Chee Tian Mun, O'Farrell Hannah E, Lima Luize G, Möller Andreas, Fong Kwun M, Yang Ian A, Bowman Rayleen V
The University of Queensland Thoracic Research Centre The Prince Charles Hospital Chermside Queensland Australia.
Tumour Microenvironment Laboratory QIMR Berghofer Medical Research Institute Herston Queensland Australia.
J Extracell Biol. 2023 Oct 16;2(10):e119. doi: 10.1002/jex2.119. eCollection 2023 Oct.
Pleural effusion occurs in both benign and malignant pleural disease. In malignant pleural effusions, the diagnostic accuracy and sensitivity of pleural fluid cytology is less than perfect, particularly for the diagnosis of malignant pleural mesothelioma, but also in some cases for the diagnosis of metastatic pleural malignancy with primary cancer in the lung, breast or other sites. Extracellular vesicles (EVs) carry an enriched cargo of microRNAs (miRNAs) which are selectively packaged and differentially expressed in pleural disease states. To investigate the diagnostic potential of miRNA cargo in pleural fluid extracellular vesicles (PFEVs), we evaluated methods for isolating the extracellular vesicle (EV) fraction including combinations of ultracentrifugation, size-exclusion chromatography (SEC) and ultrafiltration (10 kDa filter unit). PFEVs were characterized by total and EV-associated protein, nanoparticle tracking analysis and visualisation by transmission electron microscopy. miRNA expression was analyzed by Nanostring nCounter® in separate EV fractions isolated from pleural fluid with or without additional RNA purification by ultrafiltration (3 kDa filter unit). Optimal PFEV yield, purity and miRNA expression were observed when PFEV were isolated from a larger volume of pleural fluid processed through combined ultracentrifugation and SEC techniques. Purification of total RNA by ultrafiltration further enhanced the detectability of PFEV miRNAs. This study demonstrates the feasibility of isolating PFEVs, and the potential to examine PFEV miRNA cargo using Nanostring technology to discover disease biomarkers.
胸腔积液可发生于良性和恶性胸膜疾病。在恶性胸腔积液中，胸腔积液细胞学检查的诊断准确性和敏感性并不理想，尤其是对于恶性胸膜间皮瘤的诊断，在某些情况下对于诊断伴有肺、乳腺或其他部位原发性癌症的转移性胸膜恶性肿瘤也是如此。细胞外囊泡（EVs）携带丰富的微小RNA（miRNAs），这些miRNAs在胸膜疾病状态下被选择性包装并差异表达。为了研究胸腔积液细胞外囊泡（PFEVs）中miRNA成分的诊断潜力，我们评估了分离细胞外囊泡（EV）组分的方法，包括超速离心、尺寸排阻色谱（SEC）和超滤（10 kDa过滤单元）的组合。通过总蛋白和与EV相关的蛋白、纳米颗粒跟踪分析以及透射电子显微镜观察对PFEVs进行表征。通过Nanostring nCounter®分析从胸腔积液中分离的单独EV组分中的miRNA表达，这些胸腔积液经过或未经过超滤（3 kDa过滤单元）进行额外的RNA纯化。当通过超速离心和SEC技术联合处理从更大体积的胸腔积液中分离PFEV时，观察到了最佳的PFEV产量、纯度和miRNA表达。通过超滤纯化总RNA进一步提高了PFEV miRNAs的可检测性。本研究证明了分离PFEVs的可行性，以及使用Nanostring技术检测PFEV miRNA成分以发现疾病生物标志物的潜力。
