Fig. 4

High cytoplasmic LIF enhances cancer vascular invasion. a Real-time impedance analysis. HUVEC cells were grown on E-plates until confluence. Cancer cells were added on top of the HUVEC layer at the indicated times (black arrow). data are presented as means and SD of triplicate experiments. b Representative images of HUVEC layer replacement assay. Equal numbers of cancer cells were plated on top of the confluent HUVEC layer and co-cultivated for 24 h. Cells were labeled with antibodies against pan-cytokeratin (red) and VE-cadherin (green). Yellow closed polygons indicate damaged HUVEC areas. Scale bars, 20 μm. c Quantification of displaced areas in b. Data are presented with scatter dot plot (mean ± SEM). Each black dot represents one captured image. Invaded areas were calculated using CellSens imaging software (Olympus). ***p < 0.001, Mann–Whitney test. d Live-cell imaging of the HUVEC replacement assay. Cancer cells stably expressing LiveAct-RFP were plated onto confluent HUVEC cells expressing LiveAct-GFP2. Live interactions were continuously monitored for 30 hours (see also Supplementary movies 4-6). e Addition of cancer cells to the HUVEC layer breaks down endothelial junctions. An equal number of cancer cells was added on the top of HUVEC layer and co-cultivated for 24 h before fixation. Cells were labeled with antibodies against pan-cytokeratin (red) and CD31 (green, upper panels) or pan-cytokeratin and VE-cadherin (green, bottom panels). Scale bars, 10 μm. f Western blot of CD31 and VE-cadherin expression using GAPDH as a loading control. g Representative image of Tg (fli1a:EGFP)y1 zebrafish embryo carrying cancer cells expressing LifeAct-RFP on day 6 post-cancer cell injection. Tumor-like structures (red) that spread through the vasculature (green) were observed. h Representative images of disseminated tumor-like structures in zebrafish embryos injected with WT, LIF^+/−, or cLIF cells. Asterisks indicate the dissemination sites of tumor-like structures. i Quantification of tumor-like structures shown in h. Areas of tumor dots were calculated using CellSens imaging software. The number of counts was determined 6 days after injection with cancer cells. Data are presented with scatter dot plot (mean ± SEM). Each black dot represents one fish embryo. *p < 0.05, **p < 0.01, ***p < 0.001, Mann–Whitney test