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.
Researchers at Massachusetts General Hospital fractionated EV subpopulations in vitro and in vivo and characterized their phenotype and generic cargo. They used 5-aminolevulinic acid (5-ALA) to induce release of endogenously fluorescent tumour-specific EVs (EVPpIX ). Analysis of five different subpopulations (EVPpIX , EVCD63 , EVCD9 , EVEGFR , EVCFDA ) from glioblastoma (GBM) cell lines revealed unique transcriptome profiles, with the EVPpIX 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. The researchers propose that fractionation of EV populations facilitates detection and isolation of tumour-specific EVs for disease monitoring.
Quantification of cell line-derived EV subpopulation cargo
EVs were fluorescently labeled and sorted using nanoFACs. A workflow for EV sorting and downstream library preparation was developed for low-input, bulk RNA sequencing (A). The relative RNA landscape of EV subpopulations as defined by long and small RNA biotypes was mapped according to detection (B). Gene expression (transcripts per million; TPM) across EV subpopulations and cell lines were assessed in order of phylogenetic relation (C).