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Proteomics analysis of plasma-derived exosomes extracted by thrombin pre-treated ExoQuick isolation

ABSTRACT: Exosomes are very small nanosized, membrane-bound vesicles, ranging from 30 to 150 nm in size, are originated and released from a variety of cells from body fluids including blood plasma. The isolation of pure exosome from blood plasma is challenging since it is rich in various contaminants from diverse origins. We applied ExoQuick with thrombin for purification of plasma-derived exosome from healthy individual. Immunoassays confirmed the presence of proteins that are classically associated to exosomes (CD9, CD63 and CD81) but these proteins have not been detected by MS. We found several common proteins enriched in our data set compared with plasma-derived exosome markers from databases. Moreover, proteomics analysis and external database integration revealed an enrichment of those extracellular vesicles. The Gene Ontology data analyses determined the presence of proteins that are involved in catalytic, transporter functions and protein metabolism activities. These results allowed the detection of tetraspanin i.e. TSPAN14 in plasma where proteins were undetected by other studies. Furthermore, this study confirmed the detection of CD5L and LGALS3BP proteins. Although the undouble presence of plasma contaminants hamper MS analysis, still proteins with biomarker potential (TSPAN14, CD5L and LGALS3BP) are accessible in a reasonable sample processing frame that outperforms high-purity focused extraction methods e.g. SEC. The study suggests the use of TSPAN14, CD5L and LGALS3BP as new exosomal markers of plasma-derived exosomes in quantitative proteomics analysis.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

SUBMITTER: Johannes Schmidt

LAB HEAD: Stefan Kalkhof

PROVIDER: PXD028172 | Pride | 2022-03-04

REPOSITORIES: Pride

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Project description:Background: Docosahexaenoic acid (DHA) is a natural compound with anticancer and anti-angiogenesis activity that is currently under investigation as both a preventative agent and an adjuvant to breast cancer therapy. However, the precise mechanisms of DHA’s anticancer activities are unclear. It is understood that the intercommunication between cancer cells and their microenvironment is essential to tumor angiogenesis. Exosomes are extracellular vesicles that are important mediators of intercellular communication and play a role in promoting angiogenesis. However, very little is known about the contribution of breast cancer exosomes to tumor angiogenesis or whether exosomes can mediate DHA’s anticancer action. Results: Exosomes were collected from MCF7 and MDA-MB-231 breast cancer cells after treatment with DHA. We observed an increase in exosome secretion and exosome microRNA contents from the DHA-treated cells. The expression of 83 microRNAs in the MCF7 exosomes was altered by DHA (>2-fold). The most abundant exosome microRNAs (let-7a, miR-23b, miR-27a/b, miR-21, let-7, and miR-320b) are known to have anti-cancer and/or anti-angiogenic activity. These microRNAs were also increased by DHA treatment in the exosomes from other breast cancer lines (MDA-MB-231, ZR751 and BT20), but not in exosomes from normal breast cells (MCF10A). When DHA-treated MCF7 cells were co-cultured with or their exosomes were directly applied to endothelial cell cultures, we observed an increase in the expression of these microRNAs in the endothelial cells. Furthermore, overexpression of miR-23b and miR-320b in endothelial cells decreased the expression of their pro-angiogenic target genes (PLAU, AMOTL1, NRP1 and ETS2) and significantly inhibited tube formation by endothelial cells, suggesting that the microRNAs transferred by exosomes mediate DHA’s anti-angiogenic action. These effects could be reversed by knockdown of the Rab GTPase, Rab27A, which controls exosome release. Conclusions: We conclude that DHA alters breast cancer exosome secretion and microRNA contents, which leads to the inhibition of angiogenesis. Our data demonstrate that breast cancer exosome signaling can be targeted to inhibit tumor angiogenesis and provide new insight into DHA’s anticancer action, further supporting its use in cancer therapy. Examination of small RNA populations in MCF7 cells and exosomes after DHA treatment.

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Proteomics analysis of plasma-derived exosomes extracted by thrombin pre-treated ExoQuick isolation

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