Figure 7
Regulation of actomyosin associated with E-cadherin is crucial for stable cell aggregation in FSS. (a) Left panel: GO enrichment of specifically up-regulated genes in clone H. The p-values represent Fisher's exact test with Bonferroni correction results. Right panel: CadH genes overlapped with reported EBP genes were defined as EBP-H. (b) The number of aggregates from the indicated cells transduced with scramble shRNA (shScr), or shRNA targeting E-cadherin (shEcad) after 3 h of suspension culture under FSS conditions. Values are means ± SEM of triplicate samples. *p < 0.0001; †p < 0.005 (two-tailed t tests). (c) Results of myosin IIA immunofluorescence staining of cell aggregates at 1 h of suspension culture under FSS conditions. Representative images of cell aggregates are shown (left panels). The regions surrounded by white dotted lines are magnified in the adjacent images. Frequency of cell aggregates showing “stable aggregates pattern” are shown (right panel). *p < 0.01 (two-tailed t tests). (d,e) Number of aggregates generated by SAS parental (Par) and the clones pretreated with LatA (d, left panel), Bleb (d, right panel), 50 μM NSC23766 (e, left panel), or 10 μM Y-27632 (e, right panel) at 3 h of suspension culture under FSS conditions. Values are means ± SEM of triplicate samples. *p < 0.0001; †p < 0.005 (two-tailed t tests). (f) GO enrichment of EBP-H signature genes. The p-values represent Fisher's exact test with Bonferroni correction results. (g) Correlation between the frequency of aggregates with “stable aggregates pattern” (myosin IIA distribution) at early phase (30 min) (X-axis) and the number of aggregates detected at 12-h suspension culture in FSS (Y-axis) (left panel). Representative images of aggregates with “stable aggregates pattern” in MDA-MB231 and HOC-313 are shown (right panels). (h) Results of myosin IIA immunofluorescence staining of CTC clusters derived from the SAS-GL xenografts. Representative images of a CTC cluster (left panels) and average frequency of clusters (right panel) with “stable aggregates pattern” are shown. Arrowheads indicate the myosin IIA (MyoIIA) on the cortex at free external surfaces. Cell–cell contact areas are surrounded by white dotted lines. (i) Diagram showing the experimental setup for the selection of FA cells, and subsequent injection of the mixed tumor cells into mice. Inserted picture shows the aggregates captured on the metal sheet filter. Scale bar, 20 μm. (j) Proportions of total cells (Total) and cell clusters positive for only GFP (green) or mCherry (red) in CTCs (left panels) and BM-DTCs (right panels) were calculated for each pooled sample. Relative average numbers of total cells and clusters from each mouse are given above each bar. *p < 0.0001; †p < 0.0005; §p < 0.05 (Pearson's χ2 test).
