Breast Cancer Side Population Versus CD44 + /CD24 − Cells: Functional and Tumorigenic Differences

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Background/Aim: Cancer stem cells (CSCs) are heterogeneous and exhibit distinct phenotypic and functional characteristics. The side population (SP) and CD44+/CD24− phenotypes are widely used to enrich breast cancer cells with CSC-associated properties; however, whether these approaches identify functionally equivalent populations remains unclear. Building on our previous establishment of a DyeCycle Violet (DCV)-based method for isolating SP cells from MCF-7 breast cancer cells, this study compared the biological characteristics of DCV-defined SP cells and a CD44+/CD24−-enriched fraction. Materials and Methods: SP cells were isolated from MCF-7 cells by fluorescence-activated cell sorting using DCV and fumitremorgin C. A CD44+/CD24−-enriched fraction was isolated by magnetic-activated cell sorting. Parental MCF-7 cells, SP cells, and the CD44+/CD24−-enriched fraction were compared for proliferation and invasion. Tumor-forming ability was evaluated in nude mice following subcutaneous inoculation of 100 or 500 cells. Xenograft tumors were examined histopathologically and immunohistochemically. Results: No significant differences in proliferative activity were observed among parental MCF-7, SP, and the CD44+/CD24−-enriched fraction at either 48 or 72 h. Invasion was highest in the CD44+/CD24−-enriched fraction (87.63±20.29%), followed by SP cells (51.86±6.83%) and parental MCF-7 cells (32.52±9.95%; p=0.001). Tumors developed in all three mice receiving the CD44+/CD24−-enriched fraction at both cell doses, whereas tumor formation was less frequent in the other groups. Xenograft tumors showed morphological and immunohistochemical characteristics consistent with MCF-7 cells. Conclusion: DCV-defined SP cells and the CD44+/CD24−-enriched fraction exhibited distinct biological characteristics. The CD44+/CD24−-enriched fraction showed high invasive activity and frequent tumor formation, whereas SP cells showed relatively high proliferative activity and retained tumor-forming capacity under the experimental conditions examined. These findings suggest that SP and CD44+/CD24− enrichment methods select populations with different functional properties.