Modeling human tumor angiogenesis in a three-dimensional culture system

Abstract

Key Points• Human arterial ring assay is an innovative system for the three-dimensional study of tumor angiogenesis.• This assay can be exploited for antiangiogenic drug screening and gene function analysis on human vessels.The intrinsic complexity of the process of vessel formation limits the efficacy of cellular assays for elucidation of its molecular and pharmacologic mechanisms. We developed an ex vivo three-dimensional (3D) assay of sprouting angiogenesis with arterial explants from human umbilical cords. In this assay, human arterial rings were embedded in basement membrane extract gel, leading to a network of capillarylike structures upon vascular endothelial growth factor (VEGF) A stimulation. The angiogenic outgrowth consisted of endothelial cells, which actively internalized acetylated-low-density lipoprotein, surrounded by pericytes. Computer-assisted quantification of this vascular network demonstrated considerable sensitivity of this assay to several angiogenic inhibitors, including kinase inhibitors and monoclonal antibodies. We also performed targeted gene knockdown on this model by directly infecting explanted umbilical arteries with lentiviruses carrying short-hairpin RNA. Downregulation of VEGFR2 resulted in a significant reduction of the sprouting capability, demonstrating the relevance of human vascular explants for functional genomics studies. Furthermore, a modification of this assay led to development of a 3D model of tumor-driven angiogenesis, in which angiogenic outgrowth was sustained by spheroids of prostate cancer cells in absence of exogenous growth factors. The human arterial ring assay bridges the gap between in vitro endothelial cell and animal model, and is a powerful system for identification of genes and drugs that regulate human angiogenesis. (Blood. 2013;121(21):e129-e137) IntroductionSignificant progress in angiogenesis research has been made after observations that a neoplastic mass does not grow above a few millimeters in diameter without recruiting new vessels. 1,2 Numerous cellular and animal models have been developed to design robust and exhaustive assays able to mechanistically decipher the angiogenic cascade and to validate new antiangiogenic drugs. Endothelial cell (EC) assays can mimic individual steps of the angiogenic cascade, such as cell migration, proliferation, and capillarylike formation. [3][4][5] However, different cell types-such as fibroblasts, pericytes, and smooth muscle cells-play a crucial role during new vessel formation by releasing soluble factors and establishing homotypic and heterotypic interactions. In addition, collective cell migration and extracellular matrix invasion are critical steps in the angiogenic process but are difficult to investigate with cultured cells. 6 Characterization of angiogenesis has therefore been established using mainly animal models. However, although powerful insights into the molecular and cellular mechanisms of angiogenesis have been elucidated by means of transgenic and knockout mouse models, 2,6 the complexity o...

Cathepsin K-mediated notch1 activation contributes to neovascularization in response to hypoxia

“…Microvascular sprouting was characterized by the incorporation of 1,1 0 -dioctadecyl-3,3,3 0 3 0 -tetramethylidocarbocyanine perchloratelabelled acetylated low density lipoprotein (Dil-acLDL, 10 mg ml À 1 , Molecular NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4838 ARTICLE Probes, Eugene, OR, USA). To further confirm that outgrowing structures have capillaries features, as previously studied in human aorta-ring culture assays 22 , we also examined sprouting cells by immunostaining and confocal microscopy using fluorescein isothiocyanate-labelled CD31 monoclonal antibody (BM Pharmingen). Sprout length was quantified using BZ-II Analyser, Exe 1.42 software (Keyence, Osaka, Japan).…”

“…To further verify that outgrowing structures feature capillaries, as previously demonstrated in human aorta-ring assays 22 , we examined sprouting cells by confocal microscopy and immunohistochemistry, and observed the presence of lumens with a mean diameter of 3.6±1.8 mm in a restricted number of outgrowing structures ( Supplementary Fig. 10).…”

Heralding a new paradigm in 3D tumor modeling

“…The predominant mechanism for tumor angiogenesis is thought to be sprouting angiogenesis, characterized by the migration, proliferation, 3D organization and tube formation of endothelial cells from pre-existing vessels [120]. This neo-vascularization process is in turn, tightly regulated by various cell types (including fibroblasts, pericytes, and smooth muscle cells) that secrete soluble factors and establish cell-cell interactions [121]. The immense complexity of this multistep process therefore has confined much of the characterization to animal models [121].…”

“…This neo-vascularization process is in turn, tightly regulated by various cell types (including fibroblasts, pericytes, and smooth muscle cells) that secrete soluble factors and establish cell-cell interactions [121]. The immense complexity of this multistep process therefore has confined much of the characterization to animal models [121]. To address this problem, Seano et al developed an ex vivo 3D assay of sprouting angiogenesis using arterial explants from human umbilical cords [121].…”

The “cord of life” serving antiangiogenic therapy

“…1 Finally, the ease of genetic modification of hARs in vitro bypasses the difficulties that scientists have encountered when knocking out genes that are required during the embryonic or adult life of the animal. 5 Although hARs have advantages, there are still some limitations (see figure).…”