4504 IRX1 inﬂuences peritoneal spreading and metastasis J Jiang et al Figure 7 Observing the effect of interfering PAK1, an effector of BDKRB2 in IRX1-overexpressed cancer cells. (a) Overexpression of IRX1 downregulated PAK1 expressing level, whereas knockdown of PAK1 level did not alter the IRX1 level signiﬁcantly. Overexpression of IRX1 combined with siRNA-PAK1 also downregulated PAK1 expressing level signiﬁcantly by western blot. (b) The cell proliferation activity was suppressed signiﬁcantly in 7901/IRX1 or 7901/siRNA-PAK1 groups, compared with controls (*Po0.001). The cell proliferation activity in 7901/IRX1/siRNA-PAK1 co-transfected group was nearly the same as that observed in 7901/IRX1 alone group (P ¼ 0.061). (c) By transwell assays, the migrated cell numbers (top) were signiﬁcantly reduced in 7901/IRX1 group (77.33±4.25) or 7901/siRNA-PAK1 group (84±1), compared with control (206.66±8.5, *Po0.001). Whereas in 7901/IRX1/ siRNA-PAK1 co-transfected group, the migrated cell numbers were nearly the same as that observed in 7901/IRX1 alone group (70.66±1.52 vs 77.33±4.25, P ¼ 0.202). The invasive cell numbers (down) were signiﬁcantly decreased in 7901/IRX1 group (79.66±5.03) or 7901/siRNA-PAK1 group (71±6), compared with control (178±4.58, *Po0.001). Whereas in 7901/IRX1/siRNA- PAK1 co-transfected group, the invasive cell numbers were nearly the same as that observed in 7901/siRNA-PAK1 alone group 69±5.57 vs 71±6, *P ¼ 0.113). The data represent the mean±s.d. of three independent experiments. (Lu et al., 2005; Bennett et al., 2008, 2009; Guo et al., 2010). However, the detailed biological functions of restoring IRX1 expression on gastric cancer remain to be explored. In the present study, we further demon- strate that peritoneal spreading and long distance metastasis were arrested by restoring IRX1 expression on gastric cancer cells. These functions are mediated by interference of angiogenesis and VM formation both in vitro and in vivo. On previous microarray analysis, we found out BDKRB2, a downregulated gene, which involves in the regulation of blood vessels. Along with the important clue, we explored the association between IRX1 expression and angiogenesis, VM and ECM adhesive capacity. Rich blood supply is the basis of tumor growth, spreading and metastasis. Tube formation assay is based on the ability of endothelial cells to form 3D capillary- like structures when cultured on a gel of basement membrane extract (Masamune et al., 2008; Nogueras et al., 2008). VM is a newly identiﬁed phenomenon different from traditional tumor angiogenesis. VM was ﬁrst described as the ﬂuid-conducting channels formed in highly aggressive melanoma cells by Maniotis et al. (1999). The channels of VM are lack of endothelial cells of liner with abundance of PAS-positive substances in the ECM. Either red blood cells or plasma could appear in those channels (Sood et al., 2001; Sun et al., 2004, 2006; van der Schaft et al., 2005). Importantly, one report suggested that SGC-7901 gastric cancer cells can form VM in 3D culture (Zhang, 2008). Our experiments conﬁrmed that overexpression of IRX1 on SGC-7901 cells effectively inhibited the formation of capillary and VM structures. In chick CAM assay, another in vivo model for studying the angiogenic activity (Deryugina and Quigley, 2008a, b), we further conﬁrmed that overexpression of IRX1 signiﬁcantly inhibited angio- genesis on avian embryo. Another factor that inﬂuences the process of tumor spreading and metastasis is tumor-ECM adhesive ability. During the process of metastasis, tumor cells leave the primary site, travel via blood and/or lymphatic circulatory systems, attach to the substratum of ECM at a distant site, and establish a secondary tumor, accompanied by angiogenesis of the newly formed neoplasm (Gupta and Massague, 2006; Lorch et al., 2007; Veeravalli et al., 2010). Therefore, examination of adhesive ability of tumor cells to ECM will become an important indicator for invasiveness. Our experiments Oncogene