Cell culture

Primary CAFs and NPFs cultures were isolated from surgical specimens of three PCa-bearing patients (Gleason score 4 + 5) who underwent radical prostatectomy. Specimens were collected upon informed consent, and the study was approved by the Ethics committee of IRCCS Istituto Nazionale dei Tumori of Milano (INT n. 154/16). Intra-tumor areas or non-tumor regions of radical prostatectomy specimens were identified by an expert uro-pathologist (M.C.), selected and digested overnight at 37 °C and 5% CO2 in DMEM medium (Lonza, Basel, Switzerland) supplemented with 300 units/ml collagenase and 100 units/ml hyaluronidase solution (Stemcell Technologies Vancouver, Canada), 1% penicillin–streptomycin (Lonza, Basel, Switzerland) and 2.5 μg/ml of Amphotericin B. The cell suspension was centrifuged at 1,500 × g for 5 min. The resulting fibroblast-rich pellet was suspended and plated in DMEM medium (Lonza) containing 10% FBS (Thermo Fisher Scientific Inc., Waltham, MA, US), 4 mM L-glutamine and 1% penicillin–streptomycin (Lonza). CAFs or NPFs were maintained in culture for 3 passages and the absence of epithelial markers expression was verified before being used in the experiments. All established primary cultures negative for epithelial markers and expressing fibroblast markers were used until the 15th passage and maintained in DMEM medium (Lonza) containing 10% FBS (Thermo Fisher Scientific Inc.) and 4 mM L-glutamine (Lonza). PCa cell lines (DU145, PC3 and LNCaP) and the prostate myofibroblast cell line WPMY-1 were purchased from American Type Tissue Culture Collection (ATCC, VA, USA). PCa cell lines were maintained in RPMI-1640 medium (Lonza) supplemented with 10% FBS (Thermo Fisher Scientific), at 37 °C and 5% CO2. WPMY-1 cells were maintained in DMEM (Lonza) supplemented with 10% FBS (Gibco, Thermo Fisher Scientific Inc.). All the cell lines were authenticated and periodically monitored by genetic profiling using short tandem repeat analysis AmpFISTR Identifier PCR amplification kit (Thermo Fisher Scientific Inc.).

Conditioned medium

For indirect co-culture and in vivo experiments, conditioned medium (CM) was collected from NPFs, antiarrhythmic-treated or untreated CAFs and DU145 cells. To obtain CM, a total of 7 × 105 cells were seeded in T-75 cm2 culture flask. CAFs were treated with 2.5 μM of amiodarone (Hikma Pharmaceuticals, London, UK), 2.5 μM of verapamil (Abbott Laboratories, Chicago, US), 2.5 μM of nifedipine (Meda AB, Solna, Sweden) or 2.5 μM flecainide (Meda AB) for 24 h. Upon treatment, the culture medium was removed, cells were washed 2 times with PBS (Lonza) and 6 ml of serum-free medium was added for starvation. Twenty-four hours later, the CM was collected, clarified for 5 min at 1,500 × g and used freshly to treat PCa cells (DU145, PC-3 or LNCaP), or to activate NPFs or WPMY-1 or concentrated for western blotting analysis.

Electrophysiological recordings and analyses

Whole cell patch-clamp recordings were performed on activated and control WPMY-1 fibroblasts using an Axopatch 200B amplifier (Molecular Devices) and data were sampled with a Digidata -1440 (Molecular Devices) interface (sampling time = 250 ms for voltage clamp recordings). Patch pipettes were pulled from borosilicate capillaries (Hingelberg, Malsfeld, Germany) and had 3–5 MΩ resistance before a seal was formed. Cells were recorded in a bath solution containing (in mM): NaCl 140, KCl 5, Hepes 10, glucose 5, CaCl2 2, MgCl2 1, pH 7.4. The filling solution contained (in mM): KCl 135, NaCl 10, Hepes 10, MgCl2 1, EGTA 1, CaCl2 0.1 and GTP 0.1. The pH was adjusted to 7.2 with KOH. Membrane passive properties were recorded in voltage clamp configuration. A hyperpolarizing step from -70 mV to -80 mV, normally used for evaluating the passive properties of the recorded cell [23], elicited an inward transient current used to estimate the series resistance, input resistance and membrane capacitance. Series resistance and input resistance were monitored throughout each experiment. Cells were rejected if these parameters deviated by more than 20% from the beginning of the recording. Outward currents were measured in voltage clamp by applying subsequent voltage steps of + 20 mV from a holding potential of -60 mV up to + 140 mV. Sustained outward currents (K+ steady) were recorded as an average of the last 15 ms of each voltage step. Transient outward currents (K + inactivated) were calculated by subtracting the sustained outward current from peak outward currents. The amplitude of the transient currents measured at the membrane potential of + 140 mV was used to report the current density. Transient inward currents (inward) were calculated as the peak outward current for each applied voltage step. For the depolarizing protocols, the PN leak subtraction of the Clampex program was used to eliminate the effects of the leakage current on the whole-cell responses [24]. The extracellular medium containing flecainide or nifedipine was bath perfused using a peristaltic pump. Pipette capacitance was compensated, and the bridge was balanced during each recording. All data were reported as the mean ± standard error of the mean (SEM). Each protocol was averaged digitally 5 times before being analyzed. All the recorded data were analyzed off-line with pCLAMP10.7 (Axon Instruments).

