All animal experiments were approved by the University of Cincinnati Ethics Committee and the Institutional Animal Care and Use Committee.

All experiments were performed according to the FELASA regulations and we also followed the ARRIVE guidelines.

Eight- to 12-week-old, wild-type C57BL/6 J mice were employed. Pancreas cancer was established in the peritoneum by intraperitoneal injection of 50,000 KPC cells resuspended in PBS. Mice were treated i.v. 6 times starting at day 3 and then every 2nd day with 4 nmol/g PAPTP + 50 mg/kg ABC294640. PAPTP was injected i.v. in a total volume of 125 µL DMSO and 0.9% NaCl (1:4, v/v); ABC294640 was injected i.p. Controls were injected with the solvent only. Survival was determined over 65 days.

Tumor-injected mice were randomly divided into experimental groups.

Cell culture and treatment

Human PDAC cell lines BxPc3, Panc-1, and MIA Paca-2 were obtained from the pHionic consortium. KPC tumor cells were originally generated from KRASLSL−G12D/+, Trp53loxP/+, and Pdx1-Cre (KPC) mice in C57BL/6 mice background (Jackson Laboratories). Rodent pathogen status of the tumor cell lines was originally verified by Charles River Laboratory diagnosis services.

All cells were cultured in modified Eagle medium (MEM) supplemented with 10 mM HEPES (pH 7.4; Carl Roth GmbH, Karlsruhe, Germany), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 µM nonessential amino acids, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% fetal calf serum (MEM/K10).

If indicated, cells were treated with 10 µM XY-14 (Echelon, #L9218) for 30 min prior to the addition of PAPTP.

Transfections

Panc-1 and MIA Paca-2 cells expressing wild-type or oncogenic RAS were transfected with shRNA targeting KRAS or sphingosine kinase 2 (all from Santa Cruz Inc., #sc-35731-SH for KRAS, sc-39225-SH for sphingosine kinase 2). Cells were stably transfected by electroporation.

Kv1.3 was downregulated by transient transfection using shRNA targeting Kv1.3 (Santa Cruz Inc., #sc-42712-SH) using a BTX electroporator. Cells were employed 48 h after transient transfection.

For transfections, cells were trypsinized and washed 3 times in HEPES/saline (H/S; 132 mM NaCl, 20 mM HEPES [pH 7.4], 5 mM KCl, 1 mM CaCl2, 0.7 mM MgCl2, 0.8 mM MgSO4). Cells were then resuspended in H/S at 20 × 106 cells/400 µL, aliquoted into electroporation cuvettes, the plasmids were added, and samples were incubated on ice for 15 min and electroporated at 500 V with 5 pulses each for 1 ms. Cells were incubated for 15 min on ice and finally resuspended in MEM/K10. Transfected cells were then selected by incubation with 3.5 µg/mL puromycin for 3–4 weeks. We used bulk cultures in the present experiments to avoid any clonal artifacts.

Reactive oxygen species (ROS)

Cells were grown in MEM/K10 as above, removed from the plates using cell dissociation buffer (ThermoFisher, #13,151,014), washed in PBS, incubated with 2.5 µM mitoSOX Red (ThermoFisher, # M36008) for 20 min at 37 °C, and stimulated with 0.25 or 1 µM PAPTP or left untreated. Cells were then analyzed by flow cytometry employing a FACSCalibur.

Cell death

Cells were treated as indicated, trypsinized, washed in H/S, resuspended in a Ca2+-containing staining buffer as provided by the vendor, and stained for 15 min with FITC-Annexin V (1:1000, Roche). Samples were analyzed by flow cytometry on a FACSCalibur. Positive controls were permeabilized for 5 min with 0.1% Triton X-100 at room temperature before incubation with FITC-Annexin V.

Protein measurements

Protein concentrations were measured employing the BioRad Protein Assay Dye (cat. no. #500,006) and served to normalize the samples.

