Cell culture and reagents

Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Lonza, Basel, Switzerland) or in Rosewell Park Memorial Institute Medium (Lonza), supplemented with 10% Fetal Bovin Serum (FBS, Lonza). The A-673/TR/shEF cell line, is a stably transduced A-673 cell line with a doxycycline-inducible shRNA targeting EWS::FLI1 [43]. A list of ES cell lines and their fusion type is provided in Table S1. For experiments in high potassium (high K+) condition, the medium was supplemented with 40 mM of KCl (Sigma, St. Quentin Fallavier, France). GW542573X and doxycycline were purchased from Tocris (Bristol, United Kingdom) and Sigma-Aldrich, respectively. All cell lines were subjected to a complete validation process and were authenticated by Short Tandem Repeat (STR) profiling analysis (Eurofins, Germany). Experiments were performed using mycoplasma-free cell lines.

Bioinformatics analyses

The “R2: Genomics Analysis and Visualization Platform” website provides free access datasets from patient biopsies. Multiple datasets were used to compare KCNN1 expression and to study the survival rate of ES patients (Table S2). The GTEx (https://gtexportal.org/home/) and the Cancer Cell Line Encyclopedia (CCLE, https://sites.broadinstitute.org/ccle/) were used to compare KCNN1 expression levels in healthy tissues (GTEx version 8) and tumorous cell lines (CCLE - DepMap 22Q4), respectively.

RNA-seq analyses

RNA-seq was performed for ES and osteosarcoma cells and gene-level quantification was realized as previously described [44].

For transcript-level quantification, RNA-seq experiments for the A-673, EW-3, EW-24, MHH-ES-1, RD-ES, SK-ES-1, and TC-71 cell lines were performed by ActivMotif Inc. Libraries were prepared in triplicate using the Illumina TruSeq Stranded mRNA Sample Preparation Kit. Sequencing was then performed on an Illumina NextSeq 500 in a 2 × 42 bp (PE42) format. RNA-seq data for A-673/TR/SHEF without induction (day 0) and with induction of the shRNA targeting EWS::FLI1 (day 7) came from Gene Expression Omnibus (GEO) GSE164373 [45], also in triplicates for each condition. These data were filtered and aligned to the same reference genome, and quantified based on the annotation file from the RefSeq associated with that genome. Packages used for the analyses have been detailed in Table S3.

ChIP-seq analyses

ChIP-Seq data were derived from the GSE176400 [46], GSE129155 [47], and GSE94278 [34] entries in the GEO database. GSE176400 contains ChIP-seq data from 14 ES lines with inducible fusion-type specific EWS::ETS knockdown. GSE129155 contains ChIP-seq data for inducible EWS::FLI1 knockdown in the cell line A673/TR/shEF. GSE94278 contains Bigwig files of normalized ChIP-Seq values of MSCs transfected with a control plasmid, a FLI1 plasmid, and an EWS::FLI1 plasmid. These data were filtered and aligned to the GRCh38.p14 reference genome analysis set from NCBI. Packages used for the analyses have been detailed in Table S4.

