Identification of ENO-1 positive extracellular vesicles as a circulating biomarker for monitoring of Ewing sarcoma

The lack of circulating biomarkers for tumor monitoring is a major problem in Ewing sarcoma management. The development of methods for accurate tumor monitoring is required, considering the high recurrence rate of drug- resistant Ewing sarcoma. Here, we describe a sensitive analytical technique for tumor monitoring of Ewing sarcoma by detecting circulating extracellular vesicles secreted from Ewing sarcoma cells. Proteomic analysis of Ewing sarcoma cell-derived extracellular vesicles identi-fied 564 proteins prominently observed in extracellular vesicles from three Ewing sarcoma cell lines. Among these, CD99, SLC1A5, and ENO-1 were identified on extra-cellular vesicles purified from sera of patients with Ewing sarcoma before treatment but not on extracellular vesicles from those after treatment and healthy individuals. Notably, not only Ewing sarcoma-derived extracellular vesicles but also Ewing sar-coma cells demonstrated proteomic expression of CD99 and ENO-1 on their surface membranes. ENO-1(+)CD6(3+) extracellular vesicle detection was reduced after tumor resection while both CD99+CD63+ and ENO-1(+)CD6(3+) extracellular vesicles were detected in serum from Ewing sarcoma- bearing mice. Finally, the accuracy of liquid biopsy targeting these candidates was assessed using extracellular vesicles from the sera of patients with Ewing sarcoma. Elevated ENO-1+CD81+ extracellular vesicles in the serum of patients before treatments distinguished patients with Ewing sarcoma from healthy individuals with an area under the curve value of 0.92 (P< 0.001) and reflected the tumor burden in patients with Ewing sarcoma during multidisciplinary treatments. Collectively, circulating ENO-1(+)CD81(+) extracellular vesicle detection could represent a novel tool for tumor monitoring of Ewing sarcoma.