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Glioblastoma biomarkers in urinary extracellular vesicles reveal the potential for a ‘liquid gold’ biopsy

ABSTRACT: Background: Biomarkers that reflect glioblastoma tumour activity and treatment response are urgently needed to help guide clinical management, particularly for recurrent disease. As the urinary system is a major clearance route of circulating extracellular vesicles (EVs; 30-1000 nm nanoparticles) we explored whether sampling urinary-EVs could serve as a simple and non-invasive liquid biopsy approach for measuring glioblastoma-associated biomarkers. Methods: Fifty urine specimens (15-60 ml) were collected from 24 catheterised glioblastoma patients immediately prior to primary (n=17) and recurrence (n=7) surgeries, following gross total resection (n=9), and from age/gender-matched healthy participants (n=14). EVs isolated by differential ultracentrifugation were characterised and extracted proteomes were analysed by high-resolution data-independent acquisition liquid chromatography tandem mass spectrometry (DIA-LC-MS/MS). Results: Overall, 6857 proteins were confidently identified in urinary-EVs (q-value≤0.01), including 94 EV marker proteins. Glioblastoma-specific proteomic signatures were determined, and putative urinary-EV biomarkers corresponding to tumour burden and recurrence were identified (FC≥|2|, adjust p-val≤0.05, AUC>0.9). Conclusion: In-depth DIA-LC-MS/MS characterisation of urinary-EVs substantiates urine as a viable source of glioblastoma biomarkers. The promising ‘liquid gold’ biomarker panels described here warrant further investigation.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Urine

DISEASE(S): Brain Glioblastoma Multiforme

SUBMITTER: Susannah Hallal

LAB HEAD: Kimberley Alexander

PROVIDER: PXD046511 | Pride | 2024-04-18

REPOSITORIES: Pride

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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.

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Glioblastoma biomarkers in urinary extracellular vesicles reveal the potential for a ‘liquid gold’ biopsy

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