For adhesion assays, 96-well flat bottom culture plates were coated with 50 μl of gelatin (0.2%; Sigma-Aldrich) and matrigel (0.9 mg/ml; Corning, Bedford, USA) or kept uncoated. Cells were labeled with 10 μM calcein acetoxymethyl (AM) (Life Technologies Inc., Schwerte, Germany) for 30 min at 37°C and washed three times in PBS. Labeled cells (2.5 × 104) were seeded in 50 μl of medium per well and kept for 2 hours of adhesion time. Thereafter, plates were washed twice with PBS (input control was not washed). Before measurement of calcein AM fluorescence in a Victor Wallac instrument, cells were lysed in 2% Triton X-100 in distilled water. For endothelial cell adhesion assay, 105 bEnd.3 cells were plated in 96-well culture plates in 100 μl of medium and cultured 24 to 48 hours to generate a monolayer. Endothelial cells were stimulated with tumor necrosis factor–α (TNF-α; 10 ng/ml; Thermo Fisher Scientific, Bleiswijk, The Netherlands) for 5 hours before the assay. After removal of TNF-α–containing medium, 2.5 × 104 labeled tumor cells were seeded in 50 μl of medium per well and kept for 2 hours of adhesion time. Further measurements were performed as mentioned above.

Transwell invasion assay was performed using Transwell chambers (8 μm; Falcon, Durham, USA). A total of 105 cells were seeded in the upper chamber of a 24-well plate and coated with growth factor–reduced matrigel (Corning, Bedford, USA) in 200 μl of serum-free medium. The lower chamber was filled with 800 μl of medium containing 10% FBS. The chamber was incubated at 37°C for 16 hours. At the end of incubation, cells in the upper surface of the membrane were removed with a cotton swab. Migrated cells on the lower surface of the membrane were stained with 1% crystal violet/70% methanol solution. Then, membranes were transferred to empty 96 wells, adding 200 μl of acetic acid to dissolve crystal violet–stained cells. After incubating for 10 min on an orbital shaker, OD at 590 nm was measured in a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

For scratch assay, cells were seeded in six-well plates and cultured to a density of 80%. The standard culture medium was replaced by a medium without FBS, and 8 hours later, a scratch was set with a sterile pipette tip. Cells were washed thrice with PBS, and three random sections of two scratches per experimental group were marked. Pictures were taken at the indicated time points under an Axiovert 25 microscope (ZEISS, Jena, Germany) with a Samsung WB750 camera (Samsung, Schwalbach, Germany). To calculate the migration velocity, the gap area was calculated using ImageJ software.

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