Li Yanping, Li Yonglin, Xiang Bin, Tian Xiaomao, Shi Qinlin, Liu Feng, Lin Tao, Wei Guanghui
Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Pediatrics, Chongqing Key Laboratory of Children Urogenital Development and Tissue Engineering, China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Pediatric Research Institute, Children's Hospital of Chongqing Medical University, Chongqing 400014, China.
Department of Pediatric Urology Surgery, Children's Hospital of Chongqing Medical University, Chongqing 400014, China.
J Oncol. 2022 Sep 30;2022:4544773. doi: 10.1155/2022/4544773. eCollection 2022.
The dysfunction of immune surveillance, a hot spot in cancer research, could lead to the occurrence and development in multicancers. However, the potential mechanisms of immunity in Wilms' tumor (WT) remain unclear on Wilms' tumor (WT). In this study, we aim to investigate the immune cell in WT and explore the underlying treatment strategy.
We quantified stromal and immune scores by using ESTIMATE algorithm based on gene expression matrix of WT patients in TCGA and GEO databases. Different expression genes (DEGs) and functional enrichments were analyzed by R studio and DAVID tools. Flow cytometry, immunofluorescence staining, ELISA assay, and qRT-PCR were used for detecting the NK cells, cytotoxic cytokines (INF-, PRF, and GZMB), and NK cell receptor expression, respectively. WT patient autologous NK cells were stimulated by IL-2 and IL-15, and the cytotoxicity of NK cells against WT cell lines was detected by LDH assay. Western blot experiment was used for measuring the MAPK signaling pathway protein maker in NK cells.
ESTIMATE indicated that WT tissue had a lower immune score than adjacent kidney tissue. Meanwhile, the low immune score group was associated with poorly outcomes. DEG functional enrichment analysis showed that NK cell-mediated cytotoxicity was significantly different in low and high immune score groups. Although few of proportion of NK cells in WT patients were increased, most of that were significantly lower than normal children. Moreover, the proportion of NK cells and the expression level of INF-, PRF, and GZMB in WT tissue were lower than adjacent kidney tissue. Importantly, the NKG2D expression level of NK cells was significantly lower in WT tissue. Furthermore, in vitro, compared with uncultured NK cells, IL-2 and IL-15 could effectively enhance the cytotoxicity of NK cells on killing the WT cell lines. The FACS and WB results showed that the NKG2D and p-PI3K ratio PI3K, MEK1/2, and p-ERK1/2 ratio ERK1/2 were significantly increased in IL-2 and IL15 group compared with uncultured groups.
The abnormal NK cell-mediated cytotoxicity may cause the occurrence of WT. Costimulation of WT patients autologous NK cells could effectively enhance the antitumor reaction which involved in activation of NKG2D-mediated MAPK signaling pathway.
免疫监视功能障碍是癌症研究的热点，可导致多种癌症的发生和发展。然而，肾母细胞瘤（WT）的免疫潜在机制仍不清楚。在本研究中，我们旨在研究WT中的免疫细胞并探索潜在的治疗策略。
我们使用ESTIMATE算法基于TCGA和GEO数据库中WT患者的基因表达矩阵对基质和免疫评分进行量化。通过R studio和DAVID工具分析差异表达基因（DEG）和功能富集情况。分别使用流式细胞术、免疫荧光染色、ELISA检测和qRT-PCR检测NK细胞、细胞毒性细胞因子（INF-、PRF和GZMB）以及NK细胞受体的表达。WT患者自体NK细胞用IL-2和IL-15刺激，通过LDH检测法检测NK细胞对WT细胞系的细胞毒性。蛋白质印迹实验用于检测NK细胞中MAPK信号通路蛋白标志物。
ESTIMATE表明WT组织的免疫评分低于相邻肾组织。同时，低免疫评分组与较差的预后相关。DEG功能富集分析表明，NK细胞介导的细胞毒性在低免疫评分组和高免疫评分组中有显著差异。虽然WT患者中NK细胞比例有所增加，但大多数仍显著低于正常儿童。此外，WT组织中NK细胞比例以及INF-、PRF和GZMB的表达水平均低于相邻肾组织。重要的是，WT组织中NK细胞的NKG2D表达水平显著降低。此外，在体外，与未培养的NK细胞相比，IL-2和IL-15可有效增强NK细胞对WT细胞系的杀伤细胞毒性。流式细胞术和蛋白质印迹结果显示，与未培养组相比，IL-2和IL-15组中NKG2D以及p-PI3K与PI3K的比值、MEK1/2和p-ERK1/2与ERK1/2的比值显著增加。
异常的NK细胞介导的细胞毒性可能导致WT的发生。WT患者自体NK细胞的共刺激可有效增强抗肿瘤反应，这涉及NKG2D介导的MAPK信号通路的激活。
