The hanging drops technique according to Insphero (InSphero AG) manufacturer’s instructions was used. Single DHSF-BR16 and MCF-7 cells were seeded at various cell densities (ranging from 1000 to 8000) into special hanging drop culture plates. In order to obtain optimal spheroids (200–300 µm) without central necrotic area after 4 days of culture, an inoculum of 2000 cells/well was used. MCF-7 culture conditions are described in Supplementary Methods.

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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