Sphingosine measurements

Cells were grown in 24-well plates overnight, washed, and stimulated with 1 µM PAPTP for 30 min in H/S or left untreated, the medium was removed, and cells were washed once with cold H/S and shock-frozen in liquid nitrogen. Cells were then lysed in 250 µL 0.5% NP40 in H2O and 200 µL was extracted in 600 µL CHCl3:CH3OH:1N HCl (100:100:1, v/v/v). Phases were separated, and an aliquot of the lower phase was dried in a Speedvac and resuspended in 20 µL of a detergent solution (7.5% [w/v] n-octyl glucopyranoside, 5 mM cardiolipin in 1 mM diethylenetriaminepentaacetic acid [DTPA]). The samples were sonicated in a bath sonicator for 10 min and the kinase reaction was performed by the addition of 80 µL of 0.001 units sphingosine kinase in 50 mM HEPES (pH 7.4), 250 mM NaCl, 30 mM MgCl2 1 mM ATP, and 10 µCi [32P]γATP. The kinase reaction was performed for 30 min and terminated by extraction of the samples in 20 µL 1N HCl followed by the addition of 800 µL CHCl3:CH3OH:1N HCl (100:200:1, v/v/v), and 240 µL each of CHCl3 and 2 M KCl. Samples were vortexed between additions. Phases were separated, and the lower phase was collected, dried, dissolved in 20 µL CHCl3:CH3OH (1:1, v/v), and separated on Silica G60 TLC plates with CHCl3:CH3OH:acetic acid:H2O (90:90:15:5, v/v/v/v) as developing solvent. The TLC plates were analyzed with a phosphorimager. Sphingosine levels were determined with a standard curve of C18-sphingosine.

Ceramide measurements

Cells were treated and extracted as described for sphingosine. The dried samples were resuspended in 20 µL of a detergent solution (7.5% [w/v] n-octyl glucopyranoside and 5 mM cardiolipin in 1 mM diethylenetriamine-pentaacetic acid [DTPA]), and micelles were obtained by bath sonication for 10 min. The kinase reaction was initiated by the addition of 70 µL of a reaction mixture containing 10 µL diacylglycerol (DAG) kinase (GE Healthcare Europe, Munich, Germany), 0.1 M imidazole/HCl (pH 6.6), 0.2 mM DTPA, 70 mM NaCl, 17 mM MgCl2, 1.4 mM ethylene glycol tetraacetic acid, 2 mM dithiothreitol, 1 µM adenosine triphosphate (ATP), and 5 µCi [32P]γATP. The kinase reaction was performed for 30 min at room temperature under shaking at 300 rpm, terminated by the addition of 1 mL CHCl3:CH3OH:1N HCl (100:100:1, v/v/v), 170 µL buffered saline solution (135 mM NaCl, 1.5 mM CaCl2, 0.5 mM MgCl2, 5.6 mM glucose, and 10 mM HEPES; pH 7.2), and 30 µL of a 100 mM ethylenediaminetetraacetic acid (EDTA) solution. The samples were vortexed, phases were separated, and the lower phase was collected, dried, dissolved in 20 µL CHCl3:CH3OH (1/1, v/v), separated on Silica G60 thin-layer chromatography (TLC) plates using chloroform/acetone/methanol/acetic acid/H2O (50:20:15:10:5, v/v/v/v/v) as solvent, and developed employing a Fuji phosphorimager. Ceramide levels were determined by comparison with a standard curve; C16 to C24 ceramides were used as substrates.

Sphingosine-1-phosphate-phosphatase activity

Cells were stimulated with 1 µM PAPTP for 30 min or left untreated, and mitochondria were prepared as above and incubated with 10 µM [3H]S1P complexed with fatty acid–free BSA for 30 min at 37 °C. The samples were organically extracted as described above for sphingosine measurements, separated by TLC, and consumption of S1P to sphingosine was determined.

Sphingosine kinase activity

Cells were stimulated with 1 µM PAPTP for 30 min or left untreated; mitochondria were prepared as above and resuspended after purification in a buffer consisting of 50 mM HEPES, pH 7.4, 150 mM NaCl, 30 mM MgCl2 in the presence of 500 pmol sphingosine (i.e., excess substrate), 10 µM ATP, and 10 µCi[32P]γATP. The kinase reaction was allowed to proceed for 60 min at 30 °C and stopped by the addition of 20 µL 1N HCl followed by the addition of 800 µL CHCl3:CH3OH:1N HCl (100:200:1, v/v/v), and 240 µL each of CHCl3 and 2 M KCl. Samples were then processed as described above for sphingosine measurements.

