Cell-lines and cell culture
Human PANC-1 and MIA-PaCa2 pancreatic cancer cells (ATCC, Manassas, VA) were cultured in high glucose (4.5 g/mL) DMEM (Gibco, Thermo Fischer Scientific, Waltham, MA) and human THP-1 cells were cultured in RPMI-1640 (Gibco). All media were supplemented with 10% fetal bovine serum (FBS), L-glutamine (2 mM), except RPMI-1640 media for conditioned media experiments, which was supplemented with 1X GlutaMAX (Gibco), penicillin (100 units/mL), and streptomycin (500 µg/mL) (Lonza, Basel, Switzerland) according to routine cell culture procedures. Cells were incubated in 5% CO2 incubators at 37oC. All cell lines were authenticated by STR profiling (Promega PowerPlex, Leiden, Netherlands), and tested for mycoplasma by PCR monthly.
Generation of M0, M1, M2 and TAM macrophages from THP-1 cells
THP-1 cells were treated with 150 nM phorbol 12-myristate 13-acetate (PMA, Sigma, St. Louis, MO, USA) for 24 hours in RPMI-1640 medium. Next, activated THP-1 cells were washed with fresh medium to remove PMA, after which cells were cultured in fresh medium for another 24 hours, after which the medium was refreshed once more. Without any other additions, macrophages were considered M0 macrophages at this stage (see Fig. 1 for validation). To generate M1 macrophages, M0 macrophages were treated with 1 ng/ml LPS (Ultrapure, Invivogen, Toulouse, France) for 24 hours. To generate M2 macrophages, M0 macrophages were treated with 20 ng/ml recombinant IL-4 (Peprotech, Rocky Hill, NJ) and IL-13 (Peprotech) each for 72 hours. To generate tumor-associated macrophages (TAM), M0 macrophages were treated with a supernatant mix collected from PANC-1 and MIA PaCa-2 cells at ~ 80% confluence, at a 1:1 dilution with fresh complete RPMI-1640 media. At the end of each incubation, macrophages were washed twice with complete medium to remove the cytokines and other factors from the flask. Media collections were done after 48 hours of incubation. The media were centrifuged at 1200 rpm for 4 minutes to remove cell debris, filtered using 0.2 µm syringe filters (Corning, Corning, New York), and stored at 4oC. For experimental procedures, all conditioned media were diluted 1:1 with fresh media (DMEM) to ascertain the appropriate nutrient content of the medium.
Quantitative real-time PCR
Total RNA was isolated using a NucleoSpin RNA miniprep kit (Macherey Nagel, Düren, Germany). cDNA was synthesized from DNase-treated total RNA using M-MLV-RT (Promega, Leiden, Netherlands) and random hexamers (Qiagen, Hilden, Germany). Quantitative RT-PCR was performed using a Sensifast SYBR No-Rox Kit (Bioline, London, UK) on a LightCycler 480 II (Roche, Basel, Switzerland). Relative expression levels were calculated using the comparative threshold cycle (dCt method) and normalized for expression of the reference gene TBP. Primer sequences of the analyzed genes are shown in Supplementary Table 1.
BrdU proliferation assay
PANC-1 and MIA PaCa2 cells were seeded in black, clear-bottom 96-well plates (Corning) in DMEM without serum. The following day, macrophage media (M0, M1, MM2, and TAM) were added in a 1:1 dilution with complete DMEM. BrdU labeling solution was given to cells after 48 hours of conditioned media treatment. The BrdU assay was performed according to the manufacturer’s instructions (Chemiluminescent BrdU assay, Roche). Luminescence was measured on a Synergy HT Biotek Microplate Reader (Biotek Instruments, Winooski, VT). For testing the functional importance of TNF-α in M0 conditioned medium, we included the commercially available TNF-α inhibitor infliximab (Remicade, MSD, Kenilworth, NJ) at 25 ug/ml.
TNF-α ELISA
A commercially available ELISA kit (R&D Systems, Minneapolis, MN) was used to determine TNF-α levels in M0, M1, M2, and TAM conditioned media. Samples were measured in 4 replicates. The experimental procedure was followed according to the manufacturer’s instructions. Absorbance was measured with an i-Mark Microplate Reader (Bio-Rad, Hercules, CA) at 450 and 655 nm.
Flow cytometry detection of Annexin V
Cells were treated with different macrophage CM and 1:1 DMEM-RPMI mix as the control for 48 hours. Cell supernatant was collected in a tube to include non-adherent cells. Cells were detached using trypsin, and this cell fraction was pooled with the cell supernatant in a 15 mL tube. The mix was pelleted by centrifugation at 1200 rpm for 5 minutes. The pellet was re-suspended in Annexin V binding buffer (BD, Franklin Lakes, NJ) and distributed to 96-well plates for staining. Each well had 100 µl of cell mix and 1 µl Annexin V FITC antibody (BD). The plate was incubated on ice, in the dark, for 1 hour. After incubation, the plate was washed twice with Annexin V binding buffer and re-suspended in 200 µL in the same buffer for measuring. The measurements were performed on a FACS Canto II (BD). Data were analyzed using FLOWJO v10 (FlowJo LLC, Ashland, OR). Cells were gated initially based on FCS and SSC for the main cell population, and then on FCS-H and FCS-W to obtain single cells. FITC-positive populations were gated based on isotype antibody control samples. For analysis, Geometric Mean Fluorescent Intensity (gMFI) on FITC values were used.
Statistical Analysis
Data were presented as mean ± SEM. Statistical analyses were performed using GraphPad PRISM 7.0 (Graphpad Software Inc., La Jolla, CA). Differences were considered statistically significant at a p-value of less than 0.05. For further details of the statistical analysis, see figure legends. P-values are indicated by asterisks with * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.