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A Nanoparticle-Based Approach for the Detection of Extracellular Vesicles.

ABSTRACT: The analysis of extracellular vesicles (EVs) typically requires tedious and time-consuming isolation process from bio-fluids. We developed a nanoparticle-based time resolved fluorescence immunoassay (NP-TRFIA) that uses biotinylated antibodies against the proteins of tetraspanin family and tumor-associated antigens for capturing EVs from urine samples and cell culture supernatants without the need for isolation. The captured-EVs were detected either with Eu³⁺-chelate or Eu³⁺-doped nanoparticle-based labels conjugated either to antibodies against the tetraspanins or lectins targeting the glycan moieties on EVs surface. The NP-TRFIA demonstrated specific capturing and detection of EVs by antibodies and lectins. Lectin-nanoparticle based assays showed 2-10 fold higher signal-to-background ratio compared with lectin-chelate assays. The nanoparticle assay concept allowed surface glycosylation profiling of the urine derived-EVs with lectins. It was also applied to establish an assay showing differential expression of tumor-associated proteins on more aggressive (higher ITGA3 on DU145- and PC3-EVs) compared to less aggressive (higher EpCAM on LNCaP-EVs) PCa- cell lines derived-EVs. This NP-TRFIA can be used as a simple tool for analysis and characterization of EVs in urine and cell culture supernatants. Such approach could be useful in identification of disease-specific markers on the surface of patient-derived urinary EVs.

SUBMITTER: Islam MK

PROVIDER: S-EPMC6624270 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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A Nanoparticle-Based Approach for the Detection of Extracellular Vesicles.

Islam Md Khirul MK Syed Parvez P Lehtinen Laura L Leivo Janne J Gidwani Kamlesh K Wittfooth Saara S Pettersson Kim K Lamminmäki Urpo U

Scientific reports 20190711 1

The analysis of extracellular vesicles (EVs) typically requires tedious and time-consuming isolation process from bio-fluids. We developed a nanoparticle-based time resolved fluorescence immunoassay (NP-TRFIA) that uses biotinylated antibodies against the proteins of tetraspanin family and tumor-associated antigens for capturing EVs from urine samples and cell culture supernatants without the need for isolation. The captured-EVs were detected either with Eu<sup>3+</sup>-chelate or Eu<sup>3+</sup ...[more]

PMID: 31296879

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Project description:Among the various biomarkers that are used to diagnose or monitor disease, extracellular vesicles (EVs) represent one of the most promising targets in the development of new therapeutic strategies and the application of new diagnostic methods. The detection of circulating platelet-derived microvesicles (PMVs) is a considerable challenge for laboratory diagnostics, especially in the preliminary phase of a disease. In this study, we present a multistep approach to immobilizing and detecting PMVs in biological samples (microvesicles generated from activated platelets and human platelet-poor plasma) on functionalized silicon substrate. We describe the application of time-of-flight secondary ion mass spectrometry (TOF-SIMS) and spectroscopic ellipsometry methods to the detection of immobilized PMVs in the context of a novel imaging flow cytometry (ISX) technique and atomic force microscopy (AFM). This novel approach allowed us to confirm the presence of the abundant microvesicle phospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) on a surface with immobilized PMVs. Phosphatidylcholine groups (C5H12N+; C5H15PNO4+) were also detected. Moreover, we were able to show that ellipsometry permitted the immobilization of PMVs on a functionalized surface to be evaluated. The sensitivity of the ISX technique depends on the size and refractive index of the analyzed microvesicles. Graphical abstract Human platelets activated with thrombin (in concentration 1IU/mL) generate population of PMVs (platelet derived microvesicles), which can be detected and enumerated with fluorescent-label method (imaging cytometry). Alternatively, PMVs can be immobilized on the modified silicon substrate which is functionalized with a specific IgM murine monoclonal antibody against human glycoprotein IIb/IIIa complex (PAC-1). Immobilized PMVs can be subjected to label-free analyses by means ellipsometry, atomic force microscopy (AFM) and time-of-flight secondary ion mass spectrometry (TOF-SIMS).

Project description:IntroductionOptical techniques are routinely used to size and count extracellular vesicles (EV). For comparison of data from different methods and laboratories, suitable calibrators are essential. A suitable calibrator must have a refractive index (RI) as close to that of EV as possible but the RI of EV is currently unknown. To measure EV, RI requires accurate knowledge of size and light scattering. These are difficult to measure as most EVs cannot be resolved by light microscopy and their diameter is smaller than the wavelength of visible light. However, nanoparticle tracking analysis (NTA) provides both size and relative light scattering intensity (rLSI) values. We therefore sought to determine whether it was possible to use NTA to measure the RI of individual EVs.MethodsNTA was used to measure the rLSI and size of polystyrene and silica microspheres of known size and RI (1.470 and 1.633, respectively) and of EV isolated from a wide range of cells. We developed software, based on Mie scattering code, to calculate particle RI from the rLSI data. This modelled theoretical scattering intensities for polystyrene and silica microspheres of known size (100 and 200 nm) and RI. The model was verified using data from the polystyrene and silica microspheres. Size and rLSI data for each vesicle were processed by the software to generate RI values.ResultsThe following modal RI measurements were obtained: fresh urinary EV 1.374, lyophilised urinary EV 1.367, neuroblastoma EV 1.393, blood EV 1.398, EV from activated platelets 1.390, small placental EV 1.364-1.375 and 1.398-1.414 for large placental EV (>200 nm). Large placental EV had a significantly higher RI than small placental EV (p<0.0001). The spread of RI values was narrower for small EV than for the more heterogeneous large EV.DiscussionUsing NTA and Mie scattering theory, we have demonstrated that it is possible to estimate the RI of sub-micron EV using NTA data. EV typically had a modal RI of 1.37-1.39, whereas values of >1.40 were observed for some large (>200 nm) microvesicles.ConclusionThis method for measuring EV RI will be useful for developing appropriate calibrators for EV measurement.

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.

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A Nanoparticle-Based Approach for the Detection of Extracellular Vesicles.

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A Nanoparticle-Based Approach for the Detection of Extracellular Vesicles.

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