Diagnosing renal cell cancer (RCC) faces limitations such as distinguishing indolent from aggressive tumors, highlighting the need for improved non-invasive diagnostic methods. Urine contains extracellular vesicles (EVs), which are secreted by various cell types originating from the kidneys, including RCC cells. In this study, we performed mass spectrometry-based proteomics of urinary EVs (uEVs) with the aim to identify and characterize protein markers within uEVs and kidney tissue of RCC patients that could serve also non-invasive biomarkers for the detection of RCC.
uEVs were isolated from urine samples using the heat shock protein binding peptide Vn96. We included 15 control urine samples and 9 from RCC patients. RCC tissue (n=48) was obtained from formalin fixed samples and micro-dissected to enrich for specific cancer tissues. The proteome was measured using mass spectrometry-based proteomics.
In total 3560 uEV proteins (of which 1593 with 30% data presence) were identified. Over 10,000 proteins were identified in RCC tissue. Supervised heatmap of uEV protein profiles showed complete separation of RCC patients from controls, based on p-value (p<0.05 for 518 proteins). Of the 461 known human atlas kidney specific proteins, 25% were associated with uEVs, indicating that kidneys excrete specific proteins into the urine via EVs. Differential analysis yielded 94 proteins significantly increased by at least two-fold in uEVs and 347 in RCC tissue. Notably, 19 proteins were elevated in both uEVs and RCC tissue, making these potential biomarkers for RCC detection. The significantly upregulated uEV proteins exhibited high connectivity in STRING network analysis, which indicates that the identified proteins have strong interconnections and likely work together in biological networks or pathways. Functional enrichment analysis revealed these proteins to be involved in immune respons and mTOR signaling and oxidative phosphorylation, mirroring key pathways altered in RCC tissue. Among the 19 significant pathways altered in uEVs, 11 were also disrupted in RCC tissue, suggesting the altered tissue biology is reflected within the uEVs.
Our study demonstrates that uEVs contain RCC-specific proteins with potential as non-invasive biomarkers for RCC diagnosis and prognosis, offering insights into immune-related and cell-regulatory pathways pertinent to RCC pathology.