Sinawang Prima Dewi, Multani Priyanka, Ozen Mehmet O, Wong Jodie, Akin Demir, Hanson Claire, Larsen Matt, Ampaw Enos, Rondina Matthew T, Tolley Neal D, Wang Liang, Cunningham Brian T, Kohli Manish, Demirci Utkan
Department of Chemical Engineering Stanford University Stanford California USA.
Bio-Acoustic MEMS in Medicine (BAMM) Laboratories, Department of Radiology Stanford University Palo Alto California USA.
J Extracell Biol. 2025 Jul 31;4(8):e70071. doi: 10.1002/jex2.70071. eCollection 2025 Aug.
Extracellular vesicles (EVs) offer a minimally invasive approach for cancer detection and monitoring. However, the lack of standardized methods for clinical biospecimen preparation and EV isolation limits the clinical utility of EV-based biomarker assessments. A targeted need exists for detailed analysis of plasma EV content. Our study investigates the impact of clinical sample preparation and our ExoTIC device on the quality of plasma-derived EVs and their RNA/protein cargo in metastatic castration-resistant prostate cancer (mCRPC) patients. We assessed sample preparation variables: blood anti-coagulant choice (EDTA or sodium citrate), type of plasma platelet fraction (platelet-rich or platelet-poor), and use of protease inhibitors. EVs were isolated via ExoTIC device, followed by EV characterization and biomarker analysis using nanoparticle tracking analysis (NTA), cryogenic electron microscopy, Western blot, and digital PCR (dPCR). We detected mCRPC-relevant proteins (ARv7 and PSMA) in EVs from all plasma sample types with different sample preparation variables. Additionally, our findings indicate that platelet-poor plasma (PPP) is optimal for detecting EV- and biologically associated mCRPC biomarker miR-375. In this pilot study ( = 3), elevated EV miR-375 levels in PPP samples from mCRPC patients experiencing disease progression during docetaxel treatment were associated with poor therapeutic response to docetaxel chemotherapy, which aligns with our preceding in vitro and in vivo study. Optimal biospecimen preparation for EV analysis could enhance detection accuracy and patient management, highlighting detection of plasma EV-associated mCRPC-specific marker proteins (ARv7 and PSMA) and microRNA miR-375.
细胞外囊泡（EVs）为癌症检测和监测提供了一种微创方法。然而，临床生物样本制备和EV分离缺乏标准化方法限制了基于EV的生物标志物评估的临床应用。目前迫切需要对血浆EV含量进行详细分析。我们的研究调查了临床样本制备和我们的ExoTIC设备对转移性去势抵抗性前列腺癌（mCRPC）患者血浆来源的EVs及其RNA/蛋白质含量质量的影响。我们评估了样本制备变量：血液抗凝剂的选择（乙二胺四乙酸或柠檬酸钠）、血浆血小板组分类型（富含血小板或血小板贫乏）以及蛋白酶抑制剂的使用。通过ExoTIC设备分离EVs，随后使用纳米颗粒跟踪分析（NTA）、低温电子显微镜、蛋白质免疫印迹和数字PCR（dPCR）对EVs进行表征和生物标志物分析。我们在所有具有不同样本制备变量的血浆样本类型的EVs中检测到了与mCRPC相关的蛋白质（ARv7和前列腺特异性膜抗原）。此外，我们的研究结果表明，血小板贫乏血浆（PPP）最适合检测与EV和生物学相关的mCRPC生物标志物miR-375。在这项初步研究（n = 3）中，多西他赛治疗期间疾病进展的mCRPC患者的PPP样本中EV miR-375水平升高与多西他赛化疗的治疗反应不佳相关，这与我们之前的体外和体内研究一致。用于EV分析的最佳生物样本制备可以提高检测准确性和患者管理水平，突出了血浆EV相关的mCRPC特异性标记蛋白（ARv7和前列腺特异性膜抗原）和微小RNA miR-375的检测。
