Non adherent round bottom 96 well plates

After 8 h from transfection HUVECs were suspended at a density of 4000 cells/mL in EBM culture medium containing 0.4% Methylcellulose and 10% FBS. 800 cells were seeded into non-adherent round bottom 96-well plates (Corning, NY, USA) and cultured overnight at 37 °C. The spheroids were harvested by gently pipetting and centrifuged at 300× g for 15 min. The spheroids were then suspended and embedded in fibrin in the presence of 10 µg/mL aprotinin to prevent the dissolution of the substrate. The sprouting was stimulated with VEGF-A, or with vehicle (PBS) as control. After 16 h at 37 °C, sprouts were counted and phase-contrast images were captured with an inverted microscope (Leica).

Primary and metastatic breast cancer cells (5 × 10³) were plated into non-adherent round bottom 96-well plates (Corning) in full growth media and cultured for 3 days. At this point, the tumorspheres were physically transferred with 50 μl of residual media and 150 μl of fresh media to a flat bottom 96-well plate coated with 50 μl of growth factor reduced basement membrane hydrogel (Trevigen) in the presence or absence of trametinib and EGF. These structures were allowed to grow for an additional 3 days for metastatic NM-LM1 cells or 10 days for non-metastatic NME cells at which point structure size was quantified using image J analyses or invasive tips were enumerated.