Quantification of Circulating Cell Free Mitochondrial DNA in Extracellular Vesicles with PicoGreen™ in Liquid Biopsies: Fast Assessment of Disease/Trauma Severity.
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ABSTRACT:
SUBMITTER: Marcatti M
PROVIDER: S-EPMC8067453 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Extracellular vesicles (EVs) are secreted by all living cells and are ubiquitous in every human body fluid. They are quite heterogeneous with regard to biogenesis, size, and composition, yet always reflect their parental cells with their cell-of-origin specific cargo loading. Since numerous studies have demonstrated that EV-associated proteins, nucleic acids, lipids, and metabolites can represent malignant phenotypes in cancer patients, EVs are increasingly being discussed as valuable carriers of cancer biomarkers in liquid biopsy samples. However, the lack of standardized and clinically feasible protocols for EV purification and characterization still limits the applicability of EV-based cancer biomarker analysis. This review first provides an overview of current EV isolation and characterization techniques that can be used to exploit patient-derived body fluids for biomarker quantification assays. Secondly, it outlines promising tumor-specific EV biomarkers relevant for cancer diagnosis, disease monitoring, and the prediction of cancer progression and therapy resistance. Finally, we summarize the advantages and current limitations of using EVs in liquid biopsy with a prospective view on strategies for the ongoing clinical implementation of EV-based biomarker screenings.
| S-EPMC9953862 | biostudies-literature
Project description:Substantial research has been devoted to elucidate the roles that extracellular vesicles (EVs) play in the regulation of both normal and pathological processes, and multiple studies have demonstrated their potential as a source of cancer biomarkers. However, several factors have slowed the development of liquid biopsy EV biomarkers for cancer diagnosis, including logistical and technical difficulties associated with reproducibly obtaining highly purified EVs suitable for diagnostic analysis. Significant effort has focused on addressing these problems, and multiple groups have now reported EV analysis methods using liquid biopsies that have the potential for clinical translation. However, there are still important issues that must be addressed if these discoveries and technical advances are to be used for clinical translation of EV cancer biomarkers from liquid biopsies. To address these issues, this review focuses on the potential application of EV biomarkers for diagnosis of major cancer types, discussing approaches for EV biomarker discovery and verification, EV clinical assay development, analytical and clinical validation, clinical trials, regulatory submission, and end user utilization for the intended clinical application. This review also discusses key difficulties related to these steps, and recommendations for how to best accomplish steps in order to translate EV-based biomarkers into clinical settings.
| S-EPMC6469512 | biostudies-literature
Project description:Purpose: Aseptic tissue injuries including major surgeries are associated with structural and functional changes in the brain suggesting the existence of periphery-to-brain communication. However, the nature of the carriers of such signaling remains largely unknown. Analyzis of the miRNAome and proteome of circulating extracellular vesicles (EVs) to evaluate their potential contribution to the periphery-to-brain signaling. Methods: EVs were isolated from the serum of the mice subjected to a tibia surgery after 6, 24, and 72 h, using Exoquick ULTRA commercial ki. Proteome and miRNAome were mapped using mass spectrometry and RNA-seq approaches. Results: Next-generation sequencing of small RNA libraries prepared from the total RNA isolated from the mouse serum EVs detected 252 miRNAs. Differential expression analysis using DESeq2 statistical package revealed 50 dysregulated miRNAs in the 24 h post-surgery EVs. The surgery effect declines after 72 h as judged by the lower number of dysregulated miRNAs. The differentially expressed miRNAs were examined by the DIANA-miRPath online tool that predicted dysregulation of several KEGG pathways including pathways involved in the lipid metabolism, ECM-receptor interaction, gap junction, various signal transduction pathways. Conclusions: The main question of the current project, is the surgery capable of modifying the expression of cargo molecules in circulating EVs, can be answered positively. Moreover, differential expression of a specific set of miRNAs in the “surgical” EVs suggests that they can be involved in the regulation of several metabolic pathways, such as ECM-receptor interaction and lipid metabolism in the recipient tissues, which is consistent with our data on hippocampal metabolic and functional dysregulation after surgery We conclude that surgery alters the EV composition in the blood, which paves the way for further studies on the functional effects of these EVs on the brain.
