Xie Hang, Wu Yujie, Huang Jingyao, Shen Quan, Li Xiaoyan, Wang Lili, Lin Junqing, Chi Zhen, Ke Kun, Lin Xin, Chen Rong, Liao Rihua, Li Yong, Huang Ning
Department of Interventional Radiology, Fujian Medical University Union Hospital, Fuzhou, China.
Pathology Department, Fujian Medical University Union Hospital, Fuzhou, China.
Immunol Invest. 2025 Apr;54(3):382-395. doi: 10.1080/08820139.2024.2445608. Epub 2025 Jan 2.
Liver cancer (LC) is a deadly malignancy with limited therapeutic options in recent years. Natural killer cell-derived exosomes (NK-exo), as an important bridge of information transmission between cells, also have a certain killing effect on tumor cells. On this basis, this study investigated the specific regulatory mechanism of NK-exo on LC cells.
NK-exo was collected by differential centrifugation. The diameter and size distribution were characterized by dynamic light scattering (DLS), respectively. Western Blot (WB) assay detected the expression levels of exosome marker protein, PD-L1, and PI3K-AKT-mTOR signal-related proteins. The effect of NK-exo treatment on LC cell viability was measured by the CCK-8. With the use of CFDA·SE, we assessed the proliferation ability of CD8T cells in direct co-culture with LC cells. The content of cytokines secreted by CD8T cells in each treatment group was determined by enzyme-linked immunosorbent assay (ELISA) kits. We employed flow cytometry to analyze the expression of PD-L1 protein on the surface of LC cells and CD8 level in mice tumor tissues.
CCK-8 assay demonstrated that NK-exo repressed the cell viability of LC cells. WB uncovered that the protein expressions of PD-L1, p-AKT, and p-mTOR in NK-exo treated LC cells were decreased, which was returned to the control level after the addition of PI3K agonist. When NK-exo-treated LC cells were directly co-cultivated with CD8T cells, the proliferation ability and cytokine secretion content of T cells were considerably elevated, and the expression of PD-L1 on LC cell surface was considerably reduced. However, these effects were restored to control levels by PI3K agonists.The in vivo experiments also confirmed that NK-exo could effectively inhibit the progression of LC, and the PI3K agonist could restore this effect to the level of the control group.
This study provided the first evidence that exosomes derived from NK cells inhibited the PI3K-AKT-mTOR signaling pathway in LC cells, and reduced PD-L1 expression, thereby promoting tumor immunity. In comparison to traditional immune checkpoint inhibitors, NK-exo possessed unique mechanisms of action and potential advantages. NK-exo holds the promise of becoming an innovative immunotherapy for the treatment of LC.
肝癌（LC）是一种致命的恶性肿瘤，近年来治疗选择有限。自然杀伤细胞衍生的外泌体（NK-exo）作为细胞间信息传递的重要桥梁，对肿瘤细胞也有一定的杀伤作用。在此基础上，本研究探讨了NK-exo对肝癌细胞的具体调控机制。
采用差速离心法收集NK-exo。分别用动态光散射（DLS）表征其直径和大小分布。蛋白质免疫印迹法（WB）检测外泌体标志物蛋白、程序性死亡受体配体1（PD-L1）以及磷脂酰肌醇-3激酶-蛋白激酶B-哺乳动物雷帕霉素靶蛋白（PI3K-AKT-mTOR）信号相关蛋白的表达水平。用细胞计数试剂盒（CCK-8）检测NK-exo处理对肝癌细胞活力的影响。利用羧基荧光素二乙酸琥珀酰亚胺酯（CFDA·SE），我们评估了与肝癌细胞直接共培养的CD8⁺T细胞的增殖能力。通过酶联免疫吸附测定（ELISA）试剂盒测定各治疗组中CD8⁺T细胞分泌的细胞因子含量。我们采用流式细胞术分析肝癌细胞表面PD-L1蛋白的表达以及小鼠肿瘤组织中CD8水平。
CCK-8检测表明，NK-exo抑制了肝癌细胞的活力。WB显示，经NK-exo处理的肝癌细胞中PD-L1、磷酸化蛋白激酶B（p-AKT）和磷酸化哺乳动物雷帕霉素靶蛋白（p-mTOR）的蛋白表达降低，添加PI3K激动剂后恢复到对照水平。当经NK-exo处理的肝癌细胞与CD8⁺T细胞直接共培养时，T细胞的增殖能力和细胞因子分泌量显著升高，肝癌细胞表面PD-L1的表达显著降低。然而，这些作用通过PI3K激动剂恢复到对照水平。体内实验也证实，NK-exo能有效抑制肝癌的进展，PI3K激动剂可将这种作用恢复到对照组水平。
本研究首次证明，自然杀伤细胞来源的外泌体抑制肝癌细胞中的PI3K-AKT-mTOR信号通路，并降低PD-L1表达，从而促进肿瘤免疫。与传统免疫检查点抑制剂相比，NK-exo具有独特的作用机制和潜在优势。NK-exo有望成为治疗肝癌的创新免疫疗法。
