Urinary peptidome and proteome alterations related to tumor progression and invasion in RCC

INTRODUCTION AND OBJECTIVES: Up to now, for renal cell carcinoma (RCC), tumor size and growth rate are the most used prognostic factors at diagnosis likely being related with potential progressive neoplasms. Moreover, the degree of the free tumor extension into renal vessel seems to contribute to disease-free survival and to influence prognosis. Thus, firstly, in order to highlight urinary peptidome signatures that might reflect kidney cancer progression, a systematic investigation by MALDI profiling was carried out identifying endogenous peptides that show a correlation between their abundance in urine and pT, tumor size and grade. Secondly, in order to explore proteome changes reflecting the local tumor infiltration through vena cava, a labelfree nLC MS/MS was performed in trypsinized urine from subjects affected by RCC at different level of invasion (vascular endothelium/ vein/ thrombosis). METHODS: Peptidomic evaluation of tumor progression was performed in 117 RCC urine samples, through the application of C8 functionalized magnetic beads purification followed by MALDI-TOF and statistical analysis for group comparisons and correlations. nLC-ESI-MS/MS was used for peptide identification. Urine proteome investigation correlated to different renal vein invasion extent was carried out by label-free nLC MS/MS strategy using FASP digested pools of 3 groups. Statistical evaluation was obtained by Progenesis QI for Proteomics. RESULTS: 15 endogenous peptides showed a statistically significant correlation between their urinary concentration and tumor size (only 3 negatively), 26 with pT (only 6 negatively), and only 5 with grade (only 1 positively). Most of them were differentially represented in urine of RCC patients compared to controls and for some of them varied according to pT or stage. Identity of several of them are likely to confirm their possible role in tumor progression. Proteomic labelfree approach regarding tumor invasion allowed to select 671 protein IDs significantly altered in at least one of the 3 group comparisons (fold change¼2). 12 of them showed a progressive increase from the endothelium wall infiltration to thrombosis CONCLUSIONS: These methodological approaches applied to urine may provide useful keys to highlight alterations triggered by RCC aggressiveness or vascular neoplastic infiltration from endothelial layer to vein ostium obstruction, better understand tumor protein handling aspects, and describe such a dynamic system as growing cancer cells are.