Figure 4 (IMAGE)
Caption
Tumor-secreted ANGPTL2 promotes osteosarcoma metastasis by mediating endothelial monolayer disruption, lung capillary permeability, and trans-endothelial tumor cell migration. (A) HUVEC monolayers were grown to confluence on fibronectin-coated slides and then treated with either vehicle (PBS) or human recombinant ANGPTL2 protein (rhANGPTL2) for 24h. Slides were subsequently fluorescently stained with anti-phalloidin, and anti-β-catenin antibody. (B) HUVEC monolayers were treated for 24 h with media conditioned by control OS17 osteosarcoma cells that express ANGPTL2 (ctrl) or knockdown OS17-shANGPTL2 (sh) cells. Samples were stained for β-catenin and phalloidin. (C) Administration of ANGPTL2 receptor inhibitor, ATN-161, prevents disruption of vascular endothelial cell-cell junctions. HUVEC monolayers were treated for 24 h with media conditioned by OS-17 cells in the presence of drug. Subsequently, samples were stained for β-catenin and phalloidin. ((A-B) Quantitative analysis of paracellular gap formation is shown in Supplementary Figure 7) (D) GFP-labeled indicated OS17 cells were injected via the tail vein and allowed to lodge in the lungs. One day post injection, a rhodamine-dextran dye was injected into circulation. Three hours after dye injection, lungs were extracted and frozen sections were obtained. Representative confocal images are shown here of cells with and without accumulation of dye in the lung parenchyma. (E) Images were obtained as described in (D) with control or ANGPTL2 knockdown OS17 cells. A region of interest was drawn around the GFP-labeled cells and the amount of dextran dye was quantified based on rhodamine emissions. n = 40 cells; error bars indicate s. e. m; p-values calculated using the one-tailed unpaired t-test. (F) OS17-shCtr (1), OS17-sh1ANGPTL2 (2) or OS17-sh2ANGPTL2 (3) were seeded into trans-well inserts that were previously covered with a HUVEC monolayer. Cells that migrated cross the endothelial layer into the bottom side of the transwell membrane were quantified with Volocity software. n = 15, error bars indicate s. e. m; p-values calculated using the one-tailed unpaired t-test.
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Correspondence to - Hakan Cam - hcam@cscc.edu
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