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. Anti-MT1-MMP, Anti-MMP2 and Anti-MMP9 antibody were produced in rabbit and purchased from Cell Signalling Technology, U.S.A.. Anti-GAPDH antibody was purchased from Millipore, U.S.A..
For the invadopodia 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.
Spheroids were formed in black walled 96-wells clear black round bottom ultra-low attachment spheroid microplates (Corning). First, 1000 cancer cells were seeded in 200µl DMEM F-12 growth media (10% FBS, 1 mM penicillin/streptomycin), Then, the was centrifuged at 200g for 8min at room temperature. Cells were then cultured at 37°C with 5% CO2 for 72 hours. After spheroid assembly was achieved, 170ul medium was removed by multichannel pipetting. A collagen mixture was prepared on ice with a final concentration of 1.3 mg/ml rat tail collagen I (Corning). A collagen matrix of 100µl was then added to each well with a multichannel pipette. The spheroid plate was then incubated at 37°C with 5% CO2 for 2 hrs. A 1:1 mixture of serum-free RPMI and DMEM-F12 with 10% FBS and 1 mM penicillin/streptomycin was then added on top of the collagen matrix to initiate the assay. Brightfield images were obtbained with a 4x objective at 0hrs and 48hrs, respectively.
MTT viability assay
For the MTT assay, Patu8902 cells in 100µl full growth media (DMEM supplemented with 10% FBS and 1 mM penicillin/streptomycin) containing 4000 cells were seeded in a triplicates in three 96 well plates. Plate 1 was used as a treatment-naïve control. Growth medium in plate 2 and 3 were removed from the wells and replaced with a) normal growth medium (DMEM supplemented with 10% FBS and 1 mM penicillin/streptomycin), b) DMEM supplemented with 10% FBS and 1 mM penicillin/streptomycin mixed with serum-free DMEM (1:1), c) (DMEM supplemented with 10% FBS and 1 mM penicillin/streptomycin mixed with frozen and rewarmed THP1-CM (1:1), d) DMEM supplemented with 10% FBS and 1 mM penicillin/streptomycin containing rTIMP2 (50ng/ml). The MTT assay is performed after 24 hrs (plate 1, control), 72 hrs (plate 2) and 120 hrs (plate 3).
The MTT powder was made up to a working concentration of 500μg/ml in DMEM media prior to each experiment. First, media was removed from each well. Cells were then washed with sterile PBS twice and then 1ml MTT:DMEM solution was added to the wells. Cells were incubated for four hours. The MTT:DMEM was removed and 500uL of DMSO was added, the plates were then incubated at 37°C for 10 minutes. Absorbance was measured at a wavelength of 562nm using a Nanodrop.
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). Cell lysates were seeded in 6-well plates and cultured for 24 hours. Lysates were generated when cells were 70-80% confluent. First, cells were washed with PBS and then lysates were generated with 100µl NP40 based lysis buffer per well. Lysates were scrapped and centrifuged at 13 000 x g for 15min at 4°C. The Supernatant was then transferred to an Eppendorf tube and boiled for 3 min at 95°C in 6x laemmli buffer. Samples were stored at -20°C. Equal amounts of protein were then electrophoresed on 7.5% SDS-polyacrylamide gels and immunoblotting was performed as described above.
Determination of rTIMP1 and rTIMP2 concentrations
Since there are no comparable studies using rTIMP1 and 2, respectively, in an invadopodia model, the approximate concentration had to be estimated. Physiologic TIMP1 and 2 levels, respectively, range between 5 and 1000 ng/ml [47, 48]. In a second step, CM and RPMI control media containing either TIMP1 or TIMP2 in various concentrations ranging from 5 to 6000 ng/ml was concentrated as described above and immunoblotted for TIMP1 and TIMP2, respectively. Autoradiographs were quantified using ImageJ software. The concentration of TIMP2 in the conditioned medium was calculated from the intensity values of the TIMP2 signal in the conditioned medium against the intensity values for recombinant TIMP2 signal. TIMP1 and TIMP2 levels in the CM varied between the experiments and were ranging between 5 and 50 ng/ml. Therefore, these concentrations were used for the later rTIMP1 and rTIMP2 experiments.
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 the spheroid assay, the invasive front was determined as the area of all cells invading from the spheroid. The invasive front was calculated as the area of the invading cells relative to the size of the spheroid after 48 hrs. Relative spheroid growth was determined by measuring the total area of the spheroids at 0 hrs and 48 hrs, respectively.
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.