Fig. 7: SOCE formation is necessary for the dissemination of amoeboid single cells from cancer spheroid in 3D environment.

a–e Role of SOCE in cell migration. a Transwell migration assays using non-transfected DLD1 cells and cells expressing either dominant negative Orai1 (E106Q) or constitutive active Ezrin (T567E). Individual data points are plotted with the means ± SD based on the values from N = 5 independent experiments. b 4T1 cell spheroids embedded in type I collagen were treated with DMSO (left panel) or DMOG (1 mM), an inhibitor of prolyl hydroxylase, to activate HIF-1, (right panel). Scale bar, 100 μm (left panel) or 200 μm (right panel). c Representative 4T1 cell spheroid embedded in type I collagen was treated with DMOG only (1 mM, left panel), or with a combination of DMOG and SOCE inhibitors AnCoA4 (50 µM, center panel) or SKF96365 (10 µM, right panel) for 72 h. Scale bar, 200 μm (left panel) or 100 μm (center and right panels). d Number of single cells migrating away from the spheroid were quantified based on the experiment shown in c. N = 10 independent spheroids per condition from three independent experiments. Individual data points are plotted with the means ± SD. e Representative amoeboid migration of 4T1 cells expressing Lifeact-GFP and stained with ER-tracker (red). after spheroid treatment with DMOG as in b from three independent experiments. Arrowheads indicate regions where the ER flows into blebs. Times shown are relative to the first image. Scale bar, 10 μm. f Schematic showing the proposed mechanism of SOCE-dependent cytoplasm solation. In the expansion phase of membrane blebbing, the influx of calcium ions via SOCE inhibits actin polymerization and promotes solation of the cytoplasm, which supports rapid bleb expansion. By contrast, during the retraction phase, activated Ezrin inhibits SOCE by directly binding to Orai1, allowing the reassembly of the actin cytoskeleton by decreasing the calcium ion concentration in the cytoplasm of the bleb. a, d ****P < 0.0001 (One-way ANOVA with Tukey’s post-hoc multiple comparison test). Source data are provided as a Source Data file.