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Comparative Study
. 2002 Aug 20;99(17):11399-404.
doi: 10.1073/pnas.172398399. Epub 2002 Aug 12.

Potent VEGF blockade causes regression of coopted vessels in a model of neuroblastoma

Eugene S Kim  1 , Anna Serur, Jianzhong Huang, Christina A Manley, Kimberly W McCrudden, Jason S Frischer, Samuel Z Soffer, Laurence Ring, Tamara New, Stephanie Zabski, John S Rudge, Jocelyn Holash, George D Yancopoulos, Jessica J Kandel, Darrell J Yamashiro
Affiliations
Comparative Study

Potent VEGF blockade causes regression of coopted vessels in a model of neuroblastoma

Eugene S Kim et al. Proc Natl Acad Sci U S A. .

Abstract

Vascular endothelial growth factor (VEGF) plays a key role in human tumor angiogenesis. We compared the effects of inhibitors of VEGF with different specificities in a xenograft model of neuroblastoma. Cultured human neuroblastoma NGP-GFP cells were implanted intrarenally in nude mice. Three anti-VEGF agents were tested: an anti-human VEGF(165) RNA-based fluoropyrimidine aptamer; a monoclonal anti-human VEGF antibody; and VEGF-Trap, a composite decoy receptor based on VEGFR-1 and VEGFR-2 fused to an Fc segment of IgG1. A wide range of efficacy was observed, with high-dose VEGF-Trap causing the greatest inhibition of tumor growth (81% compared with controls). We examined tumor angiogenesis and found that early in tumor formation, cooption of host vasculature occurs. We postulate that this coopted vasculature serves as a source of blood supply during the initial phase of tumor growth. Subsequently, control tumors undergo vigorous growth and remodeling of vascular networks, which results in disappearance of the coopted vessels. However, if VEGF function is blocked, cooption of host vessels may persist. Persistent cooption, therefore, may represent a novel mechanism by which neuroblastoma can partly evade antiangiogenic therapy and may explain why experimental neuroblastoma is less susceptible to VEGF blockade than a parallel model of Wilms tumor. However, more effective VEGF blockade, as achieved by high doses of VEGF-Trap, can lead to regression of coopted vascular structures. These results demonstrate that cooption of host vasculature is an early event in tumor formation, and that persistence of this effect is related to the degree of blockade of VEGF activity.

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Figures

Fig 1.
Fig 1.
Inhibition of neuroblastoma tumors by anti-VEGF agents. Neuroblastoma xenografts were treated with the anti-VEGF agents (NX1838, n = 5; anti-VEGF antibody, n = 19; VEGF-Trap LD, n = 10; and VEGF-Trap HD, n = 10), with the results expressed as percent of control tumors (control NX1838, n = 5; control anti-VEGF antibody, n = 21; control VEGF-Trap LD, n = 10; and control VEGF-Trap HD, n = 10), with error bars representing SEM for treated tumors. Statistical analysis was done by Kruskal–Wallis analysis, with NX1838 (P = 0.08), anti-VEGF antibody (P = 0.12), VEGF-Trap LD (P = 0.10), and VEGF-Trap HD (P = 0.0009).
Fig 2.
Fig 2.
Fluorescein angiography. Tumor vasculature was evaluated by fluorescein–dextran angiography at 4 (A–H) and 6 weeks (I–N). At 4 weeks, standard fluorescent microscopy (A–D, ×10 original magnification) revealed cooption of renal glomeruli in Control (A), anti-VEGF antibody (B), VEGF-Trap LD (C), and VEGF-Trap HD (D). Close-up view of coopted glomeruli by confocal microscopy: Control (E), anti-VEGF antibody (F), VEGF-Trap LD (G), and VEGF-Trap HD (H), demonstrates connection of glomeruli to afferent and efferent vessels, seen in best in E and G. At 6 weeks, standard fluorescent microscopy (I–L, ×4 original magnification) demonstrates vascular remodeling with abundant vessels in control tumors (I), but persistent cooption in the anti-VEGF antibody (J) and VEGF-Trap LD (K), with glomeruli indicated by arrowheads. In VEGF-Trap HD (L), sparse vasculature and little cooption were seen at 6 weeks. Pseudodepth coloring of Control (M) and anti-VEGF antibody (N), demonstrates the abundant vasculature in Control tumors, and the persistent cooption in the anti-VEGF antibody-treated tumors. The coopted glomeruli are approximately 80 μm in size (Bar = 100 μm.).
Fig 3.
Fig 3.
Cooption of renal glomeruli at 4 weeks. Early in tumor growth, all tumors demonstrate cooption of renal glomeruli (arrowheads) as seen by hematoxylin/eosin staining (A–D, ×20; E–F, ×10): Control (A and E), anti-VEGF antibody (B and F), VEGF-Trap LD (C and G), and VEGF-Trap HD (D and H). In control (E) and VEGF-Trap HD (H), coopted glomeruli (arrowheads) encased by tumor tissue (t) are seen adjacent to renal tissue (r).
Fig 4.
Fig 4.
Decreased vascularity (reflected by diminished recruitment of perivascular cells) is seen by αSMA staining. Four (A–D) and 6 weeks (E–H), ×10 original magnification. Control (A and E) tumors have abundant vasculature and are associated with numerous perivascular cells. There was marked decrease in neoangiogenesis with only a few larger-caliber vessels in tumors after injection of anti-VEGF antibody (B and F), VEGF-Trap LD (C and G), and VEGF-Trap HD (D and H).
Fig 5.
Fig 5.
Apoptosis in renal glomeruli. Apoptosis was evaluated at 4 (A–D) and 6 weeks (E–H) by TUNEL. Control (A and E), anti-VEGF antibody (B and F), VEGF-Trap LD (C and G), and VEGF-Trap HD (D and H). Endothelial apoptosis was seen in anti-VEGF antibody (Inset B), VEGF-Trap LD (Inset C), and VEGF-Trap HD (D) but rarely in control tumors (A). Both control tumors and VEGF-Trap HD demonstrated apoptosis within glomeruli, suggesting that in tumors where cooption is transient, glomeruli undergo early apoptosis. Red blood cells are seen in the glomeruli of anti-VEGF antibody (B) and VEGF-Trap LD (C) tumors, but there is little apoptosis within the glomeruli. Apoptosis within glomeruli is seen at a later time point (6 weeks) in the anti-VEGF antibody (F) and VEGF-Trap LD (G) tumors.
Fig 6.
Fig 6.
Anti-VEGF treatment results in up-regulation of VEGF expression. In situ hybridization demonstrated a low-level diffuse expression of VEGF in control (A) tumors at 6 weeks, but marked up-regulation of VEGF in anti-VEGF antibody (B), VEGF-Trap LD (C), and VEGF-Trap HD (D) tumors at the same time point. In D, renal tissue is present (r) and contains glomeruli that express VEGF.
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