Selective enrichment of plasma cell-free messenger RNA in cancer-associated extracellular vesicles

Cell-free messenger RNA (cf-mRNA) has shown promising diagnostic potentials. Unlike well-characterized extracellular miRNA, how cf-mRNA is protected and its potential biological significance remains unknown. To characterize the carriers of cf-mRNA in human plasma and their association with cancers, we performed transcriptome analysis of cf-mRNA in size-fractionated plasma from patients with lung cancer, liver cancer and multiple myeloma, and healthy donors. We found that the majority of cf-mRNA was enriched in extracellular vesicles (EVs) and was protected in EVs with remarkable stability in RNase-rich environments. We observed specific enrichment patterns of cf-mRNA in each vesicular and non-vesicular subpopulation in cancers. These EV-enriched differentiating genes are associated with specific biological pathways, such as immune systems, liver function, and oxygen transport in lung cancer, liver cancer, and multiple myeloma, respectively. Our results suggest that dissecting the complexity of EVs subpopulations illuminates their biological significance and becomes a promising approach for novel biomarker development. We sequenced total cell-free plasma RNA from 120 size-fractionated extracellular carriers (medium EVs, small EVs, non-EV particles, and soluble plasma protein fractions associated with early-, middle-, and late-eluting proteins) using lung cancer, liver cancer, multiple myeloma and healthy control sample set.