Imaging flow cytometry facilitates multiparametric characterization of extracellular vesicles in malignant brain tumours

Ricklefs, Franz; Maire, Cécile L.; Reimer, Rudolph; Dührsen, Lasse; Kolbe, Katharina; Holz, Mareike; Schneider, Enja; Rissiek, Anne; Babayan, Anna; Hille, Claudia; Pantel, Klaus; Krasemann, Susanne; Glatzel, Markus; Heiland, Dieter Henrik; Flitsch, Jörg; Martens, Tobias; Schmidt, Nils Ole; Peine, Sven; Breakefield, Xandra O.; Lawler, Sean E.; Chiocca, E. Antonio; Fehse, Boris; Giebel, Bernd; Görgens, André; Westphal, Manfred; Lamszus, Katrin

doi:10.1080/20013078.2019.1588555

Cited by 106 publications

(127 citation statements)

References 54 publications

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“…By using these eGFP‐labelled EVs as biological reference material, we further show that IFCM can be used to analyse sEVs in EV‐containing samples without any preparation, even with cells being present. Single sEVs can be labelled with fluorophore‐conjugated antibodies, which surely is of high interest for studies aiming at biomarker discovery, as we have further elaborated in our approach to specifically detect glioblastoma derived EVs [78]. Here, at the example of MSC‐derived CM we demonstrate that the ISX facilitates robust multicolour analyses of sEVs without the prior need for EV isolation or enrichment.…”

Section: Discussionmentioning

confidence: 95%

Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence‐tagged vesicles as biological reference material

Görgens

Bremer

Ferrer-Tur

et al. 2019

J of Extracellular Vesicle

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307

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Section: Discussionmentioning

confidence: 95%

Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence‐tagged vesicles as biological reference material

Görgens

Bremer

Ferrer-Tur

et al. 2019

J of Extracellular Vesicle

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260

307

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“…Ricklefs et al [49] previously showed that circulating EV concentrations in GBM patients are elevated. Tumor-specific EVs were detected in a syngeneic mouse tumor model [49].…”

Section: Evs In Glioblastomamentioning

confidence: 97%

“…Ricklefs et al [49] previously showed that circulating EV concentrations in GBM patients are elevated. Tumor-specific EVs were detected in a syngeneic mouse tumor model [49]. Osti et al [35] ( Table 1) analyzed plasma EVs (microvesicles) in preoperative GBM samples.…”

Section: Evs In Glioblastomamentioning

confidence: 97%

Monitoring Therapy Efficiency in Cancer through Extracellular Vesicles

Stevic

Buescher

2020

Cells

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Extracellular vesicles (EVs) are a heterogeneous group of membrane-enclosed vesicles made of a phospholipid bilayer and are secreted by all cell types. EVs are present in a variety of body fluids containing proteins, DNA, RNA species, and lipids, and play an important role in cell-to-cell communication and are worth being considered as biomarkers for both early diagnosis of cancer patients and real-time monitoring of treatment response. Recently, emerging evidence verified EVs to have crucial roles in cancer progression and metastasis and a great potential in therapeutic applications. In this review, we discuss the potential of EVs in monitoring the efficacy of cancer therapies.

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“…Other potential limitations may be that the fluorescence intensity is dependent on protein expression level, the efficiency of EV membrane labeling, and excitation strength of the light source (42). However, IFC is sensitive, detecting low fluorescence intensity with accurate visualization of CD63-GFP particles at the 100 nm range (27,31).…”

Section: Ev In Vitro Uptake Standardization Processmentioning

confidence: 99%

“…Previously, researchers have used standard flow cytometry along with various forms of low throughput microscopy including confocal microscopy to analyze EV uptake (25)(26)(27). However, these technologies have several limitations.…”

mentioning

confidence: 99%

Kinetics and Specificity of HEK293T Extracellular Vesicle Uptake using Imaging Flow Cytometry

Jurgielewicz¹,

Yao²,

Stice³

2020

Preprint

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Background : Extracellular vesicles (EVs) are nanosized vesicles naturally secreted from cells responsible for intercellular communication and delivery of proteins, lipids, and other genetic material. Ultimately, EVs could provide innate therapeutic contents and loaded therapeutic payloads such as small molecules and gene therapy vectors to recipient cells. However, comparative kinetic measures that can be used to quantify and ultimately optimize delivery and uptake of EV payloads are lacking. We investigated both dose and time effects on EV uptake and evaluated the potential specificity of EV uptake to better understand the kinetics and uptake of human embryonic kidney (HEK293T) derived EVs. Results : Utilizing an imaging flow cytometry platform (IFC), HEK293T EV uptake was analyzed. HEK293T EV uptake was dose and time dependent with a minimum threshold dose of 6,000 EVs per cell at 4 hours of co-culture. HEK293T EV uptake was inhibited when co-cultured with recipient cells at 4°C or with pre-fixed recipient cells. By co-culturing HEK293T EVs with cell lines from various germ layers, HEK293T EVs were taken up at higher quantities by HEK293T cells. Lastly, human neural stem cells (hNSCs) internalized significantly more HEK293T EVs relative to mature neurons. Conclusions : Imaging flow cytometry is a quantitative, high throughput, and versatile platform to quantify the kinetics of EV uptake. Utilizing this platform, dose and time variables have been implicated to affect EV uptake measurements making standardization of in vitro and in vivo assays vital for the translation of EVs into the clinic. In this study, we quantified the selectivity of EV uptake between a variety of cell types in vitro and found that EVs were internalized at higher quantities by cells of the same origin. The characterization of HEK293T EV uptake in vitro, notably specificity, dose response, and kinetic assays should be used to help inform and develop EV based therapeutics.

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Imaging flow cytometry facilitates multiparametric characterization of extracellular vesicles in malignant brain tumours

Abstract: Lamszus (2019) Imaging flow cytometry facilitates multiparametric characterization of extracellular vesicles in malignant brain tumours,

Cited by 106 publications

References 54 publications

Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence‐tagged vesicles as biological reference material

Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence‐tagged vesicles as biological reference material

Monitoring Therapy Efficiency in Cancer through Extracellular Vesicles

Kinetics and Specificity of HEK293T Extracellular Vesicle Uptake using Imaging Flow Cytometry

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