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

Extracellular vesicles (EVs) have been shown as key mediators of extracellular small RNA transport. However, carriers of cell-free messenger RNA (cf-mRNA) in human biofluids and their association with cancer remain poorly understood. Researchers at Oregon Health and Science University performed a transcriptomic analysis of size-fractionated plasma from lung cancer, liver cancer, multiple myeloma, and healthy donors. Morphology and size distribution analysis showed the successful separation of large and medium particles from other soluble plasma protein fractions. The researchers developed a strategy to purify and sequence ultra-low amounts of cf-mRNA from particle and protein enriched subpopulations with the implementation of RNA spike-ins to control for technical variability and to normalize for intrinsic drastic differences in cf-mRNA amount carried in each plasma fraction. They found that the majority of cf-mRNA was enriched and protected in EVs with remarkable stability in RNase-rich environments. They observed specific enrichment patterns of cancer-associated cf-mRNA in each particle and protein enriched subpopulation. The EV-enriched differentiating genes were associated with specific biological pathways, such as immune systems, liver function, and toxic substance regulation in lung cancer, liver cancer, and multiple myeloma, respectively. These results suggest that dissecting the complexity of EV subpopulations illuminates their biological significance and offers a promising liquid biopsy approach.

Selective Enrichment of Multiple Cancer Differentiating cf-mRNA

a Heatmap of gene expression in multiple cancers (lung cancer, multiple myeloma, and liver cancer) relative to healthy across fractions. Annotation row indicates fraction of DE genes identified for specific cancer types compared to the healthy controls. Representative images were generated using BioRender illustration Software and PowerPoint. b Enrichment map for multiple cancer DE genes found in individual fractions using Gene Ontology (Biological Properties) and Reactome with lung cancer, multiple myeloma, and liver cancer color coded by green, purple and yellow, respectively. Cluster represents (i) steroid hormone corticosteroid, (ii) cellular response chemical, (iii) sphingolipid metabolism glycosphingolipid, (iv) localization nucleus nucleolus, (v) temperature homeostasis, (vi) regulation myeloid cell, (vii) alpha beta cell, (viii) toll cell receptor 4, (ix) hemidesmosome assembly, (x) negative regulation response, and (xi) g1 specific transcription. Cluster of nodes were automatically labeled using the AutoAnnotate from Cytoscape.