Hydrocortisone

Four adherent LA-derived primary cell lines were seeded for tumorsphere formation. StemXVivo serum-free tumorsphere media (cat# CCM012, R&D Systems, Minneapolis, MN, USA) was supplemented with 2U/mL Heparin (cat# 2812, Tocris, Auckland, New Zealand) and 0.5 µg/mL Hydrocortisone (cat# 4093, Tocris Bioscience, Bristol, UK). T75 Nunclon™ Sphera™ EasYFlasks (cat# 174952, ThermoFisher Scientific, Waltham, MA, USA) were seeded with 24 mL of media containing 1 × 10⁴ live cells per mL as determined by cell counting using the Trypan Blue Exclusion method. They were incubated at 37 °C with 5% CO₂ for 10 days or until initial signs of dark necrotic centers were observed. Feeding occurred every three to four days by the addition of 12 mL of tumorsphere media. Daily measuring was performed from day three by taking three representative images for each cell line. Five typical sphere-like structures from each image were measured in the longest and shortest dimension, and measurements used to establish a nominal average size for each sphere.
A cell line was considered positive for initial tumorsphere formation if the average sphere size was greater than 50 µm, and if at least 50% of measured spheres were greater than 50 µm, according to previously established criteria [44 (link)].

Hepatoblasts generated from progenitor media without 100 ng/ml HGF were maintained in maturation media, modified from Touboul et al.'s protocol [11 (link)] and is called Touboul's hepatocyte maturation media in this manuscript. RPMI-1640 was used as the basal media, along with 25 ng/ml HGF, 20 ng/ml oncostatin M, 1% insulin, 0.1% BSA (Sigma, Cat. no. A6003), 10 μM hydrocortisone, and 5 μM SB 431542 (Tocris, Cat. no. 1614). The progenitor cells were maintained for 5 days, with media changed every day.

Single cell was plated in ultralow attachment 6-well plates (Corning) at 25,000 cells per well for primary tumorsphere formation. After incubation for 7 days, mammospheres were collected and dissociated by trypsin. As the secondary tumorsphere formation, 200 cells per well were plated in ultralow attachment 96-well plates. Cells were grown in StemXVivo Serum-Free Media (R&D) containing 2 U/ml Heparin (Tocris) and 0.8 μg/ml Hydrocortisone (Tocris). Mammospheres were harvested 7 days later, and tumorsphere was calculated under inverted phase-contrast light microscope (Olympus). The experiment was repeated three times. Statistical difference on secondary tumorsphere formation was assessed using student T-test with p ≤ 0.05 as the significance criterion.
When the effect of IL6 on tumorsphere formation was examined, recombinant human IL6 (Peprotech) was supplemented at concentration of 0, 100, 200 ng/μl in the beginning of the primary and secondary tumorsphere formation stages.