Isolation and identification of cancer stem cells from human LACC cell line

Abstract

To find ways to isolate and culture lacrimal adenoid cystic carcinoma (LACC) stem cells from human LACC cell line and to identify their biological properties. Experimental research. LACC cells were digested, centrifuged and suspended in specific serum free medium (SFM) to get LACC cancer stem cell spheres. Limiting dilution analysis and monoclonal formation assay were performed to determine the proportion of cancer stem cells in LACC cell line. MTT assay was used to determine the proliferation and chemotherapeutic drug resistance of stem cell spheres. The expressions of cell surface markers CD44⁺/CD24⁻ were detected by flow cytometry. Stem cell spheres differentiation were induced by dropping serum medium into SFM and the morphologic changes were observed. The IC₅₀ and UV optical density of two kinds of cells were tested by Student's t-test. About (0.92 ± 0.02) % cells were clonogenic in LACC cell line. They could survive and proliferate to form free-floating cell spheres in SFM, which could stably passaged. The IC₅₀ values of cancer stem cells and LACC cell line were 22.53 mg/L and 11.06 mg/L (t = 37.94, P < 0.05), which suggested that cancer stem cells were more resistant to cis-platinum than LACC cell line. In flow cytometry assay, 84.25% stem cell spheres were CD44⁺/CD24⁻ cells, comparing with 23.77% in LACC cell line. Stem cell spheres could differentiate into normal adenoid cystic carcinoma cells in medium with serum. LACC stem cell spheres, containing a large number of cancer stem cells, could be obtained by serum free suspension culture. This provide us an ideal model system for cancer stem cell research.