Figure 2.

TTField-mediated inhibition of cancer stem cell features is enhanced by hyperthermia. (A) The cells were treated as described in Figures 1A, B , (B) After 3 days, the cells were detached from the cell culture plates by trypsinization and reseeded at clonal density (AsPC-1: 1,500 cells/well; BxPC-3 and Bx-GEM: 1,000 cells/well) in 6-well plates. The cells were cultured under regular conditions at 37°C without a medium change for 2 weeks, resulting in first-generation colonies (1^st GEN). The number of colonies was evaluated by fixing and Coomassie staining, followed by counting colonies with at least 50 cells using a dissecting microscope. The survival fraction and representative images are shown on the left. For the formation of second-generation (2^nd GEN) colonies, surviving cells from each group of first-generation colonies were collected, reseeded and analyzed as described above. (B) After treatment, as described in Figure 1A , the cells were seeded at a clonal density of 500 cells/well in ultralow-attachment 24-well plates in cell growth factor-supplemented serum-free culture medium to induce spheroid formation. Six days later, the first generation of spheroids developed, and the percentage of viable spheroids was evaluated by microscopy at 100× magnification and counting. Representative photographs and the means are shown on the left. For the formation of second-generation spheroids (2^nd GEN), surviving cells were collected from each group of first-generation spheroids and reseeded and analyzed as described above. The data are presented as the means ± SDs. *P < 0.05, **P < 0.01.