enhance the efficacy of VEGF inhibitors, particularly in DLL4-upregulated tumors, and thus provide a rational base for the development of novel strategies to overcome antiangiogenic resistance in the clinic

Diffusion Limits of an in Vitro Thick Prevascularized Tissue

Although tissue engineering promises to replace or restore lost function to nearly every tissue in the body, successful applications are currently limited to tissue less than 2 mm in thickness. In vivo capillary networks deliver oxygen and nutrients to thicker ( 2 mm) tissues, suggesting that introduction of a preformed in vitro vascular network may be a useful strategy for engineered tissues. This article describes a system for generating capillary-like networks within a thick fibrin matrix. Human umbilical vein endothelial cells, growing on the surface of microcarrier beads, were embedded in fibrin gels a known distance ( 1.8–4.5 mm) from a monolayer of human dermal fibroblasts. The distance of the growth medium, which contained vascular endothelial growth factor and basic fibroblast growth factor, from the beads, C, was varied from 2.7 to 7.2 mm. Capillaries with visible lumens sprouted in 2–3 days, reaching lengths that exceeded 500 m within 6–8 days. On day 7, capillary network formation was largely independent of C; however, a strong inverse correlation with was observed, with the maximum network formation at 1.8 mm. Surprisingly, the thickness of the gel was not a limiting factor for oxygen diffusion as these tissue constructs retained a relatively high oxygen tension of 125 mmHg. We conclude that diffusion of oxygen in vitro is not limiting, allowing the development of tissue constructs on the order of centimeters in thickness. In addition, diffusion of fibroblast-derived soluble mediators is necessary for stable capillary formation, but is significantly impeded relative to that of nutrients present in the medium.This project was supported in part by seed grants from the the Council on Research, Computing, and Library Resources (CORCLR) and the Department of Biomedical Engineering at the University of California, Irvine, as well as the National Institutes of Health (HL60067 and AI40710). We thank Mr. Adrian Fernandez, Mr. Brandon Lee, and Mr. Elbert Jacinto for assistance in quantifying the capillary network. In addition, we also thank Dr. Robert L. Newcomb, Director of the Center for Statistical Consulting at UCI, for assistance in the statistical analysis of the data