DataSheet_1_Proteomic profiling of cell line-derived extracellular vesicles to identify candidate circulatory markers for detection of gallbladder cancer.xls

Gallbladder cancer (GBC) is the sixth most common gastrointestinal tract cancer with a very low overall survival and poor prognosis. Profiling of cancer-derived extracellular vesicles (EVs) is an emerging strategy for identification of candidate biomarkers for the detection and prognosis of the disease. The aim of the study was to analyse the protein content from GBC cell line- derived EVs with emphasis on proteins which could be used as candidate biomarkers for the detection of GBC. NOZ and OCUG-1 cell lines were cultured and EVs were isolated from conditioned media. LC-MS/MS analysis of total EV proteins led to the identification of a total of 268 proteins in both the cell lines. Of these, 110 proteins were identified with ≥2 unique peptides with ≥2 PSMs in at least two experimental and technical replicate runs. STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) database was used to perform bioinformatics analysis of 110 proteins which showed ‘cell adhesion molecule binding’, ‘integrin binding’, ‘cadherin binding’ among the top molecular functions and ‘focal adhesion’ to be among the top pathways associated with the EV proteins. A total of 42 proteins including haptoglobin (HP), pyruvate kinase (PKM), annexin A2 (ANXA2), thrombospondin 1 (THBS1), were reported to be differentially abundant in GBC tissue. Of these, 16 proteins were reported to be differentially abundant in plasma and plasma-derived EVs. We infer these proteins to be highly important to be considered as potential circulatory biomarkers for the detection of GBC. To check the validity of this hypothesis, one of the proteins, haptoglobin (HP) as a representative case, was analysed in plasma by quantitative Enzyme- linked immunosorbent assay (ELISA) and we observed its increased levels in GBC in comparison to controls (p value= 0.0063). Receiver operating characteristic (ROC) curve analysis for GBC vs controls showed an Area under the ROC Curve (AUC) of 0.8264 for HP with 22% sensitivity against 100% specificity. We propose that HP along with other candidate proteins may be further explored for their clinical application.

胆囊癌（Gallbladder cancer, GBC）是第六大常见胃肠道恶性肿瘤，总体生存率极低且预后不良。对癌症来源细胞外囊泡（extracellular vesicles, EVs）进行组学分析，是筛选该疾病检测与预后候选生物标志物的新兴策略。本研究旨在分析胆囊癌细胞系来源细胞外囊泡的蛋白质组成，重点关注可作为胆囊癌检测候选生物标志物的蛋白。
研究培养了NOZ与OCUG-1细胞系，并从条件培养基中分离细胞外囊泡。对总细胞外囊泡蛋白进行液相色谱-串联质谱（LC-MS/MS）分析后，在两种细胞系中共鉴定出268种蛋白。其中，至少两次实验与技术重复实验中，有110种蛋白经≥2个独特肽段、≥2个肽谱匹配数（Peptide Spectrum Matches, PSMs）鉴定得到。本研究使用STRING（蛋白质相互作用检索数据库，Search Tool for the Retrieval of Interacting Genes/Proteins）对这110种蛋白进行生物信息学分析，结果显示其核心分子功能包括「细胞黏附分子结合」「整联蛋白结合」「钙黏蛋白结合」，「黏着斑」为其富集的核心通路之一。
共42种蛋白在胆囊癌组织中呈差异表达，包括结合珠蛋白（haptoglobin, HP）、丙酮酸激酶（pyruvate kinase, PKM）、膜联蛋白A2（annexin A2, ANXA2）、血小板反应蛋白1（thrombospondin 1, THBS1）等。其中，16种蛋白在血浆及血浆来源细胞外囊泡中呈差异表达。本研究推断，这些蛋白可作为极具潜力的循环生物标志物用于胆囊癌的检测。
为验证该假说，本研究以其中一种蛋白——结合珠蛋白（HP）为代表，通过定量酶联免疫吸附试验（Enzyme-linked immunosorbent assay, ELISA）对血浆样本进行分析，结果显示胆囊癌患者血浆中HP水平较对照组显著升高（p值=0.0063）。针对胆囊癌与对照组的受试者工作特征（Receiver Operating Characteristic, ROC）曲线分析显示，HP的ROC曲线下面积（Area Under the ROC Curve, AUC）为0.8264，灵敏度为22%，特异度为100%。本研究提出，HP与其他候选蛋白可进一步开展临床应用探索。