*3.2. Radiation-Enriched CD44+ Cells Exhibited the Properties of CSCs Including an Increase in SOX2 Expression*

To delineate the role of radiation-induced CD44 expression in radioresistant colorectal cancer cells, we isolated both CD44 positive (CD44+) and negative (CD44−) cells in HCT116 and DLD1 cells following irradiation using anti-CD44-FITC antibodies by FACS, and the expression of CD44 in both CD44+ and CD44− cells is shown in Figure 2A. Since the CD44 marker correlated with the features of CSCs in colorectal cancers [19,20], we evaluated the properties of colorectal CSCs including metastatic potential and self-renewal. We observed an increase in colony formation, migration and invasion in the sorted CD44+ cells after irradiation and not in CD44− cells in both cell lines (Figure 2B–D). Interestingly, immunoblotting of stemness-related proteins revealed significant elevation in SOX2 levels among stemness-related proteins [21,22] on sorted CD44+ cells (Figure 2A). Given the evidence that SOX2 was aberrantly expressed and involved in the maintenance of CSCs in colorectal cancer [14,15], these results indicated the possibility of a functional relationship between SOX2 expression and CD44-mediated CSC property in radioresistant cells upon radiation exposure.

sistant cells upon radiation exposure.

SOX2 levels among stemness-related proteins [21,22] on sorted CD44+ cells (Figure 2A). Given the evidence that SOX2 was aberrantly expressed and involved in the maintenance of CSCs in colorectal cancer [14,15], these results indicated the possibility of a functional relationship between SOX2 expression and CD44-mediated CSC property in radiore-

**Figure 1.** Enriched population of CD44 positive (CD44+) cells was found in radioresistant cells and not in radiosensitive cells of colorectal cancer in response to radiation. (**A**) The apoptotic cells on day 2 after irradiation with 10 Gy were measured by annexin V staining and flow cytometry analysis in various types of colorectal cancer cell lines including both radioresistant cells (HT29, DLD1, and HCT116) and radiosensitive cells (SW480, RKO, and LoVo). Data are shown as mean ± SD (*n* = 3) with \* *p* < 0.05 for the pairwise comparisons between radioresistant cells and radiosensitive cells. (**B**) Colony formation assay was performed with indicated cells treated with 4 Gy (left panel). Graph showing quantification of relative colony numbers at different doses of IR (right panel). (**C**) Cell populations for the CD44+, CD133+, or ALDH+, which are known markers of cancer stem-like cells (CSC) in these indicated cells after radiation exposure were measured by flow cytometric analysis. The percentage of each CSC marker-expressing cell is shown as a bar graph. Data are shown as mean ± SD (*n* = 3) with \* *p* < 0.05 for the pairwise comparisons between radioresistant cells and radiosensitive cells. (**D**) Cells were stained with an anti-CD44 antibody (green) and anti-CD133 antibody (red). Nuclei were counterstained with DAPI ((blue). CSCs: cancer stem-like cells. **Figure 1.** Enriched population of CD44 positive (CD44+) cells was found in radioresistant cells and not in radiosensitive cells of colorectal cancer in response to radiation. (**A**) The apoptotic cells on day 2 after irradiation with 10 Gy were measured by annexin V staining and flow cytometry analysis in various types of colorectal cancer cell lines including both radioresistant cells (HT29, DLD1, and HCT116) and radiosensitive cells (SW480, RKO, and LoVo). Data are shown as mean ± SD (*n* = 3) with \* *p* < 0.05 for the pairwise comparisons between radioresistant cells and radiosensitive cells. (**B**) Colony formation assay was performed with indicated cells treated with 4 Gy (left panel). Graph showing quantification of relative colony numbers at different doses of IR (right panel). (**C**) Cell populations for the CD44+, CD133+, or ALDH+, which are known markers of cancer stem-like cells (CSC) in these indicated cells after radiation exposure were measured by flow cytometric analysis. The percentage of each CSC marker-expressing cell is shown as a bar graph. Data are shown as mean ± SD (*n* = 3) with \* *p* < 0.05 for the pairwise comparisons between radioresistant cells and radiosensitive cells. (**D**) Cells were stained with an anti-CD44 antibody (green) and anti-CD133 antibody (red). Nuclei were counterstained with DAPI ((blue). CSCs: cancer stem-like cells.

#### *Colorectal CSCs Following Irradiation*  We further determined whether the expression of either CD44 or SOX2 in response *3.3. Modulation of SOX2 Expression in Colorectal Cancer Cells Is Associated with Induction of Colorectal CSCs Following Irradiation*

*3.3. Modulation of SOX2 Expression in Colorectal Cancer Cells Is Associated with Induction of* 

to radiation is dependent on radioresistant colorectal cancer cells. These proteins were upregulated by irradiation in radioresistant, but not radiosensitive colorectal cancer cells (Figure 3A). To further clarify the role of radiation induced SOX2 in regulating colorectal CSCs, we examined the effect of SOX2 siRNA on the properties of CSCs in both HCT116 and DLD1 cells. Immunoblotting analysis in Figure 3B showed the efficient knockdown of SOX2 expression in both cells with SOX2 siRNA treatment. In addition, the knockdown of SOX2 attenuated the radiation-induced properties of CSCs, including the enhanced ability to migrate, invade, and form tumourspheres, and reduced CD44+ population We further determined whether the expression of either CD44 or SOX2 in response to radiation is dependent on radioresistant colorectal cancer cells. These proteins were upregulated by irradiation in radioresistant, but not radiosensitive colorectal cancer cells (Figure 3A). To further clarify the role of radiation induced SOX2 in regulating colorectal CSCs, we examined the effect of SOX2 siRNA on the properties of CSCs in both HCT116 and DLD1 cells. Immunoblotting analysis in Figure 3B showed the efficient knockdown of SOX2 expression in both cells with SOX2 siRNA treatment. In addition, the knockdown of SOX2 attenuated the radiation-induced properties of CSCs, including the enhanced ability to migrate, invade, and form tumourspheres, and reduced CD44+ population growth (Figure 3B–D). Next, we examined the effects of SOX2 overexpression. Upon irradiation, the overexpression of SOX2 facilitated the acquisition of the properties of colorectal CSCs in radiosensitive colorectal cancer cells (SW480 and LoVo) due to an increase in CD44+ population, cell survival, migration, invasion, and tumoursphere-formation (Figure 4). Taken together, these results suggested that SOX2 regulated population growth **Figure 2** 

and properties of CSCs in colorectal cancer following irradiation, and SOX2 may be a potential target for studies involving resistance to radiation.

**Figure 2.** CD44+ cells induced by radiation exhibited the properties of cancer stem-like cells (CSCs) with an increase in SOX2 levels. (**A**) CD44+ CD44− cells on day 2 after irradiation with 10 Gy in radioresistant colorectal cancer cells (HCT116 and DLD1) were sorted (left panel). Immunoblotting for the expression of CSC-related proteins in CD44+ (positive) and CD44− (negative) in radioresistant cells (right panel). (**B**) Colony formation assay was performed with CD44+ (or CD44−) cells, and the bar graphs show the quantification of relative colony numbers in indicated cells. Data are shown as mean ± SD (*n* = 3) \* *p* < 0.05 compared to control. (**C**,**D**) The migration and invasion analysis (left panel) and quantification of cells involved in migration and invasion (right panel) in CD44+ and CD44− cells sorted from HCT116 and DLD1 cells, respectively. All experiments were performed in triplicates. Data are shown as mean ± SD. \* *p* < 0.05 compared to CD44− cell. CD44−: negative, CD44+: positive, CSCs: cancer stem-like cells.
