Gastrin

Liver organoids were seeded and kept 7–10 days under the liver medium explained above (EM, expansion medium) supplemented with BMP7 (25 ng/ml). Then, the cultures were split and seeded accordingly in this EM supplemented with BMP7 for at least 2–4 days. Then, medium was changed to the differentiation medium (DM): AdDMEM/F12 medium supplemented with 1% N2 and 1% B27 and containing EGF (50 ng/ml), gastrin (10 nM, Sigma), HGF (25 ng/ml), FGF19 (100 ng/ml), A8301 (500 nM), DAPT (10 uM), BMP7 (25 ng/ml), and dexamethasone (30 uM). Differentiation medium was changed every 2–3 for a period of 11–13 days.
To assess hepatocyte function, culture medium was collected 24 hr after the last medium change. Functional studies were performed in the collected supernatant or in whole organoids, as described in the Extended Experimental Procedures.

For the immunohistochemical study, the tissue sections after dewaxing and rehydration were heated for 15 min in citrate buffer (0.01 mol/L, pH = 6.0) to recover the antigens. Subsequently, the tissue sections were placed in 3% H₂O₂ for 15 min to incubate, and then washed with phosphate-buffered saline (PBS, 0.01 mol/L). Afterward, the tissue sections were incubated in goat serum blocking solution at room temperature for 30 min, and then incubated overnight at 4 °C with the primary antisera (anti-serotonin, anti-glucagon, and anti-gastrin; dilution ratio = 1:200). Then, the sections were incubated with a biotinylated secondary antibody, followed by staining with DAB colorimetric solution (dilution ratio = 1:20) at room temperature and re-staining with hematoxylin staining solution [18 (link),31 (link)]. Finally, the sections were subjected to dehydration, immersed in a clearing agent to enhance their transparency, and sealed with neutral gum. All of the above reagents were from Beijing Zhongshan Golden Bridge Biological Company, China. Three microphotographs (400× magnification) were randomly acquired from each part of each sample by using a Digital trinocular microscope camera system (BA400Digital, Motic, Xiamen, China). The distribution characteristics of 5-HT-immunoreactive cells, Gas-immunoreactive cells, and Glu-immunoreactive cells were observed and their positive expression levels (the proportion of DAB positive tissue = DAB positive area/tissue area × 100%) were calculated using the Data Image Analysis System (Halo 101-WL-HALO-1, Indica labs, Albuquerque, NM, USA).

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.