One-Pot Sequential Enrichment of Urinary Extracellular Vesicle and miRNAs Identifies a Noninvasive Biomarker Panel for Prostate Cancer Diagnosis

Extracellular vesicles (EVs) offer promising noninvasive alternatives for convenient and noninvasive prostate cancer (PCa) diagnosis, but inefficient EV enrichment and cargo extraction hinder discovery and validation for their clinical applications. Here, we present an integrated pipeline based on functionalized magnetic beads to streamline and enhance the efficiency of urinary EV miRNA analysis. EVs are first enriched on amphiphilic magnetic beads through chemical affinity, followed by EV lysis and the isolation of miRNAs through solid phase extraction. The new pipeline demonstrated a more than 10-fold increase in urine EV miRNA extraction efficiency compared to the traditional ultracentrifugation combined TRIzol method while reducing the sample processing time to within 1 h. The one-bead strategy further allowed us to automate the procedure on a 96-channel instrument. We applied the pipeline to analyze urine samples from 108 benign prostatic hyperplasia (BPH) controls and 92 PCa cases. Among 195 miRNA biomarkers from the literature, we prioritized 18 miRNAs for quantification and successfully validated 12 miRNAs with a ratio-based normalized method. The quantification data from BPH controls and PCa cases in the training set were subjected to a machine learning analysis of Random Forest, through which we generated a five-miRNA panel consisting of miR-148a-5p, miR-21-5p, miR-181a-5p, miR-222-3p, and miR-100-5p. This panel showed high sensitivity (89%) and specificity (72%) in the test set, highlighting immense potential of this streamlined pipeline for noninvasive diagnosis.