Figure 4.

Evaluation of stemness-associated properties of tumorspheres. (A) Tumorsphere formation frequency of 5–8F and H460 cell lines in the traditional and the novel three-dimensional culture systems. (B) Frequency of cancer stem cells demonstrated no significant differences (5–8F, P=0.544; H460, P=0.591). An extreme limiting dilution algorithm was used to calculate this frequency. (C) Viability was measured using a cell counting kit-8 assay (mean ± standard deviation; n=3). The sphere-forming cells were more resistant to chemotherapy compared with the adherent cells. (D) In vivo tumorigenicity of sphere-forming cells cultured in the novel culture system. Sphere-forming and adherent cells were subcutaneously injected into the left and right thighs of male nude mice, respectively. The volumes of tumors developed from the sphere-forming cells (red circles) were greater compared with those of the adherent cells (black circle) in the same mouse.