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.