ABSTRACT

Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers.

摘要

细胞外囊泡（EV）越来越被认为是细胞间通讯的重要载体，并有望成为癌症中诊断和预后的生物标志物。尽管具有巨大的临床潜力，但从生物流体中分离出EV的方法过多，提供了高度可变的纯度的材料，并且缺乏有关方法可重复性的知识，这为临床翻译带来了障碍。由于尿液与泌尿生殖系统直接接触，因此尿液被认为是研究泌尿系统癌症的EV的理想近端液体。我们证明了通过自下而上的Optiprep密度梯度离心法对尿液进行基于密度的分级分离，可分离出具有高特异性和可重复性的EV和可溶性蛋白。对患有良性和恶性前列腺疾病的男性的尿液EV（uEV）进行基于蛋白质组学的蛋白质组学分析，这使我们能够以高特异性显着扩展已知的人类uEV蛋白质组，并确定了可溶蛋白分析中未发现的前列腺癌独特的生物学特征。另外，对与前列腺肿瘤条件培养基分离并匹配的uEV的EV蛋白质组进行谱分析，证实了所鉴定的uEV蛋白质组对前列腺癌的特异性。最后，来自膀胱癌和肾癌患者的uEV的比较蛋白质组学分析提供了额外的证据，表明uEV中蛋白质标记的选择性富集反映了其各自的癌组织。结论，