Cell culture and chemical reagents
PL-45 and Capan-1 human pancreatic cancer cell lines were obtained from LGC-ATCC (Barcelona, Spain), and LC5 embryonic fibroblasts cell line was kindly provided by Dr. G. Criado (Instituto de Investigación Hospital 12 de Octubre, Madrid). Differential genetic characteristics between tumor cell lines included in the study are shown in Supplementary Table 1. Cell lines were grown according to ATCC instructions, at 37ºC in humidified atmosphere with 5% CO2. IMDM and RPMI cell media, fetal bovine serum (FBS), penicillin, streptomycin and L-glutamine were obtained from Lonza Ltd. (Verviers, BE).
Establishment of LC5 cell line constitutively expressing EGFP
Lentivirus was generated by co-transfection of pRRL.SIN-EF1a-PGK-EGFP (encoding green fluorescent protein; #12252) with pRSV-Rev (#12253), pMD2.VSVg (#12259), and pMDL.gag/pol.RRE (#12251) plasmids in 293T cells. After 48h, viral supernatants were collected, filtered and frozen at -80 until use. LC5 cell line was infected for 24h with 4 mL of virus-containing medium in presence of 10 µg/mL polybrene. Selection and isolation of fibroblasts-GFP+ was performed with Sony Synergy Cell Sorter separation device (Sony Biotechnology Inc.), and based of GFP expression, and incorporation of Propidium Iodide (PI) for the exclusion of dead cells. All plasmids required were from non-profit plasmid repository Addgene (http://www.addgene.org/).
Co-cultures of fibroblasts and pancreas cancer cell lines
For direct co-cultures between pancreatic tumor cell lines and fibroblasts, cells were cultured in a ratio 1:1, in RPMI medium supplemented with 10% FBS. Tumor pancreatic cells were seeded 72 hours before fibroblasts were added to the culture. The optimum time for these direct co-cultures was experimentally established in 72h, to obtain a final ratio of tumor cells:fibroblasts of 20:80, reflecting what is described in vivo. For functional analysis, cells were separated by cell sorting based of GFP expression and incorporation of PI.
Conditioned media (CM) were prepared from fibroblasts and pancreatic cancer cell lines. Cells were grown in 75cm flasks until semiconfluent conditions, washed three times with phosphate-buffered saline (PBS), and then incubated in 1% of FBS medium for another 48 h. Next, supernatants were collected, centrifuged at 2000rpm for 10 min, sterile filtered (0.45 µm pore), and immediately used for assays of indirect co-cultures.
Wound healing, migration, and invasion assays
For wound-healing assay, cells were seeded on 6-well plates, and left to reach 80% confluence. A single wound per well was scratched with a sterile 200 µL pipette tip within the monolayers and then washed with PBS to remove detached cells. CM from tumor cells was added to fibroblasts, and similarly CM from fibroblasts was added to tumor cells, and the rate of wound closure was observed in the following 24, 48, and 72 hours. Wound closure was monitored using a Nikon TE2000-S microscope.
Transwell migration assays were carried out using 8.0 µm polycarbonate cell culture inserts (Transwell® Permeable Supports, Corning Inc., NY). Cells were serum-starved 24h before the start of the experiments. Tumor cells were seeded at 1x10e5 per well in 2 mL growth medium in the upper chamber (cell culture inserts) of 6-well plates. The same number of fibroblasts was seeded in the lower wells. Cells were allowed to migrate for 72 hours toward the underside of the membrane to the lower chamber. After that, cells attached to the upper side of the filter were removed by wiping with a cotton swab, and the migrated cells on the lower surface were fixed with formaldehyde 4% in PBS and permeabilized with absolute methanol in darkness. After several washes with PBS, cell nuclei were stained with 4'-6-diamino-2-phenylindole (DAPI).
We used Ibidi culture-insert 2 well in µ-dish 35 mm plates, with a defined 500 µm cell free gap (Inycom Biotech, Zaragoza, ES) for migration assays. Tumor cells were seeded on one side of the insert and fibroblasts-GFP+ on the opposite side. After 24h for cells adhesion, the insert was removed, and migration was allowed. This system allows us to observe the migratory behavior of each cell type under the indirect influence of the other.
Western blot analysis
Whole cell lysates were prepared using MCL1 lysis buffer in the presence of protease and phosphatase cocktail inhibitor (Sigma-Aldrich, St Louis, MO) following the manufacter's protocol. Protein lysates were subjected to SDS-PAGE on 10% polyacrylamide gel and transferred onto PVDF membranes. Membranes were probed for the protein levels of E-cadherin, and α-tubulin as loading control, using specific primary antibodies (#4065, Cell Signaling Tech. Inc., Danvers, MA, and #sc-5286, Santa Cruz Biotechnology, Inc., Dallas, TX, respectively). Specific bands for each protein were visualized by WesternBright™ kit (Advansta, San Jose, CA).
Enzyme-linked Immunosorbent Assay (ELISA) and human cytokine array.
Culture supernatant was collected from each growth condition, and keratinocyte growth factor (KGF-7) concentration was determined using Quantikine human KGF/FGF-7 Immunoassay (R&D Systems Inc., Minneapolis, MN). ExoQuick-TC® ULTRA EV Kit and ExoELISA-ULTRA CD63 Kit (System Biosciences, Palo Alto, CA) were used for extracellular vesicles isolation and quantification, respectively. For the semi-quantitative detection of 23 human protein in cell culture media, the human cytokine antibody array C1 (AAH-CYT-1; RayBiotech, Norcross, GA), was used (Supplementary Figure 2). All determinations were performed according to the manufacturer's instructions.
Confocal immunofluorescence microscopy
Direct and indirect co-cultures of fibroblasts-GFP+ and tumor pancreatic cells were seeded on glass coverslips. Cells were washed twice with warm PBS, fixed in 4% paraformaldehyde and processed for immunofluorescence staining. First, cells were incubated overnight with mouse anti-α-Smooth Muscle Actin (α-SMA) antibody (n1584, Dako, Carpinteria, CA) at 4ºC. After thorough washing with PBS, cells were incubated for 1h with Alexa Fluor® 594-conjugated goat anti-mouse antibody and DAPI. Cells were mounted with Fluorsave™ Reagent (Calbiochem, San Diego, CA) and examined in a Zeiss Axioplan 2 confocal microscope.
Transmission electron microscopy (TEM)
Cells were fixed in 2.5% glutaraldehyde at pH 7.2 for 24h, and later in 1% OsO4 in a 0.1 M cacodylate buffer for 1h. Then, the samples were spinned to obtain pellets. The pellets were fixed in 1% uranyl acetate for 30 minutes, were then dehydrated in a series of graded ethanol steps, and finally embedded in epoxy resin. Thin sections were performed and stained with toluidine blue. Ultrathin sections were obtained from representative areas and were double stained with lead citrate and uranyl acetate and viewed under a JEOL JEM-1011 microscope.
Each experiment was performed in triplicate in at least three independent experiments. The analysis of differences was performed using Mann-Whitney U test and variance analysis for repeated measurement data. Results were analyzed using software GraphPad Prism version 6.0 (GraphPad software, San Diego, CA) and SPSS v21.0 (SPSS Inc., Chicago, IL). The value P<0.05 was considered as significant.