Detection of Extracellular Vesicles with Colocalized Surface Markers via a Capture–Release–Capture Strategy for Treatment Monitoring in Ewing Sarcoma

Ewing Sarcoma (ES) is a rare but aggressive malignancy of bone tissue in adolescents and young adults, where early detection of progression and real-time treatment monitoring remain unmet clinical needs. Tumor extracellular vesicles (EVs) carry surface markers and nucleic acid cargo that can serve as minimally invasive biomarkers, but single-marker EV assays often lack specificity, and colocalized-marker approaches may suffer from low sensitivity. Here, we report the ES EV Capture-Release-Capture (CaReCa) assay, a two-step enrichment strategy that combines desthiobiotin (DTB)-mediated capture/release of CD99⁺ EVs with click chemistry-mediated recapture of CD99⁺/B7-H3⁺ EVs, introducing molecular specificity to suppress background signals. To overcome limited yield from EVs with colocalized markers, we incorporated RT-digital PCR quantification of encapsulated ACTB mRNA, a stable housekeeping transcript, as a sensitive proxy for EV abundance. Using only 100 µL of plasma, the ES EV CaReCa assay distinguished ES patients (n = 20) from healthy donors (n = 20) with an AUROC of 0.98. Longitudinal analysis further demonstrated that dynamic changes in the assay readouts paralleled disease progression and treatment response, consistent with PET/CT findings. Together, these results establish CaReCa as a sensitive, specific, and scalable liquid biopsy platform with translational potential for noninvasive monitoring of ES patients.