Single-step size exclusion chromatography enriches for plasma vesicle-associated miRNAs that are directly applicable for cancer biomarker discovery using small RNAseq

Introduction: Circulating microRNAs are generally believed to have biomarker potential for diagnosis, prognosis and monitoring of treatment response in multiple diseases, including cancer. Extracellular vesicles (EVs) are actively secreted by tumour cells and normal cells, and they encapsulate miRNAs, protecting them from degradation by RNases. Circulating miRNAs can also be associated with and protected by proteins and HDL; moreover, dying cells release biomolecules (i.e. RNA, DNA, protein) into circulation. The use of tumour-derived EV as biomarker has many advantages: 1) they reflect living tumour cells, 2) EV-associated miRNAs are stable in archived material, 3) EV-secreted miRNAs outnumber other types, 4) EV-associated miRNAs promote tumourigenic processes, 5) tumour vesicles carry proteins on their surface that trace them back to the tumour cells. Thus for circulating miRNA biomarker research it maybe of critical importance to isolate (tumour) EV from circulation. Methods: We performed size exclusion chromatography using sepharose CL-2B and commercially available qEV columns (iZON™) on 1.5 ml plasma to separate EV from protein/HDL. Results: Electron Microscopy showed the presence of EV in the vesicle fractions but not in the protein/HDL fractions. Particle analysis using qNano (iZON™) showed that the vesicle fractions are highly enriched in particles with a size-distribution that corresponds to the EM images. Moreover, western analysis showed the presenceof exosome-marker CD63in the vesicle fractions, but not the protein/HDL fractions. RNA was isolated using TRIzol from the EV and protein/HDL fractions separated by SEC, followed by RT-PCR. The EV fractions were highly enriched for vtRNA1-1, let7a and miR142-3p, whereas protein/HDL fractions are enriched for miR92a, miR21 and miR451. EV isolated using SEC contained sufficient RNA of suitable quality for screening using RNAseq. Classical Hodgkin lymphoma plasma EVs had a distinct smallRNA profile compared to healthy donor plasma EVs. We validated potential miRNA biomarker candidates by RT-PCR. Summary/conclusion: All together these results indicate that EVs isolated using SEC are useful for miRNA biomarker discovery in cancer patients.