Complement-dependent cytotoxicity assay

Exosomes applied in CDC assays were isolated using the density gradient floatation approach described above and underwent buffer exchange (in serum free RPMI) using Zeba™ Desalt Spin Columns according to manufacturer’s instructions (Thermo Scientific). A pool of pre-diagnostic PDAC patient and matched healthy control (n = 13 per each group; set #2 in Supplementary Table ⁶) serum samples from the CARET study were used to mediate the CDC.

To test the dose-dependent decoy function of PDAC exosome in inhibiting CDC mediated by PDAC serum, PANC-1 cells were seeded in a 96-well plate (5 × 10³ per well) in RPMI + 1% FBS overnight for adhesion. Cells were washed with 200 µL pre-warmed serum free RPMI, incubated in presence of increasing amount of exosomes (0, 1, and 2.5 µg; 25 µL per well) and with equal volume of sera diluted 1:10 in PBS for 15 min at RT, followed by incubation with 50 µL of fresh reconstituted rabbit complement (Low-Tox rabbit complement; Cedarlane) for 2 h at 37 °C. Total lysis of the cells was achieved by solubilizing a non-treated control sample with 4% CHAPS. Antibody-independent (spontaneous) lysis was controlled for each experimental condition (0, 1, and 2.5 µg of exosomes) by incubating cells with complement and without serum in presence of equal amount of exosomes. Viability was evaluated with the CellTiter 96 Aqueous One Solution (Promega). The percentage of cell viability was calculated as: (experimental maximum/spontaneous maximum) × 100.

Live cell imaging cytotoxicity assay was performed to test in real time the decoy function of PDAC or non-neoplastic exosomes in inhibiting CDC mediated by PDAC or matched healthy control sera. PANC-1 or Pa03C cells were seeded in a 96-well plate (10 × 10³ per well) in RPMI + 1% FBS overnight for adhesion. Cells were washed with 200 µL pre-warmed serum free RPMI, incubated in presence or absence of exosomes (5 µg per well; 50 µL per well) with equal volume of sera diluted 1:2 in PBS for 30 min at RT. Cells were washed with 200 µL serum free RPMI, incubated with 50 µL per well of IncuCyte™ Cytotox Green Reagent (Essen Bioscience) diluted 1:1000 in serum free RPMI, followed by incubation at 37 °C with equal volume of fresh reconstituted rabbit complement (Cedarlane). Green fluorescence (dead cells) and cell confluency was measured on an Incucyte™ ZOOM instrument (Essen Bioscience), and the number of green dead cells was calculated using ZOOM software (Essen Bioscience). To control for antibody-independent (spontaneous) lysis, the number of green dead cells obtained by incubating the cells with complement and without serum was subtracted to each experimental condition. All measurements were performed in triplicates.