Figure 4: Multicellular adhesion assays.
(a) Both MV3 control and MV3 NHE1-overexpressing cells form tumour spheroids using the hanging-drop assay that is presented in Fig. 2a. However, spheroids of NHE1-overexpressing cells are less regular and circular. (b) For the cell aggregation assays, MV3 cells were incubated in experimental medium on a shaker overnight. Here, NHE1-overexpressing cells do not form stable tumour spheroids thus pointing towards weaker cell-cell adhesion. MV3 control cells formed spheroids with an average diameter of 466 μm (399/526 μm, N = 3 experiments, n = 84 spheroids) and a projected cross-sectional area of 0.16 mm2 (0.12/0.19 mm2, N = 3, n = 79). (c,d) Left: Increasing the extracellular proton concentration reduces the spheroid size of MV3 control cells (cross-sectional area detected by light microscopy: pHe7.4: 0.081 mm2 (0.053/0.013 mm2), N = 4, n = 154; pHe7.0: 0.052 mm2 (0.033/0.076 mm2), n = 356; pHe6.8: 0.034 mm2 (0.023/0.056 mm2), n = 517; pHe6.4: 0.022 mm2, (0.016/0.037 mm2), n = 890)) and increases the spheroid number. Right: MV3 NHE1-overexpressing cells form fewer cell aggregates at pHe7.4 than control cells. However, the adhesive strength between two cells slightly increases upon acidification as shown by a small rise of cell aggregate size. Quantification of pHe-dependent spheroid formation in MV3 NHE1-overexpression cells: pHe7.4: 0.013 mm2 (0.009/0.019 mm2), N = 3, n = 636; pHe7.0: 0.018 mm2 (0.013/0.025 mm2), n = 387; pHe6.8: 0.017 mm2 (0.012/0.028 mm2), n = 398; pHe6.4: 0.016 mm2 (0.011/0.023 mm2), n = 413. # = significant difference (p < 0.001) to all groups. Statistical significance was tested using Kruskal-Wallis ANOVA and Mann-Whitney U test.
