Selective isolation of extracellular vesicles from minimally processed human plasma as a translational strategy for liquid biopsies.

Fortunato D, Giannoukakos S, Giménez-Capitán A, Hackenberg M, Molina-Vila MA, Zarovni N.
Biomark Res. 2022 Aug 7;10(1):57. doi: 10.1186/s40364-022-00404-1.

The study addresses central aspects of EV immunoaffinity isolation from simple and complex matrices, such as plasma. Fluorescently-labelled spike-in EVs emerged as reliable, high-throughput and easily measurable readouts, which were employed to optimize our EV immunoprecipitation strategy and evaluate its performance. Plasma-derived EVs were captured and detected using this straightforward protocol, sequentially combining isolation and staining of specific surface markers, such as CD9 or CD41. Multiplexed digital transcript detection data was generated using the Nanostring nCounter platform and evaluated through a dedicated bioinformatics pipeline. mRNA profiling experiments proved that distinct EV subpopulations can be captured by directly targeting different surface markers. Furthermore, EVs isolated with anti-CD61 beads enclosed mRNA expression patterns that might be associated to early-stage lung cancer, in contrast with EVs captured through CD9, CD63 or CD81. The differential clinical value carried within each distinct EV subset highlights the advantages of selective isolation.

Integrated isolation and quantitative analysis of exosome shuttled proteins and nucleic acids using immunocapture approaches.

Zarovni N, Corrado A, Guazzi P, Zocco D, Lari E, Radano G, Muhhina J, Fondelli C, Gavrilova J, Chiesi A.
Methods. 2015 Oct 1;87:46-58. doi: 10.1016/j.ymeth.2015.05.028. Epub 2015 Jun 2.

This early study features an evaluation of several commercially available solutions for immunoplate- and immunobead-based affinity isolation and have further optimized protocols to decrease non-specific binding due to exosomes complexity and matrix contaminants. In order to identify best molecular targets for total exosome capture from diverse biological sources, as well as for selective enrichment in populations of interest (e.g. tumor derived exosomes) several exosome displayed proteins and respective antibodies have been evaluated for plate and bead functionalisation. Moreover, we have optimized and directly implemented downstream steps allowing on-line quantification and characterization of bound exosome markers, namely proteins and RNAs. Thus assembled assays enabled rapid overall quantification and validation of specific exosome associated targets in/on plasma exosomes, with multifold increased yield and enrichment ratio over benchmarking technologies. Assays directly coupling selective immobilization of exosomes to a solid phase and their immune- and or molecular profiling through conventional ELISA and PCR analysis, resulted in easy-to-elaborate, quantitative readouts, with high low-end sensitivity and dynamic range, low costs and hands-on time, minimal sample handling and downscaling of a working plasma volumes to as few as 100 μl.

Extraction and Analysis of Extracellular Vesicle-Associated miRNAs Following Antibody-Based Extracellular Vesicle Capture from Plasma Samples.

Zocco D, Zarovni N.
Methods Mol Biol. 2017;1660:269-285. doi: 10.1007/978-1-4939-7253-1_22.

In this book chapter we propose a protocol for antibody-based isolation of blood-derived EVs followed by extraction and expression analysis of small-RNA species (miRNA) by reverse transcriptase quantitative PCR (RT-qPCR). The advantages of immunoaffinity approaches over other isolation methods are multiple and include: (1) the selective enrichment of specific EV subpopulations with restricted tissue/cell origin, (2) reduction of matrix effects and blood contaminants that may confound miRNA profiling from complex biological fluids and (3) easy coupling to conventional quantitative assays (e.g., RT-qPCR). In conclusion, we describe a protocol for standard enrichment and quantitative analysis of EV-miRNAs from blood and we warrant for technological improvements, such as the use of novel biomaterials, surface chemistries, binding agents and assay/sensor design that may further improve it.

TM9SF4 expression in tumor tissues: a novel diagnostic biomarker for gastrointestinal tumors.

Guazzi P, Zocco D, Isajevs S, Zarovni N, Bianciardi L, Toots M, Sivins A, Leja M, Chiesi A, Lozupone F.
Transl Cancer Res. 2020 Nov;9(11):6652-6659. doi: 10.21037/tcr-20-516.

TM9SF4 is a newly described V-ATPase interacting protein involved in the malignant progression of cancer cells. In this study, we detected by immunohistochemistry (IHC) in tumor and surrounding healthy tissues TM9SF4, in comparison with clinically adopted biomarkers CEA and CA 19-9 to evaluate TM9SF4 potential as a novel tissue marker for early detection and monitoring of GC and CRC cancers. TM9SF4 as highly specific cancer biomarker, exploitable for disease detection and staging of gastrointestinal cancers patients.

Cancer-ID: Toward Identification of Cancer by Tumor-Derived Extracellular Vesicles in Blood

L G Rikkert , P Beekman , J Caro , F A W Coumans , A Enciso-Martinez , G Jenster , S Le Gac , W Lee , T G van Leeuwen , G B Loozen , A Nanou , R Nieuwland , H L Offerhaus , C Otto , D M Pegtel , M C Piontek , E van der Pol , L de Rond , W H Roos , R B M Schasfoort 1, M H M Wauben , H Zuilhof , L W M M Terstappen 
Front Oncol. 2020 Jun 4;10:608.  doi: 10.3389/fonc.2020.00608. eCollection 2020.

Extracellular vesicles (EVs) have great potential as biomarkers since their composition and concentration in biofluids are disease state dependent and their cargo can contain disease-related information In the blood of cancer patients, a subpopulation of EVs originate from tumor cells, but these EVs are outnumbered by non-EV particles and EVs from other origin. In the Dutch NWO Perspectief Cancer-ID program, we developed and evaluated detection and characterization techniques to distinguish EVs from non-EV particles and other EVs. Despite low signal amplitudes, we identified characteristics of these small tdEVs that may enable the enumeration of small tdEVs and extract relevant information.

