Cell migration was studied by using tumor spheroids. 200–300 μm-spheroids were transferred into gelatin-coated flat-bottomed 96-well plates (a single spheroid/well) with addition of culture medium (T0). Cell migration was recorded every 24 h for a total period of 144 h. We obtained images using an inverted Leitz microscope (Leitz Corporation, Stuttgart, Germany), equipped with a 6MP Digital Camera (CCD vision sensor, square pixels of 4.4 µm side length, 1600 × 1200 pixel resolution, 8-bit grey level) (TiEsseLab, Italy). The ISCapture software (version 3.6.9.1; http://www.tiesselab.com) was used to obtain 2D morphological parameters (diameter, perimeter, area) as well as to select morphologically homogeneous spheroids and to measure degree of the migration in the time22.

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