Cyprofloxacine

HCT116, SW620, and HT29 CRC cell lines were a kind gift from Prof. Kari Alitalo, University of Helsinki, Finland. SW1222 cells and normal human colon fibroblasts (ATCC-1459) were from ECACC and ATCC, respectively. Thp-1 cells were from ATCC. Cells were cultured in DMEM high glucose (Gibco) supplemented with 10% FCS (Gibco), cyprofloxacine, antibiotic/antimycotic mix, and glutamine (Sigma). Cells were tested for mycoplasma contamination with Hoechst staining and only negative cultures were used in our experiments. Two days before EV collection, cells were washed with PBS three times and they were further cultured in either medium without FBS or containing 2.5% EV-free FBS. EV-free FBS was prepared by overnight ultracentrifugation at 100,000g or purchased from Gibco (exosome depleted One-Shot FBS). For 3D cultures, cells were treated with TrypLE (Gibco), embedded into Matrigel with 5000–10,000 cells depending on the cell line and cultured for 12-14 days.

500 cells per 500 μl Serum-Free Medium (SFM: DMEM/F12 (Thermo Fisher Scientific, Saint Aubin, France), Insulin (20 μg/ml) (Sigma-Aldrich, Lyon, France), 1 % N-2 supplement (Thermo Fisher Scientific, Saint Aubin, France), EGF (20 ng/ml) (R&D systems), FGF (10 ng/ml) (R&D systems), Cyprofloxacine (2 ug/ml) (Sigma-Aldrich, Lyon, France), Gentamicine (5 ug/ml) (Thermo Fisher Scientific, Saint Aubin, France) and D-Glucose (3 mg/ml) (Sigma-Aldrich, Lyon, France) were seeded in 24 wells Corning^® Costar^® ultra-low attachment plates (Sigma-Aldrich, Lyon, France). Subcultures were made by centrifugation at 1000 rpm for 5 minutes and dissociation to single cells using Accumax (Millipore, Molsheim, France). Analyses were performed on colonospheres cultured for 11 days. Cell viability was assayed using the Cell Titer non-radioactive cell proliferation assay (Promega, Charbonnières-les-Bains, France). Colonosphere counting was performed with cultures grown from a starting concentration of 5 cells per 100 μl SFM per well in 96 plates.

We used PDAC cell lines (derived from primary tumors) Panc02.03 (ATCC® CRL-2553), Panc08.13 (ATCC® CRL-2551), Panc10.05 (ATCC® CRL-2547) and Panc-1 (ATCC® CRL-1469). Panc02.03, Panc08.13 and Panc10.05 were cultured in RPMI-1640 (Gibco) supplemented with 15% FBS (Gibco), cyprofloxacine, antibiotic/antimycotic mix, glutamine (Sigma) and 10 U/ml human recombinant insulin (Santa-Cruz). Panc-1 cells were cultured in DMEM with 10% FBS (Gibco) and antibiotics. The control immortalized Human Pancreatic Duct Epithelial Cell Line (H6c7, HPDEC, Kerafast) was maintained in keratinocyte serum-free medium that contained EGF and bovine pituitary extract (Thermo Fisher, 17005042), antibiotic/antimycotic mix and glutamine (Sigma). Cells were tested for mycoplasma contamination with Hoechst staining and only negative cultures < 9 passage numbers were used in our experiments. Cells were washed three times with phosphate buffered saline (PBS) 2 days after culturing them in serum-free medium, the medium was then replaced and EVs were collected after 2 days. For 3D cultures, cells were removed from culture dishes (Eppendorf) with TrypLE (Gibco), embedded into Matrigel with 20,000 cells/droplet (25 μl) and cultured in 2.5% EV-free FBS (exosome depleted One-Shot FBS, Gibco) prior change to serum-free medium 2 days before starting EV collection.