Study subjects
Surgically resected iCCA specimens were obtained from patients admitted to Fondazione IRCCS Policlinico San Matteo, Pavia, IRCCS Humanitas Research Hospital, and ASST Santi Paolo e Carlo Hospital, Milan, Italy. The main patient characteristics are listed in Table 1.
PBMC were isolated as previously described [38] from HC (3 females and 5 males). A written informed consent was obtained from each individual. The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Fondazione IRCCS Policlinico San Matteo, Pavia (protocol numbers: P-20140031379, P-20190104922).
Cell Cultures
NHC cells were isolated from normal liver tissue specimens and maintained as previously described [22].
Primary tumor cell cultures were established from patients affected by iCCA as previously described [38]. Briefly, tumor samples were treated by enzymatic and mechanical dissociation with the human Tumor Dissociation Kit and gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer's instructions. The cell suspension was filtered and centrifuged to obtain a cell pellet that was plated in tissue culture flasks (Corning, NY, USA) with Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% foetal bovine serum (FBS, HyClone, GE Healthcare, South Logan, Utah, USA), 1% antibiotic antimycotic solution (Merck, Darmstadt, Germany) and 1% non-essential amino acids (Thermo Fisher Scientific).
CK19 was evaluated in primary iCCA cell cultures by flow cytometry. Briefly, iCCA cells were fixed with BD Cytofix/Cytoperm (BD Biosciences, San Diego, CA, USA) and permeabilized with the BD Perm/Wash buffer (BD Biosciences) in the presence of FITC mouse anti-human monoclonal antibody (mAb) (clone SB39g, Abcam, Cambridge, UK) for 30 min at 4°C, according to the manufacturer’s instructions. Flow cytometry analysis was performed using a 12-color FACSCelesta (BD Biosciences, San Diego, CA, USA) instrument. Kaluza™ software (Beckman Coulter, Brea, CA, USA) was used for data analysis.
HuCCT-1 cells (kindly provided by professor M. Cadamuro, Dept. of Molecular Medicine, University of Padua, Italy) were maintained as previously described [38].
Ev Isolation And Characterization
Supernatants from low-passage primary iCCA and NHC cells were collected after 72h of culture in medium without serum, bovine pituitary extract and lipid and cryopreserved at -80°C until EV isolation. Supernatants from HuCCT-1 cells were collected after 16h of culture in medium without serum with 0.8% bovine serum albumin (Merck). When indicated, HuCCT-1 cells were treated with Myr at 10µM (Merck) and DMSO (Merck) for 16h before collecting supernatants to isolate EVs. After serial centrifugations (1000, 2000 and 3000g for 10 min at 4°C), EVs were obtained by ultracentrifugation (100,000g for 75 min at 4°C). EVs were then resuspended in PBS with protease inhibitors and stored at − 20°C until LC–MS/MS evaluation. Freshly isolated EV from HuCCT-1 cells were resuspended in PBS and used to treat PBMC. EVs characterization was performed by DLS, NTA and Western Blotting, as previously described [17]. Briefly, NTA was carried out with the Malvern NanoSight NS300 system (Malvern Panalytical ltd., Malvern, UK), used to visualize EVs by laser light scattering. For each sample, three 60-sec records were registered. NTA output was then analyzed with integrated NTA software (Malvern Panalytical ltd.), providing high-resolution particle size distribution profiles and EV concentration measurements. For TEM, samples were fixed in 2.5% glutaraldehyde in 0.13 M phosphate buffer pH 7.2–7.4 for 2h, post-fixed in 1% osmium tetroxide, dehydrated and embedded in epoxy resin. A Jeol JEM 1010 transmission electron microscope (Jeol, Tokyo, Japan) was used.
Characterization Of Sphingolipids By Lc-ms/ms
SPLs extraction and analysis were performed as previously described [17]. Briefly, SPLs were extracted with a cold methanol/chloroform mixture (850µL, 2:1, v/v) coupled with alkaline methanolysis (75µL KOH 1M, 2h at 38°C). The lipid content was normalized to the inorganic phosphorus amount that was determined by the phosphomolybdate and malachite green assay [39]. Briefly, for the inorganic phosphorus determination an aliquot (10µL) of the lipid extract was mineralized in concentrated perchloric acid (50µL, 100°C, >1h) and added with a solution made of 4.2% ammonium molybdate in 5M HCl/0.2% malachite green/H2O (700µL, 1:1:5, v/v/v). The complex of phosphomolybdate and malachite green was detected and quantified by spectrophotometry at 630 nm [39]. The LC-MS/MS consisted of an LC Dionex 3000 UltiMate (Thermo Fisher Scientific) coupled to a tandem mass spectrometer AB Sciex 3200 QTRAP (AB Sciex, Concord, ON, Canada) equipped with electrospray ionization TurboIonSpray™ source operating in positive mode (ESI+).
Pbmc Treatment
PBMCs were cultured in RPMI supplemented with 10% FBS, 1% antibiotic antimycotic solution and 1% glutamine (Merck) for 16h in the presence of BD GolgiPlug™ Protein Transport Inhibitor (BD Biosciences). When indicated, PBMC were treated with HuCCT-1-derived EVs at a PBMC:EVs ratio of 1:50 for 16h. After incubation, 5 × 105 PBMC were harvested and stained with anti-human CD14 BB700, CD3 BV421, HLA-DR BV605 and CD16 APC-H7 mAbs (all from BD Biosciences) for 30 min at 4°C. Cells were subsequently fixed with BD Cytofix/Cytoperm (BD Biosciences) and permeabilized with the BD Perm/Wash buffer (BD Biosciences) in the presence of anti-human MIP-1α FITC, IL-1α PE and IL-8 BV510 mAbs according to the manufacturer’s instructions. FACSCelesta (BD Biosciences) instrument and Kaluza™ software (Beckman Coulter) were used for data acquisition and analysis.
Statistical analysis
This was performed using the GraphPad Prism 8.4.3 software (GraphPad, La Jolla,CA, USA). Data distribution among groups was checked for normality prior to analysis. We used parametric and non-parametric tests as detailed in the legend. A p value ≤ 0.05 was deemed statistically significant. Pearson correlation test was used to assess correlations between EV content of SPLs and clinical features of the patients.
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.