Sw 620

Human colorectal adenocarcinoma cells (CaCo-2), metastatic SW-620 colon cancer cells, hepatocellular carcinoma cell lines (Hep-3B, HLF and HLE cells), liver hepatoma cells (PLC-5), and gastric carcinoma (derived from metastatic site) N-87 cells were purchased from ATCC. Human gastric carcinoma (derived from metastatic site) cell line (HGC-27) was purchased from Sigma-Aldrich. Human intrahepatic cholangiocellular carcinoma cell line (HUCCT-1) was purchased from JCRB Cell Bank derived from ascitic fluid. All cell lines were cultivated in according to retailer protocols. Briefly, colon cancer cell lines (CaCo-2 and SW-620) were cultured in 10% of Inactivated Fetal Bovine Serum (FBS) exosomes free (Euroclone) in Dulbecco’s Modified Eagle’s Medium (DMEM) with sodium pyruvate, 4.5 g/L glucose (GIBCO), 4mM L-Glutamine (GIBCO) and 5 mL Pen-Strep (penicillin 10,000 u/mL, streptomycin 10,000 u/mL, Lonza Biowhittaker). Gastric (N-87 and HGC-27), hepatocellular carcinoma (Hep-3B, HLE and HLF), hepatoma (PLC-5), and intrahepatic cholangiocellular carcinoma (HUCCT-1) cell lines were cultured with Roswell Park Memorial Institute (RPMI) medium (GIBCO) supplemented with 10% of FBS exosomes free, sodium pyruvate, 4.5 g/L glucose, 4mM L-Glutamine, and 5 mL Pen-Strep.Cells were grown until reaching semi-confluence, in a humidified incubator at 37 °C with an atmosphere containing 5% of CO₂.

To validate our method, we chose two cancer cell lines harbouring known point mutations in homozygous state (see Table 1), i.e., the pancreatic cancer PANC-1 and the colon cancer SW-620. These cell lines and the BEAS-2B normal bronchial epithelial cell line were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). BEAS-2B was used as WGA positive control (see paragraph 2.6). PANC-1 was cultured in DMEM medium (EuroClone, Pero, Milan, Italy), while SW-620 and BEAS-2B in RPMI-1640 medium (EuroClone), both supplemented with 10% fetal bovine serum (EuroClone) and Penicillin-Streptomycin mixture (Sigma-Aldrich). All cells were incubated at 37°C and 5% CO₂. For spike-in and WGA experiments, cells were gently detached, resuspended in DPBS (Sigma-Aldrich), and counted with Trypan blue (Sigma-Aldrich). Genomic DNA was extracted from BEAS-2B cells using the gSYNC^TM DNA Extraction Kit (Geneaid Biotech Ltd., New Taipei City, Taiwan) according to the manufacturer’s instructions and measured using the Qubit™ Fluorometer (Life Technologies, Carlsbad, USA).

Colon cancer cell lines, HCT116, SW480 and SW620 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). HCT116 and SW620 were cultured at 37 °C in DMEM medium containing 10% fetal bovine serum (FBS), 100 U/mL penicillin/streptomycin and 2 mM L-glutamine (EuroClone, Pero, Italy). SW480 cell line was propagated in RPMI-1620 (EuroClone), supplemented with 10% FBS, 2 mM L-glutamine and 100 U/mL penicillin/streptomycin. For the experiments, cells at sub-confluence (70%), were starved using a medium with 0.2% FBS (serum-free medium) cells overnight in order to reduce basal cellular activity [37 (link)]. Then, the starved cells were acutely stimulated with H₂O₂ (Sigma-Aldrich, Milan, Italy) as an oxidizing agent, for the times and concentrations described below.