Isolating cancer stem cells in breast cancer models: a study of CD marker expression and Rhodamine 123 as predictors of chemoresistance

It is believed that CSCs, which are resistant to chemotherapy and radiation, develop in certain situations, stay in the tissue niche after treatment, and cause disease recurrence. Targeting these cells, which are thought to be chemoresistant, may enable treatment to eliminate metastasis or recurrence. For this reason, we aimed to isolate CSCs from various breast cancer cell lines using a drug selection method in this study and then identify them via flow cytometry and fluorescent staining. MDA-123, MCF-7, and 4T1 were used as in vitro models, with HUVEC as controls. After isolation of stem cells from these cells by incubation with 5-fluorouracil (5-FU) and cisplatin (Cis), CSCs were identified by flow cytometry using CD24/CD44/CD133/EPCAM by assessing the rhodamine 123 (Rho 123). The results showed that 5-FU and Cis reduced the CD24 receptor in both cancer and normal cells, while the others showed varying expression levels. Administration of 5-FU and Cis significantly decreased Rho 123 fluorescence intensity in MDA-MB-231, MCF-7, 4T1, and HUVEC cells compared to the untreated group. A subpopulation of breast cancer cells with low CD24 and Rho123 expression was cancer stem cells. The 4T1 cells, which expressed low levels of CD24/CD133/EpCAM expression, had significantly lower Rho 123 levels than MDA-MB-231 and MCF-7 cells. This suggests that 4T1 cells may have greater chemoresistance and more aggressive properties. In conclusion, studying surface receptors and rhodamine uptake for the identification of CSCs is a powerful and valuable method for evaluating the efficacy of chemotherapy. The discovery of CSC markers that indicate therapeutic strategies could lead to clinical trials of medicines with potentially better therapeutic outcomes, particularly for recurring malignancies. © The Author(s), under exclusive licence to Springer Nature B.V. 2026.