2022-01-08 | GSE189972 | GEO
Project description:Surgical interventions rapidly trigger a cascade of molecular, cellular, and neural signaling responses that ultimately reach remote organs, including the brain. Using a mouse model of orthopedic surgery, we have previously demonstrated hippocampal metabolic, structural, and functional changes associated with cognitive impairment. However, the nature of the underlying signals responsible for such periphery-to-brain communication remains hitherto elusive. Here we present the first exploratory study that tests the hypothesis of extracellular vesicles (EVs) as potential mediators carrying information from the injured tissue to the distal organs including the brain. The primary goal was to investigate whether the cargo of circulating EVs after surgery can undergo quantitative changes that could potentially trigger phenotypic modifications in the target tissues. EVs were isolated from the serum of the mice subjected to a tibia surgery after 6, 24, and 72 h, and the proteome and miRNAome were investigated using mass spectrometry and RNA-seq approaches. We found substantial differential expression of proteins and miRNAs starting at 6 h post-surgery and peaking at 24 h. Interestingly, one of the up-regulated proteins at 24 h was α-synuclein, a pathogenic hallmark of certain neurodegenerative syndromes. Analysis of miRNA target mRNA and corresponding biological pathways indicate the potential of post-surgery EVs to modify the extracellular matrix of the recipient cells and regulate metabolic processes including fatty acid metabolism. We conclude that surgery alters the cargo of circulating EVs in the blood, and our results suggest EVs as potential systemic signal carriers mediating remote effects of surgery on the brain.
| S-EPMC8804340 | biostudies-literature
Project description:Cancer cells secrete small membranous extracellular vesicles (EVs) into their microenvironment and circulation. Although their potential as cancer biomarkers has been promising, the identification and quantification of EVs in clinical samples remains challenging. Here we describe a sensitive and rapid analytical technique for profiling circulating EVs directly from blood samples of patients with colorectal cancer. EVs are captured by two types of antibodies and are detected by photosensitizer-beads, which enables us to detect cancer-derived EVs without a purification step. We also show that circulating EVs can be used for detection of colorectal cancer using the antigen CD147, which is embedded in cancer-linked EVs. This work describes a new liquid biopsy technique to sensitively detect disease-specific circulating EVs and provides perspectives in translational medicine from the standpoint of diagnosis and therapy.
| S-EPMC3988821 | biostudies-literature
Project description:Extracellular vesicles (EVs) are key mediators of intercellular communication. Part of their biological effects can be attributed to the transfer of cargos of diverse types of RNAs, which are promising diagnostic and prognostic biomarkers. EVs found in human biofluids are a valuable source for the development of minimally invasive assays. However, the total transcriptional landscape of EVs is still largely unknown. Here we develop a new method for total transcriptome profiling of plasma-derived EVs by next generation sequencing (NGS) from limited quantities of patient-derived clinical samples, which enables the unbiased characterization of the complete RNA cargo, including both small- and long-RNAs, in a single library preparation step. This approach was applied to RNA extracted from EVs isolated by ultracentrifugation from the plasma of five healthy volunteers. Among the most abundant RNAs identified we found small RNAs such as tRNAs, miRNAs and miscellaneous RNAs, which have largely unknown functions. We also identified protein-coding and long noncoding transcripts, as well as circular RNA species that were also experimentally validated. This method enables, for the first time, the full spectrum of transcriptome data to be obtained from minute patient-derived samples, and will therefore potentially allow the identification of cell-to-cell communication mechanisms and biomarkers.
| S-EPMC5663969 | biostudies-literature
Project description:Over the past decade, extracellular vesicles (EVs) have emerged as a promising source of cancer-derived RNAs for liquid biopsies. However, blood contains a pool of heterogeneous EVs released by a variety of cell types, making the identification of cancer RNA biomarkers challenging. Here, we performed deep sequencing of plasma EV RNA cargo in 32 patients with locally advanced breast cancer (BC) at diagnosis and 7 days after breast surgery and in 30 cancer-free healthy controls (HCs). To identify BC-derived RNA biomarkers, we searched for RNAs that had higher levels in BC EVs at the time of diagnosis compared with HCs and decreased after surgery. Data analysis showed that the fractions of miRNAs, snRNAs, snoRNAs, and tRFs were increased, but the fraction of lncRNAs was decreased in BC EVs as compared to HCs. BC-derived biomarker candidates were identified across various RNA biotypes. Considered individually, they had very high specificity but moderate sensitivity for the detection of BC, whereas a biomarker model composed of eight RNAs: SNORD3H, SNORD1C, SNORA74D, miR-224-5p, piR-32949, lnc-IFT-122-2, lnc-C9orf50-4, and lnc-FAM122C-3 was able to distinguish BC from HC EVs with an AUC of 0.902 (95% CI = 0.872-0.931, p = 3.4 × 10-9) in leave-one-out cross-validation. Furthermore, a number of RNA biomarkers were correlated with the ER and HER2 expression and additional biomarker models were created to predict hormone receptor and HER2 status. Overall, this study demonstrated that the RNA composition of plasma EVs is altered in BC patients and that they contain cancer-derived RNA biomarkers that can be used for BC detection and monitoring using liquid biopsies.
