Leucovorin

To measure the sensitivity to chemotherapy, 6000 cells of LuPanc-1 and 8000 cells of LuPanc-2 growing in a log phase were seeded per well in 96-well plates, and chemotherapeutics were added 24 h after cell seeding. For gemcitabine treatment, gemcitabine (Sigma Aldrich, St. Louis, MO, USA) was added to a final concentration in a range of 2.5–320 nmol/L for LuPanc-1 and 10–12,800 nmol/L for LuPanc-2. For FOLFIRINOX treatment, 5-FU, irinotecan, leucovorin, and oxaliplatin were combined, and the final concentration ranged as follows: 5-FU (Medac) 0.005–426 µmol/L, irinotecan (Medac) 0.07 nmol/L–5.78 µmol/L, oxaliplatin (GRY) 0.062 nmol/L–4.96 µmol/L, and leucovorin (Sigma Aldrich) 0.078 nmol/L–6.19 µmol/L. After 72 h, cell metabolism was measured by a Resazurin reduction assay. Resazurin (STEMCELL) was diluted in DPBS to a concentration of 10 mg/mL. This stock solution was further diluted in the medium, 20 µL was added per well to a final concentration of 40 µg/mL, and the cells were incubated for 2 h at 37 °C in a humidified incubator with 5% CO₂. Extinction was quantified with a plate photometer, according to the manufacturer’s instructions. The excitation wavelength was set to 545 nm, and the emission wavelength was set to 600 nm. Each experiment was performed in triplicate and repeated three times.

Metformin (#D150959), 5-FU (#F6627), and leucovorin (#F7878) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Metformin was dissolved in phosphate-buffered saline (PBS) to prepare a 1 M stock solution. In addition, 5-FU and leucovorin were diluted in PBS to obtain 5 mg/mL stock solution. Oxaliplatin was obtained from Sanofi (Sanofi, Gentilly, France). All drugs were filtered through a 0.22 µM pore filter before use in the experiment.