Figure 6.

ALDH^hiCD44⁺CD24⁻ and ALDH^hiCD44⁺CD133⁺ breast cancer cells demonstrate enhanced adhesion, migration and invasion in vitro. (A) Cell adhesion of ALDH^hiCD44⁺CD24⁻ versus ALDH^lowCD44^low/−CD24⁺ cells isolated from the MDA‐MB‐231 cell line (left panel); and ALDH^hiCD44⁺CD133⁺ versus ALDH^lowCD44^low/− CD133⁻ cells isolated from the MDA‐MB‐468 cell line (right panel). Cells were isolated by FACS as described in Figs 3 and 4 and plated onto sterile 96‐well non‐tissue culture plates treated with either 10 μg/ml of fibronectin (MDA‐MB‐231 cells), 5 μg/ml of vitronectin (MDA‐MB‐468 cells) (black bars) or PBS (negative control) (white bars), using 1 × 10⁴ cells/well in triplicate wells for each sorted cell population. Cells were allowed to adhere for 5 hrs and adhered cells were quantified by manual counting of five high‐powered fields (HPF) per well. Data are presented as the mean ± S.E.M. *= significantly different than respective population of ALDH^lowCD44^low/− cells adhered to vitronectin or fibronectin (P < 0.001). (B) Cell migration and (C) Cell invasion of ALDH^hiCD44⁺CD24⁻ versus ALDH^lowCD44^low/−CD24⁺ cells isolated from the MDA‐MB‐231 cell line (left panels); and ALDH^hiCD44⁺CD133⁺ versus ALDH^lowCD44^low/− CD133⁻ cells isolated from the MDA‐MB‐468 cell line (right panels). Transwells (8 μm) were pre‐coated with either gelatin (migration assays; 6 μg/well) or Matrigel (invasion assays; 10 μg/well) and FACS‐isolated cells (3.5 × 10⁴ cells/well; n= 3 for each cell population) were allowed to migrate or invade for 24 hrs towards chemoattractant media (5% foetal bovine serum) (black bars) or control media (0.01% bovine serum albumin) (white bars). Migrated or invaded cells were quantified by manual counting of five HPF per well. Data are presented as the mean ± S.E.M. δ= significantly different than respective population of ALDH^lowCD44^{low/− cells} migrating towards FBS (P < 0.001). φ= significantly different than respective population of ALDH^lowCD44^{low/− cells} invading through Matrigel towards FBS (P < 0.001).