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EV-miRome-wide identification of noncoding RNAs for detecting metastatic colorectal cancer and monitoring the therapeutic response

ABSTRACT: Molecular composition of circulating small extracellular vesicles (EVs) does not merely reflect the cells of origin, but also is enriched in specific biomolecules directly associated with the cellular transformation. Given their functional and clinical relevance, small EVs have emerged as a promising target among the liquid biopsy approaches for cancer detection. However, while most of the currently identified EV-miRs are only geared towards one-dimensional disease detection, their application for long-term tracking and treatment response monitoring has been largely elusive. In this study, we established and optimized a rapid, sensitive and robust liquid biopsy sampling method, and further used small RNA sequencing to comprehensively catalogue EV-miRomes in association with the progression and outcome of metastatic colorectal cancer (mCRC). By cross-comparison of EV-miRomes (n = 290) from multi-stage and longitundial cohorts, we uncovered 28 EV-miRs for detecting mCRC, and 131 EV-miRs for long-term monitoring of tumor size progression. Among these candidates, we further pinpointed a 15-EV-miR signature with dual detection and monitoring functions for mCRC. From this panel, EV-miR-320c was uncovered as a strong clinical marker – aside from its diagnostic power, its high expression has also been linked to lower overall survival and a greater likelihood of disease recurrence. Furthermore, target spectrum of this 15-EV-miR signature of mCRC suggested an involvement of small EVs in programming the mesenchymal–epithelial transition (MET) transition for distant localization of the metastasized cells and also in creating a tumor-favoring metastatic niche. Our clinically-oriented delineation of the mCRC-associated circulating EV-miRomes revealed the functional significance of these liquid biopsy markers and further strengthen their translational potential in tumor therapeutic monitoring.

ORGANISM(S): Homo sapiens

PROVIDER: GSE188627 | GEO | 2022/09/14

REPOSITORIES: GEO

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Project description:Circulating extracellular vesicles (EVs) contain molecular footprints - lipids, proteins, metabolites, RNA, and DNA - from their cell of origin and may provide non-invasive access for detection, characterization, and monitoring of diseases. Consequently, EV-associated RNA and proteins have gained widespread interest in developing liquid-biopsy assays for cancer. Yet, an integrative proteo-transcriptomic landscape of EVs and a direct comparison with their cell of origin remains relatively unexplored. Here, we report that EVs enrich for distinct molecular cargo and their proteo-transcriptome does not linearly correlate with their cell of origin. Our sub-cellular compartment analyses revealed that EV enrich 2-6 times more cytoskeletal and extracellular proteins, while their donor cells cargo 3-10 times more nuclear and mitochondrial proteins. EVs predominantly enrich (40-60%) for small RNA between ~15-200 nucleotides, which include miRNAs, siRNAs, tRNA, 5S rRNAs, and snRNAs, associated with cell differentiation, development, and Wnt signaling signatures. Finally, our integrated proteo-transcriptomic analyses reveal that EVs are enriched with specific small RNAs (RNY3, vtRNA, and MIRLET-7) and their complementary proteins (YBX1, IGF2BP2, SRSF1/2). These analyses suggest that RNA-protein complexes may constitute a functional interaction network inside EVs to protect and regulate access to EV-RNA. To ensure that our analyses are unbiased and independent of the cell of origin, EV isolation methods, site of experimentation, site of data generation, and methodologies applied for RNAseq and mass spectrometry, we have generated and curated comprehensive datasets of proteomics and transcriptomics from a total of 12 cancer cell lines, their EVs, and prostate cancer patientsÕ serum EVs (A detailed description is provided in Fig. 1). These combined analyses demonstrated that irrespective of the cell of origin and other aforementioned conditions, EVs enrich for distinct RNA and protein signatures that do not linearly correlate with their cell of origin.

Project description:Lifestyle disorders like obesity, type 2 diabetes (T2D), and cardiovascular diseases can be prevented and treated by regular physical activity. During exercise, skeletal muscles release signaling factors that communicate with other organs and mediate beneficial effects of exercise. These factors include myokines, metabolites, and extracellular vesicles (EVs). In the present study, we have examined how electrical pulse stimulation (EPS) of myotubes, a model of exercise, affects the cargo of released EVs. Chronic low frequency EPS was applied for 24 h to human myotubes isolated and differentiated from biopsy samples from 6 morbidly obese females with T2D, and EVs, both exosomes and microvesicles (MV), were isolated from cell media 24 h thereafter. Size and concentration of EV subtypes were characterized by nanoparticle tracking analysis, surface markers were examined by flow cytometry and Western blotting, and morphology was confirmed by transmission electron microscopy. Protein content was assessed by high-resolution proteomic analysis (LC-MS/MS), non-coding RNA was quantified by Affymetrix microarray, and selected microRNAs (miRs) validated by real time RT-qPCR. The size and concentration of exosomes and MV were unaffected by EPS. Of the 400 miRs identified in the EVs, EPS significantly changed the level of 15 exosome miRs, of which miR-1233-5p showed the highest fold change. The miR pattern of MV was unaffected by EPS. Totally, about 1000 proteins were identified in exosomes and 2000 in MV. EPS changed the content of 73 proteins in exosomes, 97 in MVs, and of these 4 were changed in both exosomes and MV (GANAB, HSPA9, CNDP2, and ATP5B). By matching the EPS-changed miRs and proteins in exosomes, 31 targets were identified, and among these several promising signaling factors. Of particular interest were CNDP2, an enzyme that generates the appetite regulatory metabolite Lac-Phe, and miR-4433b-3p, which targets CNDP2. Several of the regulated miRs, such as miR-92b-5p, miR-320b, and miR-1233-5p might also mediate interesting signaling functions.

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EV-miRome-wide identification of noncoding RNAs for detecting metastatic colorectal cancer and monitoring the therapeutic response

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