Glioblastoma‐derived extracellular vesicle subpopulations following 5‐aminolevulinic acid treatment bear diagnostic implications

Abstract

Liquid biopsy is a minimally invasive alternative to surgical biopsy, encompassing different analytes including extracellular vesicles (EVs), circulating tumour cells (CTCs), circulating tumour DNA (ctDNA), proteins, and metabolites. EVs are released by virtually all cells, but at a higher rate by faster cycling, malignant cells. They encapsulate cargo native to the originating cell and can thus provide a window into the tumour landscape. EVs are often analysed in bulk which hinders the analysis of rare, tumour-specific EV subpopulations from the large host EV background. Here, we fractionated EV subpopulations in vitro and in vivo and characterized their phenotype and generic cargo. We used 5-aminolevulinic acid (5-ALA) to induce release of endogenously fluorescent tumour-specific EVs (EV PpIX ). Analysis of five different subpopulations (EV PpIX , EV CD63 , EV CD9 , EV EGFR , EV CFDA ) from glioblastoma (GBM) cell lines revealed unique transcriptome profiles, with the EV PpIX transcriptome demonstrating closer alignment to tumorigenic processes over the other subpopulations. Similarly, isolation of tumour-specific EVs from GBM patient plasma showed enrichment in GBM-associated genes, when compared to bulk EVs from plasma. We propose that fractionation of EV populations facilitates detection and isolation of tumour-specific EVs for disease monitoring.

Rigorous Comparison of Extracellular Vesicle Processing to Enhance Downstream Analysis for Glioblastoma Characterization

“…We hypothesize that the differences in NTA and IFC‐derived EV concentrations may be due to variation in instrument quantification, as previously reported, [ 27 ] but can primarily be attributed to CFDA + EV‐specific fluorescent quantification. Following a previously established IFC gating strategy, [ 28 ] EVs were gated to exclude large EVs identified via brightfield to account for the high rate false‐positives caused by debris of similar sizes. However, improved strategies are warranted to differentiate EVs from noise at larger sizes, before and after imaging, to prevent gating bias.…”

Deciphering the causal relationship between plasma and cerebrospinal fluid metabolites and glioblastoma multiforme: a Mendelian Randomization study

“…Metabolomics, the extensive study of small molecules within biological systems, stands at the forefront of this challenge, offering promising potential to decipher the altered metabolic pathways associated with cancer phenotypes [ 6 , 7 ]. Plasma metabolites, as downstream products of genetic, enzymatic, and cellular processes, reflect the dynamic state of GBM’s pathophysiology and therefore hold promise for revealing new facets of its biology [ 8 – 10 ]. The identification and in-depth analysis of these metabolites could unveil novel biomarkers that not only deepen our understanding of GBM’s underpinnings but also pave the way for targeted treatment strategies.…”

Plasma extracellular vesicle sampling from high grade gliomas demonstrates a small RNA signature indicative of disease and identifies lncRNA RPPH1 as a high grade glioma biomarker.

“…While EVs are produced by almost all cell-types [32], malignant HGG cancer cells have been shown to increase production of EVs and their secretion into plasma [33]. Despite the blood-brain barrier, EVs derived from HGG tumours have been detected in the plasma, most notably in the well-designed experiment using surgically resected tissue and plasma collected from patients treated with 5-aminolevulinic acid (5-ALA) [34]. Thus, the isolation of plasma EVs and elucidation of their cargo is an appealing non-invasive methodology (liquid biopsy) for informing on HGG.…”

“…Thus, the isolation of plasma EVs and elucidation of their cargo is an appealing non-invasive methodology (liquid biopsy) for informing on HGG. Numerous methods of EV isolation have been described in the literature to analyze various cancer pathologies [34][35][36][37]. The Vn96 peptide is a peptide a nity capture method developed for the capture of EVs in clinically relevant bio uids [18-20, 33, 38-41].…”

“…Non-coding RNAs (such as miRNA, snoRNA, lncRNA, piRNA, and others) contained in EVs of HGG plasma and those of cancers outside of the brain, have already been shown to be effective forms of tumour-associated cell-to-cell communication [27,29,34,[45][46][47][48]. For example, the long non-coding RNA (lncRNA) ribonuclease P RNA component H1 (RPPH1) has been shown to play an important role in multiple cancers (e.g., lung, colorectal cancer) [49,50].…”