PO.CL01.21 · 临床研究
通过SEC-免疫亲和整合高纯度分离肿瘤来源细胞外囊泡可提高乳腺癌诊断灵敏度
High-purity isolation of tumor-derived extracellular vesicles via SEC-immunoaffinity integration improves diagnostic sensitivity in breast cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：细胞外囊泡（EVs）因其富集的分子货物和结构稳定性，为液体活检提供了一种非侵入性方法。然而，EV的异质性以及当前EV分离方法受非囊泡细胞外颗粒（NVEPs）尤其是脂蛋白的污染，仍是可靠生物标志物分析的重大障碍。若干已提出的EV生物标志物在NVEP组分中富集的报道，凸显了高纯度EV分离的必要性。
方法：为改进血浆来源的小EV（sEV）纯化，我们使用代表sEV、大EV、脂蛋白（HDL、LDL、VLDL）和白蛋白的纯化对照材料，系统评估了尺寸排阻色谱（SEC）树脂。绘制了组分分布模式以预测血浆处理过程中的NVEP共洗脱。基于这些观察，将SEC与基于apoB100的免疫亲和去除相结合，以减少脂蛋白污染。随后使用靶向乳腺癌相关表面表位的免疫捕获富集肿瘤来源EV（tdEVs），从而实现下游多组学分析。
结果：CL-6B对单个对照材料产生尖锐峰，但VLDL与部分LDL在第6组分中出现汇聚，表明应用于血浆时与sEV相关组分高度重叠。相反，CL-4B产生更宽的洗脱模式，LDL在sEV相关组分中基本缺失，VLDL分布于第5-15组分，减少了与sEV的重叠程度。这些发现表明CL-4B为血浆sEV纯化提供了更有利的分离特性。ApoB100介导的免疫亲和去除进一步降低了VLDL和HDL信号，同时保持了sEV回收率。所得组分显示出改善的颗粒-蛋白比、降低的ApoA1/ApoB特征，以及经典sEV标志物的保留。乳腺癌特异性免疫捕获富集了携带既往在我们队列中报道的致癌miRNA特征升高水平的tdEVs。值得注意的是，与我们此前基于总EV的方法相比，这一聚焦tdEV的miRNA检测组合表现出改善的诊断灵敏度，提示高纯度tdEV富集更准确地反映了肿瘤来源的分子谱。
结论：我们开发了一套分析稳定的EV纯化工作流程，将优化的SEC与序贯免疫亲和策略相结合，在保留sEV完整性的同时最大限度减少NVEP干扰。这一高纯度、聚焦肿瘤的平台可实现对循环tdEVs的可靠多组学分析，并支持开发适用于乳腺癌早期检测和疾病监测的临床可用生物标志物。
查看英文原文 English abstract
Background: Extracellular vesicles (EVs) provide a non-invasive approach for liquid biopsy due to their enriched molecular cargo and structural stability. However, EV heterogeneity and contamination of current EV isolation methods by non-vesicular extracellular particles (NVEPs), particularly lipoproteins, remain substantial obstacles to reliable biomarker analysis. Reports showing that several proposed EV biomarkers are enriched in NVEP fractions underscore the need for high-purity EV isolation.
Methods: To improve plasma-derived small EV (sEV) purification, we systematically evaluated size-exclusion chromatography (SEC) resins using purified control materials representing sEVs, large EVs, lipoproteins (HDL, LDL, VLDL) and albumin. Fraction distribution patterns were mapped to predict NVEP co-elution during plasma processing. Based on these observations, SEC was combined with apoB100-based immunoaffinity depletion to reduce lipoprotein contamination. Tumor-derived EVs (tdEVs) were then enriched using immunocapture targeting breast cancer-associated surface epitopes, enabling downstream multi-omics analysis.
Results: CL-6B generated sharp peaks for individual control materials but showed convergence of VLDL and a portion of LDL in fraction 6, indicating a high likelihood of overlap with sEV-associated fractions when applied to plasma. In contrast, CL-4B produced broader elution patterns, with LDL largely absent from sEV-associated fractions and VLDL distributed across fractions 5-15, reducing the degree of overlap with sEVs. These findings indicate that CL-4B provides more favorable separation characteristics for plasma sEV purification. ApoB100-mediated immunoaffinity depletion further reduced VLDL and HDL signals while maintaining sEV recovery. The resulting fractions showed improved particle-to-protein ratios, decreased ApoA1/ApoB signatures, and retention of canonical sEV markers. Breast cancer-specific immunocapture enriched tdEVs carrying elevated levels of an oncogenic miRNA signature previously reported in our cohorts. Notably, this tdEV-focused miRNA panel demonstrated improved diagnostic sensitivity compared with our earlier total-EV-based approach, suggesting that high-purity tdEV enrichment more accurately reflects tumor-derived molecular profiles.
Conclusion: We developed an analytically stable EV purification workflow that combines optimized SEC with sequential immunoaffinity strategies to minimize NVEP interference while preserving sEV integrity. This high-purity, tumor-focused platform enables reliable multi-omics profiling of circulating tdEVs and supports the development of clinically applicable biomarkers for early breast cancer detection and disease monitoring.
利益披露 Disclosure
Y. Kim, None..
M. Kim, None..
S. Moon, None..
S. Lee, None..
J. Kim, None..
S. Kim, None..
J. Kim, None.