Wang Ling, Skotland Tore, Berge Viktor, Sandvig Kirsten, Llorente Alicia
Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital-The Norwegian Radium Hospital, 0379 Oslo, Norway; Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, 0379 Oslo, Norway.
Department of Urology, Oslo University Hospital, 0586 Oslo, Norway.
Eur J Pharm Sci. 2017 Feb 15;98:80-85. doi: 10.1016/j.ejps.2016.09.023. Epub 2016 Sep 21.
Exosomes have recently appeared as a novel source of non-invasive cancer biomarkers since tumor-specific molecules can be found in exosomes isolated from biological fluids. We have previously analyzed the proteome of urinary exosomes by mass spectrometry, and identified proteins differentially expressed in prostate cancer patients compared to healthy males. Since mass spectrometry is so far not commonly used in clinical laboratories, we have here investigated whether antibody-based methods such as Western blot or ELISA can be used to validate the use of the identified proteins as prostate cancer biomarkers. Western blot experiments designed to detect flotillin 2, TMEM256, Rab3B and LAMTOR1 showed that the level of these proteins was higher in urinary exosomes from prostate cancer patients compared to healthy males. Furthermore, a receiver operating characteristic curve of flotillin 2 in samples from 16 controls and 16 patients showed an area under the curve of 0.91, and 88% sensitivity at a threshold set to give 94% specificity. In addition, ELISA-based detection of flotillin 2 and PARK7 showed that the combination of these proteins was able to distinguish prostate cancer patients and healthy controls with 68% sensitivity and 93% specificity. Several promising biomarkers identified by mass spectrometry could not be evaluated by Western blot or ELISA due to their low exosomal amount and/or lack of good antibodies. In conclusion, our results show that several urinary exosomal proteins identified as prostate cancer biomarkers by mass spectrometry have a high diagnostic value also when analyzed by immunology-based methods, thus bringing these biomarkers closer to a potential clinical use.
由于在从生物流体中分离出的外泌体中可以发现肿瘤特异性分子，外泌体最近已成为非侵入性癌症生物标志物的新来源。我们之前通过质谱分析了尿外泌体的蛋白质组，并鉴定了前列腺癌患者与健康男性相比差异表达的蛋白质。由于目前质谱法在临床实验室中并不常用，我们在此研究了基于抗体的方法（如蛋白质印迹法或酶联免疫吸附测定法）是否可用于验证所鉴定的蛋白质作为前列腺癌生物标志物的用途。旨在检测 flotillin 2、TMEM256、Rab3B 和 LAMTOR1 的蛋白质印迹实验表明，与健康男性相比，前列腺癌患者尿外泌体中这些蛋白质的水平更高。此外，对 16 名对照和 16 名患者样本中的 flotillin 2 进行的受试者工作特征曲线分析显示，曲线下面积为 0.91，在设定为 94% 特异性的阈值下灵敏度为 88%。此外，基于酶联免疫吸附测定法检测 flotillin 2 和 PARK7 表明，这些蛋白质的组合能够以 68% 的灵敏度和 93% 的特异性区分前列腺癌患者和健康对照。由于外泌体含量低和/或缺乏优质抗体，质谱法鉴定的几种有前景的生物标志物无法通过蛋白质印迹法或酶联免疫吸附测定法进行评估。总之，我们的结果表明，通过质谱法鉴定为前列腺癌生物标志物的几种尿外泌体蛋白质在通过基于免疫学的方法分析时也具有很高的诊断价值，从而使这些生物标志物更接近潜在的临床应用。
