In vitro angiogenesis by human umbilical vein endothelial cells (HUVEC) induced by three-dimensional co-culture with glioblastoma cells

Abstract

Glioblastoma multiforme (GBM) is one of the most highly vascularized of all human tumors. Our objective was to characterize a 3-dimensional (3-D) in vitro angiogenesis model by co-culturing HUVEC and GBM cells, and to study the role of VEGF in mediating capillary tubule formation in this model. HUVEC-coated dextran beads were suspended in fibrin gel with human glioma cells on top. The number of sprouts and the length of the processes were measured. HUVEC can be induced to form sprouts and longer processes with lumens, in co-culture with glioma cells that secrete VEGF. Addition of exogenous VEGF enhances this effect. In the absence of glioma cells, many single HUVEC migrate away from the beads, without significant tubule formation. Hypoxia further stimulated sprout formation by 50-100%. Anti-VEGF neutralizing antibody suppressed HUVEC sprouting by 75% in co-culture with glioma cells. This 3-D in vitro co-culture system provides a robust and useful model for analysis of the major steps of glioma-induced angiogenesis.

A Metabolic Shift Favoring Sphingosine 1-Phosphate at the Expense of Ceramide Controls Glioblastoma Angiogenesis

“…6A). This in vitro model closely reproduced all the key features of physiological angiogenesis (31,44). In the absence of U87MG cells, there was very little sprouting of endothelial cells after 4 days in culture.…”

“…Angiogenesis was assessed in a three-dimensional fibrin gel coculture model (31). Briefly, near-confluent HMEC-1 cells were incubated with microcarrier beads for 72 h. The microcarriers were then embedded into fibrin gel in 24-well plates, and U87MG cells were seeded on top (25,000 cells/well) in 500 l of minimal essential medium, 2% FBS, and 2 mM L-glutamine.…”

In VitroAngiogenesis Induced by Tumor-Endothelial Cell Co-Culture in Bilayered, Collagen I Hydrogel Bioengineered Tumors

“…The emergence of tumor engineering, defined as the development of complex three-dimensional (3D) in vitro tumor models that reproduce the phenotypes and physiological responses of the in vivo tumor microenvironment, 22 is facilitating the establishment of new platforms for identifying and testing more efficacious anti-angiogenic drugs. These systems are beginning to successfully incorporate multiple cell types (endothelial, tumor and/or stromal) into controlled 3D in vitro environments, demonstrating reproducible angiogenic sprouting [23][24][25][26][27][28] as well as inhibition in response to known angiogenic blockers. 25,26,28 However, many of these models are unable to recapitulate co-culture induced angiogenic sprouting from a confluent monolayer, 24,26,28 which is important for maintaining proper endothelial cell polarity.…”

“…These systems are beginning to successfully incorporate multiple cell types (endothelial, tumor and/or stromal) into controlled 3D in vitro environments, demonstrating reproducible angiogenic sprouting [23][24][25][26][27][28] as well as inhibition in response to known angiogenic blockers. 25,26,28 However, many of these models are unable to recapitulate co-culture induced angiogenic sprouting from a confluent monolayer, 24,26,28 which is important for maintaining proper endothelial cell polarity. Furthermore, most co-culture angiogenesis models have been unsuccessful in achieving invasive sprouting without the contribution of exogenous angiogenic growth factors 26,27 or fibroblasts.…”

“…25,26,28 However, many of these models are unable to recapitulate co-culture induced angiogenic sprouting from a confluent monolayer, 24,26,28 which is important for maintaining proper endothelial cell polarity. Furthermore, most co-culture angiogenesis models have been unsuccessful in achieving invasive sprouting without the contribution of exogenous angiogenic growth factors 26,27 or fibroblasts. [23][24][25]28 To address this deficiency, we introduce an in vitro angiogenesis model based on microvascular endothelial cells cultured as a confluent monolayer on the surface of previously characterized pro-angiogenic bioengineered tumors 29 in the absence of exogenous angiogenic growth factors.…”