FIGURE 6.

Caffeic acid suppresses the clonogenicity and pluripotency of colorectal CSCs by disrupting the Akt signaling axis in the PDTX model. Schematic representation of the experimental protocol as described in the section “Materials and Methods.” Anesthetized 7 weeks old male NSG mice were inoculated with a 1:1 mixture of Matrigel and 3 × 10⁶ HCT116 cells into the subcutaneous tissue. Next, primary CSC spheres derived from two PDTX mice were exposed to radiation (2Gy) and dissociated into single cells. These cells were subsequently replated onto culture dishes without additional caffeic acid exposure to form secondary CSC spheres. Caffeic acid treatment successfully attenuated radiation-induced sphere formation of colorectal CSCs (A). The percentage of CD44⁺CD133⁺ subpopulations was evaluated by FACS analysis. The treatment of HCT116 cells with caffeic acid (0.3 mM) for 48 h decreased the percentage of CD44⁺ CD133⁺ cells in the total cell population (B). Colorectal CSC spheres derived from two PDTX mice were exposed to radiation (2Gy) with or without additional caffeic acid exposure and the activities of Akt signaling were determined by western blotting (C). The sphere sizes greater than 100 μm were enumerated, and a representative image of a tumor sphere is shown. Abbreviations: TSFE, tumor sphere-forming efficiency. β-actin was used as the internal control. The data represent the mean ± SD of three independent experiments.