Extended Data Fig. 7: KCa3.1 drives periodic Ca²⁺ oscillations and their proliferation-stimulating effects in glioblastoma cell networks.

a—d, After gap junction inhibition via MFA, pharmacological inhibition, and genetic perturbation of KCa3.1 strongly reduce the fraction of cells displaying periodic Ca²⁺ activity but do not affect the remaining sporadic non-periodic Ca²⁺ activity, demonstrating a specific effect on periodic Ca²⁺ activity that – due to gap junction inhibition – is unlikely to be mediated by nonspecific effects on global Ca²⁺ communication. a,b, Fraction of cells displaying periodic (a) and non-periodic (b) Ca²⁺ activity of all cells after treatment with 100 µM MFA and with both MFA and 1 µM Senicapoc in vitro; S24 line; n = 10 recordings (Control), n = 11 recordings (MFA), and n = 13 recordings (MFA+Senicapoc) from 2 biologically independent experiments, respectively; One-way ANOVA, Dunnett’s test. c,d, Fraction of cells displaying periodic (c) and non-periodic (d) Ca²⁺ activity after treatment with MFA and after both genetic knockout of KCa3.1 and treatment with MFA in vitro; n = 5 recordings (S24 KO control line, no treatment; “Control”), n = 6 recordings (S24 KO control line, MFA treatment; “MFA”), and n = 6 recordings (S24 KCa3.1 KO-2 line, MFA treatment; “MFA+KCa3.1 KO”) from 2 biologically independent experiments, respectively; One-way ANOVA, Dunnett’s test. e—h, Intercellular coactivity is not reduced in Ca²⁺ recordings of control versus 1 µM TRAM-34 and 1 µM Senicapoc treatment (e), S24 KO control versus S24 KCa3.1 KO-1 and KO-2 (f), S24 KD control versus S24 KCa3.1 KD (g), and P3 control versus P3 KCa3.1 KD (h) in vitro; intercellular coactivity was calculated by dividing the number of coactive cells of a recording by the number of coactive cells of its corresponding null control data, representing the overall degree to which active cells are communicating their Ca²⁺ activity to other cells; increased intercellular coactivity after KCa3.1 inhibitor treatment might be because the strongly reduced number of periodic cells leads to less interference of different signals and therefore an even stronger synchronization between cell pairs. e, n = 11 recordings (S24, control treatment), n = 6 recordings (S24, TRAM-34), n = 15 recordings (S24, Senicapoc), n = 10 recordings (BG5, control treatment), n = 7 recordings (BG5, TRAM-34), and n = 11 recordings (BG5, Senicapoc) from 2 biologically independent experiments; Kruskal-Wallis test, Dunn’s test. f—h, n = 5 recordings (S24 KO control, S24 KCa3.1 KO-2), n = 4 recordings (S24 KCa3.1 KO-1), n = 6 recordings (S24 KD control), n = 5 recordings (S24 KCa3.1 KD), and n = 5 recordings (P3 KD control, P3 KCa3.1 KD) from 2 biologically independent experiments per group, respectively. f, One-way ANOVA, Dunnett’s test; g, two-sided t-test; h, two-sided Mann-Whitney test. i, Representative images of cells in adherent and spheroid conditions; in spheroid conditions tumor cells do not form networks (j) and do not display periodic activity (l), resulting in a much lower KCa3.1 expression (k), and therefore also do not show any Ca²⁺ activity (m). j, Number of TMs per cell in adherent versus spheroid conditions; n = 45 cells from 3 recordings from 3 biologically independent experiments (adherent) and n = 1087 cells from 5 recordings from 2 biologically independent experiments (spheroid); two-sided Mann-Whitney test. k, Relative expression of KCa3.1 in adherent versus spheroid conditions as determined via qPCR; n = 2 technical replicates; two-sided t-test. l, Fraction of periodic cells and m, global Ca²⁺ activity in adherent versus spheroid conditions; n = 11 recordings from 3 biologically independent