One-Pot Sequential\nEnrichment of Urinary Extracellular\nVesicle and miRNAs Identifies a Noninvasive Biomarker Panel for Prostate\nCancer Diagnosis

Extracellular\nvesicles (EVs) offer promising noninvasive alternatives\nfor convenient and noninvasive prostate cancer (PCa) diagnosis, but\ninefficient EV enrichment and cargo extraction hinder discovery and\nvalidation for their clinical applications. Here, we present an integrated\npipeline based on functionalized magnetic beads to streamline and\nenhance the efficiency of urinary EV miRNA analysis. EVs are first\nenriched on amphiphilic magnetic beads through chemical affinity,\nfollowed by EV lysis and the isolation of miRNAs through solid phase\nextraction. The new pipeline demonstrated a more than 10-fold increase\nin urine EV miRNA extraction efficiency compared to the traditional\nultracentrifugation combined TRIzol method while reducing the sample\nprocessing time to within 1 h. The one-bead strategy further allowed\nus to automate the procedure on a 96-channel instrument. We applied\nthe pipeline to analyze urine samples from 108 benign prostatic hyperplasia\n(BPH) controls and 92 PCa cases. Among 195 miRNA biomarkers from the\nliterature, we prioritized 18 miRNAs for quantification and successfully\nvalidated 12 miRNAs with a ratio-based normalized method. The quantification\ndata from BPH controls and PCa cases in the training set were subjected\nto a machine learning analysis of Random Forest, through which we\ngenerated a five-miRNA panel consisting of miR-148a-5p, miR-21–5p,\nmiR-181a-5p, miR-222–3p, and miR-100–5p. This panel\nshowed high sensitivity (89%) and specificity (72%) in the test set,\nhighlighting immense potential of this streamlined pipeline for noninvasive\ndiagnosis.