Culturing TNBC Cell Line Mammospheres

The human TNBC cell lines, SUM159 and 2LMP, were cultured under adherent conditions, as previously described [56 (link)]. Nonadherent mammospheres (“CIC” conditions) were cultured with MEGM medium (Lonza, Walkersville, MD, USA) in ultra-low attachment plates (Corning Costar, Corning, NY, USA). After 3 days of culture in CIC conditions, SUM159 mammospheres grew as irregular clusters (60–120 μm) of tightly joined cells; 2LMP mammospheres grew as non-uniform aggregates (90–230 μm) of cells. Mammospheres from both cell lines maintained high viability (>95%) under these conditions. Cell culture and in vitro experiments were performed at 37 °C in a 5% CO₂ humidified atmosphere unless specified otherwise.