Dataset Information

Extracellular vesicle membrane protein profiling and targeted mass spectrometry unveil novel plasma biomarkers for detection of colorectal cancer

ABSTRACT: Extracellular vesicles (EVs) are valuable sources for the discovery of useful cancer biomarkers. This study explores the potential usefulness of tumor cell-derived EV membrane proteins as plasma biomarkers for early detection of colorectal cancer (CRC). EVs were isolated from the culture supernatants of four CRC cell lines by ultracentrifugation, and their protein profiles were analyzed by LC-MS/MS. Bioinformatics analysis of identified proteins revealed 518 EV membrane proteins in common among at least three CRC cell lines. We next used accurate in-clusion mass screening (AIMS) in parallel with iTRAQ-based quantitative proteomic analysis to highlight candidate proteins and validated their presence in pooled plasma-generated EVs from 30 healthy controls and 30 CRC patients. From these, we chose 14 potential EV-derived targets for further quantification by targeted MS assay in a separate individual cohort comprising of 73 CRC and 80 healthy subjects. Quantitative analyses revealed significant increases in ADAM10, CD59 and TSPAN9 levels (2.19- to 5.26-fold, p <0.0001) in plasma EVs from CRC patients, with AUC values of 0.83, 0.95 and 0.87, respectively. Higher EV CD59 levels were significantly corre-lated with distant metastasis (p = 0.0475), and higher EV TSPAN9 levels were significantly asso-ciated with lymph node metastasis (p = 0.0011), distant metastasis at diagnosis (p = 0.0104) and higher TNM stage (p = 0.0065). A two-marker panel consisting of CD59 and TSPAN9 outper-formed the conventional marker CEA in discriminating CRC and stage I/II CRC patients from healthy controls, with AUC values of 0.98 and 0.99, respectively. Our results identify EV mem-brane proteins in common among CRC cell lines and altered plasma EV protein profiles in CRC patients, and suggest plasma EV CD59 and TSPAN9 as a novel biomarker panel for detecting early-stage CRC.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

DISEASE(S): Colon Cancer

SUBMITTER: Chia-Chun Wu

LAB HEAD: Jau Song Yu

PROVIDER: PXD038871 | Pride | 2023-03-11

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	0_0488fH-10fL-1ugEV-1.msf	Msf
	0_0488fH-10fL-1ugEV-1.raw	Raw
	0_0488fH-10fL-1ugEV-2.msf	Msf
	0_0488fH-10fL-1ugEV-2.raw	Raw
	0_0488fH-10fL-1ugEV-3.msf	Msf

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Publications

Extracellular Vesicle Membrane Protein Profiling and Targeted Mass Spectrometry Unveil CD59 and Tetraspanin 9 as Novel Plasma Biomarkers for Detection of Colorectal Cancer.

Dash Srinivas S Wu Chia-Chun CC Wu Chih-Ching CC Chiang Sum-Fu SF Lu Yu-Ting YT Yeh Chien-Yuh CY You Jeng-Fu JF Chu Lichieh Julie LJ Yeh Ta-Sen TS Yu Jau-Song JS

Cancers 20221228 1

Extracellular vesicles (EVs) are valuable sources for the discovery of useful cancer biomarkers. This study explores the potential usefulness of tumor cell-derived EV membrane proteins as plasma biomarkers for early detection of colorectal cancer (CRC). EVs were isolated from the culture supernatants of four CRC cell lines by ultracentrifugation, and their protein profiles were analyzed by LC-MS/MS. Bioinformatics analysis of identified proteins revealed 518 EV membrane proteins in common among ...[more]

PMID: 36612172

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Project description:Background: Acute coronary syndromes (ACS) are associated with aberrant gene expression and epigenetic mechanisms. In particular, de novo DNA methylation is typically linked to gene silencing, but its role in heart disease remains not fully understood. Extracellular vesicles (EVs) are active components in cellular communication for their ability to carry a plethora of signalling biomolecules, thus representing a promising new diagnostic/therapeutic approach in cardiovascular diseases (CVDs). Indeed, there is the need of novel biomarkers for ACS prediction and timely detection. Purpose: We hypothesized that specific epigenetic signals can be carried by EVs. In this regard, we isolated and characterized circulating EVs from ACS patients and evaluated their potential role to influence DNA methylation in target cells. Methods: Circulating EVs were recovered, by ultracentrifugation, from plasma samples of 19 ACS patients and 50 healthy subjects (HS). Nanoparticle tracking analysis (NTA) and western blot (WB) were used to confirm the EVs integrity and purity. Peripheral blood mononuclear cells (PBMCs) of volunteer donors were treated with both ACS and HS derived EVs and genomic DNA was extracted to perform epigenome wide analysis through Reduced Representation Bisulfite Sequencing. ShinyGO, PANTHER, and STRING tools were interrogated to perform GO and PPI network analyses. Flow Cytometry, qRT-PCR, and WB analysis were also performed to evaluate and validate both intra-vesicular and intra-cellular signals. Results: Plasma ACS-derived EVs showed a significant up-regulation of DNA methyltransferases (DNMTs) gene expression levels as compared to HS (P<0.001). Specifically, de novo methylation transcripts, as DNMT3A and DNMT3B were significantly increased in plasma ACS-EVs. DNA methylation analysis of PBMCs from volunteer donors treated with HS- and ACS-derived EVs showed that RNF166 and CCND3 genes resulted the most hyper- and hypo-methylated, respectively, after by ACS-EV treatment. In addition, PPI network analysis specifically evidenced the subnetwork with SRC, as a hub gene, connecting it to NOTCH1, FOXO3, CDC42, IKBKG, RXRA, DGKG, known as important genes already involved in the onset of CVDs. Surprising, other novel genes, BAIAP2, SYP, CHL1, and SHB, which were hypomethylated, were found significantly overexpressed in PBMCs (P<0.005), underlying the fundamental modulating properties of EV cargo in atherosclerosis. Conclusions: These findings support the significant role of ACS plasma-derived EVs, inducing de novo DNA methylation signals, and modulating specific signaling pathways in target cells.