Cell growth assay

PCa cells (DU145, PC-3 or LNCaP) were seeded in 12-well plates (2 × 104 cells/well) and after 24 h were exposed to CM of NPFs, CM of CAFs or CM of CAF-treated with antiarrhythmics (as described above). Upon starvation with appropriate CM, cells were harvested with Trypsin–EDTA (Lonza) and counted with an automated cell counter (Beckman, Coulter, Brea, CA, US).

Migration assay

Cell were seeded at 4 × 104 cells/well into a 12-well culture plate. After 2 days, the monolayer of cells was wounded by manual scratching with a pipet tip, washed with PBS 1 × (Lonza), photographed (t0 point) and media were replaced with serum-free DMEM containing 2.5 μM of amiodarone (Hikma Pharmaceuticals), 2.5 μM of verapamil (Abbott Laboratories), 2.5 μM nifedipine (Meda AB), or 2.5 μM flecainide (Meda AB) for CAF migration experiments experiments or with CM of CAFs treated or not with antiarrhythmics (as described above) for DU145 cell migration experiments. Images of cell movement were captured at regular time intervals until 48 h by using EVOS XL – Core microscope system (Thermo Fischer Scientific Inc.).

3D gel collagen contraction assay

Type I collagen from rat-tail (Sigma-Aldrich, St. Louis, MO, US) was dissolved at 2 mg/ml in 0.1% acetic acid to create a stock solution. The collagen matrix was quickly prepared on ice by adding 6 ml of collagen stock solution to 3.6 ml of 0.1% acetic acid, 1.2 ml of 10 × concentrated DMEM, and 1.2 ml of sodium bicarbonate solution (11.76 mg/ml) for a final concentration of 1 mg/ml collagen. The pH was adjusted to 7.2–7.4 by adding 0.1 mol/l NaOH. CAFs or NPFs cells were then added to achieve a final concentration of 5 × 105 cells/ml; gel-cells suspension was aliquoted into each well of a 24-well culture plate. After polymerization for 30 min at 37 °C, the gel in each well was overlayed with 500 μl of complete growth medium. Twenty-four hours later, CAFs were treated with 2.5 μM amiodarone (Hikma Pharmaceuticals), 2.5 μM verapamil (Abbott Laboratories), 2.5 μM nifedipine (Meda AB) or 2.5 μM flecainide (Meda AB), in serum free medium. Then, the gels were mechanically released from the wall and bottom of the wells with a sterile spatula. Gel contraction was monitored for 48 h and scanned by standardized photography at time 0 and at sequential time points.

Gene expression profile

After RNA quality check, transcriptomic profiles of CAFs, treated or not with nifedipine (2.5 µM) or flecainide (2.5 µM), and NPFs were assessed using the Clariom™ S Human Microarray (Thermo Fisher Scientific Inc.). Raw data were normalized according to the Robust multiarray averaging (RMA) algorithm, implemented into the oligo package [25]. Normalized data were filtered removing probes with no associated official gene symbol; for probes mapping on the same gene symbol, the one with highest variance was selected. In addition, an empirical Bayes approach was applied to adjust gene expression for batch effect, using ComBat function implemented into the sva package [26]. To verify the efficacy of these analyses, the t-distributed Stochastic Neighbor Embedded (t-SNE) statistical method was employed to visualized data in a low-dimensional space since it adopts a non-linear reduction of high-dimensional transcriptomic data maintaining the similarity among samples. Differential expression analysis was performed applying a linear model implemented into the limma package [27]. A pre-ranked Gene Set Enrichment Analysis (GSEA) was performed on gene sets of the Molecular Signature Database (MSigDB), selecting Reactome pathway in the C2 collection [28]. Ranking was defined according to the t-statistic and normalized enrichment score (NES) was calculated using the functions implemented into the fgsea package [29].

Three different normalized datasets (GSE68164, GSE85606, and GSE86256) were retrieved from the Gene Expression Omnibus database [30] and for each, a pre-ranked GSEA analysis was conducted on custom selected gene sets of MSigDB, using “ion channel activity” as query in the C5 collection and using the t-statistics, obtained by linear model implemented into the limma package, as the measure for ranking gene expression in the comparison CAF vs NPF samples. A FDR threshold of 0.05 was applied to assess significant enrichments.

