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Zhaohua Xiao,1 Xumei Feng,2 Yongjia Zhou,1 Peiwei Li,3 Junwen Luo,1 Wenhao Zhang,1 Jie Zhou,1 Jiangfeng Zhao,1 Dong Wang,4 Yongjie Wang,5 Zhongxian Tian,1,6 Xiaogang Zhao1,6 1Department of Thoracic Surgery, The Second Hospital of Shandong University, Jinan, 250033, People’s Republic of China; 2Health Management Center, The Second Hospital of Shandong University, Jinan, People’s Republic of China; 3Institute of Medical Sciences, The Second Hospital of Shandong University, Jinan, People’s Republic of China; 4Department of Thoracic Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, 250021, People’s Republic of China; 5Department of Thoracic Surgery, The Affiliated Hospital of Qingdao University, Qingdao, 266000, People’s Republic of China; 6Key Laboratory of Chest Cancer, Shandong University, The Second Hospital of Shandong University, Jinan, People’s Republic of ChinaCorrespondence: Zhongxian Tian; Xiaogang Zhao, Department of Thoracic Surgery, The Second Hospital of Shandong University, Jinan, 250033, People’s Republic of China, Tel +86-17660082365 ; +86-053185875009, Email tianzhongxian@email.sdu.edu.cn; zhaoxiaogang@sdu.edu.cnBackground: Cancer cell-derived exosomal microRNAs (miRNAs) play critical role in orchestrating intercellular communication between tumor cells and tumor microenvironmental factors, including lymphatic endothelial cells (LECs). Nevertheless, the functions and underlying mechanisms of exosomal miRNAs in lymphatic metastasis and lymphangiogenesis in esophageal squamous cell carcinoma (ESCC) remain unclear.Methods: Small RNA sequencing, Gene Expression Omnibus (GEO) analysis and qRT‒PCR were performed to identify the candidate exosomal miRNAs involved in ESCC metastasis. Receiver operating characteristic curve analysis was conducted to evaluate the diagnostic potential of exosomal miR-10527-5p in predicting lymph node metastasis (LNM) status. An in vitro coculture system was used to investigate the effects of exosomal miR-10527-5p on ESCC cells and human LECs (HLECs), followed by a popliteal LNM assay in vivo. The relationship between miR-10527-5p and Rab10 was identified by dual-luciferase reporter, fluorescence in situ hybridization and qRT‒PCR assays. Then, a series of rescue assays were performed to further investigate whether Rab10 is involved in exosomal miR-10527-5p mediated ESCC metastasis.Results: MiR-10527-5p was found to be notably reduced in both the plasma exosomes and tumor tissues of ESCC patients with LNM, and plasma exosomal miR-10527-5p had a high sensitivity and specificity for discrimination of LNM status. Moreover, exosome-shuttled miR-10527-5p suppressed the migration, invasion and epithelial-to-mesenchymal transition (EMT) of ESCC cells as well as the migration and tube formation of HLECs via Wnt/β-catenin signaling in vitro and in vivo. Further investigation revealed that Rab10 was a direct target of miR-10527-5p, and re-expression of Rab10 neutralized the inhibitory effects of exosomal miR-10527-5p.Conclusion: Our study demonstrated that exosomal miR-10527-5p had a strong capability to predict preoperative LNM status and anti-lymphangiogenic effect. Exosomal miR-10527-5p inhibited lymphangiogenesis and lymphatic metastasis of ESCC in a vascular endothelial growth factor-C (VEGF-C)-independent manner, showing potential as a therapeutic target for ESCC patients.Keywords: esophageal squamous cell carcinoma, exosomes, miR-10527-5p, biomarker, lymphangiogenesis