Patu8902 and Capan2 were obtained from “Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH” [DSMZ], Germany), Capan1 and MiaPaCa2 were kindly provided by Prof. H. Kocher at Barts Cancer Institute, UK. The human breast cancer cell line MDA-MB-231 was purchased from ATCC. All these cells were maintained in Dulbecco’s modified Eagle’s media (DMEM, Sigma Aldrich UK) supplemented with 10% v/v fetal bovine serum (FBS) and 1 mM penicillin/streptomycin. CFPAC-1 cells (kindly provided by Dr. A. Pessina at Universita degli studi di Milano, Italy) were cultured in Iscove’s modified Eagle’s media (IMEM, Sigma Aldrich UK) supplemented with 10% v/v fetal bovine serum (FBS) and 1 mM penicillin/streptomycin. Colo-357 (kindly provided by Prof. Michalski University Hospital Heidelberg, Germany) and AsPC1 (kindly provided by Dr Stéphanie Kermorgant, Barts Cancer Institute, UK) were cultured in Roswell Park Memorial Institute-1640 (RPMI-1640, Sigma Aldrich UK) media supplemented with 10% v/v FBS with 1 mM penicillin/streptomycin. THP-1 cells (obtained by the European cell collection bank) were tagged with enhanced green fluorescent protein (eGFP) using lentiviral vector technique as described before . Cells were maintained in RPMI-1640 media supplemented with 10% v/v FBS with 1 mM penicillin/streptomycin. For inhibitor treatment cells were incubated with 10 mM GM6001 as previously described . All cell lines were regularly screened for mycoplasma contamination using DAPI staining.
THP-1-conditioned media (CM)
THP-1 cells were seeded in serum-free, antibiotic-free RPMI media at a density of 200,0000 cells/ml and incubated at 37°C and a 5% CO2 atmosphere. After 24 hours, the cell suspension was centrifuged at 1,500 rpm at room temperature for 10 minutes. CM was either directly used for experiments (fresh CM), stored at -20°C and rewarmed to 37°C (frozen CM) or heat-inactivated at 95°C for 10 mins (boiled CM).
Anti-TIMP1 antibody (D10E6) produced in rabbit was purchased from Cell Signalling Technology, U.S.A., anti-TIMP2 antibody produced in rabbit antibody (SAB4502972) was purchased from Sigma.
For the invasion assays, the QCM™ Gelatin Invadopodia Assay (Red) (Chemicon® / Millipore) was used. Briefly, coverslips were inverted onto poly-L-lysine in deionized water for 20 minutes at room temperature (RT). The slides were then washed with PBS three times before incubation with glutaraldehyde: PBS for 15 minutes at RT. After washing three times with PBS, each coverslip was placed on gelatin in PBS in a 1:5 ratio of fluorescently-labelled - unlabelled gelatin and incubated for 10 minutes at RT and subsequently washed in PBS three times. The. Patu8902 and CFPAC-1 cells were detached using non-enzymatic Cell Dissociation Solution (Sigma Aldrich UK), resuspended in DMEM F-12 growth media (10% FBS, 1 mM penicillin/streptomycin) and seeded onto the prepared coverslips. For co-culture experiments
cells were seeded in the presence of control media (serum-free RPMI media and DMEM-F12 media in a 1:1 ratio), in the presence of THP1-CM (frozen CM mixed with DMEM-F12 media with 10% FBS and 1% P/S in a 1:1 ratio) or in the presence of 50,000 THP1 cells. Alternatively, PDAC cells were co-cultured with THP1 cells for 24 hours prior to dissociation and seeding on prepared coverslips.
Serum-free THP-1 conditioned media (CM), serum-free RPMI containing recombinant TIMP1 and TIMP2 (expressed in CHO cells Sigma Aldrich UK) in various concentrations ranging from 5 to 6000 ng/ml, respectively, and serum-free RPMI control media, respectively, were filled in a Spin-X® UF concentrators (Spin-X UF 6 10K MWCO, Corning) and centrifuged at 4,000 rpm for 18 minutes at RT. Subsequently, gel sample buffer was added to the concentrated CM and the sample was boiled at 90°C for 3 min. Equal amounts of protein were electrophoresed on 10% SDS-polyacrylamide gels then transferred to nitrocellulose membranes as described elsewhere . Nitrocellulose were incubated with primary antibodies using the recommended concentrations and HRP-conjugated secondary antibodies (Dako Ltd).
THP1 CM was screened for proteins using the RayBio® C-Series Human Cytokine Antibody Array C5 (RayBiotech Norcross, USA) according to the manufacturer’s protocol.
Following 24h-incubation, cells were fixed and stained as previously described . Cells were stained for F-actin (Alexa fluor 488-conjugated phalloidin,Invitrogen),DAPI (Sigma Aldrich UK) and cortactin (Anti-Cortactin (p80/85) Antibody, clone 4F11; Millipore).
Images were analysed using ImageJ 1.51h (National Institutes of Health, USA). For gelatine degradation analysis, the total amount of degradation per image was measured in a total of 10 images per tested condition. The amount of degradation was computed automatically using ImageJ.
For data collection and statistical analysis, Microsoft Excel (Microsoft, Redmond, USA) and Prism 5.0 (GraphPad Software, La Jolla, CA, USA) were used. To test for significant differences, the two-tailed Student’s t-tests was used. Data are presented as mean ± standard error of the mean (SEM). A difference was considered significant at p < 0.05.