Isolation and Characterization of Primary Breast Cancer Stem Cells

Primary human breast cancer sphere cells (BCSC) were obtained through mechanical and enzymatical digestion of breast cancer tissues, collected at the Department of Surgical, Oncological and Stomatological Sciences, in accordance with the ethical standards of the University of Palermo institutional committee, as previously described [38 (link)]. BCSCs were cultured in serum-free DMEM/F12 medium, supplemented with 2% B27 (50 ×, Gibco), basic fibroblast growth factor (bFGF; 10 ng/mL) and EGF (20 ng/mL) in ultra-low attachment flask (Corning). In these culture conditions the breast cancer cells grow as aggregates conventionally defined as “spheres”. In order to assess the presence of cancer stem cells in our BCSC population, BCSCs were characterized in terms of ALDH1 activity, and then subcutaneously injected in NOD/SCID mice to test their ability to form tumor xenografts [39 (link)].