Priya Ratna, Jain Vaishali, Akhtar Javed, Saklani Neeraj, Sakhuja Puja, Agarwal Anil Kumar, Polisetty Ravindra Varma, Sirdeshmukh Ravi, Kar Sudeshna, Gautam Poonam
Laboratory of Molecular Oncology, Indian Council of Medical Research (ICMR)- National Institute of Pathology, New Delhi, India.
Jamia Hamdard- Institute of Molecular Medicine, Jamia Hamdard, New Delhi, India.
Front Oncol. 2022 Nov 23;12:1027914. doi: 10.3389/fonc.2022.1027914. eCollection 2022.
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（GBC）是第六大常见的胃肠道癌症，总体生存率极低，预后较差。分析癌症来源的细胞外囊泡（EVs）是一种新兴策略，用于识别该疾病检测和预后的候选生物标志物。本研究的目的是分析来自GBC细胞系的EVs的蛋白质含量，重点关注可作为GBC检测候选生物标志物的蛋白质。培养NOZ和OCUG-1细胞系，并从条件培养基中分离出EVs。对总EV蛋白质进行液相色谱-串联质谱（LC-MS/MS）分析，在两个细胞系中总共鉴定出268种蛋白质。其中，110种蛋白质在至少两次实验和技术重复运行中被鉴定出具有≥2个独特肽段且≥2个肽段谱匹配（PSMs）。使用STRING（检索相互作用基因/蛋白质的搜索工具）数据库对110种蛋白质进行生物信息学分析，结果显示在顶级分子功能中存在“细胞粘附分子结合”“整合素结合”“钙粘蛋白结合”，并且在与EV蛋白质相关的顶级通路中存在“粘着斑”。据报道，共有42种蛋白质，包括触珠蛋白（HP）、丙酮酸激酶（PKM）、膜联蛋白A2（ANXA2）、血小板反应蛋白1（THBS1），在GBC组织中丰度存在差异。其中，有16种蛋白质据报道在血浆和血浆来源的EVs中丰度存在差异。我们推断这些蛋白质对于作为检测GBC的潜在循环生物标志物非常重要。为了检验这一假设的有效性，作为一个代表性案例，通过定量酶联免疫吸附测定（ELISA）分析了血浆中的一种蛋白质——触珠蛋白（HP），我们观察到与对照组相比，其在GBC中的水平升高（p值 = 0.0063）。GBC与对照组的受试者工作特征（ROC）曲线分析显示，HP的曲线下面积（AUC）为0.8264，敏感性为22%，特异性为100%。我们建议，HP以及其他候选蛋白质可能需要进一步探索其临床应用价值。
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