A Single-Step Immunocapture Assay to Quantify HCC Exosomes Using the Highly Sensitive Fluorescence Nanoparticle-Tracking Analysis.
A Single-Step Immunocapture Assay to Quantify HCC Exosomes Using the Highly Sensitive Fluorescence Nanoparticle-Tracking Analysis.
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DOI：
10.2147/jhc.s423043
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发表时间：
2023
影响因子：
4.1
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中科院分区：
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细胞外囊泡可以作为早期癌症检测的非侵入性生物标志物。然而，定量天然状态下的癌症衍生囊泡的方法有限，仍然是临床转化的重大障碍。本研究旨在开发一种快速、一步式免疫亲和方法，直接从少量血清中定量 HCC 外泌体。使用针对 GPC3 和甲胎蛋白 (AFP) 的荧光藻红蛋白 (PE) 偶联抗体捕获血清中 HCC 衍生的外泌体。然后使用荧光纳米颗粒跟踪分析 (F-NTA) 对培养物上清液或患者血清中的总外泌体和 HCC 特异性外泌体进行定量。将血清中 HCC 外泌体定量的性能与 MRI 确定的肿瘤大小进行比较。最初，我们使用合成荧光和非荧光珠测试了 F-NTA 的检测限。该测定显示出可接受的灵敏度，检测范围为 104-108 个颗粒/mL。此外，免疫捕获和尺寸排阻柱纯化相结合，可以分离较小尺寸的 EV，并通过 F-NTA 进行定量。我们的检测表明，与非 HCC 培养物（10-40 个颗粒/细胞）相比，HCC 细胞培养物释放的 GPC3 或 GPC3+AFP 阳性 EV（100-200 个颗粒/细胞）数量显着更高（分别为 p<0.01 和 p<0.05）。 F-NTA 能够对临床样本中 HCC 特异性外泌体进行绝对计数，并保留生物免疫反应性。 F-NTA 的性能在肝硬化和确诊 HCC 患者的血清中进行了临床验证。 F-NTA 定量数据显示，与无恶性肝硬化相比，HCC 血清中 AFP 和 GPC3 阳性 EV 选择性富集（GPC3、AFP 和 GPC3/AFP 的 AUC 值分别为 0.79、0.71 和 0.72）。 MRI 证实的患者队列表明，肿瘤总大小与 GPC3 阳性外泌体浓度之间存在正相关性（r：0.78 和 p<0.001）。我们开发了一种免疫捕获测定法，可用于从少量血清中同时高精度地分离和定量 HCC 衍生的外泌体。
Extracellular vesicles could serve as a non-invasive biomarker for early cancer detection. However, limited methods to quantitate cancer-derived vesicles in the native state remain a significant barrier to clinical translation. This research aims to develop a rapid, one-step immunoaffinity approach to quantify HCC exosomes directly from a small serum volume. HCC-derived exosomes in the serum were captured using fluorescent phycoerythrin (PE)-conjugated antibodies targeted to GPC3 and alpha-fetoprotein (AFP). Total and HCC-specific exosomes were then quantified in culture supernatant or patient-derived serums using fluorescence nanoparticle tracking analysis (F-NTA). The performance of HCC exosome quantification in the serum was compared with the tumor size determined by MRI. Initially we tested the detection limits of the F-NTA using synthetic fluorescent and non-fluorescent beads. The assay showed an acceptable sensitivity with a detection range of 104–108 particles/mL. Additionally, the combination of immunocapture followed by size-exclusion column purification allows the isolation of smaller-size EVs and quantification by F-NTA. Our assay demonstrated that HCC cell culture releases a significantly higher quantity of GPC3 or GPC3+AFP positive EVs (100–200 particles/cell) compared to non-HCC culture (10–40 particles/cell) (p<0.01 and p<0.05 respectively). The F-NTA enables absolute counting of HCC-specific exosomes in the clinical samples with preserved biological immunoreactivity. The performance of F-NTA was clinically validated in serum from patients ± cirrhosis and with confirmed HCC. F-NTA quantification data show selective enrichment of AFP and GPC3 positive EVs in HCC serum compared to malignancy-free cirrhosis (AUC values for GPC3, AFP, and GPC3/AFP were found 0.79, 0.71, and 0.72 respectively). The MRI-confirmed patient cohort indicated that there was a positive correlation between total tumor size and GPC3-positive exosome concentration (r:0.78 and p<0.001). We developed an immunocapture assay that can be used for simultaneous isolation and quantification of HCC-derived exosomes from a small serum volume with high accuracy.