A total of 1.5 × 103 HCC1937 cells/well was seeded in 24-well plates covered with 1% agarose in DMEM:F12 supplemented with 20 ng ml−1 bFGF, 40 ng ml−1 EGF (Bio-techne, Citomed Lda, Lisboa, Portugal), 1 × B27 (Life Technologies, Porto, Portugal), 10 μg ml−1 insulin (Sigma-Aldrich, Sintra, Portugal) and 2 mM L-Glutamine (Sigma) and treated with BBIT20 at the seeding time (Bessa et al., 2018 (link)). Mammospheres also grown in compound-free medium for 3 followed by BBIT20 treatment (Bessa et al., 2018 (link)).
Using 3-day-old mammospheres, BBIT20 synergistic potential was evaluated through analysis of its effect, alone and in combination withcisplatin (CDDP) or olaparib , on spheroid growth for additional 14 days. New medium with the drugs (or vehicle only) was added to the wells at days 1, 3, 6, 9 and 12. Mammospheres were monitored using an inverted Nikon TE 2000-U microscope at 100X magnification, with DXM1200F digital camera and Nikon ACT-1 software (Raimundo et al., 2018 (link)). Spheroid diameters were quantified using Fiji Software (Schindelin et al., 2012 (link)).
Using 3-day-old mammospheres, BBIT20 synergistic potential was evaluated through analysis of its effect, alone and in combination with
