P-245 Evaluation of colon-specific plasma nanovesicles as new markers of colorectal cancer

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Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide, which is associated with low detection efficiency in the early stages of its development. The traditional methods of CRC screening are not effective enough. The liquid biopsy, based on the identification of extracellular nanovesicles (ENVs) in plasma, appears to be a promising diagnostic technology. Current approaches ENV-based cancer diagnostics suppose either quantification of tumor-derived ENVs or detection of cancer-associated components (e.g. miRNA) in total population of plasma ENVs. The first approach lacks specificity due to an absence of well-established vesicular membrane-associated markers of CRC. The second approach lacks sensitivity due to the extremely heterogeneous composition of plasma ENVs derived mostly from endothelium and blood cells. We hypothesized that development of a well-differentiated adenocarcinoma from colon epithelium should result in increased release of ENVs bearing markers of the intestinal differentiation. If this hypothesis is true, these vesicles might serve as markers of CRC. Plasma samples from CRC patients (n=48) and healthy donors (n.=50) were used. Design of study was confirmed by the local ethics committee, each participant signed a form of informed consent. The total population of ENVs was isolated from plasma using a two-phase polymer system and analyzed in terms of size, concentration, morphology and surface markers using NTA, Cryo-EM and on-bead flow cytometry. To isolate tissue-specific exosomes, we used streptavidin-coated super-paramagnetic particles (SPMP) and biotinylated antibodies to CLRN3, GPA33, GCNT3, PIGY, REG4, and other ten potential markers. The efficiency of the immune-sorption of such exosomes to SPMP was assessed by flow cytometry after labeling with CD63 FITC and/or CD9 PerCP-Cy5.5 antibodies. Potential markers were selected on the basis of their colon-specific expression pattern and cell surface membrane-associated localization. We selected 16 potential markers and evaluated the amount of ENVs positive for these markers in plasma of CRC patients and healthy donors. The amount of ENVs bearing CLRN3, GPA33, GCNT3, PIGY and REG4 differed between groups of patients and donors, difference was statistically significant. The value of AUC varied between 0,6 and 0,81. Multiplexed analysis allowed us to observe an increased difference between compared groups (CRC vs HD) and to achieve AUC = 0.72. The presented study is the first attempt to explore intestine-specific ENVs as a marker of cancer arising from this tissue. Our results confirmed the initial hypothesis and showed the effectiveness of the proposed approach. Further investigations are still required to identify the best markers of colon epithelium cell-derived ENVs, to better understand the link between CRC characteristics and the profile of tumor-released ENVs and to optimize techniques of quantification of the intestine-specific ENVs in plasma.