Hypoxia Enhances TIC Invasion.

<p>A) Characterization of SK-3rd TICs: SK-3rd and SK-BR3 cells were cultured on ultra-low attachment culture dishes for 3 days and mammosphere formation was examined by microscopy (Upper panel). Bar, 100 µm. These cells were collected and incubated with APC-conjugated anti-CD44 antibodies and PE-conjugated anti-CD24 antibodies followed by flow cytometry analysis (Lower panel). B–C) Determination of invasive ability of SK-3rd TICs: SK-BR3 and SK-3rd cells were pretreated with or without CoCl₂ (250 µM) or cultured under hypoxia (1% O₂) for 24 hours followed by examination of invasive ability of the cells using the 3-D invasion assay (B). After 24 hours, the cells were fixed and invaded cells in six different fields in the invasion zone were microscopically counted (C). D) Evaluation of hypoxic conditions by examining HIF-1α in SK-3rd cells: Total cell lysate from SK-3rd TICs pretreated with or without CoCl₂ (250 µM) or under 1% O₂ for 24 hours were examined by Western blotting using anti-HIF-1α antibody. NT refers to cells without pre-hypoxia culture.