Decoding Extracellular Vesicle-mediated organ crosstalk in vivo

Project Information

CROSSTALK

Grant agreement ID: 101075975

DOI

10.3030/101075975

EC signature date 20 December 2022

Start date 1 July 2023

End date 30 June 2028

Funded under

• European Research Council (ERC)

Total cost

€ 1 500 000,00

EU contribution

€ 1 500 000,00

Coordinated by

UNIVERSITEIT UTRECHT
Netherlands

Periodic Reporting for period 1 - CROSSTALK (Decoding Extracellular Vesicle-mediated organ crosstalk in vivo)

Reporting period: 2023-07-01 to 2025-12-31

Summary of the context and overall objectives of the project

To coordinate activities between different tissues, multicellular organisms critically rely on intercellular communication.
Whereas homeostatic signalling within organs is studied extensively, inter-organ crosstalk is only starting to be
unravelled. In recent years, Extracellular Vesicles (EVs) have emerged as central mediators of this pathway by shuttling
specific bioactive cargo including proteins between cells. These nano-sized lipid vesicles are released by virtually every
cell type, are transported via the blood and combine the intricacy of cell-cell contact with the long-acting range of
cytokines. However, due to their small size and a lack of suitable approaches, to what extent endogenous EVs mediate
inter-organ crosstalk and support custom cargo delivery is unclear.
I recently developed a unique transparent zebrafish embryo model expressing optical EV-reporters to examine
endogenous EV biology at unprecedented detail in vivo.
The aim of this proposal is to understand endogenous EV-mediated organ crosstalk by studying EVs secreted by the
liver, the central metabolic hub in vertebrates. To this end, I will decode hepatic EV communication routes in vivo in
health and liver pathology by exploiting my model system combined with novel tools and high-resolution microscopy.
I will pursue three key-objectives: 1) Targets: mapping hepatic EV target organs; 2) Contents: analysing custom cargo
delivery by hepatic EVs to specific target organs. 3) Function: determining the function of endogenous hepatic EVs by
developing and applying molecular tools to interfere with their release in vivo.
This project will provide the first advanced mapping of endogenous EV inter-organ crosstalk in a living model organism.
By studying how pathology impacts EV mediated organ crosstalk and gaining precise control over EV release in vivo
I will obtain unique insights into EV pathophysiology. In turn, this will open avenues for optimized diagnostics and
therapeutic intervention.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The main objectives of CROSSTALK are to understand inter-organ communication by small extracellular vesicles. We are using the liver as main EV-secreting organ, due to its main metabolic role in body homeostasis. For this, we will map target organs of hepatic EVs, to analyse hepatic EV cargo, and to decipher their function. The technological achievements needed to reach this point are
Task 1) the generation of zebrafish lines expressing EV-reporters in the liver.
Task 2) the development of proximity-biotinylation tools to specifically label EV cargo from donor organs, the liver in particular.
Task 3) the development of tools to inhibit EV release in a tissue specific manner, the liver in particular
Regarding Task 1, we did the following: we performed an extensive analysis of the zebrahub expression data (https://zebrahub.sf.czbiohub.org) for EV marker expression in the liver of zebrafish larvae to select the highest expressing marker for our tagging strategies. We selected the top 4, and cloned the zebrafish genes fused to mStayGold and UltraID (for Task 2). We first expressed these transiently, to confirm correct expression and sorting into EVs in vivo. Indeed, we observed fluorescent EVs in the circulation of zebrafish coming from the liver. Based on these encouraging results, we started with the generation of stable integrated EV-marker lines, using the conventional Tol2 strategy as well as a newly develop strategy (pIGLET), that are now growing up in the facility. In the mean time, we have performed preliminary target organ analysis where we observe both expected target organs/tissues (CVP) as target tissues that seem specific for a subpopulation of liver EVs.
Regarding Task 2, we did the following: the 4 selected EV markers based on the zebrahub expression dataset were fused to UltraID. To broaden the applicability and in vitro optimization, we did this both for the zebrafish as well as their human ortholog proteins. In addition, we developed ER-anchored UltraID constructs, as well as tested candidates as mentioned in the proposal (including NPC1). These were tested for best EV-labelling properties, and lowest presence in the soluble secretome (based on SEC separation of EVs and the soluble secretome). In collaboration with the Mass Spec department (see also below) we are now assessing the sensitivity of this approach.
Regarding Task 3, we have almost completed the set-up of the tool in vitro, and are now planning its application in vivo. Thanks to a parallel project in the lab in which we have investigated the balance between EV secretion and degradation, we unravelled the underpinning mechanism that naturally prevents EV release from cells. This mechanism uses a machinery that is conserved in yeast, and therefore has the potential to be widely active. For the EV-inhibition tool, we now utilise the insights into this natural pathway to create an EV-inhibition tool that can be used in a tissue specific manner with as little as possible side-effects that are associated with traditional approaches, such as Rab27a interference.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Being able to interfere with EV secretion in a cell-type specific manner with little side-effects can be considered beyond state-of-the-art. This development of an EV-inhibition tool per se was not unplanned, but the mechanism we are settling on to achieve this is fundamentally different and based on new insights obtained during the course of this project.
Likewise, the development of the tool to track newly biosynthesized proteins in an unperturbed manner was not anticipated but serendipitously stumbled upon. Current tools that study biosynthetic protein trafficking either utilize a temperature switch, a general block of protein exit, or a specific block of protein exit from the ER, that are all to a smaller or larger extent associated with unwanted side-effects.