Evaluating TNBC Cell Anchorage-Independence

To investigate the ability of TNBC cells to grow in an anchorage-independent manner, soft agar colony formation assay was performed as previously described by our group (25 (link)). Briefly, TNBC cells were seeded in 6-well plates (1 × 10⁴ cells/well) with/without 100 and 200 μl/ml of EA extract (treated/control, respectively) placed in DMEM medium containing 0.3% agar and plated over a layer of DMEM medium with 10% FBS and containing 0.4% agar. The growing colonies were examined every 2 days for a period of 3 weeks. Colonies in each well were counted under a light microscope, Leica SP8 UV/Visible Laser confocal microscope (Leica Microsystems, Wetzlar, Germany), in five predetermined fields.