Comparative Analysis of Tumor-Associated microRNAs and Tetraspanines from Exosomes of Plasma and Ascitic Fluids of Ovarian Cancer Patients

Abstract

Ovarian cancer (OC) is one of the most common and fatal types of gynecological cancer. In the early phase of OC detection, the current treatment and diagnostic methods are not efficient and sensitive enough. Therefore, it is crucial to explore the mechanisms of OC metastasis and discover valuable factors for early diagnosis of female cancers and novel therapeutic strategies for metastasis. Exosomes are known to be involved in the development, migration, and invasion of cancer cells, and their cargo could be useful for the non-invasive biopsy development. CD151- and Tspan8-positive exosomes are known to support the degradation of the extracellular matrix, and are involved in stroma remodeling, angiogenesis and cell motility, as well as the association of miR-24 and miR-101 with these processes. The objective of this study was to explore the relationship of these components of exosomal cargo, in patients with OC, to clarify the clinical significance of these markers in liquid biopsies. The levels of tetraspanins Tspan8+ and CD151+ exosomes were significantly higher in plasma exosomes of OC patients compared with healthy females (HFs). The relative levels of miR-24 and miR-101 in plasma exosomes of HFs were significantly higher than in plasma exosomes of OC patients, while the levels of these microRNAs in exosomes from plasma and ascites of ill females showed no difference. Our study revealed a strong direct correlation between the change in the ascites exosomes CD151+Tspan8+ subpopulation level and the expression levels of the ascites (R = 0.81, p < 0.05) and plasma exosomal miR-24 (R = 0.74, p < 0.05) in OC patients, which confirms the assumption that exosomal cargo act synergistically to increase cellular motility, affecting cellular processes and signaling. Bioinformatics analysis confirmed the involvement of CD151 and Tspan8 tetraspanins and genes controlled by miR-24-3p and miR-101 in signaling pathways, which are crucial for carcinogenesis, demonstrating that these tetraspanins and microRNAs are potential biomarkers for OC screening, and predictors of poor clinicopathological behavior in tumors.

Keywords: CD151; Tspan8; exosomes; miR-101; miR-24-3p; ovarian cancer; tumor-specific microRNAs.

Figure 1

Identification of isolated exosomes. (A) TEM demonstrated the presence of exosome vesicles with typical cup-shape morphology (size bar 100 nm) and absence vesicles more than 100 nm. Arrows indicate exosomes, exosomes are on the insets. (B) Expression of CD63, CD81, and CD24 on CD9-positive exosomes of blood plasma of HFs, blood plasma, and ascites of OC patients. For flow cytometry representative median fluorescence intensity (MFI) are shown (see Table S1 for details). Each study is done in triplets. For isotype controls (histograms in the center and on the left), CD9-labeled beads-exosome complexes were incubated with FITC mouse IgG1, k Isotype control (Cat N555748, BD, Heidelberg, Germany) or FITC mouse IgG2a, k Isotype control (Cat N553456, BD, Heidelberg, Germany). For the negative control (histogram on the right), latex particles labeled with CD9 antibodies were incubated with FITC labeled antibodies anti-CD63, anti-CD81, and anti-CD24 antibody. One of the representative negative controls is shown.

Figure 2

Flow cytometry analysis of plasma exosome subpopulations. (A) Forward scatter area (FSC-A) versus side scatter area (SSC-A) dot plot representing exosomes samples adsorbed on aldehyde/sulphate latex beads labeled anti-CD9 antibody (red), debris is marked in green. (B) Tspan8-positive and (C) CD151-positive plasma exosomes populations in OC patients. (D) Double labeling Tspan8 versus CD151 of OC patients’ ascites exosomes. Quantitative data on the expression of tetraspanins Tspan8 and CD151 on the surface of CD9-positive exosomes from blood plasma of HFs and blood plasma and ascites of OC patients is represented in Table 1.

Figure 3

RNA content in exosomes. Size distribution of RNA extracted from pooled samples: (A) plasma exosomes of HFs, (B) plasma exosomes of OC patients, and (C) ascites exosomes of OC patients. The data from Agilent 2100 Bioanalyzer with 25 nt RNA fragment as an internal standard are shown. Quantification of exosomal miR-24-3p (D) and miR-101 (E) from the blood plasma of HFs and from the blood plasma and ascites fluid of OC patients. Tukey box plots of exosomal miRNAs. The statistically significant p-values are indicated. Correlation of miR-24-3p level (F) and miR-101 level (G) in blood plasma exosomes with ascites exosomes in OC patients. Solid red line—trend line, dotted red line—95% confidence interval.

Figure 4

Correlation of miR-24-3p level in blood plasma exosomes (A) and in ascites exosomes (B) with level of CD151+Tspan8+ CD9+ exosomes in OC patients ascites. Solid red line–trend line, dotted red line–95% confidence interval.

Figure 5

Venn-Euler diagram showing the involvement of miRNA-regulated genes in the development of OC (DIANA database).

Figure 6

The interactions of proteins coded by genes that are regulated by miR-24 and miR-101, which are involved in oocyte meiosis, progesterone-dependent oocyte maturation and cancer pathways and exosome-associated tetraspanins (STRING Database).

Figure 7

Biological processes, controlled by 14 genes (the following data is done using PANTHER (www.pantherb.org, (accessed on 30 May 2022)).