Improving Reproducibility to Meet Minimal Information for Studies of Extracellular Vesicles 2018 Guidelines in Nanoparticle Tracking Analysis

Abstract Background ErbB2/HER2 oncoprotein often drives breast cancers (BCs) which are treated with the anti-ErbB2 antibody trastuzumab. The efficacy of trastuzumab-based metastatic BC therapies is routinely assessed by imaging studies. Trastuzumab typically becomes ineffective in the case of this disease and is then replaced by other drugs. Biomarkers of BC trastuzumab response could allow imaging studies and the switch to other drugs to occur earlier than is now possible. Moreover, bone-only BC metastases can be hard to measure, and biomarkers of their trastuzumab response could facilitate further treatment decisions. Such biomarkers are presently unavailable. In this study, we searched for proteins whose levels in BC cell-emitted extracellular vesicles (EVs) potentially correlate with BC trastuzumab sensitivity. Methods We isolated EVs from cultured trastuzumab-sensitive and trastuzumab-resistant human BC cells before and after trastuzumab treatment and characterized these EVs by nanoparticle tracking analysis and electron microscopy. We found previously that ErbB2 drives BC by downregulating a pro-apoptotic protein PERP. We now tested whether trastuzumab-induced PERP upregulation in EVs emitted by cultured human BC cells correlates with their trastuzumab sensitivity. We also used mass spectrometry to search for additional proteins whose levels in such EVs reflect BC cell trastuzumab sensitivity. Once we identified proteins whose EV levels correlate with this sensitivity in culture, we explored the feasibility of testing whether their levels in the blood EVs of trastuzumab-treated metastatic BC patients correlate with patients’ response to trastuzumab-based treatments. Results We found that neither trastuzumab nor acquisition of trastuzumab resistance by BC cells affects the size or morphology of EVs emitted by cultured BC cells. We established that EV levels of proteins PERP, GNAS2, GNA13, ITB1, and RAB10 correlate with BC cell trastuzumab response. Moreover, these proteins were upregulated during trastuzumab-based therapies in the blood EVs of a pilot cohort of metastatic BC patients that benefited from these therapies but not in those derived from patients that failed such treatments. Conclusions Upregulation of a protein set in EVs derived from cultured breast tumor cells correlates with tumor cell trastuzumab sensitivity. It is feasible to further evaluate these proteins as biomarkers of metastatic BC trastuzumab response.

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Evaluation of plasmatic extracellular vesicles by size

10.21203/rs.3.pex-1287/v1 ◽

2021 ◽

Author(s):

Laura Cantone ◽

Mirjam Hoxha ◽

Chiara Favero ◽

Luca Ferrari ◽

Valentina Bollati

Keyword(s):

Plasma Membrane ◽

Blood Plasma ◽

Extracellular Vesicles ◽

High Precision ◽

Extracellular Vesicle ◽

Nanoparticle Tracking Analysis ◽

Research Needs ◽

Plasma Samples ◽

Constant Flow ◽

Syringe Pump

Abstract Extracellular vesicles (EVs) play a key role in many physiological and pathological processes [1]. EVs are a heterogeneous group of membrane-confined particles including endosome-derived exosomes and plasma membrane-originated microvesicles. The expanding field of extracellular vesicle research needs reproducible and accurate methods to characterize EVs [2]. EV profiling can be challenging due to the small size and heterogeneity. This protocol aims to provide a method to isolate EVs and facilitate high-precision particle quantitation by Nanoparticle Tracking Analysis (NTA)[3, 4]. NTA is commonly used to determine EV concentration and diameter [5, 6]. The protocol here described refers to the isolation of EVs from blood-plasma samples by using ultracentrifugation and then quantification and sizing of EVs with NTA by NanoSight NS300 system (Malvern Panalytical Ltd., Malvern, UK) provided with a syringe pump module enabling analysis in constant flow for improved sample statistics.

