Qin Yan, Wang Fang, Ni Hengli, Liu Yao, Yin Yuan, Zhou Xinyi, Gao Guihua, Li Qing, Qi Xiaowei, Li Jianming
Department of Pathology, Medical College of Soochow University, Soochow University, Suzhou, China.
Department of Pathology, the Affiliated Hospital of Jiangnan University, Wuxi, China.
J Cancer. 2021 Mar 19;12(10):3011-3023. doi: 10.7150/jca.49707. eCollection 2021.
Cancer-associated fibroblasts (CAFs) are principal constituents of the tumor microenvironment (TME) and play a critical role in tumor progression. The CXCL12/CXCR4 axis regulates multiple facets of the TME. The aim of this study was to determine the relationship between CXCL12 expression in CAFs and the malignant progression of gastric cancer (GC). In the GEO (Gene Expression Omnibus) database, we performed transcriptome analysis on paired gastric cancer RNA sequencing samples, and scRNA analysis was performed on advanced malignant GC samples from the scRNA sequencing data set. Fibroblast cells were co-cultured with GC cells, and invasion, migration, epithelial-mesenchymal transformation (EMT) were determined. After blocking the expression of fibroblast CXCL12, cells were co-cultured with a GC cell line. Detection of GC cell line invasion, migration, EMT and CXCR4, Wnt5a and β-Catenin expression levels was performed. Primary CAFs and gastric normal fibroblasts were isolated and CXCL12 mRNA and protein expression were determined. In addition, a cohort of 285 GC cases was established, protein expression was evaluated immunohistochemically, and prognostic results were analyzed. GC transcriptome analysis suggested that cytokine-cytokine receptor interaction and the Wnt signaling pathway in GC tissues were significantly up-regulated. scRNA analysis of advanced malignant GC samples showed that severe intestinal metaplasia (SIM) in GC specimens of different malignant grades had obvious fibroblast clusters compared to non-atrophic gastritis (NAG) and early gastric cancer (EGC). In the SIM group, fibroblast cluster, CXCL12, CXCR4, and Wnt5a were overexpressed. Co-culturing with fibroblast cells significantly increased the invasion, migration, and EMT of GC cells, and blocking CXCL12 in CAFs disturbed the expression of Wnt5a and β-catenin. In our cohort of GC patients, high CXCL12 expression in CAFs significantly correlated with histological grade (P = 0.012) and TNM stage (P = 0.014), as well as with poor overall survival (p = 0.0107). High expression of CXCL12 in CAFs in a GC microenvironment can affect the migration, invasion, and EMT of GC cells. Furthermore, it can cause poor prognosis in patients with GC.
癌症相关成纤维细胞（CAFs）是肿瘤微环境（TME）的主要组成部分，在肿瘤进展中起关键作用。CXCL12/CXCR4轴调节TME的多个方面。本研究的目的是确定CAFs中CXCL12表达与胃癌（GC）恶性进展之间的关系。在基因表达综合数据库（GEO）中，我们对配对的胃癌RNA测序样本进行了转录组分析，并对来自单细胞RNA测序数据集的晚期恶性GC样本进行了单细胞RNA分析。将成纤维细胞与GC细胞共培养，并检测侵袭、迁移和上皮-间质转化（EMT）情况。在阻断成纤维细胞CXCL12的表达后，将细胞与GC细胞系共培养。检测GC细胞系的侵袭、迁移、EMT以及CXCR4、Wnt5a和β-连环蛋白的表达水平。分离原发性CAFs和胃正常成纤维细胞，测定CXCL12 mRNA和蛋白表达。此外，建立了一个包含285例GC病例的队列，通过免疫组织化学评估蛋白表达，并分析预后结果。GC转录组分析表明，GC组织中的细胞因子-细胞因子受体相互作用和Wnt信号通路显著上调。对晚期恶性GC样本的单细胞RNA分析显示，与非萎缩性胃炎（NAG）和早期胃癌（EGC）相比，不同恶性程度的GC标本中的严重肠化生（SIM）有明显的成纤维细胞簇。在SIM组中，成纤维细胞簇、CXCL12、CXCR4和Wnt5a均过表达。与成纤维细胞共培养显著增加了GC细胞的侵袭、迁移和EMT，而阻断CAFs中的CXCL12会干扰Wnt5a和β-连环蛋白的表达。在我们这个GC患者队列中，CAFs中CXCL12的高表达与组织学分级（P = 0.012）和TNM分期（P = 0.014）显著相关，也与总生存期较差（P = 0.0107）相关。GC微环境中CAFs中CXCL12的高表达可影响GC细胞的迁移、侵袭和EMT。此外，它可导致GC患者预后不良。
