Leukemia inhibitory factor

The tibias and femurs from 8-week-old male C57BL/6 mice were extracted, and the bone
marrow was flushed as described previously^{43 (link)}. Bone marrow mononuclear cells (BMNCs) were then isolated by density-gradient
centrifugation with Ficoll-Paque PREMIUM 1.084 (GE Healthcare, Little Chalfont, United
Kingdom). CD133⁺ cells were separated from BMNCs with anti-mouse-prominin-1
microbeads (Miltenyi MACS; Miltenyi Biotec, Auburn, CA, USA) using a magnetically
activated cell sorter (Miltenyi Biotec). Freshly isolated cells were seeded at
2×10⁵ cells/well in 24-well plates (NEST, China) pre-coated with 0.015 mg/ml
poly-L-lysine (PLL; Sigma-Aldrich) and vitronectin (Gibco, Invitrogen, Carlsbad, CA, USA).
The cells were then cultured in alpha minimal essential medium (αMEM) supplemented with
10% fetal bovine serum (FBS), 100 IU/ml penicillin, 100 mg/ml streptomycin (Gibco), 100
ng/ml stem cell factor, 100 ng/ml FMS-like tyrosine kinase-3 ligand, 20 ng/ml
interleukin-6 and 20 ng/ml leukemia inhibitory factor (PeproTech, Rocky Hill, NJ, USA)
according to previous studies with slight modifications^39,44–46 at 37°C in a
humidified atmosphere containing 5% CO₂. The cells were harvested with 0.05%
trypsin/ethylenediaminetetraacetic acid (EDTA; Invitrogen) before passaging.

NSC were derived and cultured as described [22 (link)]. After harvesting from rosettes, NSC were grown on Matrigel (Corning, CB40,234)-coated dishes in Neural Proliferation Medium (Life Technologies,21,103–049). Neurobasal medium was supplemented with 1XB27 (Life Technologies, 17,504–044), 2 mM L-glutamine (Gibco, 35,050–061), 10 ng/mL leukemia inhibitory factor (Peprotech, 300–05), 25 ng/mL bFGF (Peprotech, 100–18B), and 1% penicillin-streptomycin (Corning, 15,140–122). NSC were passaged with Accutase (A11105-01) every 7 d, passaging at a 1:3 ratio. Lysates were harvested by cell scrapping in MPER (Thermo Fisher Scientific, 78,501) with Complete Mini protease inhibitor cocktail (MilliporeSigma, 11,836,170,001), 1% phosphatase Inhibitor Set II (EMD Millipore, 524,625), 1% phosphatase Inhibitor Set III (EMD Millipore, 539,134), 50 μM tricostatin A (MilliporeSigma, T8552), 30 μM sodium butyrate (MilliporeSigma, 303,410), and 30 mM nicotinamide (MilliporeSigma, 72,340). Lysate was sonicated once at 40 mA with 5 s pulses, 5X, with 5 s breaks between each pulse. Sonicated lysate was then centrifuged at 15,294 g for 20 min at 4°C. The supernatant was transfer to a new tube. Protein concentration of the supernatant was determined using the BCA assay (Thermo Fisher Scientific, 23,252).

The SKOV3 human EOC cell line was purchased from the China Center for Type Culture Collection. A previously generated subline marked with green fluorescent protein (SKOV3-GFP) was also used (19 (link)). For MCA formation, tumor cells (SKOV3 or primary ascitic tumor cells) were seeded into 6-well low-adherence plates (cat. no. 3471; Corning Inc.) at 2×l0⁴ cells per well, either alone or co-cultured with CAFs (tumor cells:CAFs = 8:1). The cells were cultured in serum-free DMEM/F12 (2 ml per well) containing 2% B27 (cat. no. 12587010; Gibco; Thermo Fisher Scientific, Inc.), 20 ng/ml fibroblast growth factor (cat. no. 100-66-500, PeproTech, Inc.), 20 ng/ml epidermal growth factor (cat. no. AF-100-15-100; PeproTech, Inc.), 10 ng/ml leukemia inhibitory factor (cat. no. 300-05-100; PeproTech, Inc.) and 1% insulin-transferrin-selenium (cat. no. 1933661; Gibco; Thermo Fisher Scientific, Inc.). The MCAs were observed under an inverted microscope (IX73; Olympus Corporation) once daily for 5 days. Images were acquired at a magnification of ×100 and five regions were randomly selected for quantitative evaluation of MCA (three-dimensional cell clusters with a diameter of >50 µm) count and size. All assays were performed in triplicate.