br Author Contributions br Acknowledgments This work was sup

Author Contributions
Acknowledgments This work was supported, in part, by a grant from the National Cancer Institute of the National Institutes of Health under Grant U01CA202241 and National Institutes of Health under Grant R01NS083856.
Introduction The Eph (erythropoietin-producing human hepatoma) family is the largest among transmembrane receptor tyrosine kinases (RTKs). Fifteen Eph RTKs are classified into two subclasses based on their structural features and binding affinities to various ephrin ligands (Lemke, 1997). The Eph receptors and their membrane-anchored ephrin ligands regulate tumorigenesis and angiogenesis (Heroult et al., 2006). Among the EphB receptors, increased SC 560 mg of EphB4 has been reported in various tumour types, including colon (Kumar et al., 2009), prostate (Li et al., 2015), breast (Brantley-Sieders et al., 2011), oesophageal (Hu et al., 2014), pancreatic (Bai et al., 2014), lung (Ferguson et al., 2013), and mesothelioma (Liu et al., 2013), but with low or no expression in most normal tissues. As for CRC tissue, EphB4 was shown to be expressed in all 102 CRC specimens analysed, but was absent in normal colon mucosae, and its expression level was correlated with higher tumour grade and stage. Additionally, EphB4 inhibition decreased tumour growth and metastasis (Kumar et al., 2009). Another study analysing expression of all Eph and ephrin genes in 153 clinical specimens and CRC cell lines showed that EphA1, EphA2, EphB2, and EphB4 were all significantly overexpressed in CRC (Herath et al., 2012). Other studies showed that EphB4 inactivation resulted in higher proliferation of both intestinal tumours and normal epithelium (Dopeso et al., 2009). These opposing conclusions illustrate why the functional role of EphB4 in colorectal tumours remains undefined. EphB4 and EphrinB2 play critical roles in the development of the vascular system and contribute to vascular function (Pitulescu and Adams, 2010). Research concerning the angiogenic role of EphB4 mainly focuses on embryonic and retinal vasculogenesis (Katsuta et al., 2013, Pitulescu et al., 2010, Xue et al., 2014). The role of EphB4 signalling in CRC has only recently been elucidated, and there are limited studies focusing on the relationship between microvessel density in human CRC and EphB4 expression. In a preliminary study, we stably transfected EphB4 overexpression and knockdown lentiviral vectors into CRC cell line SW480, and in vitro experiments showed that EphB4 overexpression promoted cell proliferation and stimulated migration and invasion, while EphB4 knockdown resulted in reduced cell viability, migration, and invasion. Here, we evaluated the expression of EphB4 and EphrinB2 using tissue microarrays from 200 colorectal tumours by immunohistochemistry. Furthermore, we performed in vivo experiments using xenograft models to investigate the role of EphB4 in CRC proliferation, invasion, and angiogenesis.