Cell cultures and induction of TNBCSCs

The TNBC cell line (MDA-MB-231) was procured from the Shanghai Cell Bank at the Academy of Sciences (China). The culture conditions of MDA-MB-231 were dulbecco’s modified eagle medium (DMEM; Gibco, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin–streptomycin (Gibco), in a humidified incubator at 37 °C with 5% CO2. To ensure optimal cell growth and to prevent over-confluency, we conducted our experiments before primary cell line passages reached 40 doublings. Subsequently, enzymatic digestion was employed to convert well-adhered MDA-MB-231 cells in a robust growth state into a single-cell suspension using pancreatin. The supernatant was discarded after centrifugation. The cells were resuspended in a specific medium based on DMEM/F12 (1:1) media (C11330500CP, Gibco), which was supplemented with 0.4% BSA, 20 ng/ml EGF, 10 ng/ml bFGF, and 5 μg/ml recombinant human insulin, then Cells (1*10⁵ per well) are grown in 6-well low adsorption culture plate. The resuspended cells were then cultured in a CO2-regulated incubator at 37 °C for 7–10 days, with the addition of 500 μl specific medium every other day. Throughout this cultivation period, the induction of sphere formation was observed under a microscope, and images were captured. The growth of cells in suspended spherical aggregates were identified as triple-negative breast cancer stem cells (TNBCSCs). Every condition was measured three times. Additional MDA-MB-231 cells (1*10^5 per well) were seeded as a control in a standard 6-well plate and cultured in a complete medium (DMEM supplemented with 10% FBS and 1% penicillin–streptomycin). In the logarithmic growth phase, MDA-MB-231 adherent cells were cultured and conventionally digested at 1000 rpm for 5 min. Following centrifugation, the cells were resuspended in complete culture medium, and the cell density was adjusted to 1 × 10^5 cells/mL, resulting in a single-cell suspension. Subsequently, the cells were seeded into a 6-well plate, with each well receiving 2.5 mL of the single-cell suspension. This process was replicated for each of the three samples. The plate was then placed in a 37 °C incubator with 5% CO2 and left overnight. After the cells adhered to the surface, culture medium was added, and further incubation occurred for an additional 5 days.