Wang Mei, Yu Wanjun, Cao Xiaoli, Gu Hongbing, Huang Jiaying, Wu Chen, Wang Lin, Sha Xin, Shen Bo, Wang Ting, Yao Yongliang, Zhu Wei, Huang Feng
Key Laboratory of Medical Science and Laboratory Medicine of Jiangsu Province, School of Medicine, Jiangsu University, Zhenjiang, China.
Department of Laboratory Medicine, Affiliated Tumor Hospital of Nantong University, Nantong, China.
Front Oncol. 2022 Mar 10;12:860175. doi: 10.3389/fonc.2022.860175. eCollection 2022.
Lymph node metastasis (LNM) commonly occurs in gastric cancer (GC) and is tightly associated with poor prognosis. Exosome-mediated lymphangiogenesis has been considered an important driver of LNM. Whether exosomes directly transmit the LNM phenotype between GC cells and its mechanisms remain elusive.
A highly lymphatic metastatic GC cell line (HGC-27-L) was established by serial passage of parental HGC-27 cells in BALB/c nude mice. The capacities of migration, invasion and LNM; fatty acid oxidation (FAO) levels; and the role of exosome-transferred LNM phenotype were compared among HGC-27-L, HGC-27 and primary GC cell line AGS. Exosomes derived from GC cells and sera were separately isolated using ultracentrifugation and ExoQuick exosome precipitation solution, and were characterized by transmission electron microscopy, Nanosight and western blotting. Transwell assay and LNM models were conducted to evaluate the capacities of migration, invasion and LNM of GC cells and . β-oxidation rate and CPT1 activity were measured to assess FAO. CPT1A inhibitor etomoxir was used to determine the role of FAO. Label-free LC-MS/MS proteome analysis screened the differential protein profiling between HGC-27-exosomes and AGS-exosomes. Small interference RNAs and YAP inhibitor verteporfin were used to elucidate the role and mechanism of exosomal CD44. TCGA data analysis, immunochemistry staining and ELISA were performed to analyze the expression correlation and clinical significance of CD44/YAP/CPT1A.
FAO was increased in lymphatic metastatic GC cells and indispensable for sustaining LNM capacity. Lymphatic metastatic GC cell-exosomes conferred LNM capacity on primary GC cells in an FAO-dependent way. Mechanistically, CD44 was identified to be enriched in HGC-27-exosomes and was a critical cargo protein regulating exosome-mediated transmission, possibly by modulating the RhoA/YAP/Prox1/CPT1A signaling axis. Abnormal expression of CD44/YAP/CPT1A in GC tissues was correlated with each other and associated with LNM status, stages, invasion and poor survival. Serum exosomal CD44 concentration was positively correlated with tumor burden in lymph nodes.
We uncovered a novel mechanism: exosomal CD44 transmits LNM capacity between GC cells YAP-CPT1A-mediated FAO reprogramming from the perspective of exosomes-transferred LNM phenotype. This provides potential therapeutic targets and a non-invasive biomarker for GC patients with LNM.
淋巴结转移（LNM）在胃癌（GC）中普遍存在，且与预后不良密切相关。外泌体介导的淋巴管生成被认为是LNM的重要驱动因素。外泌体是否直接在GC细胞之间传递LNM表型及其机制仍不清楚。
通过将亲本HGC-27细胞在BALB/c裸鼠中连续传代建立高淋巴转移GC细胞系（HGC-27-L）。比较了HGC-27-L、HGC-27和原发性GC细胞系AGS之间的迁移、侵袭和LNM能力；脂肪酸氧化（FAO）水平；以及外泌体转移的LNM表型的作用。使用超速离心和ExoQuick外泌体沉淀溶液分别从GC细胞和血清中分离出外泌体，并通过透射电子显微镜、纳米可视技术和蛋白质印迹进行表征。进行Transwell实验和LNM模型以评估GC细胞的迁移、侵袭和LNM能力。测量β-氧化速率和CPT1活性以评估FAO。使用CPT1A抑制剂依托莫昔芬确定FAO的作用。无标记液相色谱-质谱/质谱蛋白质组分析筛选了HGC-27-外泌体和AGS-外泌体之间的差异蛋白质谱。使用小干扰RNA和YAP抑制剂维替泊芬阐明外泌体CD44的作用和机制。进行TCGA数据分析、免疫化学染色和ELISA以分析CD44/YAP/CPT1A的表达相关性和临床意义。
FAO在淋巴转移GC细胞中增加，并且对于维持LNM能力是必不可少的。淋巴转移GC细胞来源的外泌体以FAO依赖的方式赋予原发性GC细胞LNM能力。机制上，CD44被鉴定在HGC-27-外泌体中富集，并且是调节外泌体介导的传递的关键货物蛋白，可能是通过调节RhoA/YAP/Prox1/CPT1A信号轴。GC组织中CD44/YAP/CPT1A的异常表达相互关联，并与LNM状态、分期、侵袭和不良生存相关。血清外泌体CD44浓度与淋巴结中的肿瘤负荷呈正相关。
我们发现了一种新机制：外泌体CD44从外泌体转移的LNM表型的角度通过YAP-CPT1A介导的FAO重编程在GC细胞之间传递LNM能力。这为具有LNM的GC患者提供了潜在的治疗靶点和非侵入性生物标志物。
