Isolation and Characterization of Extracellular Vesicles from Gastric Juice

Abstract

EVs are involved in local and distant intercellular communication and play a vital role in cancer development. Since EVs have been found in almost all body fluids, there are currently active attempts for their application in liquid diagnostics. Blood is the most commonly used source of EVs for the screening of cancer markers, although the percentage of tumor-derived EVs in the blood is extremely low. In contrast, GJ, as a local biofluid, is expected to be enriched with GC-associated EVs. However, EVs from GJ have never been applied for the screening and are underinvestigated overall. Here we show that EVs can be isolated from GJ by ultracentrifugation. TEM analysis showed high heterogeneity of GJ-derived EVs, including those with exosome-like size and morphology. In addition to morphological diversity, EVs from individual GJ samples differed in the composition of exosomal markers. We also show the presence of stomatin within GJ-derived EVs for the first time. The first conducted comparison of miRNA content in EVs from GC patients and healthy donors performed using a pilot sampling revealed the significant differences in several miRNAs (-135b-3p, -199a-3p, -451a). These results demonstrate the feasibility of the application of GJ-derived EVs for screening for miRNA GC markers.

Keywords: cancer markers; exosomes; extracellular vesicles; gastric cancer; gastric juice; miRNA; stomatin.

Figure 1

The characterization of GJ-derived EVs by nanoparticle tracking analysis. T1–T7—samples obtained from GC patients. Clinical and morphological characteristics are shown in Supplementary Materials, Table S1. N1-N6—samples obtained from non-cancer individuals. (A) Examples of size spectra of a CD9-positive (CD9(+)) and a CD9-negative (CD9(−)) EV samples isolated from GJ of GC patients (samples T1, T4). (B) NTA data for EV size distribution among all CD9(+) and CD9(−) EV samples studied. (C) Main NTA characteristics of CD9(+) EVs, CD9(−) EVs and across the entire sample. (D) Comparison of the mean size of CD9(+) and CD9(−) EVs (** p < 0.01).

Figure 2

Analysis of EV morphology and exosomal marker composition. (A) Western-blot analysis of exosomal markers Alix, flotillin-2 (Flot-2), TSG-101, and CD9 as well as stomatin protein (Stom) in EVs from GJ of GC patients (T1–T7, clinical and morphological characteristics are shown in Supplementary Materials, Table S1) and non-cancer individuals (N1–N6). Full Western blot images can be found in Figure S1. The PCNA protein was used to confirm the absence of cellular proteins of non-vesicular origin in EV preparations. Protein lysates of GIST-T1 cells (Cntrl 1) and GC tissue (Cntrl2) were used as molecular weight controls and to compare levels of proteins in cells and EVs. Two bands of CD9 protein correspond to full-size form (24 kDa) and lower molecular weight form (of about 20 kDa). (B) TEM analysis of EV morphology. Examples of CD9(+) EVs (samples N1, T2, T6) and CD9(−) EVs (samples T1, N3, N4) isolated from GJ of GC patients (T) and non-cancer individuals (N); scale bar 500 nm.

Figure 3

Examples of vesicles with atypical morphology visualized by TEM: (a–c)—elongated (tubular) EVs; (d–f)—multilayered EVs. The scale bars correspond to 100 nm.

Figure 4

Analysis of miRNA levels in EVs isolated from GJ of GC patients and non-cancer individuals. (A) An example of an electropherogram of small RNA from Agilent 2100 Bioanalyzer. (B) The relative expression of miR-451a, miR-199a-3p, miR-135b-3p (* p < 0.05); miR-204-3p and miR-135b-5p (p > 0.05) in EVs of GC patients (Tumor) and non-cancer individuals (Normal) from RT-qPCR data. Gene expression data were normalized to miR-23a. Fold change (FC) was determined using the ΔΔCt method.