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Urinary Extracellular Vesicles for Renal Tubular Transporters Expression in Patients With Gitelman Syndrome

Frontiers in Medicine ◽

10.3389/fmed.2021.679171 ◽

2021 ◽

Vol 8 ◽

Author(s):

Chih-Chien Sung ◽

Min-Hsiu Chen ◽

Yi-Chang Lin ◽

Yu-Chun Lin ◽

Yi-Jia Lin ◽

...

Keyword(s):

Extracellular Vesicles ◽

Membrane Transporters ◽

Gitelman Syndrome ◽

Healthy Controls ◽

Nanoparticle Tracking Analysis ◽

Non Invasive ◽

Transmission Electron ◽

Renal Tubular ◽

Inactivating Mutations

Background: The utility of urinary extracellular vesicles (uEVs) to faithfully represent the changes of renal tubular protein expression remains unclear. We aimed to evaluate renal tubular sodium (Na+) or potassium (K+) associated transporters expression from uEVs and kidney tissues in patients with Gitelman syndrome (GS) caused by inactivating mutations in SLC12A3.Methods: uEVs were isolated by ultracentrifugation from 10 genetically-confirmed GS patients. Membrane transporters including Na+-hydrogen exchanger 3 (NHE3), Na+/K+/2Cl− cotransporter (NKCC2), NaCl cotransporter (NCC), phosphorylated NCC (p-NCC), epithelial Na+ channel β (ENaCβ), pendrin, renal outer medullary K1 channel (ROMK), and large-conductance, voltage-activated and Ca2+-sensitive K+ channel (Maxi-K) were examined by immunoblotting of uEVs and immunofluorescence of biopsied kidney tissues. Healthy and disease (bulimic patients) controls were also enrolled.Results: Characterization of uEVs was confirmed by nanoparticle tracking analysis, transmission electron microscopy, and immunoblotting. Compared with healthy controls, uEVs from GS patients showed NCC and p-NCC abundance were markedly attenuated but NHE3, ENaCβ, and pendrin abundance significantly increased. ROMK and Maxi-K abundance were also significantly accentuated. Immunofluorescence of the representative kidney tissues from GS patients also demonstrated the similar findings to uEVs. uEVs from bulimic patients showed an increased abundance of NCC and p-NCC as well as NHE3, NKCC2, ENaCβ, pendrin, ROMK and Maxi-K, akin to that in immunofluorescence of their kidney tissues.Conclusion: uEVs could be a non-invasive tool to diagnose and evaluate renal tubular transporter adaptation in patients with GS and may be applied to other renal tubular diseases.

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Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

Journal of Visualized Experiments ◽

10.3791/62447 ◽

2021 ◽

Author(s):

Nicole Comfort ◽

Kunheng Cai ◽

Tessa R. Bloomquist ◽

Madeleine D. Strait ◽

Anthony W. Ferrante ◽

...

Keyword(s):

Extracellular Vesicles ◽

Nanoparticle Tracking Analysis ◽

Size Determination

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Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

Journal of Extracellular Vesicles ◽

10.1080/20013078.2018.1535750 ◽

2018 ◽

Vol 7 (1) ◽

pp. 1535750 ◽

Cited By ~ 1464

Author(s):

Clotilde Théry ◽

Kenneth W Witwer ◽

Elena Aikawa ◽

Maria Jose Alcaraz ◽

Johnathon D Anderson ◽

...

Keyword(s):

Extracellular Vesicles ◽

International Society ◽

Position Statement ◽

Minimal Information

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Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation

Scientific Reports ◽

10.1038/s41598-020-75817-9 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Zivile Useckaite ◽

Anindya Mukhopadhya ◽

Barry Moran ◽

Lorraine O’Driscoll

Keyword(s):

Flow Cytometry ◽

Conditioned Medium ◽

Density Gradient ◽

Cell Lines ◽

Extracellular Vesicles ◽

Solid Tumour ◽

Reliable Data ◽

Nanoparticle Tracking Analysis ◽

Cells Of Origin ◽

Extensive Range

Abstract MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary.

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