EV Separation: Release of Intact Extracellular Vesicles Immunocaptured on Magnetic Particles

Dario Brambilla , Laura Sola , Anna Maria Ferretti , Elisa Chiodi , Natasa Zarovni , Diogo Fortunato , Mattia Criscuoli , Vincenza Dolo , Ilaria Giusti , Valentina Murdica , Katarzyna KluszczyƄska , Liliana Czernek , Markus Düchler , Riccardo Vago , Marcella Chiari 
Anal Chem. 2021 Apr 6;93(13):5476-5483. doi: 10.1021/acs.analchem.0c05194. Epub 2021 Mar 26.

Methods that exploit immunoaffinity capture provide high-purity samples and overcome the issues of currently used separation methods. However, the release of captured nanovesicles usually requires harsh conditions that hinder their use in certain types of downstream analysis. A novel capture and release approach for small extracellular vesicles (sEVs) is presented based on DNA-directed immobilization of antiCD63 antibody. The flexible DNA linker increases the capture efficiency and allows for releasing EVs by exploiting the endonuclease activity of DNAse I. This separation protocol works under mild conditions, enabling the release of vesicles suitable for analysis by imaging techniques.

Standardization of Blood Collection and Processing for the Diagnostic Use of Extracellular Vesicles

Venturella, M., Carpi, F.M. & Zocco, D.
Curr Pathobiol Rep 7, 1–8 (2019). 

A number of preanalytical variables during blood collection and processing greatly impact the levels of blood derived EVs, thus affecting sample quality. So far, lack of standard protocols for blood collection and processing as well as quality control metrics has limited the clinical validation and adoption of EV-based diagnostic assays. In this review, we describe the preanalytical variables that affect sample quality and suitability for EV-based diagnostic approaches and suggest biochemical and molecular quality control (QC) metrics to minimize intra- and interstudy variability and improve data robustness and reproducibility.

When Less Is More: Specific Capture and Analysis of Tumor Exosomes in Plasma Increases the Sensitivity of Liquid Biopsy for Comprehensive Detection of Multiple Androgen Receptor Phenotypes in Advanced Prostate Cancer Patients

Chiara Foroni , Natasa Zarovni , Laura Bianciardi , Simona Bernardi , Luca Triggiani , Davide Zocco , Marta Venturella , Antonio Chiesi , Francesca Valcamonico , Alfredo Berruti
Biomedicines. 2020 May 22;8(5):131. doi: 10.3390/biomedicines8050131.

We evaluated the advantages and the reliability of novel protocols for the enrichment of tumor extracellular vesicles (EVs), enabling a blood-based test for the noninvasive parallel profiling of multiple androgen receptor (AR) gene alterations. Three clinically relevant AR variants related to response/resistance to standard-of-care treatments (AR-V7 transcript, AR T878A point mutation and AR gene amplification) were evaluated by digital PCR in 15 samples from patients affected by Castration-Resistant Prostate Cancer (CRPC). Plasma was processed to obtain circulating RNA and DNA using protocols based on tumor EVs enrichment through immuno-affinity and peptide-affinity compared to generic extraction kits. Our results showed that immuno-affinity enrichment prior to RNA extraction clearly outperforms the generic isolation method in the detection of AR-V7, also allowing for a distinction between responder (R) and non-responder (NR) patient.

Isolation of extracellular vesicles improves the detection of mutant DNA from plasma of metastatic melanoma patients

Davide Zocco , Simona Bernardi , Mauro Novelli , Chiara Astrua , Paolo Fava , Natasa Zarovni  Francesco M Carpi , Laura Bianciardi , Ottavia Malavenda , Pietro Quaglino , Chiara Foroni , Domenico Russo , Antonio Chiesi , Maria Teresa Fierro
Sci Rep. 2020 Sep 25;10(1):15745. doi: 10.1038/s41598-020-72834-6.

The value of extracellular vesicle-(EV)-associated-DNA (EV-DNA) as an alternative source of circulating BRAFV600E  is showcased in this proof-of-concept study. To do so, a clinical practice-compatible protocol was proposed for the isolation of EV-DNA and assessed BRAF gene status on plasma samples from metastatic melanoma patients at the beginning and during BRAFi therapy. This protocol uses a peptide with high affinity for EVs and it has been found to recover more mutant DNA from plasma than standard ultracentrifugation. It improves the detection of BRAFV600E gene copies in comparison to the reference protocol for ctDNA isolation.

Standardization and Commercialization of Extracellular Vesicles

N. Zarovni, F. Loria, R. Zenatelli, D. Mladenovic, L. Paolini, G. Adamo, A. Redighieri, A. Bongiovanni and P. Bergese
Chapter 12, Extracellular Vesicles Science Book, RCSI

The first commercial grade EV-based Diagnostic test, Prostate (IntelliScore) (EPI), was released in 2018, and has obtained a Breakthrough Device Designation from the Food and Drug Administration (FDA) in 2019, as a first tribute to the benefit from an EV-based approach to provide rapid, patient-specific information to guide diagnosis and treatment decisions. This precedent in oncology flagged up the enthusiasm in other top diagnostic niches such as cardiology, transplantation and neurology while paradoxically some of the most-likely- to- benefit- from areas such as infective diseases and prenatal diagnostics are still on hold. The same high-resolution and sensitivity technologies, whose development was fundamental for the realization of diagnostic and prognostic promise of EV biomarkers, proved to be key enablers of the characterization of therapeutic EV formulations and the bioprocess development. This offers huge requalification opportunities pursued today by EV pre-analytics and analytics solution providers.

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