Figure 7. Knockout of EphA2 or RCP reduces invasion and metastasis in pancreatic cancer in vivo.

(a) KPC (Pdx1-Cre:Kras^G12D/+:p53^R172H/+) mice were crossed with EPHA2^+/− or EPHA2^−/− animals or those with floxed RCP (RCP^+/fl or RCP^fl/fl) or floxed α5 integrin (ITGA5^+/fl or ITGA5^fl/fl) alleles. Survival time (which is dictated by the size of the primary tumour) is represented by a Kaplan-Meier curve. Censored mice (indicated by ticks) succumbed to complications other than PDAC. (b) The proportion of animals with detectable metastases to liver, lung and other tissues was assessed by gross pathology and confirmed by histology. The number of animals in each category is indicated above the bars. *P<0.05; Comparison is between EPHA2^+/+ and EPHA2^−/−, or between RCP^+/+ and RCP^fl/fl; Chi-squared test, one-tailed. ns=not significantly different from ITGA5^+/+ control. (c,d) Primary mouse cell lines were derived from PDACs harvested from KPC (Pdx1-Cre, Kras^G12D/+, p53^R172H/+), KPC:EPHA2^−/− and KPC:RCP^fl/fl mice. At least three cell lines were derived per condition and the knockout of EphA2 and RCP was confirmed by immunoblotting (see Supplementary Fig. 3c). PDAC cells (two cell lines per condition) were plated onto the underside of transwells containing Matrigel plugs enriched with fibronectin (25 μg ml⁻¹). Cells were allowed to migrate into the plugs towards a gradient of HGF and serum for 72 h, and then visualized by Calcein-AM followed by confocal microscopy. Optical sections were taken every 10 μm and consecutive images are displayed as a series running from left to right (c; left panels). Cell invasion beyond 20 μm (to the right of the red dashed line) was quantified and expressed as a % of the total quantity of fluorescent cells in the plug (c; right panel). Data are represented as box and whiskers plots (whiskers: 10–90 percentile). ***P<0.001; one-way ANOVA (Kruskal-Wallis test, Dunn's Multiple Comparison Test). Data are from three independent experiments. The EPHA2 #1 knockout PDAC line was stably transfected with EphA2-GFP or EphA2^897A-GFP, and the RCP #2 knockout line was stably transfected with GFP-RCP or GFP-RCP^435A as indicated, and the expression of these GFP-tagged proteins was determined by western blotting. The propensity of these PDAC cells to form tight or scattered colonies was determined using phase contrast microscopy (d). Bar, 100 μm. Lower magnification images of these fields are displayed in Supplementary Fig. 7d.