Measurement of cardiolipin binding to sphingosine

Cells were treated with 1 µM PAPTP for 30 min and lysed in 125 mM NaCl, 25 mM Tris HCl (pH 7.4), 10 mM EDTA, 10 mM sodium pyrophosphate, 2% Nonidet P40, and 10 µg/mL aprotinin and leupeptin for 10 min on ice. Sphingosine was immunoprecipitated from the lysates using a monoclonal anti-sphingosine antibody (clone NHSPH, Alfresa Pharma Corporation, Japan) for 60 min at 4 °C. Immunocomplexes were immobilized by incubation with protein A/G-coupled agarose for 45 min at 4 °C. The precipitates were extensively washed 6 times in H/S and extracted in CHCl3/CH3OH/1N HCl (100:100:1, v/v/v). Samples were dried and then resuspended in 10 µM of a cardiolipin probe (Abcam, ab241036), a fluorescent probe for the detection and quantification of cardiolipin. The samples were incubated for 5 min, 250 rpm shaking and the fluorescence was measured using a fluorescence reader. Cardiolipin concentration was determined using a standard curve of cardiolipin.

Incubation of isolated mitochondria with sphingosine and measurement of mitochondrial membrane potential in vitro

Mitochondria were isolated as above, incubated with 2.5 nM TMRM (ThermoFisher M20036) for 5 min at 37 °C and incubated with 100 nM sphingosine (Avanti Polar Lipids, #860,490) or 100 nM PAPTP. If indicated, we added 10 µM Cardiolipin (Merck, #C0563). Samples were analyzed by flow cytometry using a FACSCalibur. The results were analyzed for the mean fluorescence using the BD software.

Flow cytometry

Cells were treated with sphingosine, resuspended in buffer A consisting of 0.3 M sucrose, 10 mM TES (pH 7.4), and 0.5 mM EGTA, and incubated for 30 min on ice. Cells were then pottered in a tight glass potter with 40 strokes, centrifuged at 600 × g for 5 min at 4 °C, and the supernatants were collected. The supernatants were centrifuged at 6000 × g for 10 min at 4 °C, the pellets were resuspended in buffer B consisting of 50 mM Pipes-KOH (pH 7.4), 50 mM KCl, 2 mM MgCl2, 2 mM EGTA, 10 µg/mL A/L, 2 mM ATP, 10 mM phosphocreatine, 5 mM succinate, and 50 µg/mL creatine kinase, and samples were stained for 30 min with anti-TIM23 (1:250 dilution; BD, #611,222) and anti-sphingosine antibodies (1:1000 dilution, Alfresa, clone NHSPH) or anti-Kv1.3 antibodies (1:250, Alamone, #APC-101), respectively. Samples were washed and stained with Cy3-coupled donkey anti-mouse IgG F(ab)2 fragments (1:500 dilution; Jackson Immunoresearch, #715–166-150) for the mouse anti-TIM23 IgG + Alexa Fluor 647-coupled donkey anti-mouse IgM F(ab)2 fragments (1:500 dilution; Jackson Immunoresearch, #715–606-020) for the IgM anti-sphingosine or APC-coupled donkey anti-rabbit IgG F(ab)2 fragments (1:500 dilution; Jackson Immunoresearch, #711–136-152) for the rabbit anti-Kv1.3 IgG. Samples were washed again and analyzed by flow cytometry using a FACSCalibur employing FL2 vs. FL4. The mean fluorescence was analyzed using BD software.

Statistics

Data are expressed as arithmetic means ± SD. For the comparison of continuous variables from independent groups with one variable, we used one-way ANOVA followed by post hoc Tukey test for all pairwise comparisons, applying the Bonferroni correction for multiple testing. The P values for the pairwise comparisons were calculated after the Bonferroni correction. All values were tested for normal distribution using the Kolmogorov–Smirnov test. Statistical significance was set at a P value of 0.05 or lower (two-tailed). Outliers were not removed and all data are provided. The sample size planning was based on the results of two-sided Wilcoxon-Mann–Whitney tests (free software: G*Power, Version 3.1.7, University of Duesseldorf, Germany). Investigators were blinded to the samples in microscopic studies and to animal identity. Animals were randomly assigned to cages by a technician who was not involved in the experiments; thus, the mice were purely randomly assigned for every experiment. Cages were then randomly assigned to the various experimental groups.