Isolation and Biological Evaluation of Breast Cancer Side Population Cells Using DyeCycle Violet

Background/Aim: Breast cancer is the most frequently diagnosed malignancy in women and remains a leading cause of cancer-related death. Tumor heterogeneity contributes to therapeutic resistance and recurrence, and cancer stem cells (CSCs) are implicated in tumor maintenance. As one approach to enrich CSCs, side population (SP) analysis isolates cells with high drug efflux activity. DyeCycle Violet (DCV), a less cytotoxic alternative to Hoechst 33342, has been used for SP analysis, but data in breast cancer are limited. We compared cancer stem cell-like properties of SP and main population (MP) cells isolated from a breast cancer cell line.

Materials and Methods: The human breast cancer cell line MCF-7 was analyzed using flow cytometry with the fluorescent dye DCV and the ABCG2 inhibitor Fumitremorgin C to isolate SP and MP cells. After sorting, cell proliferation was assessed by the CCK-8 assay and invasion by a cell invasion assay. Sorted cells were recultured short- or long-term and drug efflux profiles were re-analyzed.

Results: DCV analysis identified an SP fraction that disappeared upon inhibitor treatment. SP and MP cells showed comparable proliferation, whereas SP cells exhibited higher invasive ability. Short-term reculture showed a marked shift from the SP to the MP fraction; after long-term reculture, both fractions converged toward a distribution similar to the parental population.

Conclusion: SP analysis using DCV in combination with Fumitremorgin C is feasible in breast cancer cells and enables low-cytotoxic isolation of efflux-defined subpopulations for functional analyses of cancer stem cell-like properties.