Differential protein profiling of renal cell carcinoma urinary exosomes

Abstract

aRenal cell carcinoma (RCC) accounts for about 3% of all human malignancies and its incidence is increasing. There are no standard biomarkers currently used in the clinical management of patients with renal cell carcinoma. A promising strategy for new biomarker detection is comparative proteomics of urinary exosomes (UE), nanovesicles released by every epithelial cell facing the urinary space, enriched in renal proteins and excluding high-abundance plasmatic proteins, such as albumin. Aim of the work is to establish the protein profile of exosomes isolated from urines of RCC patient compared with control subjects. We enrolled 29 clear cell RCC patients and 23 control healthy subjects (CTRL), age and sex-matched, for urine collection and vesicle isolation by differential centrifugation. Such vesicles were morphologically and biochemically characterized and proved to share exosome properties.Proteomic analysis, performed on 9 urinary exosome (UE) pooled samples by gel based digestion followed by LC-MS/MS, led to the identification of 261 proteins from CTRL subject UE and 186 from RCC patient UE, and demonstrated that most of the identified proteins are membrane associated or cytoplasmic. Moreover, about a half of identified proteins are not shared between RCC and control UE.Starting from these observations, and from the literature, we selected a panel of 10 proteins, whose UE differential content was subjected to immunoblotting validation. Results show for the first time that RCC UE protein content is substantially and reproducibly different from control UE, and that these differences may provide clues for new RCC biomarker discovery.

Preparation of Urinary Exosomes: Methodological Issues for Clinical Proteomics

“…Comparison of the protein pattern of urinary exosomes of a healthy subject (UE) with the urine (U) of origin, after sediment removal. UE were isolated by the ultracentrifugation protocol, and urine proteins were concentrated by ultrafi ltration [ 11 ]. Their protein profi les are very different, as expected: albumin, responsible for the main band appearance in urine, results depleted in UE, while THP is predominant.…”

“…Many different protocols are available to prepare urinary proteins. We used ultrafi ltration by concentrator devices, as described by Raimondo et al [ 11 ].…”

The emerging role of extracellular vesicles as biomarkers for urogenital cancers

“…For instance, several proteomic studies have revealed the presence of urinary extracellular vesicles that contain candidate proteins unique to cancer types that include a broad range of urogenital diseases. 25,26,29,102,110,112,147,157,162 Such tumour-specific vesicles are easily captured from urine using established isolation procedures, which enables repeated tissue sampling.…”

“…146 Proteomic analysis of urinary extracellular vesicles has facilitated the identification of an RCC-specific 'fingerprint' , containing proteins such as MMP-9, podocalyxin (PODXL), Dickkopf-related protein 4 (DKK4), carbonic an hydrase IX (CAIX) and ceruloplasmin (Table 4). 147 These preliminary studies suggest that urinary extracellular vesicles could provide a tool for identifying new b iomarkers for RCC, although further studies are needed.…”

PUEPro: A Computational Pipeline for Prediction of Urine Excretory Proteins

“…Therefore, it is necessary to identify and remove proteins that are from the urinary system among proteins found in urine, in order to identify biomarkers for diseases in distal organs [3]. Currently, the most useful disease markers in urine have been largely for urogenital diseases, such as urothelial cancer [4], renal cell carcinoma [5], prostate cancer [6], and bladder cancer [7]. A few recent studies have demonstrated the feasibility in using urinary proteins as disease markers in distal organs, such as ovarian carcinoma [8], lung cancer [9], hepatocellular carcinoma [10], and gastric cancer [11].…”