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Quantifying and Phenotyping Extracellular Vesicles

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Quantifying and Phenotyping Extracellular Vesicles Quantifying and Phenotyping Extracellular Vesicles Sensitivity and Specificity of the ExoView® Tetraspanin Assay WHITE PAPER THEMES: • ExoView® Assay Sensitivity Using HEK293 and AsPC1 Cells • Comparative Analysis of Assay Specificity • Detection of EVs in Physiologically Relevant Plasma Introduction Quantifying extracellular vesicle (EV) biomarkers The ExoView® Tetraspanin Assay from NanoView in unpurified or unprocessed samples is of key Biosciences provides data for single EVs and is importance in several scenarios. For example, based on specific EV capture by human or mouse companies that are developing EV-based tetraspanin antibodies arrayed on a chip. therapeutics must ensure lot-to-lot consistency of their product and any engineered cargo. In clinical Data from three fluorescent channels and research, EVs derived from specific tissues UV-based interferometric (IM) images are or tumors can contain important circulating or collected in an automated workflow using the excreted biomarkers, but the population of interest ExoView R100 instrument. Thousands of EVs is likely to be rare (perhaps less than 1%)1. are quantified to determine size and surface/ cargo marker colocalization in a single assay. Due to their small size and low protein content, Standard ExoView chips include tetraspanin EVs are challenging to quantify and phenotype. markers suitable for measuring EVs from cell Many technologies and assays are currently in culture (Tetra-C) and plasma (Tetra-P). Chips use based on various features of EVs, including can also be customized to bind custom surface nanoparticle tracking, total protein content, and markers, for example, in this paper we show acetylcholinesterase activity. However, these that Tetraspanin8 (Tspan 8) positive EVs in assays are non-specific and detect non-vesicular plasma can be captured and quantified. Tspan8 proteins/factors that affect the specificity and is a biomarker associated with carcinomas sensitivity of EV characterization. There are such as pancreatic cancer2 and assaying the several antibody-based techniques that allow number of Tspan8 positive EVs in patient blood for more specific EV characterization, such as samples would be of great clinical importance Western blot, ELISA, and nano-flow cytometry. for therapeutic monitoring. It is important to note Some of these methods lack sufficient sensitivity that Tspan8 positive EVs are present at very low to detect rare EV subpopulations or low concentrations, so it is necessary to use an assay abundance EVs in biofluids, and require sample with high levels of sensitivity and specificity. concentration that can introduce unwanted experimental variables. 2 NanoView Biosciences, Boston MA For Research Use Only +1-781-365-8439 [email protected] 1 Koliha N et al. (2016) Melanoma Affects the Composition of Blood Cell-Derived Extracellular Vesicles. Front. Immunol. 7:282. 2 Buscail E. et al. (2019) Liquid Biopsy Approach for Pancreatic Ductal Adenocarcinoma. Cancers (Basel). 11(6): 852. EV Detection Methods Nanoparticle Tracking analysis (NTA) EVs from different biofluids and cell lines have is a popular method for quantifying EV size and different tetraspanin expression profiles. It is concentration by tracking single particles as they therefore common to test samples using multiple move in solution. NTA uses particle Brownian kits, introducing inter-experiment variability. motion to determine particle size, and uses particle numbers to determine concentration. Total protein assays such as the Bradford Importantly, NTA measurements are not specific assay are often used to compare relative to EVs and instead will quantify any particle amounts of EVs between samples by measuring within the size range of detection. NanoSight changes in total protein amounts. These assays (Malvern Panalytical) and Zetaview (Particle do not discriminate between non-vesicular and Metrix) are examples of commercially available vesicular proteins, so the sample must be free NTA instruments. Differences between the two of non-vesicle proteins (or have consistent and systems have been reported demonstrating that controlled expression levels) for this measurement NanoSight is more accurate regarding particle to be useful. The Bradford assay is compatible size measurements, but overestimates the particle with phenol red and other components of number, while the Zetaview reports larger than cell culture media, and has a broad dynamic expected particle size, but more accurately range that quantifies total protein ranging from reports the expected particle concentration3. ~1.5 to 2,000 µg/mL. However, the Bradford reagent does not cross the EV membrane and Fluorocet and Exocet assays