*3.2. Fractionated Doses of Radiation Induce Cellular Plasticity by Regulating EMT*

An increase in E-BCSC signature in our study prompted us to further analyze the EMT markers. Fractionated irradiation caused the induction of MET, as levels of the epithelial marker E-cadherin were observed to be increased and the levels of mesenchymal markers Vimentin, SLUG and SNAIL were found to be decreased significantly only in BCSCenriched mammospheres but not in MCF-7 cells (Figure 2A,B), thus inducing plasticity toward epithelial phenotype.

**Figure 1.** Effect of fractionated doses of radiation on breast cancer stem cell (BCSC) population induction and epithelial– mesenchymal transition (EMT). (**A**) BCSC population was identified in MCF-7 (left) and MDA-MB-231 cells (right) irradiated with a fractionated and acute dose of radiation by assessing ALDH activity and (**B**) CD44/CD24 markers using flow cytometry in MCF-7 (left) and MDA-MB-231 cells (right) after irradiation. (**C**) Expression of stem cell markers, i.e., NANOG and SOX2, was analyzed by Western blotting. (**D**) Phase-contrast images depict the effect of a fractionated and acute dose of radiation on sphere formation. All values are given as the mean ± SE, \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs.

*3.2. Fractionated Doses of Radiation Induce Cellular Plasticity by Regulating EMT*

An increase in E-BCSC signature in our study prompted us to further analyze the EMT markers. Fractionated irradiation caused the induction of MET, as levels of the epithelial marker E-cadherin were observed to be increased and the levels of mesenchymal markers Vimentin, SLUG and SNAIL were found to be decreased significantly only in BCSC-enriched mammospheres but not in MCF-7 cells (Figure 2A,B), thus inducing plas-

control. All images are representative of three independent experiments.

ticity toward epithelial phenotype.

**Figure 2.** Effect of fractionated doses of radiation on EMT. (**A**) Expression of EMT markers, i.e., E-cadherin, Vimentin, SLUG and SNAIL, were analyzed by Western blotting and (**B**) qRT-PCR of E-cadherin, Vimentin, SLUG and SNAIL. GAPDH is used as loading control. All values are given as the mean ± SE, \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\**p <0.0001* vs. control. All images are representative of three independent experiments. **Figure 2.** Effect of fractionated doses of radiation on EMT. (**A**) Expression of EMT markers, i.e., E-cadherin, Vimentin, SLUG and SNAIL, were analyzed by Western blotting and (**B**) qRT-PCR of E-cadherin, Vimentin, SLUG and SNAIL. GAPDH is used as loading control. All values are given as the mean ± SE, \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\**p* < 0.0001 vs. control. All images are representative of three independent experiments.

*Irradiation*

*3.3. BCSCs with High ALDH<sup>+</sup> Activity Display Radioresistance Upon Exposure to Fractionated* 

assay to analyze the relative radioresistance of BCSCs. A single-cell suspension of MCF-7 cells was plated and irradiated with an acute dose (6 Gy) and fractionated doses (2 Gy x 3 days) of γ-rays. Our clonogenic survival assay demonstrated significantly higher radioresistance in MCF-7 and MDA-MB-231 cells and their corresponding mammospheres upon exposure to fractionated doses of radiation compared to controls (Figure 3A,B). Not only did the number of the colonies formed increase significantly after fractionated irradiation but also proliferative capacity, as indicated by Ki67 staining, was higher in these cells (Figure 3C). Ionizing radiation significantly increased the proportion of these CSCs and also showed enhanced proliferation shortly after treatment, further resulting in rapid tumor repopulation [25]. As there was an increase in the proliferation in cancer cells and mammospheres after fractionated irradiation, we further assessed apoptosis and the expression of anti- and proapoptotic genes, BCL2 and BAX. Although there was no significant
