Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density-based fractionation of urine
Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density-based fractionation of urine
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DOI：
10.1080/20013078.2020.1736935
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发表时间：
2020-01-01
影响因子：
16
通讯作者：
Hendrix, An
中科院分区：
文献类型：
--
作者：
Dhondt, Bert;Geeurickx, Edward;Hendrix, An
细胞外小泡(EV)越来越被认为是肿瘤细胞间沟通的重要载体，也是肿瘤诊断和预后的重要生物标志物。尽管有巨大的临床潜力，但从生物液中分离EV的方法太多，提供的材料纯度高度可变，而且缺乏关于方法学重复性的知识，这对临床翻译构成了障碍。尿液与泌尿生殖系统直接接触，被认为是研究EV在泌尿系肿瘤中的理想近端液体。我们证明，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，可以以高度的特异性和重复性分离EV和可溶性蛋白质。对良性和恶性前列腺病患者尿液EV(UEV)的蛋白质组学分析使我们能够显著扩展已知的人类uEV蛋白质组，具有高度的特异性，并鉴定了前列腺癌中未被可溶性蛋白质分析发现的独特的生物学特征。此外，从前列腺癌条件培养液中分离的EV蛋白质组与匹配的uEV蛋白质组的图谱证实了所鉴定的uEV蛋白质组对前列腺癌的特异性。最后，与膀胱癌和肾癌患者的uEV进行的蛋白质组学比较分析提供了额外的证据，证明uEV中的蛋白质特征信号选择性地丰富了各自起源的癌症组织。总之，这项研究确定了前列腺癌患者uEV中数百种以前未检测到的蛋白质，并提供了一个强大的工具箱来绘制uEV含量和污染物图，最终允许在泌尿系癌症中发现生物标记物。
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.