experiments (adherent) and n = 5 recordings from 2 biologically independent experiments (spheroid); S24 line; two-sided t-test. n—q, AlamarBlue proliferation assay demonstrates that specific KCa3.1 inhibition with 1 µM TRAM-34 and 1 µM Senicapoc and genetic knockout of KCa3.1 reduces proliferation in adherent conditions (n,p) but not in spheroid conditions (o,q); S24 line; n = 12 measurements per group from 2 biologically independent experiments; one-way ANOVA, Dunnett’s test. r, Fraction of wild-type (WT) cells of all cells in Ca²⁺ recordings (Ca²⁺) displayed in Fig. 4c–f and Extended Data Fig. 7u,v and in recordings of the EdU proliferation assay (EdU) displayed in Fig. 4g–i and Extended Data Fig. 7w after coculturing S24 wild-type cells with S24 KCa3.1 KO-2 cells; n = 9 recordings (Ca²⁺) and n = 40 recordings (EdU) in 2 biological independent experiments, respectively. s, Ca²⁺ traces from representative recordings of S24 wild-type cells (WT), S24 KCa3.1 KO-2 cells and of a co-culture of 10% S24 wild-type cells with S24 KCa3.1 KO-2 cells (10% WT); traces of wild-type cells are depicted in red, traces of KCa3.1 knockout cells are depicted in green, and traces of cells displaying periodic Ca²⁺ activity are depicted thicker and darker; adding wild-type cells rescues the effect of the KCa3.1 knockout on global Ca²⁺ activity. t, Rarely detected periodic activity in knockout cells (green) after cocultivation with wild-type cells is due to close cupelling with periodically active wild-type cells (red); representative Ca²⁺ traces from s. u—w, Same data as shown in Fig. 4c–i, which originates from joint experiments with multiple experimental groups, and is shown here in one instead of two graphs to allow statistical comparison between the subpopulations (SP) of the co-culture and the control conditions (WT and KCa3.1 KO). Black p-values indicate these comparisons and grey p-values indicate comparisons that are also depicted in the respective main figure (Fig. 4c–i). P-values can differ here due to multiple testing: u, Fraction of periodic cells of all cells, v, global Ca²⁺ activity, and w, fraction of EdU-positive cells in recordings of S24 wild-type cells (WT), of S24 KCa3.1 KO-2 cells, of a co-culture of 10% S24 wild-type cells with S24 KCa3.1 KO-2 cells (10% WT) and of the subpopulations (SP) of S24 wild-type cells and S24 KCa3.1 KO-2 cells in the respective co-culture. u,v, n = 7 recordings (WT), n = 8 recordings (KCa3.1 KO), and n = 9 recordings (Co-culture) in 2 biological independent experiments; one-way ANOVA, Dunnett’s test (u) and Kruskal-Wallis test, Dunn’s test (v). w, n = 20 recordings in 2 biological independent experiments for all groups respectively; Kruskal-Wallis test, Dunn’s test. x, Fraction of wild-type (WT) cells and y, fraction of EdU-positive cells in recordings of S24 wild-type cells (100%), S24 KCa3.1 KO-2 cells (0%) and of a co-culture of 3, 5 and 10% S24 wild-type cells with S24 KCa3.1 KO-2 cells; y, for 0, 3, 5, an 10% WT cells the fraction of proliferating KO cells is shown on the left y-axis, for comparison the fraction of proliferating cells in WT-monoculture (100%) is shown on the right y-axis. While the co-culture of 5% WT cells still significantly increases the proliferation of the KCa3.1 KO, the co-culture of 3% of WT cells does not, placing the lower limit of WT cells to rescue the effects of the KCa3.1 KO somewhere between 3–5%. Kruskal-Wallis test, Dunn’s test. x,y, n = 20 recordings (0%, 10%, 100%), n = 6 recordings (3%), and n = 9 recordings (5%), in 2 biological independent experiments for all groups. Error bars show s.e.m. ns, not significant (P ≥ 0.05).

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