Expression of transient receptor potential melastatin‐4 (TRPM4) in prostate cancer (PCa) patient samples and migration and cell death in 2D cell assays. (A) Volcano plot of gene expression in matched malignant and nonmalignant prostate tissue samples from 15 PCa patients (GSE69223, mRNA microarray). (B) Relative expression of TRPM4 from data in (A). Differences between samples were analyzed using a two‐tailed paired t‐test. (C) Statistical analysis of cell migration of DU145 cells and corresponding TRPM4 knockout (KO) clones in FluoroBlok Boyden chambers 72 h postseeding (n = 4). (D) Cell death was quantified as the relative intensity of CellTox Green staining in DU145 cells and corresponding TRPM4 KO clones on day 3 postseeding (n = 4). All individual values are plotted as dots in the graph. The median is shown as a red line, whiskers represent the interquartile range. Significance levels for data from both clones in C and D are given relative to DU145 cells. Differences between samples were analyzed using one‐way ANOVA followed by Dunnett's post‐hoc test.

Transient receptor potential melastatin‐4 (TRPM4) knockout (KO) affects tumor spheroid formation, size and adhesion. (A) Representative microscopic images of tumor spheroids from DU145 cells and corresponding TRPM4 KO clones (n = 4). Scale bar indicates 100 μm. (B) Development of spheroid size over time was quantified by measuring the area of the core spheroid at the largest diameter (n = 4). (C) Statistical analysis of spheroid size on days 3, 5, and 10 postseeding. All individual values are plotted as dots in the graph. The median is shown as a red line, whiskers represent the interquartile range. Significance levels for data from both clones in C are given relative to DU145 cells. Differences between samples were analyzed using one‐way ANOVA followed by Dunnett's post‐hoc test.

Transient receptor potential melastatin‐4 (TRPM4) knockout (KO) reduces tumor spheroid outgrowth and increases cell death in tumor spheroids. (A) Representative microscopic images of tumor spheroid outgrowth from DU145 cells and corresponding TRPM4 KO clones (n = 4). Scale bar indicates 250 μm. (B) Spheroid outgrowth was quantified as the ratio of outgrowth area to core spheroid size at 48 and 72 h. All individual values are plotted as dots in the graph. The median is shown as a red line, whiskers represent the interquartile range. (C) Cell death was quantified as the relative intensity of CellTox Green staining in tumor spheroids from DU145 cells and corresponding TRPM4 KO clones on days 3, 5, and 10 postseeding (n = 4). All individual values are plotted as dots in the graph. The median is shown as a red line, whiskers represent the interquartile range. Significance levels for data from both clones in B and C are given relative to DU145 cells. Differences between samples were analyzed using one‐way ANOVA followed by Dunnett's post‐hoc test. (D) Representative fluorescence microscopic image of a tumor spheroid from DU145 TRPM4 KO2 cells on day 5 postseeding (n = 4). Live cells were stained with calcein‐AM (green), dead cells with propidium iodide (red), and the nuclei of all cells with Hoechst 33342 (blue). Scale bar indicates 100 μm.

Transient receptor potential melastatin‐4 (TRPM4) knockout (KO) reduces extravasation and metastatic burden of PCa cells in zebrafish xenograft model. (A) Representative stereomicroscopic fluorescence images of metastatic colonization of 2 day postfertilization (dpf) zebrafish larvae with endothelial reporter tg(Fli:GFP), after injection of DU145 cells (n = 50) and corresponding TRPM4 KO clones (both n = 40) stably expressing tdTomato. Scale bar indicates 50 μm. (B) Tumor foci (left), average foci size (middle) and overall tumor burden (right) were measured at day 3 postinjection. Foci number and size were determined by automatic segmentation of the tdTomato fluorescence intensity within the tail of the injected zebrafish larvae. Tumor burden was quantified as average tdTomato fluorescence intensity within the same region. Fifty larvae injected with DU145 cells and 40 larvae injected with each of the corresponding TRPM4 KO clones were analyzed. All individual values are plotted as dots in the graph. The median is shown as a red line, whiskers represent the interquartile range. Significance levels for data from both clones in B are given relative to DU145 cells. Differences between samples were analyzed using one‐way ANOVA followed by Dunnett's post‐hoc test.