ESCC cells cultured in ultralow attachment plates mimicking the matrix detachment conditions were harvested and collected to produce a single-cell suspension. For anoikis measurement, cells were stained with an Annexin V-FITC and PI kit (KeyGEN, Nanjing, China) before measurement with flow cytometer.
For anchorage-independent growth, 2 × 103 cells were plated in 96-well ultralow attachment plates (7007, Corning) with serum-free DMEM-F12 medium with 20 ng/ml epidermal growth factor (EGF) (100-47, Peprotech), 20 ng/ml basic fibroblast growth factor (bFGF) (100-18B, Peprotech), 10 μg/ml heparin (H3149, Sigma), and 2% B27 (17504044, Gibco). Then, spheres greater than 50 μm in diameter in each well were counted under a microscope after 7 days.
For anchorage-independent growth, 2 × 103 cells were plated in 96-well ultralow attachment plates (7007, Corning) with serum-free DMEM-F12 medium with 20 ng/ml epidermal growth factor (EGF) (100-47, Peprotech), 20 ng/ml basic fibroblast growth factor (bFGF) (100-18B, Peprotech), 10 μg/ml heparin (H3149, Sigma), and 2% B27 (17504044, Gibco). Then, spheres greater than 50 μm in diameter in each well were counted under a microscope after 7 days.