| S-EPMC10487078 | biostudies-literature
Project description:Extracellular vesicles (EVs) secreted from cancer cells have potential for generating cancer biomarker signatures. Fibronectin (FN) was selected as a biomarker candidate, due to the presence in surface on EVs secreted from human breast cancer cell lines. A subsequent study used two types of enzyme-linked immunosorbent assays (ELISA) to determine the presence of these proteins in plasma samples from disease-free individuals (n=70), patients with BC (n=240), BC patients after surgical resection (n=40), patients with benign breast tumor (n=55), and patients with non-cancerous diseases (thyroiditis, gastritis, hepatitis B, and rheumatoid arthritis; n=80). FN levels were significantly elevated (p< .0001) at all stages of BC, and returned to normal after tumor removal. The diagnostic accuracy for FN detection in extracellular vesicles (ELISA method 1) (area under the curve, 0.81; 95% CI, 0.76 to 0.86; sensitivity of 65.1% and specificity of 83.2%) were also better than those for FN detection in the plasma (ELISA method 2) (area under the curve, 0.77; 95% CI, 0.72 to 0.83; sensitivity of 69.2% and specificity of 73.3%) in BC. The diagnostic accuracy of plasma FN was similar in both the early-stage BC and all BC patients, as well as in the two sets. This liquid biopsy to detect FN on circulating EVs could be a promising method to detect early breast cancer.
| S-EPMC5130002 | biostudies-literature
Project description:Extracellular vesicles (EVs) are abundantly released into the systemic circulation, where they show remarkable stability and harbor molecular constituents that provide biochemical information about their cells of origin. Due to this characteristic, EVs are attracting increasing attention as a source of circulating biomarkers for cancer liquid biopsy and personalized medicine. Despite this potential, none of the discovered biomarkers has entered the clinical practice so far, and novel approaches for the label-free characterization of EVs are highly demanded. In this regard, Fourier Transform Infrared Spectroscopy (FTIR) has great potential as it provides a quick, reproducible, and informative biomolecular fingerprint of EVs. In this pilot study, we investigated, for the first time in the literature, the capability of FTIR spectroscopy to distinguish between EVs extracted from sera of cancer patients and controls based on their mid-IR spectral response. For this purpose, EV-enriched suspensions were obtained from the serum of patients diagnosed with Hepatocellular Carcinoma (HCC) of nonviral origin and noncancer subjects. Our data point out the presence of statistically significant differences in the integrated intensities of major mid-IR absorption bands, including the carbohydrate and nucleic acids band, the protein amide I and II bands, and the lipid CH stretching band. Additionally, we used Principal Component Analysis combined with Linear Discriminant Analysis (PCA-LDA) for the automated classification of spectral data according to the shape of specific mid-IR spectral signatures. The diagnostic performances of the proposed spectral biomarkers, alone and combined, were evaluated using multivariate logistic regression followed by a Receiving Operator Curve analysis, obtaining large Areas Under the Curve (AUC = 0.91, 95% CI 0.81–1.0). Very interestingly, our analyses suggest that the discussed spectral biomarkers can outperform the classification ability of two widely used circulating HCC markers measured on the same groups of subjects, namely alpha-fetoprotein (AFP), and protein induced by the absence of vitamin K or antagonist-II (PIVKA-II).
| S-EPMC9224706 | biostudies-literature
Project description:Most cancer-related deaths are caused by distant metastases, which are tumour cells that have escaped from a primary tumour and passed into the bloodstream to colonize a new organ. In this context, communication between tumour and stromal cells is essential. Indeed, tumor cells interact with cells in the tumor microenvironment and are able to modify them to their advantage. Both extracellular vesicles (EVs) and exosomes are heterogeneous populations of small vesicles present in the tumor microenvironment and in body fluids that have recently emerged as powerful mediators involved in this communication and their transport in fluids. Tumor cells release large quantities of exosomes containing tumor markers, which can then spread to distant locations.
The exosomes are of endosomal origin. They are composed of proteins, lipids, RNA and DNA, and they circulate in the bloodstream. They can be internalized by specific distant cells and thus deliver a functional message. It has recently been shown that tumor exosomes containing pro-metastatic factors form pre-metastatic niches, before the tumor cells actually arrive, while determining the metastatic organotropism of tumors. These properties are now opening up new avenues of research in tumor biomarkers. In recent years, several studies have highlighted different markers contained specifically in exosomes derived from cancer cells. Consequently, exosomes are considered as potential reservoirs of tumor biomarkers that could be clinically useful for the non-invasive diagnosis of cancer, with the advantage of being performed by liquid biopsy. The study of microRNA (miRNA) is of particular interest. Indeed, miRNAs are small non-coding RNAs (between 21 and 25 nucleotides) involved in the regulation of gene expression and which are frequently deregulated in cancer. Several studies underline that the variation of free miRNAs in the blood is correlated with the progression of the disease, particularly in colon cancer. However, the stability of free miRNAs is controversial. Therefore, exosomes represent a very advantageous means of transporting miRNAs in the blood, as they are able to protect miRNAs from degradation by RNAase.
The hypothesis of the project is that circulating exosomes derived from tumours contain markers including specific miRNAs that could be used as biomarkers of early prognosis (survival and progression), easily measured in blood samples from patients with colon cancer. But other molecules contained in exosomes could also be of interest.
| 2349603 | ecrin-mdr-crc