Dataset Information

Pre-Analytical Framework for Routine Clinical Use of Liquid Biopsies: Combining EVs and cfDNA

ABSTRACT: Liquid biopsies hold significant potential for the non-invasive diagnostics of tumors and other diseases. While the clinical application of cell-free DNA (cfDNA) methodologies is emerging, the implementation of tumor-derived extracellular vesicles (EVs) as validated biomarkers is hindered by substantial pre-analytical variations. In this work, we are taking a step towards standardizing the pre-analytical procedures of blood collection for subsequent co-isolation of plasma cfDNA and EVs from a single blood collection tube. We compare effects of blood preservation tubes and storage to enable proteomic profiling of resulting EVs in addition to cfDNA extraction and sequencing. Following a stringent method of large EV (lEV) and small EV (sEV) isolation, consisting of differential ultracentrifugation and size exclusion chromatography, we evaluate protein concentration, particle number, quality and integrity of the isolated EVs. Subsequent proteomic analyses of EV isolates unravel the complexity of the respective tube proteomes allowing the interpretation of EV origins as well as contamination sources. While ACD-A and Citrate tubes demonstrate satisfying results in preservation of EV proteomes, only Streck RNA®, Norgen® and PAX® tubes can preserve high cfDNA purity for up to 7 days. When aiming for multi-omics analyses, Streck RNA® tubes show the most stable performance across tested parameters for both bioanalytes. Furthermore, we find more variability in protein composition in sEVs than in lEVs after 7 days of storage; thus, sEVs might be more susceptible to storage effects. Our clinically applicable workflow provides the basis for informed choice of liquid biopsy tubes along with a ready-to-use protocol to retrieve both genomic and EV proteomic biomarker information for multi-omics biomarker-based liquid biopsy studies.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Extracellular Vesicle, Peripheral Blood

SUBMITTER: Katharina Clemm von Hohenberg

LAB HEAD: Katharina Clemm

PROVIDER: PXD062837 | Pride | 2026-01-23

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	2023-03-01_UP000005640_9606_one_prot_seq_per_gene.fasta	Fasta
	checksum.txt	Txt
	contaminants.fasta	Fasta
	oecf5-KM0814-01.raw	Raw
	oecf5-KM0814-02.raw	Raw

Items per page:

1 - 5 of 84

Publications

Preanalytical framework for routine clinical use of liquid biopsies: combining EVs and cfDNA.

Simon Nike K NK Volz Stefanie S Rios de Los Rios Reséndiz Jussara J Wedig Tatjana T Montigel Sophia H SH Schwarz Nathalie N Richter Karsten K Helm Dominic D Neßling Michelle M Zielske Lin L Berker Julia J Russeck Sophia S Mauermann Monika M Hofmann Wolf-Karsten WK Pfister Stefan M SM Pajtler Kristian W KW Maaß Kendra K KK Clemm von Hohenberg Katharina K

Extracellular vesicles and circulating nucleic acids 20251009 4

<b>Aim:</b> Liquid biopsies hold significant potential for the minimally invasive diagnosis of tumors and other diseases. While the clinical application of cell-free DNA (cfDNA) methodologies is emerging, the implementation of tumor-derived extracellular vesicles (EVs) as validated biomarkers is hindered by substantial preanalytical variations. In this work, we standardized the preanalytical procedures of blood collection for subsequent serial isolation of plasma cfDNA and EVs from a single bloo ...[more]

