Sphere cells from HNSCC cell lines show CSC-like characteristics. (A) HNSCC cell lines grown as monolayers with 10% FBS under standard adherent growth conditions (top), as spheres in serum-free suspension growth conditions (middle), and as sphere-derived cells (SDCs) under adherent growth conditions in the presence of 10% FBS and 75 μg/mL Matrigel (bottom). Images were taken using phase contrast microscopy at 20x objective for monolayer and SDCs, 4x for sphere cultures. Scale bars represent 100 μm in length. (B) Expression of CSC markers CD44 and BMI1 and differentiation marker CD24 in sphere cells relative to monolayer cultures by qRT-PCR. Expression was normalized to the GAPDH housekeeping gene as an internal control and shown as mean fold change ± SE (n ≥ 3). (C) Summary of flow cytometry analysis of CD44 surface protein expression showing the percentages of the CD44high populations in monolayer and sphere cells from TR146 (n = 8), SCC-58 (n = 8) and UMSCC-17B (n = 3). Representative flow cytometry data showing the gating of the CD44high cells are shown in Additional file 1: Figure S1. Data are presented as mean ± SE for the indicated total number of independent experiments. Statistical analysis was performed using two-tailed Student’s t-test with equal variance. n.s. = not significant (p > 0.05).