癌相关成纤维细胞（CAFs）是肿瘤微环境（TME）中至关重要的组成部分，在食管鳞状细胞癌（ESCC）的肿瘤起始、发展和免疫逃逸中发挥重要作用。尽管如此，来源于CAFs的特定外泌体蛋白及其在ESCC中的功能仍未知。因此，本研究旨在研究CAFs来源的外泌体蛋白对ESCC的影响和预后意义。利用超速离心法分离CAFs和正常成纤维细胞（NFs）获得的外泌体，采用液相色谱-串联质谱（LC-MS/MS）分析外泌体的蛋白表达谱。使用CCK-8和集落形成实验评估肿瘤增殖，使用Transwell实验评估细胞侵袭和迁移。采用Lasso回归分析利用TCGA数据库建立基于CAFs来源的外泌体蛋白的特征标。通过生存分析、基因集富集分析（GSEA）、免疫分析、免疫治疗反应分析和药物敏感性分析全面研究该特征标的免疫和预后作用。利用GSE160269数据集进行单细胞转录组分析，以进一步阐明该特征标在TME中的作用。此外，采用cDNA microarray分析验证了该特征标的预后价值。我们的研究发现，来源于CAFs的外泌体明显增强了食管癌细胞的增殖、侵袭和迁移能力。通过LC-MS/MS分析，我们鉴定了842个差异表达的外泌体蛋白，其中两种关键蛋白被应用于建立风险特征标。生存分析显示高风险组患者预后显著较差，多变量分析表明风险评分是独立的预后因素。此外，我们观察到风险评分与免疫细胞浸润、免疫治疗反应以及药物化疗对治疗人群的敏感性之间存在显著相关性。最后，单细胞转录组分析进一步揭示了TNFRSF10B和ILF3在不同细胞亚群中的表达模式。总之，我们的研究成功建立了基于CAFs来源的外泌体蛋白的可靠预后特征标，可用于预测ESCC预后并评估免疫微环境。版权所有© 2023 Elsevier B.V. 保留所有权利。

Cancer-associated fibroblasts (CAFs) are a crucial component of the tumor microenvironment (TME) and play significant roles in tumor initiation, progression, and immune evasion. Despite this, the specific exosomal proteins derived from CAFs and their functions in esophageal squamous cell carcinoma (ESCC) remain unknown. Therefore, this study aims to investigate the impact and prognostic significance of CAFs-derived exosomal proteins in ESCC.Exosomes obtained from CAFs and normal fibroblasts (NFs) were isolated using ultracentrifugation, and the protein expression profiles of the exosomes were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Tumor proliferation was assessed using CCK-8 and colony formation assays, while cell invasion and migration were evaluated using transwell assays. Lasso regression analysis was employed to establish a signature based on CAFs-derived exosomal proteins using the TCGA database. The immunological and prognostic roles of this signature were comprehensively investigated through survival analysis, gene set enrichment analysis (GSEA), immune analysis, immunotherapy response analysis, and drug sensitivity analysis. The GSE160269 dataset was utilized for single-cell transcriptome analysis to further elucidate the role of the signature in the TME. Additionally, cDNA microarray analysis was utilized to validate the prognostic value of the signature.Our findings demonstrate that exosomes derived from CAFs significantly enhance the proliferation, invasion, and migration of esophageal cancer cells. We identified 842 differentially expressed exosomal proteins through LC-MS/MS analysis, and two key proteins were utilized to establish a risk signature. Survival analysis revealed a significantly worse prognosis in the high-risk group, with multivariate analysis indicating that the risk score serves as an independent prognostic factor. Moreover, we observed a significant correlation between the risk score and immune cell infiltration, immunotherapy response, and sensitivity to chemotherapeutic treatments in the study population. Lastly, single-cell transcriptome analysis further revealed the expression patterns of TNFRSF10B and ILF3 in different cell subpopulations.In conclusion, our study has successfully established a robust prognostic signature based on CAFs-derived exosomal proteins, which can serve as a reliable biomarker for predicting prognosis and evaluating the immune microenvironment in ESCC.Copyright © 2023 Elsevier B.V. All rights reserved.