PMID: 41551608

Similar Datasets

Project description:Extracellular vesicles (EVs) hold the potential for elucidating the pathogenesis and serving as biomarkers of amyotrophic lateral sclerosis (ALS). Notably, the comparative and longitudinal alterations in the protein profiles of EVs in serum (sEVs) and CSF (cEVs) of sporadic ALS (SALS) patients remain unexplored. This study sought to reveal such changes by collecting serum and CSF at fixed intervals from 10 controls and 20 SALS patients participating in the Ropinirole Hydrochloride Remedy for Amyotrophic Lateral Sclerosis trial. We also aimed to reveal longitudinal changes with disease progression and the effects of ropinirole hydrochloride (ROPI, a dopamine D2 receptor agonist identified as an anti-ALS drug candidate using induced pluripotent stem cell drug discovery) on protein profiles of EVs. Comprehensive proteomic analyses of EVs extracted from these samples were conducted using liquid chromatography-mass spectrometry to trace longitudinal shifts linked to disease progression and the influence of ROPI on EV protein profiles. The findings revealed notable disparities but high congruity in sEV and cEV protein profiles concerning disease status, time, and ROPI administration. Moreover, the sEV and cEV protein profiles converged over time in SALS patients, at least in part, suggestive of a loosening of the blood–brain barrier. In SALS patients, both sEVs and cEVs presented elevated inflammation-related protein levels but reduced levels of proteins associated with the unfolded protein response. These results mirrored the longitudinal changes after disease onset and correlated with the revised ALS Functional Rating Scale at sampling time, suggesting a link to the onset and progression of SALS. ROPI appeared to counteract these changes, attenuating inflammation-related protein levels and boosting those tied to the unfolded protein response in SALS, suggesting an anti-ALS effect on EV protein profiles. Reverse translational research using induced pluripotent stem cell-derived astrocytes indicated that these changes may partly reflect the DRD2-dependent neuroinflammatory inhibitory effects of ROPI. Baseline levels of OGN in sEVs and FRMPD1 in cEVs correlated with subsequent disease progression, indicating their potential as prognostic biomarkers for SALS. Machine learning-driven biomarker searches and diagnostic classification yielded an accuracy of 90.4% for sEVs and 80.3% for cEVs. Despite the limited sample size, this study is the first to report time-series proteomic alterations in sEVs and cEVs from SALS patients, offering comprehensive insights into SALS pathogenesis, ROPI-induced changes, and potential prognostic and diagnostic biomarkers.

Project description:While primary breast cancer (BC) is often effectively managed, metastasis remains the primary cause of BC-related fatalities. Gaps persist in our understanding of the mechanisms underlying cancer cell predilection for specific organs (organotropism). Unraveling these mediators of site-specific metastasis could enhance early detection and enable more tailored interventions. Liquid biopsy represents an innovative diagnostic approach in cancer involving the non-invasive analysis of biological materials found in body fluids like blood or urine. These materials include circulating tumor cells, cell-free circulating tumor DNA, and tumor-derived extracellular vesicles (EVs), which offer valuable insights for characterizing and monitoring tumor genomes to advance personalized medicine. EVs, lipid-bound particles released from cells into the extracellular space, facilitate intercellular communication by transporting proteins and DNA (EV-DNA) implicated in cancer progression and organ-specific metastasis. The aim of this study was to conduct an in-depth analysis of cell-free DNA (cfDNA) and EVs using a murine model of BC cell lines with distinct metastatic potentials and organotropisms (lung, bone, liver, brain). We characterized the secretome of 8 different metastatic cell lines originating from the same parental non metastatic and metastatic cell line, as well as normal mammary epithelial cells to identify unique biomarkers specific to metastatic sites. Using ultracentrifigcation, we isolated small EVs ranging from 78.9 to 101nm isolated from the conditioned media of the cell lines. We confirmed the presence of established EV markers, including CD81, TSG101 and syntenin. Transmission electron microscopy revealed interesting EV secretion patterns that differed according to cell line. We demonstrated the presence of EV-DNA and cfDNA in all the cell lines, with varying concentrations. We also found that a common BC mutation in TP53 can be specifically detected in both EV-DNA and cfDNA of cancer cell cultures using droplet digital PCR. The proteomic characterization of EVs through high throughput mass spectrometry revealed common EV signatures and proteins involved in cancer processes and organotropism unique to distinct site-specific cell lines. Specifically, we found enrichment of integrin receptors in metastatic cancer EVs compared to EVs secreted from normal epithelial cells and matched tumorigenic nonmetastatic cell lines. Gene ontology (GO) pathway analysis revealed that cancer derived EVs display a cell adhesion signature and are enriched with proteins involved in extracellular matrix interaction, focal adhesion, endocytosis, and PI3K-Akt signaling pathway. Interestingly, EVs containing heat shock proteins and DNA binding proteins are released by both cancer and normal cells. A total of 460 proteins were quantified with a high confidence level, including 70% of the TOP 200 exosome markers archived by Vesiclepedia. Taken together, we characterized the quantity and cargo of EVs in a unique model of BC organotropism, suggesting that EV-DNA and EVs were informative of normal and cancer states. This work could help to identify BC biomarkers associated with site-specific metastasis as well as new therapeutic targets.