Ku Anson, Fredsøe Jacob, Sørensen Karina D, Borre Michael, Evander Mikael, Laurell Thomas, Lilja Hans, Ceder Yvonne
Department of Translational Medicine, Lund University, Malmö, Sweden.
Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark & Department of Clinical Medicine, Aarhus University, Aarhus, Denmark.
Front Oncol. 2021 Mar 25;11:631021. doi: 10.3389/fonc.2021.631021. eCollection 2021.
Molecular profiling of extracellular vesicles (EVs) offers novel opportunities for diagnostic applications, but the current major obstacle for clinical translation is the lack of efficient, robust, and reproducible isolation methods. To bridge that gap, we developed a microfluidic, non-contact, and low-input volume compatible acoustic trapping technology for EV isolation that enabled downstream small RNA sequencing. In the current study, we have further automated the acoustic microfluidics-based EV enrichment technique that enables us to serially process 32 clinical samples per run. We utilized the system to enrich EVs from urine collected as the first morning void from 207 men referred to 10-core prostate biopsy performed the same day. Using automated acoustic trapping, we successfully enriched EVs from 199/207 samples (96%). After RNA extraction, size selection, and library preparation, a total of 173/199 samples (87%) provided sufficient materials for next-generation sequencing that generated an average of 2 × 10 reads per sample mapping to the human reference genome. The predominant RNA species identified were fragments of long RNAs such as protein coding and retained introns, whereas small RNAs such as microRNAs (miRNA) accounted for less than 1% of the reads suggesting that partially degraded long RNAs out-competed miRNAs during sequencing. We found that the expression of six miRNAs was significantly different (P < 0.05) in EVs isolated from patients found to have high grade prostate cancer [ISUP 2005 Grade Group (GG) 4 or higher] compared to those with GG3 or lower, including those with no evidence of prostate cancer at biopsy. These included miR-23b-3p, miR-27a-3p, and miR-27b-3p showing higher expression in patients with GG4 or high grade prostate cancer, whereas miR-1-3p, miR-10a-5p, and miR-423-3p had lower expression in the GG4 PCa cases. Cross referencing our differentially expressed miRNAs to two large prostate cancer datasets revealed that the putative tumor suppressors miR-1, miR-23b, and miR-27a are consistently deregulated in prostate cancer. Taken together, this is the first time that our automated microfluidic EV enrichment technique has been found to be capable of enriching EVs on a large scale from 900 μl of urine for small RNA sequencing in a robust and disease discriminatory manner.
细胞外囊泡（EVs）的分子谱分析为诊断应用提供了新的机会，但目前临床转化的主要障碍是缺乏高效、可靠且可重复的分离方法。为了弥补这一差距，我们开发了一种用于EV分离的微流控、非接触且低输入体积兼容的声学捕获技术，该技术能够进行下游的小RNA测序。在本研究中，我们进一步自动化了基于声学微流控的EV富集技术，使我们能够每次运行连续处理32个临床样本。我们利用该系统从207名当天接受10芯前列腺活检的男性晨尿中富集EVs。使用自动化声学捕获，我们成功地从199/207个样本（96%）中富集了EVs。经过RNA提取、大小选择和文库制备后，共有173/199个样本（87%）提供了足够的材料用于下一代测序，每个样本平均产生2×10个映射到人类参考基因组的读数。鉴定出的主要RNA种类是长RNA片段，如蛋白质编码片段和保留的内含子，而小RNA如微小RNA（miRNA）占读数的比例不到1%，这表明在测序过程中部分降解的长RNA比miRNA更具竞争力。我们发现，与ISUP 2005分级组（GG）3或更低的患者（包括活检时无前列腺癌证据的患者）相比，从患有高级别前列腺癌（ISUP 2005分级组4级或更高）的患者中分离出的EVs中，六种miRNA的表达存在显著差异（P<0.05）。其中包括miR-23b-3p、miR-27a-3p和miR-27b-3p在GG4或高级别前列腺癌患者中表达较高，而miR-1-3p、miR-10a-5p和miR-423-3p在GG4前列腺癌病例中表达较低。将我们差异表达的miRNA与两个大型前列腺癌数据集进行交叉参考发现，假定的肿瘤抑制因子miR-1、miR-23b和miR-27a在前列腺癌中一直存在失调。综上所述，这是首次发现我们的自动化微流控EV富集技术能够以稳健且具有疾病区分能力的方式从900微升尿液中大规模富集EVs用于小RNA测序。
