Identification of extracellular vesicle interactors at the blood-brain barrier by proximity-dependent biotinylation

Adrián Klepe (Vortragende:r), Ana Spilak, Sophie Theresa Kriwanek, Andreas Brachner, Christa Nöhammer, Winfried Neuhaus

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Introduction: Small extracellular vesicles (sEVs) are released by cells and present in body fluids. Having
reached the blood-brain barrier (BBB), sEVs can trigger interactions with brain capillary endothelial cells
(BECs) [1]. At the BBB, molecular components involved in sEV internalisation and transport are
as-yet-unidentified. We aim to investigate these molecular interactors.
Material & Methods: Systemic cancer and brain cell sEVs were labelled with CellTracker™Orange and their
effects on barrier tightness and uptake were assessed. Selected cell lines were transfected with plasmid
containing GPI-anchored GFP-biotin ligase (BioID-GPI and TurboID-GPI). Western blotting detected
biotinylation in sEVs. Biotinylated sEVs were applied to immortalised BECs – hCMEC/D3. Lysates were
harvested and biotinylated proteins were captured by magnetic streptavidin beads. Interactome will be
revealed by proteomics - mass spectrometry and bioinformatic analysis.
Results: The transwell setup was optimised for sEV/BEC studies using 1.0 µm pore size inserts and 1% BSA in
the receiver compartment. Interactions of sEVs from seven different cell lines showed different effects on the
BBB model dependent on sEV cell origin and donor compartment. Western blots detected biotinylated proteins
along with recombinant protein in transfected cells and sEVs. From sEV-BBB cell lysates, proteomics will
reveal interacting cellular surface candidates whose role will be validated by pathway-blockers.
Discussion: We established and characterised stable cell lines releasing sEVs expressing BioID-GPI and
TurboID-GPI. Tracking sEV pathways in BECs will enable us to determine sEV interactors and elucidate their
fate inside cells, which could pave the way for cancer treatment or drug delivery application into the brain.
This work was founded by the Austrian Science Fund FWF (project P 34137-B).
TitelSmall New Wordl 2.0 - Abstract Book
PublikationsstatusVeröffentlicht - 4 Sept. 2023
VeranstaltungSmall New World 2.0 - Graz, Österreich
Dauer: 4 Sept. 20235 Sept. 2023


KonferenzSmall New World 2.0

Research Field

  • Molecular Diagnostics


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