The ex ovo chick embryo model

Extravasation assays were performed as previously described [2]. Briefly, stable cell lines generated by lentiviral infection of control or PAK1 shRNA were lifted in 0.5% trypsin with 0.05 mM EDTA, pelleted and washed three times with PBS. Cells were counted and resuspended at 1 × 10⁶ cells/ml. 100ul of cells was intravenously injected into the CAM using a disposable micropipette syringe as described previously using a day 13 chicken embryo and analyzed for extravasation 24 h post-injection or metastases at 5 days post-injection using an wide field epi-fluorescence microscope. Chemosensing assays were performed by diluting either EGF (50 ng/ml), GABA (100 µM), or control (water) in Matrigel. For extravasation, a 10 ul drop of either control and EGF or GABA was placed on the CAM prior to injection of cells. Cells were counted directly under the matrigel drop area post-injection and 24 h later. For metastatic colony formation, a quarter of the CAM was covered with a thin layer (100 ul) of control and either EGF or GABA and colonies were counted in these areas 5 days post-injection. For invadopodia formation/retraction, cells were imaged 4 h post-injection and cells forming invadopodia were monitored over two hours to assess retraction. To image cells and colonies in the CAM 50 ul Lectin-Rhodamine/Dylight 649 lectin (1:10 in PBS) and Hoechst (1:300 in PBS) was intravenously injected into the CAM and analyzed using a Nikon upright confocal microscope. (12–16 chicken embryos were injected for each construct; n = 3).