the assay is not compatible with detergents (System Biosciences) such as Triton-X100 or SDS at concentrations are examples of exosome-specific assays that above 0.125%. Therefore, this assay can only are available in fluorescent and absorbance quantify EV surface proteins (unless the sample is formats, respectively. These assays are based concentrated enough that the EVs can be lysed on the principle that EVs contain enzymatically and then diluted to within the acceptable range active acetylcholinesterase, though this has been of detergent). recently questioned4, and there are reports that acetylcholinesterase expression in EVs are Western blotting is a bulk method of highest in neuronal and erythrocyte cells5. characterizing EV markers and cargo. The Furthermore, it is unclear whether the EVs are lysed and proteins are separated by acetylcholinesterase activity is from the electrophoresis. The separated proteins are EVs or sera in cell culture media. then transferred to a membrane and probed with specific antibodies. This technique allows ExoELISA kits (System Biosciences) the determination of relative amounts of different are ELISA based assays used to quantify exosome biomarkers if they are present in sufficiently high numbers per well based on a standard curve concentrations. In these experiments, the lower of a known number of exosomes. The kits are limit of detection was ~2x108 EVs/well, meaning configured for specific tetraspanin proteins a minimum concentration of ~1.5x1010 EVs/mL including CD63, CD9, and CD81. The kits are is required for abundant markers like CD81 and specific to human EVs but one challenge is that CD9. 3 NanoView Biosciences, Boston MA For Research Use Only +1-781-365-8439 [email protected] 3 Bachurski et al. (2019) Extracellular vesicle measurements with nanoparticle tracking analysis – An accuracy and repeatability comparison between NanoSight NS300 and ZetaView J. Extracellular Vesicles. 8, 1596016. 4 Witwer et al. (2019) Extracellular vesicles or exosomes? On primacy, precision, and popularity influencing a choice of nomenclature J. Extracellular Vesicles. 8, 1648167. 5 Liao et al. (2019) Acetylcholinesterase is not a generic marker of extracellular vesicles J. Extracellular Vesicles 8, 1628592. Sandwich ELISA kits use a specific antibody CD63, CD9, and CD81 are available from to capture EVs that are then detected using a various providers and are used to quantitatively conjugated antibody to a different epitope of measure EVs that express these proteins. the target protein. ELISA kits that detect human FIGURE 1 Assay sensitivity in terms of minimum input requirements (number of EVs per ml). The ExoView assay is about 1 order of magnitude more sensitive than NTA and 2+ orders more sensitive than ELISA or Fluorocet assays. 1e5 1e6 1e7 1e8 1e9 1e10 1e11 1e12 1e13 ExoView CD9 ELISA CD63 ELISA NTA WB CD81 ELISA * The variation in the ELISA sensitivities is due to variable tetraspanin expression across multiple cell lines Comparative Analysis of Assay Sensitivity The sensitivity of the ExoView Tetra-C assay was HEK 293 comparison assays: performed in 2 cells lines – HEK293 and AsPC1 HEK cell basal (serum-free) or complete (with pancreatic cancer cells. Concentration curves of EVs exosome-depleted serum) media were used in media conditioned by HEK or AsPC1 cells for 48 to assay the sensitivity of the ExoView assay. hours were used to measure the detection range and Dilutions of 50x to 25600x were tested in a lower limit of detection. 9-point two-fold dilution series. The lower limit of detection of the ExoView assay Figure 2A shows the number of EVs from is considered to be the point at which the signal HEK cell basal conditioned medium that were can no longer be distinguished from background captured on CD63 spots, and detected with antibody binding events and/or autofluorescence, a cocktail of fluorescent antibodies against and the relationship between concentration and CD81, CD63, and CD9 using the ExoView number of events becomes non-linear. R100. Figure 2B shows a similar dilution series for the complete conditioned medium, which 4 NanoView Biosciences, Boston MA For Research Use Only +1-781-365-8439 [email protected] 5Liao et al. (2019) Acetylcholinesterase is not a generic marker of extracellular vesicles J. Extracellular Vesicles 8, 1628592. contained approximately twice as many EVs as a concentration of 3.5x1010 EVs/mL. In the the basal medium. In the complete conditioned HEK293 samples, the slope of the EV count media, fluorescently labeled CD9 begins to vs. dilution scatter plot was linear down to the reach saturation on the CD81 capture spots lowest concentration tested, 1:25600. Based when the sample is more concentrated than a on an NTA concentration of 3.5x1010 EV/mL, 1:200 dilution, whereas the basal conditioned the limit of detection of ExoView
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