Epidermal growth factor (egf)

Human melanoma stem cells and breast cancer stem cells were sorted from MDA-MB-435 and MCF-7 cells using aldehyde dehydrogenase 1 (ALDH1), which were further confirmed by tumorsphere formation ability and in vivo tumorigenicity in our previous study [27 (link)]. Cancer stem cells were cultured in DMEM/F-12 medium (Invitrogen, USA) supplemented with 20 ng/ml epidermal growth factor (Beyotime Biotechnology, Jiangsu, China), 10 ng/ml basic fibroblast growth factor (Beyotime, China), 5 μg/ml of insulin (Beyotime, China), and 2% of B-27 (Sigma, USA) at 37 °C in a humidified atmosphere with 5% CO₂. Melanoma and breast cancer non-stem cells were cultured in Leibovitz’s L-15 medium (Sigma, USA) supplemented with 10% fetal bovine serum (FBS) at 37 °C with 100% humidified atmosphere.

Tumorsphere formation assay was conducted under non-adherent and serum-free conditions. The cells were suspended in DMEM/F-12 medium (Invitrogen, USA). Then, a single cell was plated into an ultralow adherent 96-well plate and cultured in DMEM/F-12 medium supplemented with 20 ng/ml epidermal growth factor (Beyotime, China), 10 ng/ml basic fibroblast growth factor (Beyotime), 5 μg/ml of insulin (Beyotime), and 2% of B27 (Sigma, USA). The cells were cultured for 15 days and examined under a light microscope every 5 days.

The melanoma stem-like cells were a gift from Dr. Zhang’s lab. The sorting of melanoma stem-like cells was conducted using an ALDEFLUORTM kit (Cyagen Biosciences Inc., USA) for the detection of melanoma stem cell marker, aldehyde dehydrogenase 1 (ALDH1) [19 (link)]. Briefly, MDA-MB-435 cells were suspended in ALDEFLUOR assay buffer containing ALDH1 fluorescent substrate BODIPY-aminoacetate (BAAA, 1 μM) and incubated for 40 min at 37 °C. After incubation, the cells were centrifuged at 250×g for 5 min followed by removing the supernatant; the cell pellet was resuspended in 0.5 mL of ALDEFLUOR assay buffer and stored at 4 °C for fluorescence-activated cell sorting (FACS). The ALDH1-positive cells were termed as melanoma stem-like cells (MSLC), and the rest were noted as melanoma non-stem-like cells (non-MSLC). The sorted melanoma stem-like cells were maintained immediately in DMEM/F-12 medium supplemented with 20 ng/mL epidermal growth factor (Beyotime, China), 10 ng/mL basic fibroblast growth factor (Beyotime, China), 5 μg/mL of insulin (Beyotime, China), and 2% of B27 (Sigma, USA).

Tumorsphere formation assay was conducted under non-adherent and serum-free conditions. To perform the tumorsphere formation assay, the cells were firstly transfected with indicated siRNAs/miRNAs for 6 h. After that, the cells were counted and seeded in 6-well ultralow adherent cell culture plate, and the number of cells was 5000 in each well. The cells were cultured in DMEM/F-12 medium supplemented with 20 ng/mL epidermal growth factor (Beyotime, China), 10 ng/mL basic fibroblast growth factor (Beyotime, China), 5 μg/mL of insulin (Beyotime, China), and 2% of B27 (Sigma, USA). Seven days after seeding, the tumorspheres were (each tumorsphere should contain at least 5 cells) detected and analyzed.

A549 and H1299 human lung cancer cell lines were obtained from the Stem Cell Bank of the Chinese Academy of Sciences. Cells were cultured with Dulbecco’s modified Eagle medium (DMEM) supplemented with 1% amphotericin B, 1% penicillin–streptomycin, and 10% fetal bovine serum. A549 and H1299 CSC derivative cell lines, the A549-oncosphere, and the H1299-oncosphere were generated as previously described^{38 (link)}. Cells were cultured in the DMEM/F12 supplemented with 2% B27, 1% amphotericin B, and 1% penicillin–streptomycin, with 20 ng/ml epidermal growth factor (Beyotime) and 20 ng/ml fibroblast growth factor (Beyotime).

Cells were suspended in DMEM/F-12 medium (Gibco, New York, NY, USA) to carry out tumorsphere formation assays in a non-adherent and serum-free environment. A single cell was grown in an ultralow adherent 96-well plate containing serum-free DMEM/F-12 (Gibco, New York, NY, USA) medium supplemented with 2% B-27 (Sigma, St. Louis, MO, USA), a 100 U mixture of the pen/strep (Shijiazhuang Pharmaceutical Group Co., Ltd., Shijiazhuang, China), 20 ng/mL epidermal growth factor, 10 ng/mL essential fibroblast growth factor, and 5 μg/mL insulin factor (Beyotime Biotechnology, Shanghai, China). The cells were observed every day under a light microscope.