Imaging flow cytometry elucidates the cellular uptake of extracellular vesicles loaded with a novel therapeutic peptide

Flow cytometry remains a commonly used methodology due to its ability to characterise multiple parameters on single particles in a high-throughput manner. In order to address limitations with lacking sensitivity of conventional flow cytometry to characterise extracellular vesicles (EVs), novel, highly sensitive platforms, such as high-resolution and imaging flow cytometers, have been developed. We provided comparative benchmarks of a conventional FACS Aria III, a high-resolution Apogee A60 Micro-PLUS and the ImageStream X Mk II imaging flow cytometry platform. Nanospheres were used to systematically characterise the abilities of each platform to detect and quantify populations with different sizes, refractive indices and fluorescence properties, and the repeatability in concentration determinations was reported for each population. We evaluated the ability of the three platforms to detect different EV phenotypes in blood plasma and the intra-day, inter-day and global variabilities in determining EV concentrations. By applying this or similar methodology to characterise methods, researchers would be able to make informed decisions on choice of platforms and thereby be able to match suitable flow cytometry platforms with projects based on the needs of each individual project. This would greatly contribute to improving the robustness and reproducibility of EV studies.

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Using Imaging Flow Cytometry to Characterize Extracellular Vesicles Isolated from Cell Culture Media, Plasma or Urine

BIO-PROTOCOL ◽

10.21769/bioprotoc.3420 ◽

2019 ◽

Vol 9 (21) ◽

Author(s):

John Woollard ◽

Amrutesh Puranik ◽

Kyra Jordan ◽

Lilach Lerman

Keyword(s):

Cell Culture ◽

Flow Cytometry ◽

Extracellular Vesicles ◽

Culture Media ◽

Cell Culture Media ◽

Imaging Flow Cytometry

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Webinar Analysis of extracellular vesicles including exosomes by imaging flow cytometry

Science ◽

10.1126/science.352.6292.1479-b ◽

2016 ◽

Vol 352 (6292) ◽

pp. 1479-1479 ◽

Cited By ~ 2

Author(s):

A. Gorgens

Keyword(s):

Flow Cytometry ◽

Extracellular Vesicles ◽

Imaging Flow Cytometry

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

Journal of Extracellular Vesicles ◽

10.1080/20013078.2019.1588555 ◽

2019 ◽

Vol 8 (1) ◽

pp. 1588555 ◽

Cited By ~ 23

Author(s):

Franz L. Ricklefs ◽

Cecile L. Maire ◽

Rudolph Reimer ◽

Lasse Dührsen ◽

Katharina Kolbe ◽

...

Keyword(s):

Flow Cytometry ◽

Extracellular Vesicles ◽

Brain Tumours ◽

Imaging Flow Cytometry ◽

Malignant Brain Tumours ◽

Multiparametric Characterization

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CBIO-26. SUBPOPULATIONS OF CIRCULATING EXTRACELLULAR VESICLES OF GLIOBLASTOMA PATIENTS CAN BE DISTINGUISHED BY IMAGING FLOW CYTOMETRY

Neuro-Oncology ◽

10.1093/neuonc/nox168.144 ◽

2017 ◽

Vol 19 (suppl_6) ◽

pp. vi37-vi38

Author(s):

Franz Ricklefs ◽

Cecile Maire ◽

Katharina Kolbe ◽

Mareike Holz ◽

Rudolph Reimer ◽

...

Keyword(s):

Flow Cytometry ◽

Extracellular Vesicles ◽

Imaging Flow Cytometry

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BIOM-25. IDENTIFYING EXTRACELLULAR VESICLES FROM GLIOBLASTOMA OR NON-NEOPLASTIC CELLS VIA IMAGING FLOW CYTOMETRY

Neuro-Oncology ◽

10.1093/neuonc/noaa215.025 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii7-ii7

Author(s):

Luz Milbeth Cumba Garcia ◽

Abudumijiti Aibaidula ◽

Nazanin Yeganeh Kazemi ◽

Miyeon Jung ◽

Fabrice Lucien-Matteoni ◽

...

