Evaluation of Colon-Specific Plasma Nanovesicles as New Markers of Colorectal Cancer.

Inga Nazarova,Nadezhda Nikiforova,Maria Slyusarenko,Achim Aigner,Anastasia Malek,Elena Sidina,Vladislav Semiglazov,Tatiana Semiglazova,Evgeny Rybakov

doi:10.3390/cancers13153905

Abstract

Simple SummaryHigh mortality in patients with colorectal cancer (CRC) is one of the main problems in healthcare. This is due to the use of invasive and existing noninvasive screening methods whose resources are limited. A promising alternative is a study of circulating in plasma extracellular nanovesicles (ENVs) reflecting the same composition of biomarkers with the secreted cells, forming subpopulations of tissue-specific ENVs. During the work, we have selected potential colon biomarkers from databases. The study aims to develop a quantitative method for isolating colon-specific ENVs based on the formation of immunocomplexes «beads with antibodies to specific biomarkers». We found that the amount of ENVs carrying potential colon biomarkers was higher in the patients with an IV CRC-stage compared with the healthy donors. It showed a high coefficient of diagnostic significance of these biomarkers in the CRC prognosis. These results will give an impetus to a deeper study of the ENVs as identifiers of cancer’s development.Purpose: Developing new and efficient approaches for the early diagnosis of colorectal cancer (CRC) is an important issue. Circulating extracellular nanovesicles (ENVs) present a promising class of cancer markers. Cells of well-differentiated adenocarcinomas retain the molecular characteristics of colon epithelial cells, and the ENVs secreted by these cells may have colon-specific surface markers. We hypothesize that an increase in the number of ENVs carrying colon-specific markers could serve as a diagnostic criterion for colorectal cancer. Experimental design: Potential colon-specific markers were selected based on tissue-specific expression profile and cell surface membrane localization data. Plasma was collected from CRC patients (n = 48) and healthy donors (n = 50). The total population of ENVs was isolated with a two-phase polymer system. ENVs derived from colon epithelium cells were isolated using immune-beads with antibodies to colon-specific markers prior to labelling with antibodies against exosomal tetraspanins (CD63 and CD9) and quantification by flow cytometry. Results: The number of ENVs positive for single colon cancer markers was found to be significantly higher in the plasma of CRC patients compared with healthy donors. The efficacy of detection depends on the method of ENV labelling. The diagnostic efficacy was estimated by ROC analysis (the AUC varied between 0.71 and 0.79). The multiplexed isolation of colon-derived ENVs using immune-beads decorated with antibodies against five markers allowed for a further increase in the diagnostic potency of the method (AUC = 0.82). Conclusions: ENVs derived from colon epithelium may serve as markers of differentiated CRC (adenocarcinomas). The composition of ligands used for capturing colon-derived ENVs and their method of labelling are critical for the efficacy of this proposed diagnostic approach.

Highlights

The concept of the “liquid biopsy” is a not new; it is still an attractive alternative to traditional methods of cancer diagnostics
The Plasma Two-Phase Polymer System (PTPS) method was applied for the isolation of the exosome-enriched population of plasma extracellular nanovesicles (ENVs)
ENVs were isolated from 1.5 mL plasma and dissolved in 100 μL PBS

Summary

Introduction

The concept of the “liquid biopsy” is a not new; it is still an attractive alternative to traditional methods of cancer diagnostics. Different liquid biopsy platforms are available and are at different stages of clinical implementation These include the assessment of free-circulating plasma proteins or glycoproteins, circulating cell-free nucleic acids, extracellular nanovesicles (ENVs), and circulating tumor cells (CTC). Extracellular vesicles are an extensive heterogeneous group of membrane-covered vesicles, such as microvesicles, apoptotic bodies, and nanovesicles (exosomes), arising through the mechanism of endosomal transport of all cell types and release into the external space [1,2]. The biogenesis of these types of vesicles differs. Microvesicles with sizes 150–1000 nm [1] are formed by budding outward from the plasma membrane [2], while the formation of nanovesicles with sizes of 50–150 nm [1] takes place in several stages [2,3]: (i) the previous stage, with invagination of the plasma membrane’s domains, covered with clathrin (clathrin-coated vesicles (CCVs)); (ii) work of the ESCRT (Endosomal Sorting Complex Required for Transport) machinery, consisting of four functional complexes (ESCRT-0, -I, -II, and -III), which are at different stages involved in the sorting of ubiquitinated cargo and contributes to the development CCVs in an early-endosome (EE) carrying ubiquitinating cargo; (iii) the secondary invagination of EEs, forming intraluminal vesicles (ILVs) that accumulate and mature into multivesicular bodies; and (iv) some multivesicular bodies delivering ubiquitinated membrane proteins for their degradation in lysosomes while others release ILVs into the extracellular space, called nanovesicles (exosomes) [4,5]

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