*3.1. Fractionated Doses of Radiation Selectively Increase E-BCSC Population While Decreasing M-BCSC Population*

Recent studies indicate that BCSCs exist in two phenotypes, i.e., epithelial (E-BCSC) and mesenchymal (M-BCSC), and BCSC plasticity plays a crucial role in future strategies for therapeutic resistance [19]. E-BCSCs characterized as ALDH<sup>+</sup> population are proliferative, locate in the tumor's hypoxic region and show the MET phenotype. On the other hand, M-BCSCs that express the CD44+/24<sup>−</sup> phenotype are primarily quiescent, located on the invasive front and have the EMT phenotype. Previous studies have shown an increase in CD44+/24<sup>−</sup> cells and high ALDH<sup>+</sup> characteristics of tumor-initiating or cancer stem cells in breast tumors and established cell lines after irradiation [20–23]. In our study, fractionated irradiation with 2 Gy x 3 days of γ-rays increased the population of ALDH<sup>+</sup> cells (Figure 1A) but decreased CD44+/24<sup>−</sup> cells (Figure 1B) in MCF-7 and MDA-MB-231 cells and their corresponding mammospheres. Since mammospheres render an enriched BCSC population [24], we characterized these mammospheres by quantifying embryonic stem cell markers, SOX2 and NANOG. Compared to the MCF-7 cells, MCF-7-derived mammospheres express significantly high levels of SOX2 and NANOG, indicating the enriched BCSC population (Figure S1). Similar to ALDH activity, the expression of embryonic stem cell markers, i.e., SOX2 and NANOG in MCF-7 cells and mammospheres, and mammosphere formation efficiency (MFE) in MCF-7 cells was also increased upon exposure to fractionated doses of radiation (Figure 1C,D). Collectively, these results suggest that exposure of fractionated doses to radiation induces the E-BCSC phenotype in mammospheres, which may contribute to radioresistance and promote tumor recurrence.