Patients data and paired slide immunohistochemistry

A cohort of 18 ES diagnostic tumors were collected retrospectively in University Hospitals of Strasbourg between 2005 and 2022. Those patients were treated in or according to the past Euro-E.W.I.N.G.99 and EURO EWING 2012 protocols [48, 49]. The research protocol was validated by the local institutional ethic committee at University Hospitals of Strasbourg for human tissue experiments and a CNIL declaration 1970 390 v0 was obtained. All patients and their parents gave their written informed consent. The diagnostic anonymized paraffin-embedded (PE) tumor samples were stored in the pediatric tumor bank at the Biological Resource Centre of Strasbourg. 3-µm sections of those ES PE tumor tissues were cut and stained for SK1 with anti-human SK1 antibody (Genetex, Irvin, CA, USA). After baking slides for 1 h at 60 °C, samples were processed routinely via deparaffinization, rehydration, antigen retrieval in Tris-EDTA buffer pH9 for 20 min at 97 °C. The endogenous peroxidase activity was then quenched, and non-specific binding was subsequently blocked with 2% (v/v) normal donkey serum (Jackson ImmunoResearch) and 1% (p/v) B.S.A. in 1× TBS Tween pH 7.4 for 25 min at room temperature. Samples were then incubated with appropriate antibody diluted at 1:50 in blocking solution for 1 h at room temperature. Negative control was achieved by omitting a primary antibody. Following the primary antibody incubation, sections were washed in 1× TBS Tween pH 7.4 and samples were incubated for 45 min at room temperature using a polyclonal biotinylated donkey anti-rabbit secondary antibody (Jackson ImmunoResearch), and finally with an HRP conjugated streptavidin complex (Agilent) for another 45 min at room temperature. Revelation was conducted with DAB liquid chromogen (microm-microtech France) and counterstained with Gill-2, dehydrated through graded ethanol baths, cleared in OTTIX-plus and cover-slipped with Pertex mounting media.

Sections were analyzed using standard light microscopy. Each sample was examined twice by two experimentators blinded to the experiment. The expression and distribution of SK1 were assessed within the sections. The intensity of staining was assessed semi-quantitatively in reference to the most intensely stained slide as follows: no expression, high expression, and intermediate expression qualified as moderate.

RT-qPCR (Reverse Transcription – quantitative Polymerase Chain Reaction)

RNA was extracted using the NucleoSpin RNA Plus Kit (Macherey-Nagel, Duren, Germany). Reverse Transcription (RT) was performed from 1 µg of RNA with the “Maxima H Minus First Strand cDNA Synthesis Kit” (Macherey-Nagel). Quantitative Polymerase Chain Reaction was performed using SYBR Select Master Mix (ThermoFischer) and the primers listed in Table 1.

Table 1 Primers used for qPCR experiments.Western blot

Protein extraction and Western blots were performed as previously described [44]. The antibodies used targeted Aurora A (Cell Signaling Technology (CST), Danvers, MA, USA), p21 (CST), SK1 (Genetex, Irvin, CA, USA), FLI1 (Abcam, Cambridge, UK), β-actin (CST) and β-tubulin (CST). Antibody binding was visualized with a fluorescence system or an enhanced chemiluminescence system (SuperSignal West Dura Extended Duration Substrate, ThermoSientific, Illkirch, France).

Transienty siRNA transfection

ES cells were transfected with a pool of five siRNAs targeting EWS::FLI1 (Dharmacon, Horizon, Perkin-Elmer, Villebon-sur-Yvette, France) or KCNN1 (SantaCruz, Dallas, TX, USA) or with a pool of five control siRNAs using Lipofectamine RNAiMax (ThermoFischer). RNAs and proteins were respectively harvested after 48 h and 72 h post transfection, respectively.

shRNA stable transduction

The A-673 and SK-ES-1 cell lines were transduced with three doxycycline-inducible shRNAs targeting KCNN1 (Dharmacon) or a doxycycline-inducible non-targeting control shRNA (Dharmacon). The expression of KCNN1 was measured by RT-qPCR after 72 h or 7 days of doxycycline treatment (Sigma-Aldrich). Before each experiment, cells were pre-treated with doxycycline for 72 h or 7 days and the treatment was maintained afterwards.

Membrane depolarization measurements

Extemporaneously, 0.18% glucose was added to Physiological Salin Solution (PSS, 137 mM NaCl, 5.6 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 0.42 mM Na2HPO4 (anhydrous), 0.44 mM Na2HPO4 (hydrated), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 4.17 mM NaHCO3, pH 7.4). DiBAC4(3) stock solution was prepared following Thermofischer recommendations. Adherent cells were loaded for 15 min at 37 °C with the 1 µM DiBAC4(3) in PSS. Fluorescence was measured at 520 nm using a FlexStation Microplate Reader (Molecular Devices, San Jose, CA, USA), in response to an excitation wavelength of 485 nm. Then, a second fluorescence measurement was taken, after a 10 min incubation of the cells with 100 µL of 1 µM DiBAC4(3).

