DataSheet3_Biejiajian Pill Promotes the Infiltration of CD8+ T Cells in Hepatocellular Carcinoma by Regulating the Expression of CCL5.docx

Tumor-infiltrating CD8+T lymphocytes are mostly associated with a favorable prognosis in numerous cancers, including hepatocellular carcinoma (HCC). Biejiajian Pill (BJJP) is a common type of traditional Chinese medicine that is widely used in the treatment of HCC in China. Previous studies showed that BJJP suppressed the growth of HCC cells both in vivo and in vitro, by exerting direct cytotoxic effects on tumor cells. The present study demonstrated that in addition to direct cytotoxicity, BJJP inhibits the growth of tumor cells by promoting the infiltration of CD8+T cells into the tumor in H22-bearing mice. Mechanistically, chemokine ligand 5 (CCL5) was identified as one of the most highly expressed chemokines by tumor cells in vivo after treatment with BJJP. Additionally, CCL5 was knocked down in H22 cells and the results showed that knockdown of the gene significantly impaired the infiltration of CD8+T cells in vivo. Furthermore, the effects of BJJP on human HCC cell lines were assessed in vitro. Similarly, cells treated with BJJP had higher expression of CCL5 mRNA, which was consistent with increased levels of CCL5 protein in human tumor cells. These findings provide new insights into the anticancer effects of BJJP, which regulated the expression of CCL5 and the infiltration of CD8+T cells. The results, therefore, suggest that BJJP has great potential application in clinical practice.

肿瘤浸润性CD8+T淋巴细胞（tumor-infiltrating CD8+T lymphocytes）在包括肝细胞癌（hepatocellular carcinoma, HCC）在内的多种癌症中，大多与良好的预后密切相关。鳖甲煎丸（Biejiajian Pill, BJJP）是一类常用中药，在我国被广泛用于肝细胞癌的临床治疗。既往研究表明，鳖甲煎丸可通过对肿瘤细胞发挥直接细胞毒性作用，在体内及体外实验中均能抑制肝癌细胞的增殖。本研究证实，除直接细胞毒性外，鳖甲煎丸还可通过促进CD8+T淋巴细胞向荷H22肝癌小鼠的肿瘤组织中浸润，从而抑制肿瘤生长。从机制层面而言，经鳖甲煎丸处理后，体内肿瘤细胞所分泌的趋化因子中，趋化因子配体5（chemokine ligand 5, CCL5）为表达量最高的趋化因子之一。此外，本研究对H22细胞中的CCL5基因进行了敲低实验，结果显示该基因的敲低显著削弱了体内CD8+T淋巴细胞的浸润水平。进一步地，本研究在体外实验中评估了鳖甲煎丸对人肝癌细胞系的干预效果。与体内实验结果一致，经鳖甲煎丸处理的细胞中CCL5信使核糖核酸（messenger RNA, mRNA）的表达水平更高，这与人类肿瘤细胞中CCL5蛋白水平的升高相一致。本研究结果为鳖甲煎丸的抗癌作用机制提供了新视角：其可通过调控CCL5的表达以及CD8+T淋巴细胞的浸润发挥抗癌功效。因此，本研究结果提示鳖甲煎丸在临床实践中具有巨大的应用潜力。