Pre-Analytical Framework for Routine Clinical Use of Liquid Biopsies: Combining EVs and cfDNA

Liquid biopsies hold significant potential for the non-invasive diagnostics of tumors and other diseases. While the clinical application of cell-free DNA (cfDNA) methodologies is emerging, the implementation of tumor-derived extracellular vesicles (EVs) as validated biomarkers is hindered by substantial pre-analytical variations. In this work, we are taking a step towards standardizing the pre-analytical procedures of blood collection for subsequent co-isolation of plasma cfDNA and EVs from a single blood collection tube. We compare effects of blood preservation tubes and storage to enable proteomic profiling of resulting EVs in addition to cfDNA extraction and sequencing. Following a stringent method of large EV (lEV) and small EV (sEV) isolation, consisting of differential ultracentrifugation and size exclusion chromatography, we evaluate protein concentration, particle number, quality and integrity of the isolated EVs. Subsequent proteomic analyses of EV isolates unravel the complexity of the respective tube proteomes allowing the interpretation of EV origins as well as contamination sources. While ACD-A and Citrate tubes demonstrate satisfying results in preservation of EV proteomes, only Streck RNA®, Norgen® and PAX® tubes can preserve high cfDNA purity for up to 7 days. When aiming for multi-omics analyses, Streck RNA® tubes show the most stable performance across tested parameters for both bioanalytes. Furthermore, we find more variability in protein composition in sEVs than in lEVs after 7 days of storage; thus, sEVs might be more susceptible to storage effects. Our clinically applicable workflow provides the basis for informed choice of liquid biopsy tubes along with a ready-to-use protocol to retrieve both genomic and EV proteomic biomarker information for multi-omics biomarker-based liquid biopsy studies.

液体活检在肿瘤及其他疾病的无创诊断领域具有巨大应用潜力。尽管无细胞DNA（cell-free DNA, cfDNA）检测方法的临床应用正逐步兴起，但肿瘤来源细胞外囊泡（extracellular vesicles, EVs）作为经验证的生物标志物的应用仍受到严重的分析前变异的阻碍。本研究旨在标准化血液采集的分析前流程，以实现从单支采血管中同步分离血浆cfDNA与EVs。我们对比了不同血液保存采血管及储存条件的影响，以期在提取cfDNA并进行测序之外，还能对分离得到的EVs开展蛋白质组学分析。我们采用差速超速离心结合尺寸排阻色谱的严格流程，分离大尺寸细胞外囊泡（large EV, lEV）与小尺寸细胞外囊泡（small EV, sEV），并对分离得到的EVs的蛋白浓度、颗粒数量、质量与完整性进行评估。后续对EV分离物的蛋白质组学分析揭示了不同采血管对应的蛋白质组的复杂性，有助于解析EV的来源与污染来源。尽管ACD-A管与柠檬酸盐管在保存EV蛋白质组方面表现优异，但仅Streck RNA®、Norgen®及PAX®采血管可在长达7天内维持高纯度的cfDNA。若开展多组学分析，Streck RNA®采血管在两种生物分析物的所有检测参数上均表现出最稳定的性能。此外，我们发现在储存7天后，sEV的蛋白质组成变异程度高于lEV，因此sEV可能对储存条件更为敏感。本研究开发的可临床应用的实验流程，可为液体活检采血管的合理选择提供依据，并提供了一套现成的实验方案，可同时获取基因组学与EV蛋白质组学的生物标志物信息，用于基于生物标志物的多组学液体活检研究。