Cell viability

The cytotoxic effect of amiodarone (Hikma Pharmaceuticals), verapamil (Abbott Laboratories), nifedipine (Meda AB) or flecainide (Meda AB) was determined by the CellTiter96® AQueous One Solution Cell Proliferation Assay (MTS) (Promega Corporation, Madison Wisconsin, USA). Cells were plated for 24 h in 96-well flat-bottomed microtiter plates at a density of 1.5 × 103/50 μl, and then treated with increasing concentrations of amiodarone (Hikma Pharmaceuticals), verapamil (Abbott Laboratories), nifedipine (Meda AB) or flecainide (Meda AB) (0–5 µM) for 72 h. At the end of the treatment, MTS solution was added to each well and the plate was incubated for 3 h in a 5% CO2 incubator at 37 °C. The absorbance at 490 nm was recorded using the POLARstar optima plate-reader (VWR International, Radnor, Pennsylvania, USA).

Immunofluorescence

CAFs were seeded at 6 × 104 cell/well in a 6-well plate containing a coverslip suitable for microscopy. After 24 h, CAFs were treated with 2.5 μM amiodarone (Hikma Pharmaceuticals), 2.5 μM verapamil (Abbott Laboratories), 2.5 μM nifedipine (Meda AB) or 2.5 μM flecainide (Meda AB), in serum free medium. Twenty-four hours later cells were fixed in 4% formaldehyde dissolved in PBS for 10 min. Cells were permeabilized with cold 70% of ethanol and probed with primary antibodies for α-SMA (1:200, A2547 Sigma-Aldrich), Collagen I (1:200 ab34710; Abcam) and phospho-FAK (1:200 Y397 ab4803; Abcam) diluted in antibody diluent (S080983, Dako, Agilent Technologies) for 1 h at room temperature. Alexa Fluor594/488-labeled secondary antibody (Thermo Fisher Scientific Inc.) was used to incubate cells for 1 h at room temperature. Actin filaments were stained using phalloidin-conjugate-Fluor488 dye and nuclei were stained with DAPI (Invitrogen, Thermo Fisher Scientific Inc.). Images were acquired by Nikon Eclipse E600 microscope using ACT-1 software (Nikon, Minato City, Tokyo, Japan).

Proteome profiler array

Proteome profiling was performed using Proteome Profiler Human Cytokine Array Kit (ARY005B, R&D, Minneapolis, MN, US) according to the manufacturer’s instructions. The array was performed on 500 μl of fivefold concentrated CM from NPFs, CAFs treated or not with nifedipine or flecainide. Chemiluminescence signals were detected using Chemi Reagent Mix provided by the kit. Semi-quantitative analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

In vivo experiments

Animal studies were performed in accordance with guidelines of animal care protocols approved by Ethical Committee for animal experimentation of IRCCS Istituto Nazionale dei Tumori of Milano and Italian Ministry of Health (approval code n. 350/2017-PR). Male SCID mice were purchased from Charles River Laboratories. PCa xenografts were generated by subcutaneous injection of 1 × 107 DU145 cells into the right flank of SCID mice. When tumor burden reached ~ 100 mm3, mice were randomly assigned to control or treatment groups (n = 6 mice per group). For conditioned medium experiments mice were intratumorally treated (5 consecutive days for 2 weeks) with 250 μl of CM of NPFs, CAFs exposed or not to 2.5 μM amiodarone (Hikma Pharmaceuticals), 2.5 μM verapamil (Abbott Laboratories), 2.5 μM nifedipine (Meda AB) or 2.5 μM flecainide (Meda AB). At two different time points (after 1 week and at the end of the treatments) tumors were harvested. For co-injection experiments, 1 × 107 DU145 cells were co-injected with NPFs or CAFs treated or not with flecainide at a ratio of 1:3 into in the right flank of SCID mice. Tumor size was measured twice a week with a Vernier caliper, and the volume was calculated using the standard modified formula: Volume (mm3) = (length × height2)/2.

Ki-67 and CD31 staining

At the end of the treatment, mice were scarified and subcutaneous tumors were harvested, and formalix-fixed and paraffin-embedded. Tumor sections were then deparaffinised in xylene, rehydrated through graded alcohols to water, and subjected to immunohistochemical analysis using Ki-67 antibody (MIB-1, Dako; 1:200) or CD31 (MEC 13.3, sc-18916, Santa Cruz; 1:100, incubation over-night). Nuclei were counterstained with hematoxylin. Images were acquired by Nikon Eclipse E600 microscope using ACT-1 software (Nikon). At least 10 fields were scanned and the average number of Ki-67-positive or CD31-positive and negative cells was plotted.

Statistical analysis

Statistical analysis was performed with Mann–Whitney test and Student’s t-test, when appropriate, using GraphPad Prism software (version 9.4; GraphPad Prism Inc., San Diego, CA, USA). P ≤ 0.05 was considered statistically significant.

Additional methods

RNA extraction, RT-qPCR, protein isolation and western blotting protocol are described in detail in Additional Methods.