Research Article

Selective Activation of Cancer Stem Cells by Size-Specific Hyaluronan in Head and Neck Cancer

Figure 3

Measurement of sphere formation (a) and clone formation (b) (CSC) cells. (a) Sphere formation of cells [treated with no HA (A) or with 200 kDa-HA (B)] in a mixture of 5 mg/mL of matrigel (Corning) and defined medium (RPMI-1640 medium containing EGF and bFGF without serum) for 3 weeks (21 days) as described in Section 2. (b) Clone formation (differentiation) of cells pretreated with no HA (A) or with 200 kDa-HA (B). Specifically, clone formation and differentiation were induced by incubating cells (dissociated from spheres treated with 200 kDa-HA or without HA for 10 days as described above). After the removal of 200 kDa-HA from the cells, these sphere-derived cells were then incubated in RPMI 1640 complete culture and 10% fetal bovine serum for ~7–10 days. After most cell clones expand to >50–100 cells, they were fixed with methanol followed by staining with crystal violet to visualize clone formation as described in Section 2.
(a) Sphere formation
(b) Clone formation