The diagnosis and monitoring of Bladder cancer (BlCa) rely on invasive procedures, highlighting the urgent need for minimally invasive alternatives. Tumor-derived extracellular vesicles (EVs) offer a promising source for non-invasive biomarker discovery by the communication of tumor-specific information via biofluids. However, isolating BlCa-specific EVs from liquid biopsies remains a challenge. This study aimed to comprehensively characterize the proteome of EVs from tissue explants, plasma, and urine of BlCa patients, comparing them to healthy donors and other urological cancers (ccRCC and PCa), to identify BlCa-specific biomarkers and elucidate EV-mediated communication.

Matched tissue explants, plasma, and urine samples were collected from BlCa patients (n=6), along with normal adjacent tissues and biofluids from healthy donors (n=6) and urine from patients with ccRCC (n=6) and PCa (n=6). EVs were isolated by ultracentrifugation and characterized using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and micro BCA. EV proteomic profiles were determined by liquid chromatography-mass spectrometry in a total of 48 samples.

Tissue EVs exhibited the highest protein diversity, followed by urine and plasma. Differential proteomic analysis revealed a 14-protein signature specific to BlCa tissue EVs. Urine proved to be a particularly rich source of BlCa-derived EVs, enabling the tracking of molecular information from tissue to urine. There was a clear distinct protein profile in BlCa urine EVs compared to healthy donors, ccRCC, and PCa. In the end, we identified several candidate BlCa biomarkers, including two proteins exclusively present in BlCa tissue and urine EVs. Furthermore, six novel urinary EV proteins were exclusively found in BlCa and absent in healthy donors, ccRCC, and PCa.

This study identified a panel of BlCa-derived EV proteins with strong potential for non-invasive diagnostics and monitoring. Urine emerged as a valuable source of BlCa EV biomarkers, and further investigation of the tissue-urine EV pathway may uncover novel biomarkers and enhance our understanding of EV-mediated communication in BlCa.