Effects of thymopentin in promoting T-cell infiltration and inhibiting the growth of subcutaneous hepatocellular carcinoma in mice

This study aimed to investigate the regulatory effect and mechanism of thymopentin on the growth of subcutaneous hepatocellular carcinoma in mice. A subcutaneous tumor model of Hepa1-6 liver cancer in immunocompetent mice was constructed, with three randomly divided groups based on tumor volume: control group, low-dose thymopentin (TP5) group (10 mg/kg), and high-dose TP5 group (20 mg/kg), with 6 mice in each group. Drugs were administered, and the intervention effect of thymopentin on tumor growth was evaluated. Hepa1-6 cells were then cultured in vitro and treated with blank medium and TP5 of different concentrations (10, 100, 1000 ng/mL) for 72 hours. Cell viability was detected by sulforhodamine B (SRB) colorimetry. A subcutaneous tumor model of liver cancer LM3 in immunocompromised mice was constructed, with three randomly divided groups based on tumor volume: control group, TP5 group (20 mg/kg), and positive drug Sorafinib group (30 mg/kg). The intervention effect of thymopentin on the growth of subcutaneous tumors in immunocompromised mice was evaluated. Flow cytometry was used to analyze the changes in the proportion of T cells and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment 11 days after TP5 administration in the Hepa1-6 model. MDSCs were cultured in vitro and treated with TP5. The effect of TP5 on MDSCs was evaluated by detecting the levels of ROS, IL-6, and NO, which are effector molecules of MDSCs. The mouse subcutaneous liver cancer model was established again using C57BL/6N mice. After 10 days, they were randomly divided into four groups based on tumor volume: control group, low-dose TP5 group (10 mg/kg), high-dose TP5 group (20 mg/kg), and arginine-deficient TP5 group (15 mg/kg). Drugs were administered continuously for 11 days, and the intervention effect of arginine-deficient TP5 on tumor growth was evaluated based on tumor weight. Annexin-V staining was used to detect the impact of TP5 on T cell survival. The results showed that both low and high doses of TP5 inhibited the growth of subcutaneous liver cancer in immunocompetent mice (P < 0.05), yet TP5 had no direct inhibitory effect on the proliferation of tumor cells cultured in vitro. Besides, a high dose of TP5 could not inhibit the growth of subcutaneous liver cancer in immunocompromised mice. Furthermore, TP5 promoted the infiltration of CD4 and CD8 T cells but decreased MDSCs in the subcutaneous tumor microenvironment of immunocompetent mice. TP5 did not affect the levels of ROS, IL-6, and NO in MDSCs. Lastly, arginine-deficient TP5 could not inhibit the growth of subcutaneous liver cancer in immunocompetent mice. Accordingly, TP5 but not arginine-deficient TP5 promoted the increase in the proportion of viable CD4 and CD8 T cells cultured in vitro. These results suggest that TP5 may inhibit the growth of liver cancer by increasing T cell number in the liver cancer microenvironment.