Protein Biomarker Discovery Studies on Urinary sEV Fractions Separated with UF-SEC for the First Diagnosis and Detection of Recurrence in Bladder Cancer Patients

Urinary extracellular vesicles (EVs) are an attractive source of bladder cancer biomarkers. Here, a protein biomarker discovery study was performed on the protein content of small urinary EVs (sEVs) to identify possible biomarkers for the primary diagnosis and recurrence of non-muscle-invasive bladder cancer (NMIBC). The sEVs were isolated by ultrafiltration (UF) in combination with size-exclusion chromatography (SEC). The first part of the study compared healthy individuals with NMIBC patients with a primary diagnosis. The second part compared tumor-free patients with patients with a recurrent NMIBC diagnosis. The separated sEVs were in the size range of 40 to 200 nm. Based on manually curated high quality mass spectrometry (MS) data, the statistical analysis revealed 69 proteins that were differentially expressed in these sEV fractions of patients with a first bladder cancer tumor vs. an age- and gender-matched healthy control group. When the discriminating power between healthy individuals and first diagnosis patients is taken into account, the biomarkers with the most potential are MASP2, C3, A2M, CHMP2A and NHE-RF1. Additionally, two proteins (HBB and HBA1) were differentially expressed between bladder cancer patients with a recurrent diagnosis vs. tumor-free samples of bladder cancer patients, but their biological relevance is very limited.

Automated summary

Protein biomarkers for non-muscle-invasive bladder cancer (NMIBC) were discovered in small urinary extracellular vesicles (sEVs) through a study comparing healthy individuals with NMIBC patients and tumor-free patients with recurrent NMIBC. The sEVs were isolated using ultrafiltration (UF) and size-exclusion chromatography (SEC), and 69 differentially expressed proteins were identified in the sEV fractions of NMIBC patients compared to healthy controls based on mass spectrometry (MS) data. MASP2, C3, A2M, CHMP2A, and NHE-RF1 showed the most potential as biomarkers for distinguishing healthy individuals from first diagnosis NMIBC patients.