Keyword(s):

Flow Cytometry ◽

Extracellular Vesicles ◽

Protoporphyrin Ix ◽

Molecular Profile ◽

Cell Of Origin ◽

Double Positive ◽

Neoplastic Cells ◽

Monocytic Cell ◽

Imaging Flow Cytometry

Abstract Patients with glioblastoma (GBM) have a median survival of 15 months despite aggressive treatment. Immunosuppressive monocytes are heavily infiltrated in these tumors and in patients’ circulation. Treatment-related pseudo-progression confounds outcome assessment by MRI. Thus, there is a need for additional non-invasive methods to assess treatment response. Extracellular vesicles (EVs) contain tumor-specific microRNA (miRNA) cargo that could serve as a liquid biopsy to distinguish true progression from treatment-related pseudo-progression. We had found significant differences in plasma EVs molecular profile (i.e. miRNA signatures) between GBM patients and healthy donors. Our overall hypothesis is that these differences reflect the EVs cell of origin. Our goal in this project was to develop a fluorescent staining paradigm by flow cytometry to distinguish EVs from different cells in vitro and determine differences in EV miRNA expression profile between GBM and monocytic cell-derived EVs. Gleolan (5-ALA) is an FDA-approved orally available agent for fluorescence-guided resection of GBM tumors. It is metabolized to protoporphyrin IX (PpIX) in GBM cells but not in non-neoplastic cells and has been reported to aid in the detection of GBM-derived EVs by flow cytometry. However, distinguishing between GBM-derived EVs and EVs from other cells of origin has not been described. We co-cultured human GBM cells (dBT114 or dBT116) and CD14+ monocytes for 72 hours in the presence or absence of 5-ALA. EVs were isolated by ultracentrifugation and stained for CD11b (myeloid cell marker). ImageStream Imaging Flow Cytometry was performed showing clear differentiation between PpIX+ EVs from GBM cells and CD11b+ EVs from monocytes. Interestingly, a small number of double-positive EVs (presumably representing monocyte-derived EVs that had taken up PpIX after phagocytizing GBM cells) were also present. Taken together, we were able to optimize a technique to distinguish EVs originating from GBM and monocytes for further characterization by short non-coding RNA sequencing.

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Large Extracellular Vesicles Can be Characterised by Multiplex Labelling Using Imaging Flow Cytometry

International Journal of Molecular Sciences ◽

10.3390/ijms21228723 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8723

Author(s):

Suzanne M Johnson ◽

Antonia Banyard ◽

Christopher Smith ◽

Aleksandr Mironov ◽

Martin G. McCabe

Keyword(s):

Flow Cytometry ◽

Extracellular Vesicles ◽

Annexin V ◽

Clinical Samples ◽

Fluorescent Light ◽

Imaging Flow Cytometry ◽

Medulloblastoma Cell Line ◽

Isolation Techniques ◽

Marker Expression ◽

Reproducible Analysis

Extracellular vesicles (EVs) are heterogeneous in size (30 nm–10 µm), content (lipid, RNA, DNA, protein), and potential function(s). Many isolation techniques routinely discard the large EVs at the early stages of small EV or exosome isolation protocols. We describe here a standardised method to isolate large EVs from medulloblastoma cells and examine EV marker expression and diameter using imaging flow cytometry. Our approach permits the characterisation of each large EVs as an individual event, decorated with multiple fluorescently conjugated markers with the added advantage of visualising each event to ensure robust gating strategies are applied. Methods: We describe step-wise isolation and characterisation of a subset of large EVs from the medulloblastoma cell line UW228-2 assessed by fluorescent light microscopy, transmission electron microscopy (TEM) and tunable resistance pulse sensing (TRPS). Viability of parent cells was assessed by Annexin V exposure by flow cytometry. Imaging flow cytometry (Imagestream Mark II) identified EVs by direct fluorescent membrane labelling with Cell Mask Orange (CMO) in conjunction with EV markers. A stringent gating algorithm based on side scatter and fluorescence intensity was applied and expression of EV markers CD63, CD9 and LAMP 1 assessed. Results: UW228-2 cells prolifically release EVs of up to 6 µm. We show that the Imagestream Mark II imaging flow cytometer allows robust and reproducible analysis of large EVs, including assessment of diameter. We also demonstrate a correlation between increasing EV size and co-expression of markers screened. Conclusions: We have developed a labelling and stringent gating strategy which is able to explore EV marker expression (CD63, CD9, and LAMP1) on individual EVs within a widely heterogeneous population. Taken together, data presented here strongly support the value of exploring large EVs in clinical samples for potential biomarkers, useful in diagnostic screening and disease monitoring.

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