Figure 4
A DTC-dominating clone efficiently forms anoikis-resistant aggregates under FSS conditions. (a) Diagram showing the experimental setup of the anoikis assay. Single cells (5 × 103) obtained from SAS-GL cells (parental), clone W, and clone H were separately suspended in culture medium and then cultured on poly-HEMA-coated plates under static conditions (−FSS) or rotated (+FSS) at 37 °C. (b) Numbers of aggregates in suspension culture at 18-h under static conditions. Values are means ± SEM of triplicate samples. n.s., not significant. (c) Numbers of aggregates generated in suspension culture under FSS conditions. Values are means ± SEM of triplicate samples. *p < 0.005 (two-tailed t tests). (d) Representative images of single cells (upper panel) and aggregates (lower panel) derived from clone H cultured under FSS conditions. (e) Annexin V staining with clone H cells at 3 h of suspension culture. Arrows and arrowheads indicate annexin V-negative aggregates and -positive single cells, respectively. (f) Proportion of annexin V-positive and -negative cells in single cells and aggregates. *p < 0.0001 (Pearson's χ2 test). (g,h) Single cells (5 × 104) were suspended in culture medium and rotated for 22 h at 37 °C. Cells were centrifuged, plated in 6-well plates, and cultured for 6 days. Colonies of surviving cells with areas > 2500 μm2 were counted. (g) Representative images of colonies. (h) Number of colonies derived from parental cells and clones W, EE, and H. Values are means ± SEM of triplicate samples. *p < 0.05 (two-tailed t tests).
