A novel imaging‐based high‐throughput screening approach to anti‐angiogenic drug discovery

Abstract

The successful progression to the clinic of angiogenesis inhibitors for cancer treatment has spurred interest in developing new classes of anti-angiogenic compounds. The resulting surge in available candidate therapeutics highlights the need for robust, highthroughput angiogenesis screening systems that adequately capture the complexity of new vessel formation while providing quantitative evaluation of the potency of these agents. Available in vitro angiogenesis assays are either cumbersome, impeding adaptation to high-throughput screening formats, or inadequately model the complex multistep process of new vessel formation. We therefore developed an organotypic endothelial-mural cell co-culture assay system that reflects several facets of angiogenesis while remaining compatible with high-throughput/high-content image screening. Co-culture of primary human endothelial cells (EC) and vascular smooth muscle cells (vSMC) results in assembly of a network of tubular endothelial structures enveloped with vascular basement membrane proteins, thus, comprising the three main components of blood vessels. Initially, EC are dependent on vSMC-derived VEGF and sensitive to clinical anti-angiogenic therapeutics. A subsequent phenotypic VEGFswitch renders EC networks resistant to anti-VEGF therapeutics, demarcating a mature vascular phenotype. Conversely, mature EC networks remain sensitive to vascular disrupting agents. Therefore, candidate anti-angiogenic compounds can be interrogated for their relative potency on immature and mature networks and classified as either vascular normalizing or vascular disrupting agents. Here, we demonstrate that the EC-vSMC co-culture assay represents a robust high-content imaging high-throughput screening system for identification of novel anti-angiogenic agents. A pilot highthroughput screening campaign was used to define informative imaging parameters and develop a follow-up dose-response scheme for hit characterization. High-throughput screening using the EC-vSMC co-culture assay establishes a new platform to screen for novel anti-angiogenic compounds for cancer therapy. ' 2009 International Society for Advancement of Cytometry Key termshigh-throughput screening; endothelial/mural cell co-culture; angiogenesis INHIBITING angiogenesis is a validated therapeutic modality for cancer (1). Hence, new agents that potently inhibit pathological angiogenesis are a critical component of future combination cancer therapies (2). The identification of candidate therapeutics relies on current in vitro angiogenesis assays that measure effects on endothelial cell migration, proliferation, apoptosis and tube formation, and in vivo models such as the chick chorioallantoic membrane assay, corneal neovascularization assay, and Matrigel plug assays (3-5). Current in vitro angiogenesis assays are generally focused on specific behaviors of monocultured endothelial cells (EC) (e.g., migration, proliferation) and fail to model heterotypic perivascular interactions, whereas in vivo systems are not compatible ...

Phenotype-based high-content chemical library screening identifies statins as inhibitors of in vivo lymphangiogenesis

“…Previously, several studies were performed based on in vitro compound screens with blood vascular endothelial cells, including proliferation assays of human umbilical vein endothelial cells (HUVEC) (16), HUVEC/vascular smooth muscle cell cocultures, human microvascular endothelial cell tube formation assays (17,18), and HUVEC spheroid sprouting assays (19). While the tube formation assay has been considered as a model of vasculogenesis rather than angiogenesis (20), the other assays attempt to measure different aspects of blood vessel formation.…”

Endothelial vascular smooth muscle cell coculture assay for high throughput screening assays to identify antiangiogenic and other therapeutic molecules

“…Evensen et al 79 developed a very useful microplate co-culture system to quantify angiogenesis. Human umbilical vein ECs and pulmonary artery vascular smooth muscle cells were mixed together and then cultured in 96-well plates.…”

Pharmacologic Characterization of a Kinetic In Vitro Human Co-Culture Angiogenesis Model Using Clinically Relevant Compounds

“…When developing new methods to study angiogenesis in vitro, it is important that the model remains sensitive and responsive to a variety of cytokines, such as VEGF, bFGF, or epidermal growth factor (EGF), similar to what is observed in vivo. 6,7 Further, as antiangiogenic therapy is becoming increasingly important in the clinical setting, quantitating anti-angiogenic potential on developing tubes and/or established networks will strengthen the pharmacologic validation of an in vitro model. [5][6][7] Here, we demonstrate that a method of human umbilical vein endothelial cells (HUVEC) in co-culture with normal human dermal fibroblasts (NHDF), when used in conjunction with a compact fluorescent imaging microscope, the IncuCyte, presents a reproducible, kinetic approach to studying angiogenesis in vitro.…”

“…6,7 Further, as antiangiogenic therapy is becoming increasingly important in the clinical setting, quantitating anti-angiogenic potential on developing tubes and/or established networks will strengthen the pharmacologic validation of an in vitro model. [5][6][7] Here, we demonstrate that a method of human umbilical vein endothelial cells (HUVEC) in co-culture with normal human dermal fibroblasts (NHDF), when used in conjunction with a compact fluorescent imaging microscope, the IncuCyte, presents a reproducible, kinetic approach to studying angiogenesis in vitro. Further, the IncuCyte's automated image acquisition and integrated angiogenesis software algorithm provides a sophisticated, straightforward approach to measuring angiogenic tube formation.…”