PO.TB10.15 · 肿瘤生物学
从前列腺癌患者中分离和表征前列腺特异性细胞外囊泡
Isolation and characterization of prostate-specific extracellular vesicles from prostate cancer patients
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：血浆来源的细胞外囊泡（EV）代表了源自不同组织的囊泡的复杂混合物。选择性分离前列腺特异性细胞外囊泡（PSEV）可为诊断和预后生物标志物的开发以及监测前列腺癌（PCa）的治疗反应提供更高的特异性。本研究旨在比较从总 EV 制备物中富集的 PSEV 与直接从血浆中分离的 PSEV 的产量和质量。
材料与方法：本研究使用了 12 份血浆样本（6 份来自 PCa 患者，6 份来自非癌受试者）。按照制造商的说明使用 SmartSEC™ 柱分离总 EV。使用抗 PSMA 偶联的磁珠从分离的总 EV 中以及直接从血浆样本中富集 PSEV。进行纳米颗粒跟踪分析（NTA）以检测总 EV 和 PSEV 的浓度（颗粒/mL）、平均直径（nm）和体积（nm³）。在 JOEL JEM1400 透射电子显微镜下对分离的 PSEV 的超微结构进行成像。将平均值进行非配对 t 检验分析，p 值小于 0.05 被认为具有统计学意义。
结果：NTA 分析显示：1）用柱分离的总细胞外囊泡从每份血浆样本中获得了显著的产量（1.4E+11 至 7.52E+11 颗粒/ml，直径 149.8 至 182 nm，体积 752 至 1,360 nm³）；2）非裔美国人（AA）人群血浆中的 PSEV 浓度（178.9×10⁹/ml）高于白种美国人（CA）人群血浆中的浓度（166.6×10⁹/ml）；3）直接从血浆中使用抗 PSMA 偶联磁珠的方法（13.4×10⁹/ml）的 PSEV 浓度高于通过柱获得的总细胞外囊泡中富集的浓度（10.98×10⁹/ml）；4）直接从血浆中使用抗 PSMA 偶联磁珠的方法（198.7 nm）的平均 PSEV 直径大于通过柱获得的总细胞外囊泡中的直径（149.3 nm，p=0.002）；以及 5）在非癌受试者中 PSEV 约占总 EV 的 3.1%，在 PCa 中占总细胞外囊泡的 4.7%。TEM 分析证实了存在直径范围为 30-258 nm 的电子致密的椭圆形或圆形颗粒。然而，PSEV 在颗粒表面显示出一些棘突。
结论：使用抗 PSMA 偶联磁珠进行 PSEV 富集是一种可行的方法，可增强潜在 EV 相关生物标志物的特异性，有助于改善前列腺癌的诊断、预后、风险预测和治疗反应监测。
查看英文原文 English abstract
Background: Plasma-derived extracellular vesicles (EV) represent a complex mixture of vesicles originating from diverse tissues. The selective isolation of prostate-specific extracellular vesicles (PSEV) can offer improved specificity for diagnostic and prognostic biomarker development as well as monitoring therapeutic response of prostate cancer (PCa). This study aimed to compare the yield and quality of PSEV enriched from total EV preparations with those isolated directly from plasma.
Materials and methods: Twelve (12) plasma samples (6 from patients with PCa, and 6 from non-cancer subjects) were used in this study. The total EV were isolated with SmartSEC TM columns following the manufacturer's instructions. The PSEV were enriched with anti-PSMA-conjugated magnetic beads from isolated total EV and also directly from plasma samples. Nanoparticle tracking analysis (NTA) was performed to examine the concentration (particles/mL), average diameter (nm), and volume (nm³) for total EV and PSEV. The ultrastructure of isolated PSEV was imaged under a JOEL JEM1400 transmission electron microscope. The mean values were subjected to an unpaired t-test analysis, and a p-value of less than 0.05 was considered statistically significant.
Results: NTA analysis showed that 1) Total extracellular vesicles isolated with the column resulted in a significant yield from each plasma sample (1.4E+11 to 7.52E+11 particles/ml, 149.8 to 182 nm in diameter, and 752 to 1,360 nm 3 in volume); 2) PSEV concentration was higher in plasma from African American (AA) population (178.9x10 9 /ml) than in plasma from Caucasian American (CA) population (166.6x10 9 /ml); 3) the PSEV concentration was higher in method using anti-PSMA-conjugated magnetic beads from plasma directly (13.4x10 9 /ml) as compared to that enriched from total extracellular vesicles obtained via column (10.98x10 9 /ml); 4) the average PSEV diameter was greater in method using anti-PSMA-conjugated magnetic beads directly from plasma (198.7nm) as compared to that from total extracellular vesicles obtained via column (149.3nm, p=0.002); and 5) PSEV was about 3.1% of total EV in non-cancer subjects, and 4.7% of total extracellular vesicles in PCa. The TEM analysis demonstrated the existence of electron-dense oval or round particles in diameters ranging from 30-258 nm. However, PSEV showed some spikes on the particle surface.
Conclusion: PSEV enrichment using anti-PSMA-conjugated magnetic beads is a feasible approach that could enhance the specificity of potential EV-associated biomarkers, aiding in improved diagnosis, prognosis, risk prediction, and monitoring therapeutic responses in prostate cancer.
利益披露 Disclosure
S. Anand, None..
E. Ridden, None..
T. Turner, None..
K. Vikramdeo, None..
R. Velagapudi, None..
D. Cornelius, None..
A. Singh, None..
X. Zhou, None.