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. 2019 May;54(5):1719-1733.
doi: 10.3892/ijo.2019.4742. Epub 2019 Mar 7.

Proteomics profiling of plasma exosomes in epithelial ovarian cancer: A potential role in the coagulation cascade, diagnosis and prognosis

Wei Zhang  1 , Xiaoxuan Ou  1 , Xiaohua Wu  1
Affiliations

Proteomics profiling of plasma exosomes in epithelial ovarian cancer: A potential role in the coagulation cascade, diagnosis and prognosis

Wei Zhang et al. Int J Oncol. 2019 May.

Abstract

Ovarian cancer remains the most lethal type of cancer among all gynecological malignancies. The majority of patients are diagnosed with ovarian cancer at the late stages of the disease. Therefore, there exists an imperative need for the development of early ovarian cancer diagnostic techniques. Exosomes, secreted by various cell types, play pivotal roles in intercellular communication, which emerge as promising diagnostic and prognostic biomarkers for ovarian cancer. In this study, we present for the first time, at least to the best of our knowledge, the proteomics profiling of exosomes derived from the plasma of patients with ovarian cancer via liquid chromatography tandem mass spectrometry (LC‑MS/MS) with tandem mass tagging (TMT). The exosomes enriched from patient plasma samples were characterized by nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), transmission electron microscopy (TEM) and western blot analysis. The size of the plasma exosomes fell into the range of 30 to 100 nm in diameter. The exosomal marker proteins, CD81 and TSG101, were clearly stained in the exosome samples; however, there was no staining for the endoplasmic reticulum protein, calnexin. A total of 294 proteins were identified with all exosome samples. Among these, 225 proteins were detected in both the cancerous and non‑cancerous samples. Apart from universal exosomal proteins, exosomes derived from ovarian cancer patient plasma also contained tumor‑specific proteins relevant to tumorigenesis and metastasis, particularly in epithelial ovarian carcinoma (EOC). Patients with EOC often suffer from coagulation dysfunction. The function of exosomes in coagulation was also examined. Several genes relevant to the coagulation cascade were screened out as promising diagnostic and prognostic factors that may play important roles in ovarian cancer progression and metastasis. On the whole, in this study, we successfully isolated and purified exosomes from plasma of patients with EOC, and identified a potential role of these exosomes in the coagulation cascade, as well as in the diagnosis and prognosis of patients.

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Figures

Figure 1
Figure 1
Various characterizations of exosomes. (A) Western blot analysis of the typical exosomal proteins, TSG 101 and CD81, and the endoplasmic reticular protein, calnexin. (B) Transmission electron microscopy (TEM) of exosomes. (C) Nanoparticle tracking analysis (NTA) of exosomes. (D) Dynamic light scattering (DLS) analysis of exosomes.
Figure 2
Figure 2
Bioinformatics analysis of proteomic results. (A) Venn diagram of the number of detected proteins in cancerous and non-cancerous patient samples and their intersection. (B) Volcano plot of differently expressed proteins. Fold change (FC) values >1.5 or <2/3 and a P-value <0.05 were set as the filter criteria. Green dots represent downregulated proteins; red dots represent upregulated proteins. (C) Gene Ontology (GO) enrichment analysis of differently expressed proteins. Top 10 items are listed in biological process, cell component and molecular function, respectively based on their statistical significance (30,31). (D) KEGG pathway enrichment analysis of differently expressed proteins. Top 10 items are listed on their statistical significance (30,31). (E) Protein-protein interaction (PPI) network of the enriched proteins and their corresponding cell signaling pathways (32,33).
Figure 3
Figure 3
Procoagulation assay of exosomes based on the concentration of Factor X via a chromogenic methodology (****P<0.0001).
Figure 4
Figure 4
ELISA data of exosomal GSN, FGA, LBP and FGG expression. (A) Exosomal GSN expression in cancer and non-cancerous patients (****P<0.0001). (B) Exosomal FGA expression in cancer and non-cancerous patients (****P<0.0001). (C) Exosomal LBP expression in cancer and non-cancerous patients (**P<0.01). (D) Exosomal FGG expression in cancer and non-cancerous patients (****P<0.0001). GSN, gelsolin; FGA, fibrinogen alpha chain; LBP, lipopolysac-charide binding protein; FGG, fibrinogen gamma chain.
Figure 5
Figure 5
Receiver operating characteristic (ROC) curve assay of the selected exosomal proteins as diagnostic biomarkers. (A) ROC-AUC of GSN was 0.8309 (0.7343 to 0.9274, P<0.0001). (B) ROC-AUC of FGA was 0.8459 (0.7602 to 0.9317, P<0.0001). (C) ROC-AUC of FGG was 0.7447 (0.6323 to 0.8571, P<0.0001). (D) ROC-AUC of LBP was 0.6588 (0.5381 to 0.7794, P<0.001). GSN, gelsolin; FGA, fibrinogen alpha chain; LBP, lipopolysaccharide binding protein; FGG, fibrinogen gamma chain.
Figure 6
Figure 6
Prognostic significance of FGG and LBP estimated by simulated Kaplan-Meier analysis. Both overall survival (OS) and progression-free survival (PFS) were evaluated. The hazard ratio (HR) was utilized to describe the efficiency of prognostic markers and log-rank test was performed to demonstrate their statistical significance (34). (A) Estimated OS of FGG in ovarian cancer. (B) Estimated PFS of FGG in ovarian cancer. (C) Estimated OS of LBP in ovarian cancer. (D) Estimated PFS of LBP in ovarian cancer. FGG, fibrinogen gamma chain; LBP, lipopolysaccharide binding protein.
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