Gemoll Timo, Rozanova Svitlana, Röder Christian, Hartwig Sonja, Kalthoff Holger, Lehr Stefan, ElSharawy Abdou, Habermann Jens K
Section for Translational Surgical Oncology & Biobanking, Department of Surgery, University of Lübeck and University Hospital Schleswig-Holstein, 23562 Lübeck, Germany.
Institute for Experimental Cancer Research, University of Kiel, 24105 Kiel, Germany.
J Clin Med. 2020 May 12;9(5):1429. doi: 10.3390/jcm9051429.
Solid tumor biopsies are the current standard for precision medicine. However, the procedure is invasive and not always feasible. In contrast, liquid biopsies, such as serum enriched for extracellular vesicles (EVs) represent a non-invasive source of cancer biomarkers. In this study, we compared two EV isolation methods in the context of the protein biomarker detection in inflammatory bowel disease (IBD) and colorectal cancer (CRC). Using serum samples of a healthy cohort as well as CRC and IBD patients, EVs were isolated by ultracentrifugation and ExoQuick in parallel. EV associated protein profiles were compared by multiplex-fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) and subsequent identification by mass spectrometry. Validation of gelsolin (GSN) was performed using fluorescence-quantitative western blot. 2D-DIGE resolved 936 protein spots in all serum-enriched EVs isolated by ultracentrifugation or ExoQuick. Hereof, 93 spots were differently expressed between isolation approaches. Higher levels of GSN in EVs obtained with ExoQuick compared to ultracentrifugation were confirmed by western blot ( = 0.0006). Although patient groups were distinguishable after both EV isolation approaches, sample preparation strongly influences EVs' protein profile and thus impacts on inter-study reproducibility, biomarker identification and validation. The results stress the need for strict SOPs in EV research before clinical implementation can be reached.
实体瘤活检是精准医学的当前标准。然而，该程序具有侵入性，且并非总是可行。相比之下，液体活检，如富含细胞外囊泡（EV）的血清，代表了癌症生物标志物的一种非侵入性来源。在本研究中，我们在炎症性肠病（IBD）和结直肠癌（CRC）的蛋白质生物标志物检测背景下比较了两种EV分离方法。使用健康队列以及CRC和IBD患者的血清样本，通过超速离心和ExoQuick并行分离EV。通过多重荧光二维差异凝胶电泳（2D-DIGE）比较EV相关的蛋白质谱，并随后通过质谱进行鉴定。使用荧光定量蛋白质免疫印迹法对凝溶胶蛋白（GSN）进行验证。2D-DIGE在通过超速离心或ExoQuick分离的所有富含血清的EV中解析出936个蛋白质斑点。其中，93个斑点在分离方法之间差异表达。蛋白质免疫印迹法证实，与超速离心相比，ExoQuick获得的EV中GSN水平更高（ = 0.0006）。尽管在两种EV分离方法后患者组均可区分，但样品制备强烈影响EV的蛋白质谱，从而影响研究间的可重复性、生物标志物鉴定和验证。结果强调在达到临床应用之前，EV研究中需要严格的标准操作规程。
