Protein Profiling of Serum Extracellular Vesicles Reveals Qualitative and Quantitative Differences After Differential Ultracentrifugation and ExoQuickTM Isolation.

Timo Gemoll,Sonja Hartwig,Holger Kalthoff,Stefan Lehr,Abdou Elsharawy,Svitlana Rozanova,Christian Röder,Jens Habermann,Sarah Strohkamp

doi:10.3390/jcm9051429

Abstract

Solid tumor biopsies are the current standard for precision medicine. However, the procedure is invasive and not always feasible. In contrast, liquid biopsies, such as serum enriched for extracellular vesicles (EVs) represent a non-invasive source of cancer biomarkers. In this study, we compared two EV isolation methods in the context of the protein biomarker detection in inflammatory bowel disease (IBD) and colorectal cancer (CRC). Using serum samples of a healthy cohort as well as CRC and IBD patients, EVs were isolated by ultracentrifugation and ExoQuick™ in parallel. EV associated protein profiles were compared by multiplex-fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) and subsequent identification by mass spectrometry. Validation of gelsolin (GSN) was performed using fluorescence-quantitative western blot. 2D-DIGE resolved 936 protein spots in all serum-enriched EVs isolated by ultracentrifugation or ExoQuick™. Hereof, 93 spots were differently expressed between isolation approaches. Higher levels of GSN in EVs obtained with ExoQuick™ compared to ultracentrifugation were confirmed by western blot (p = 0.0006). Although patient groups were distinguishable after both EV isolation approaches, sample preparation strongly influences EVs’ protein profile and thus impacts on inter-study reproducibility, biomarker identification and validation. The results stress the need for strict SOPs in EV research before clinical implementation can be reached.

Highlights

Colorectal cancer (CRC) is the fourth leading cause of cancer death worldwide [1]
While ultracentrifugation is usually regarded as the “golden standard” for extracellular vesicles (EVs)’ isolation and primarily based on the size of extracellular particles, the polymer-based isolation by the ExoQuickTM kit uses a polymer solution which creates a polymer network allowing the separation of EVs by low-speed centrifugation
EV Protein Profile Depending on Isolation Method

Summary

Introduction

About 1–2% of CRCs are associated with inflammatory bowel disease (IBD) which comprises a group of chronic inflammatory disorders of the colon and small intestine. Histopathology diagnostics of IBD-associated mucosa biopsies is clearly hampered and less reproducible among experts [3]. A non-invasive, clinically reliable screening program requires disease-specific biomarkers that accurately detect IBD, precancerous colorectal neoplasia and CRC at earliest stages. Along with circulating tumor cells and circulating cell-free DNA, extracellular vesicles (EVs) are considered to be a promising source for liquid biopsy-based biomarker discovery allowing non-invasive screening, diagnostics, therapy guidance and repeated sampling for disease monitoring [4]. EVs are membrane-bound particles, secreted in vivo by cells into the extracellular environment and are detected in various biological fluids such as plasma, urine, saliva, ascites and bronchoalveolar lavage fluid [5]. EVs are classified into three groups: apoptotic bodies (1000–5000 nm), microvesicles (200–1000 nm) as well as exosomes (30–150 nm) and are found to play key roles in physiological and pathological events, e.g., intracellular communication [6,7], cell signaling [8], immune response [9,10] and carcinogenesis [5,11]

Objectives

Methods

Results

Conclusion