Abstract B04: A role for EphA3 in angiogenesis, in vivo and in vitro exploration

Abstract

Abstract The Eph family of receptor tyrosine kinases, and their ephrin ligands, are key regulators of cell migration and tissue patterning which are crucial for the growth and assembly of blood vessels during embryonic development and in tumor angiogenesis. In particular, class A Ephs and ephrins are emerging as regulators and clinical targets in the tumor vasculature. EphA3 is expressed in perivascular cells during embryogenesis but is largely absent from mature vasculature. Recently we discovered the expression of EphA3 in the perivascular mesenchymal cells and some endothelial cells of clinical and experimental solid tumors, as well as in the regenerating human endometrium. Targeting of EphA3 in experimental tumors with an activating antibody inhibited tumor growth by disrupting the stroma and vasculature. We now seek to understand at a cellular and molecular level the role of EphA3 in regulating angiogenesis, as well as the factors controlling its expression, using refined in vivo and in vitro models. In a dorsal skin diffusion chamber assay, angiogenesis induced by tumor-secreted factors was inhibited dose-dependently by an activating EphA3 antibody. This indicates that EphA3’s role in regulating tumor-induced angiogenesis does not require direct contact with tumor cells and, as with other Ephs, is likely intrinsic to the various cells comprising blood vessels. In vitro angiogenesis assays such as aortic ring sprouting and embryoid body differentiation assays were subsequently used to investigate the expression and function of EphA3 in endothelial and perivascular cells during vascular growth, in conjunction with EphA3-null and inducible EphA3-shRNA transgenic mice. In embryoid bodies undergoing vasculogenic differentiation, EphA3 was expressed at the intercellular junctions of nascent endothelial tubules, independently of perivascular cell association. 2-dimensional cultures of embryoid body-derived endothelial cells also abundantly expressed EphA3. In aortic ring sprouting assays, EphA3 was expressed in endothelial sprouts and abundantly in associated perivascular cells. Derivation of aortic mural cell cultures endogenously expressing EphA3 provides an important tool for exploring EphA3 regulation and signaling in perivascular cells. Our results indicate that expression of EphA3 in endothelial and perivascular cells characterizes a state of active growth and differentiation during angiogenesis. Notably this expression pattern is common to a number of different angiogenic settings. These data provide further support for EphA3 as an anti-angiogenic target in cancer and other pathologies. Citation Format: Rae H. Farnsworth, Mary E. Vail, Magdaline Costa, Linda Hii, Ross A. Dickins, Andrew G. Elefanty, Edouard G. Stanley, Martin Lackmann. A role for EphA3 in angiogenesis, in vivo and in vitro exploration. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B04.