Identification and Cell Cycle Analysis of Cancer Stem Cells

Cancer stem cells were identified as ALDH^highCD44^high cells, as we described [28 (link)-30 (link)]. Single-cell suspensions were obtained from the digestion of xenograft tumors or from the UM-HMC-3B cell line. Cells were counted, resuspended at 2×10⁶ cells/ml PBS, and incubated with activated Aldefluor^® substrate (BAA) or the ALDH inhibitor (DEAB) for 45 minutes at 37°C, using the Aldefluor^® kit (StemCell; Vancouver, BC, Canada). Cells were exposed to anti-CD44 antibody (APC-Cat #559942, PE-Cat #550989) for 30 minutes at 4°C. Anti-HLA-ABC (PE-Cat #560168; BD Pharmingen) was used to separate human cells from mouse cells, and 7-AAD (Cat #00-6993-50; eBiosciences, San Diego, CA, USA) staining was used to exclude dying cells. To measure the effect of drugs on cell cycle, UM-HMC-3B cells were treated with tocilizumab, cisplatin, paclitaxel, or controls diluted in cultured medium. After 24 hours, cells were retrieved, exposed to a hypotonic solution of propidium iodide containing 0.1% sodium citrate, 25 μg/ml propidium iodide, 100 μg/ml RNase A, and 0.1% Triton X-100. Cell cycle analysis was performed by flow cytometry, as described [50 (link)].