*2.3. TCS Inhibits HCC Tumor Growth In Vivo*

To investigate the therapeutic effect of TCS on HCC in vivo, an H22 HCC xenograpft model was established subcutaneously in BALB/c mice, which were treated with TCS at Day 5, 7, 9, 11, 13, 15 and 17 (Figure 3A). TCS treatment significantly inhibited the growth of HCC tumors in mice in a dose-dependent manner (Figure 3B). The application of TCS at the highest concentration of 2 μg/g achieved a tumor volume inhibition rate of about 52.91% (Figure 3C) and a tumor mass inhibition rate of about 55.01% (Figure 3D,E). Although the mice treated with TCS exhibited a decreasing trend in body weight, there was no significant

difference compared to the control group. These results suggested that TCS could inhibit HCC cell growth in vivo.

**Figure 3.** Effects of TCS on apoptosis and autophagy of HCC cells. (**A**) Timeline schedule of mice injected with HCC cells and treated with TCS; (**B**) On day 19, mice were euthanized and tumor tissues were obtained as shown in the figure; (**C**–**E**) Quantitation of data showed the volume of tumors, weight and the inhibition rate of TCS on the weight of tumor tissue; (**F**) Quantitation of data showed the weight changes in mice treated with varying doses of TCS. \*\*\*, *p* < 0.001.

Next, we assayed Ki67, a tumor proliferation marker [30], by immunohistochemical fluorescence. The number of Ki67-positive cells decreased in the TCS-treated group in a dose-dependent manner (Figure 4A). TCS significantly promoted the activation of Caspase 9, Caspase 8 and Caspase 3 in tumor tissues. The levels of Cleaved-caspase 9, Cleavedcaspase 8 and Cleaved-caspase 3 were significantly increased (Figure 4B). Therefore, TCS could inhibit the progression of HCC malignancy in mice, mainly through activation of the caspase pathway.

**Figure 4.** Effects of TCS on proliferation and apoptosis of HCC in vivo. (**A**) Immunofluorescence method detected nuclear proliferation factor Ki67 (Ki67: red, DAPI: blue). Bar = 100 μm; (**B**) Western blot method detected the protein levels of Caspase 9, Cleaved-caspase 9, Caspase 8, Cleaved-caspase 8, Caspase 3 and Cleaved-caspase 3 in HCC tissues. \*, *p* < 0.05; \*\*, *p* < 0.01; \*\*\*, *p* < 0.001.