For membrane depolarization measurements in 3D, spheroids were formed through use of the scaffold-free method cell suspensions. Cell suspensions (100 μl of cell suspension at 4.104 cells/ml) were added to Ultra-Low Attachment microplates (Corning, Glendale, AZ, USA) and the plates were incubated in standard, stationary cell culture conditions for 72 h. Spheroids membrane potentials were measured as described previously [50].

Clonogenicity assays

Five hundred A-673 cells were incubated for 8–10 days after pre-treatment under standard conditions. Cells were fixed with 1% glutaraldehyde and stained by crystal violet. The number of colonies was then counted with a Cell Counter from Fiji Software and their surface was measured using a macro created by the Cellular and Tissular Imaging Core Facility of Nantes University (MicroPICell, Nantes, France).

Viability assays

After siRNA transfection, viability assays were performed on re-seeded cells, using an Omni device from CytoSMART (Axion Biosystems, Atlanta, GA, USA). Cell viability was monitored for 72 h in time-lapse, with a scan every 4 h. Data were collected on the CytoSMART Cloud, and the analyses were performed using the same software.

TranswellTM motility

Fourty-eight hours after siRNA transfection, 60,000 cells/well were seeded onto the upper surface of transwell inserts (Falcon, Franklin Lakes, NJ) and incubated at 37 °C for 8 h. At the end of the incubation period, cells on the upper surface of the inserts were wiped off, and the cells on the underside of the membrane were fixed, stained with crystal violet and counted by bright-field microscopy in five random fields.

Thymidine synchronization

After their adhesion, cells were treated with 2 mM thymidine (Sigma-Aldrich) for 18 h and left in DMEM containing 10% FBS for 8 h. The cells were then treated for a second time with 2 mM thymidine for 16 h before being left in DMEM containing 10% FBS. Cell distribution was observed by flow cytometry on the Platform Cytocell at Nantes University (FACSymphony A5, BD, Franklin Lakes, USA), after 0, 6, 8, and 10 h or not synchronized. Proteins were also extracted for western-blot analyses.

Cell cycle analyses

After thymidine synchronization, cells were harvested and fixed with 70% ethanol before being suspended in a phospho-citrate buffer (Na2HPO4 0.2 M, citric acid C6H8O7 0.1 M, pH 7.5). Cells were then resuspended in PBS, 0.12% Triton, 0.12 mM EDTA and 100 µg/mL RNAse A (Promega A797c, Promega) and incubated for 30 min at 37 °C. Propidium Iodide (50 µg/mL) was added for 20 min in the dark at 4 °C. Cell distribution was observed by flow cytometry on the Cytocell Platform at Nantes University (FACSymphony A5). Data were analyzed with Multicycle software.

Store operated Ca2+ entry measurement by Fura-2 AM

As previously described [51], SOCE was measured after Fura-2-AM loading of cells for 45 min at 37 °C. Maximum of fluorescence (peak of Ca2+ influx F340/F380) was measured and compared to normal condition.

Constitutive Ca2+ entry measurement by Mn2+ quenching

As previously described [51], CCE was measured by manganese (Mn2+) (Manganese chloride, Sigma) quenching after loading cells with Fura-2-AM for 45 min at 37 °C.

Statistical analyses

Statistical analyses were performed using GraphPad Prism Software. Comparison of SKCa (KCNN1, KCNN2, and KCNN3) gene expression in patients suffering from ES was performed by a Brown–Forsythe and Welch ANOVA and a Games–Howell multiple comparisons tests. The comparison of KCNN1 gene expression between ES patients and MSCs coming from healthy donors was performed by an unpaired t-test and a Welch correction. The comparison of KCNN1 gene expression among multiple tumors was performed by a Kruskal–Wallis test and a Dunn’s multiple comparisons test. To compare two groups from in vitro experiments, Mann–Whitney tests were performed and ANOVA tests were administered when N > 30 and a normality test had been passed. Wilcoxon tests were used during cell cycle assays. α = 0.05 and p < 0.05 is considered statistically significant: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.