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

ABSTRACT: 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. Here, we 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. We 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. We found that the majority of cf-mRNA was enriched and protected in EVs with remarkable stability in RNase-rich environments. We 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. Our results suggest that dissecting the complexity of EV subpopulations illuminates their biological significance and offers a promising liquid biopsy approach.

SUBMITTER: Kim HJ

PROVIDER: S-EPMC10465482 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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

Kim Hyun Ji HJ Rames Matthew J MJ Goncalves Florian F Kirschbaum C Ward CW Roskams-Hieter Breeshey B Spiliotopoulos Elias E Briand Josephine J Doe Aaron A Estabrook Joseph J Wagner Josiah T JT Demir Emek E Mills Gordon G Ngo Thuy T M TTM

Communications biology 20230829 1

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. Here, we 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 ...[more]

PMID: 37644220

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Project description:BackgroundCell-free long RNAs in human plasma and extracellular vesicles (EVs) have shown promise as biomarkers in liquid biopsy, despite their fragmented nature.MethodsTo investigate these fragmented cell-free RNAs (cfRNAs), we developed a cost-effective cfRNA sequencing method called DETECTOR-seq (depletion-assisted multiplexed cell-free total RNA sequencing). DETECTOR-seq utilised a meticulously tailored set of customised guide RNAs to remove large amounts of unwanted RNAs (i.e., fragmented ribosomal and mitochondrial RNAs) in human plasma. Early barcoding strategy was implemented to reduce costs and minimise plasma requirements.ResultsUsing DETECTOR-seq, we conducted a comprehensive analysis of cell-free transcriptomes in both whole human plasma and EVs. Our analysis revealed discernible distributions of RNA types in plasma and EVs. Plasma exhibited pronounced enrichment in structured circular RNAs, tRNAs, Y RNAs and viral RNAs, while EVs showed enrichment in messenger RNAs (mRNAs) and signal recognition particle RNAs (srpRNAs). Functional pathway analysis highlighted RNA splicing-related ribonucleoproteins (RNPs) and antimicrobial humoral response genes in plasma, while EVs demonstrated enrichment in transcriptional activity, cell migration and antigen receptor-mediated immune signals. Our study indicates the comparable potential of cfRNAs from whole plasma and EVs in distinguishing cancer patients (i.e., colorectal and lung cancer) from healthy donors. And microbial cfRNAs in plasma showed potential in classifying specific cancer types.ConclusionsOur comprehensive analysis of total and EV cfRNAs in paired plasma samples provides valuable insights for determining the need for EV purification in cfRNA-based studies. We envision the cost effectiveness and efficiency of DETECTOR-seq will empower transcriptome-wide investigations in the fields of cfRNAs and liquid biopsy.KeypointsDETECTOR-seq (depletion-assisted multiplexed cell-free total RNA sequencing) enabled efficient and specific depletion of sequences derived from fragmented ribosomal and mitochondrial RNAs in plasma. Distinct human and microbial cell-free RNA (cfRNA) signatures in whole Plasma versus extracellular vesicles (EVs) were revealed. Both Plasma and EV cfRNAs were capable of distinguishing cancer patients from normal individuals, while microbial RNAs in Plasma cfRNAs enabled better classification of cancer types than EV cfRNAs.

Dataset Information

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

Publications

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

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