Health Unit, Flemish Institute for Technological Research (VITO), 2400 Mol, Belgium.
Laboratory for Soft Matter and Biophysics, KU Leuven, 3000 Leuven, Belgium.
Int J Mol Sci. 2022 Dec 16;23(24):16052. doi: 10.3390/ijms232416052.
Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RNA and DNA as potential templates for molecular diagnostics, e.g., in cancer genotyping. While it has been established that DNA templates appear on the outside of EVs, no consensus exists on which nucleic acid species inside small EVs (<200 nm, sEVs) are sufficiently abundant and accessible for developing genotyping protocols. We investigated this by extracting total intravesicular nucleic acid content from sEVs isolated from the conditioned cell medium of the human NCI-H1975 cell line containing the epidermal growth factor (EGFR) gene mutation T790M as a model system for non-small cell lung cancer. We observed that mainly short genomic DNA (<35−100 bp) present in the sEVs served as a template. Using qEV size exclusion chromatography (SEC), significantly lower yield and higher purity of isolated sEV fractions were obtained as compared to exoEasy membrane affinity purification and ultracentrifugation. Nevertheless, we detected the EGFR T790M mutation in the sEVs’ lumen with similar sensitivity using digital PCR. When applying SEC-based sEV separation prior to cell-free DNA extraction on spiked human plasma samples, we found significantly higher mutant allele frequencies as compared to standard cell-free DNA extraction, which in part was due to co-purification of circulating tumor DNA. We conclude that intravesicular genomic DNA can be exploited next to ctDNA to enhance EGFR T790M mutation detection sensitivity by adding a fast and easy-to-use sEV separation method, such as SEC, upstream of standard clinical cell-free DNA workflows.
细胞外囊泡 (EVs) 是由脂质双层包围的细胞衍生结构，携带 RNA 和 DNA，作为分子诊断的潜在模板，例如在癌症基因分型中。虽然已经确定 DNA 模板出现在 EV 外表面，但对于哪些小 EVs(<200nm,sEVs) 内的核酸种类足够丰富且可用于开发基因分型方案，尚无共识。我们通过从小鼠 NCI-H1975 细胞系条件培养基中分离的 sEV 中提取总囊内核酸含量来研究这一点，该细胞系含有表皮生长因子 (EGFR) 基因突变 T790M，作为非小细胞肺癌的模型系统。我们观察到，主要是存在于 sEVs 中的短基因组 DNA(<35-100bp) 作为模板。与 exoEasy 膜亲和纯化和超速离心相比，使用 qEV 尺寸排除色谱 (SEC) 可获得显著更低的产量和更高纯度的分离 sEV 级分。尽管如此，我们使用数字 PCR 在 sEVs 腔中检测到 EGFR T790M 突变，其灵敏度相似。当在含有 spiked 人血浆样本的游离 DNA 提取之前应用基于 SEC 的 sEV 分离时，与标准游离 DNA 提取相比，我们发现突变等位基因频率显著更高，部分原因是循环肿瘤 DNA 的共纯化。我们得出结论，除了 ctDNA 之外，囊内基因组 DNA 可以被利用，通过在标准临床游离 DNA 工作流程的上游添加快速且易于使用的 sEV 分离方法（例如 SEC），来提高 EGFR T790M 突变检测的灵敏度。
