PDOs were derived from ascites drainage samples from four patients with GCPM. Ascites drainage was filtered with a 70-um cell strainer then centrifuged at 300 g for 5 min. Pellets were resuspended into single-cell suspensions with PBS and incubated with red blood cell lysis buffer for 5 min on ice, then centrifuged again at 300 g for 5 min. Finally, the cells were embedded in Matrigel, seeded onto pre-warmed 24-well culture plates, and cultured in Advanced DMEM/F12 medium (GIBICO) with 50% L-WRN conditioned medium supplemented with 10 mM HEPES (GIBICO), 10 mM Nicotinamide (Sigma), 1X N2 (GIBICO), 1X B27 (GIBICO), 1X Glutamax (GIBICO), 1.25 mM N-Acetylcysteine (Sigma-Aldrich), 50 ng/mL EGF (Peprotech), 200 ng/mL FGF10 (Peprotech), 10 nM gastrin (R&D Systems), and 1X Primocin (Invivogen). 10 uM ROCK inhibitor (Y-27632, R&D Systems) was provided for the first generation of PDOs to prevent anoikis. PDOs were overlaid with culture medium and incubated at 37 °C in humidified air containing 5% CO2. Culture medium was changed every 3 days. For passaging, PDOs were collected by mechanically dissociating the Matrigel, centrifuged at 150 g for 5 min, incubated with pre-warmed TrypLE express for 7 min, then pipetted about 100 times. All PDOs were trypsinized to single cells under a light microscope then transferred into new matrigel with culture medium and plated in pre-warmed 24 well-plates, incubated at 37 °C in humidified air containing 5% CO2. PDOs were passaged every 7 days until at least the 15th generation. PDOs were treated with culture media and inhibitors including Hydroxychloroquine Sulfate (1uM), DC661 (4uM), TORIN1 (2uM), or Rapamycin (400 nM) for 7 days before analysis. Culture medium and drugs were changed every 3 days. To embed PDOs, culture media was removed, PDOs were washed in DPBS on ice for 15 mins, then PDOs were fixed in 10% formalin on ice for 2 h. Finally, PDOs were moved to 75% ethanol at 4 °C overnight, mounted in 3% agar, and embedded in paraffin.
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