Abstract
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).
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).
| Original language | English |
|---|---|
| Title of host publication | Small New Wordl 2.0 - Abstract Book |
| Pages | 42-42 |
| Number of pages | 1 |
| Publication status | Published - 4 Sept 2023 |
| Event | Small New World 2.0 - Graz, Austria Duration: 4 Sept 2023 → 5 Sept 2023 https://asev.at/documents/SmallNewWorld2.0%20-%202023%20-%20Abstract%20book.pdf |
Conference
| Conference | Small New World 2.0 |
|---|---|
| Country/Territory | Austria |
| City | Graz |
| Period | 4/09/23 → 5/09/23 |
| Internet address |
Research Field
- Molecular Diagnostics