L glutamine solution

We selected Huh-7 (well-differentiated human hepatocellular carcinoma cell line) among the human in vitro models of hepatocyte [49 (link)]. We obtained Huh-7 from the JCRB Cell Bank. The Huh-7 cell line was grown without antibiotics in the Dulbecco’s Modified Eagle’s Medium (DMEM; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; Biosera, Kansas City, MO, USA) and 1% L-glutamine solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The cells were cultured in a humidified incubator with 5% CO₂ at 37 °C. PA was dissolved in isopropanol at 37 °C to a concentration of 100 mM according to other studies [50 (link),51 (link)]. The dissolved PA solution was mixed with DMEM containing 1% fetal bovine serum albumin (BSA; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and the final concentration at the time of administration was adjusted to 200, 400, 600, and 800 μM in DMEM containing 10% fetal bovine serum. 5-ALA was dissolved in serum-free DMEM to a concentration of 1 mM, and the final concentration at the time of administration was adjusted to 200 μM in DMEM containing 10% fetal bovine serum. The final concentration of BSA was adjusted to 200 μM in D-MEM containing 10% fetal bovine serum. 1% BSA was administered to the normal controls in each group in equal volume with the palmitic acid solution.

In this study, we used cells from the following two COM cell lines: KMEC (primary-tumor site of origin; oral gingiva) and LMEC (metastatic site of origin; lymph node) cell lines. One co-author, Dr. Takayuki Nakagawa of Tokyo University, provided the cells. Cell culture methods and protocols accorded with the published note on these cell lines [34 (link)]. Briefly, cells were cultured using Roswell Park Memorial Institute (RPMI) media-1640 (Gibco), l-glutamine solution (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), antibiotics (penicillin-streptomycin; Sigma), and 10 % fetal bovine serum (BI, Biological Industries), and maintained at 37 °C in a controlled-humidity environment with 5 % CO₂. The cells were then stored in liquid nitrogen with a media supplement (CultureSure®, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan). Cold phosphate-buffered saline (PBS) and 0.25 % trypsin or 0.1 % EDTA were applied during the subculture. Cells were counted using an automated cell counter (LUNA II, Logos, USA).

Bilateral uterine horns were isolated from C57BL/6 J mice at approximately 10 weeks of age. The tissues were minced into 2–3 mm pieces, washed several times with cold PBS, and dissociated with 2 U/mL dispase II and 1 mg/mL collagenase P (Roche Diagnostics K.K., Tokyo, Japan) for 30 min at 37 °C. Dissociated cells were subjected to the Matrigel bilayer organoid culture (MBOC) protocol [22 (link)]. Briefly, cells were resuspended in the medium supplemented with L-glutamine solution (Wako, Osaka, Japan), penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO), amphotericin B suspension (Wako), 50 ng/mL EGF (Peprotech, Rocky Hill, NJ), 250 ng/mL R-spondin1 (R&D, Minneapolis, MN), 100 ng/mL Noggin (Peprotech), 10 μM Y27632 (Wako, Osaka, Japan), 1 μM Jagged-1 (AnaSpec, Fremont, CA), and 2.5 μM CHIR-99021 (Cayman Chemical, Ann Arbor, MI), and plated on 65 μL of solidified Matrigel (BD Biosciences, Franklin Lakes, NJ) per well in a 12-well plate. After overnight incubation at 37 °C, floating or dead cells were removed, leaving only viable cells attached onto Matrigel, which were covered with 70 μL of Matrigel and the medium. Passage was conducted every 5–10 days at 1:2 to 1:3 dilution. In each passage, organoids were dissociated using Accutase (Innovative Cell Technologies, San Diego, CA) for 5 min at 37 °C and vigorous pipetting, followed by seeding on Matrigel.