Snu 8

A total of 67 formalin-fixed, paraffin-embedded (FFPE) samples from patients diagnosed with ovarian clear cell carcinoma were retrieved from the sample archives and were anonymized. This study was approved by the Institutional Review Board of the Samsung Medical Center by an informed consent waiver using the anonymized archival tissues with retrospective clinical data. Ovarian adenocarcinoma or clear cell adenocarcinoma cell lines were obtained from 3 different institutes: SNU-8 and SNU-119 from the Korean Cell Line Bank (KCLB) (Seoul, South Korea); SKOV3, A2780, ES2, RMGI, and TOV21G from the American Type Culture Collection (ATCC, Manassas, VA); and OVMANA, OIVSE, OVSAHO, and OVTOKO from the Health Science Research Resources Bank (HSRRB) (Tokyo, Japan). OCCC cell lines are OVMANA, RMGI, TOV21G, OVTOKO, ES2 and OVISE. TOV21G was cultured in a 1∶1 mixture of MCDB115 and R119 media supplemented with 10% serum and antibiotics (100 µg/mL penicillin and streptomycin). The other cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics.

Four human HGSC cell lines (CaoV-3, NIH OVCAR-3, SNU-8, and SNU-119) were purchased from the Korean Cell Line Bank (Seoul, Korea). Cells were cultured in RPMI 1640 medium (Welgene, Daegu, Korea) containing 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA) and 1% penicillin/streptomycin (Gibco) and maintained at 37 °C in a humidified incubator with 5% CO2. Full-length cDNA encoding LGR5 (pEX-LGR5) and control vector were purchased from GeneCopoeia (Rockville, MD). Cancer cells were seeded at 1 × 10⁵ cells/well in 6-well plates after transfection with control vector or pEX-LGR5 (2.5 μg) using the Neon transfection system (Thermo Fisher Scientific). One or two days after transfection, the cells were subjected to real-time PCR, immunoblotting or functional assays.