Plasma Preparation Strategies for Extracellular Vesicle‐Based Biomarkers in Metastatic Castration‐Resistant Prostate Cancer

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 (n = 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.