Elucidating the mechanisms and pathways of extracellular vesicle uptake and intercellular stress response.
Lead Research Organisation:
Oxford Brookes University
Department Name: Faculty of Health and Life Sciences
Abstract
Billions of years ago life consisted solely of single-celled organisms; these types of creatures tend to compete with one another, and their primary goal is to grow and reproduce. When multi-cellular organisms evolved they had to solve a problem: how to stop individual cells in the organism from fighting and competing with each other, and to actually work together for the benefit of the organism.
One way that organisms solved this problem was by getting cells to communicate with one another in different ways. This communication between cells is crucially important, as it allows them to coordinate important decisions, such as when to grow and when to die. Understanding this communication is therefore a requisite for understanding how multicellular life is regulated.
One of the ways in which cells communicate is via the release of extracellular vesicles (EVs). EVs are essentially tiny 'bags' that are released by cells which carry various cellular components such as proteins and RNA molecules (these are a copied version of DNA which act as an intermediary in the formation of proteins). We have known about the existence of EVs for decades, but it was thought that they were essentially a waste disposal system that cells could use to jettison unwanted material. However, it has emerged that EVs are actually very important. After they are released they can be taken up by other cells, where they can induce a response. In other words, they are part of the communication process that cells use to coordinate their function. Scientists across the world took note and began testing to see if EVs were involved in their favourite topics of research. In our lab we have found that when cells get stressed they are able to send EVs to their neighbours. These 'bystander cells' appear to become damaged, but they also are now more protected against stress. In other words, when cells get stressed they can warn their neighbours to 'toughen up' and prepare some danger heading their way. This seems to play an important role in helping organisms to survive stressful conditions, yet little is known about how EVs control this process.
In this proposal we will aim to better understand this EV mediated communication following stress by pursuing three core objectives:
1. To study the molecular mechanisms of this intercellular stress response
Our preliminary work has revealed some of the molecular steps involved in changing the neighbouring cells to allow this adaptation to stressful conditions. Here we will perform further work to better understand how these steps fit together in controlling the overall effect in the neighbouring cells.
2. To discover genes involved in EV processing.
Nobody has ever comprehensively tested how EVs are able to stick to their target cells, enter those cells and then avoid destruction once inside the cell. Here we will attempt to tackle this difficult but important question. We have designed some experiments which will tell us what proteins are involved in controlling these different steps. The design of the experiments will also allow us to find out what genes are involved in controlling the stress response induced by EVs.
Objective 3 - To study genes identified in objective 2 in more detail.
This will allow us to better understand the mechanisms by which stressed cells are able to communicate with one another. Indeed, we will be able to characterise the whole process from the arrival of the EVs at the bystander cell, the uptake and processing of the EV and the subsequent induction of a response that helps that cell to prepare for future danger. The findings will also broadly appeal to scientists working in a range of different topics.
One way that organisms solved this problem was by getting cells to communicate with one another in different ways. This communication between cells is crucially important, as it allows them to coordinate important decisions, such as when to grow and when to die. Understanding this communication is therefore a requisite for understanding how multicellular life is regulated.
One of the ways in which cells communicate is via the release of extracellular vesicles (EVs). EVs are essentially tiny 'bags' that are released by cells which carry various cellular components such as proteins and RNA molecules (these are a copied version of DNA which act as an intermediary in the formation of proteins). We have known about the existence of EVs for decades, but it was thought that they were essentially a waste disposal system that cells could use to jettison unwanted material. However, it has emerged that EVs are actually very important. After they are released they can be taken up by other cells, where they can induce a response. In other words, they are part of the communication process that cells use to coordinate their function. Scientists across the world took note and began testing to see if EVs were involved in their favourite topics of research. In our lab we have found that when cells get stressed they are able to send EVs to their neighbours. These 'bystander cells' appear to become damaged, but they also are now more protected against stress. In other words, when cells get stressed they can warn their neighbours to 'toughen up' and prepare some danger heading their way. This seems to play an important role in helping organisms to survive stressful conditions, yet little is known about how EVs control this process.
In this proposal we will aim to better understand this EV mediated communication following stress by pursuing three core objectives:
1. To study the molecular mechanisms of this intercellular stress response
Our preliminary work has revealed some of the molecular steps involved in changing the neighbouring cells to allow this adaptation to stressful conditions. Here we will perform further work to better understand how these steps fit together in controlling the overall effect in the neighbouring cells.
2. To discover genes involved in EV processing.
Nobody has ever comprehensively tested how EVs are able to stick to their target cells, enter those cells and then avoid destruction once inside the cell. Here we will attempt to tackle this difficult but important question. We have designed some experiments which will tell us what proteins are involved in controlling these different steps. The design of the experiments will also allow us to find out what genes are involved in controlling the stress response induced by EVs.
Objective 3 - To study genes identified in objective 2 in more detail.
This will allow us to better understand the mechanisms by which stressed cells are able to communicate with one another. Indeed, we will be able to characterise the whole process from the arrival of the EVs at the bystander cell, the uptake and processing of the EV and the subsequent induction of a response that helps that cell to prepare for future danger. The findings will also broadly appeal to scientists working in a range of different topics.
Technical Summary
Extracellular vesicles (EVs) are a type of vesicle that is released by cells into the extracellular space. There has been a recent renaissance in the study of EVs which has coincided with the realisation that they are not simply routes of extracellular waste disposal as had previously been believed. Indeed, it is now widely acknowledged that EVs can play an important role in a variety of processes, including angiogenesis, cell motility and immune regulation. Despite the increasing panoply of functions that EVs are involved in no study has ever set about trying to comprehensively identify the proteins involved in mediating their uptake and processing into recipient cells. In addition it is unclear how EVs released by stress can help coordinate intercellular responses to non-physiological conditions. We will address this shortcoming by performing a range of experiments, including genome-wide RNAi screens combined with high-throughput microscopy and flow cytometry to identify proteins involved in EV uptake and stress response.
In addition to measuring uptake, our experimental design will allow us to simultaneously identify proteins involved in the release of EVs and their cargo from endosomal compartments following internalisation as well uncovering whether EVs with different functional effects on cells are taken up by different pathways. We will also be able to assess whether EVs released by stressed cells, which we have shown are qualitatively different and are involved in coordinating a homeostatic intercellular stress response, are taken up by different mechanisms to regular EVs. We will also be able to identify and test the role of different genes in regulating this intercellular stress response mediated by EVs.
Thus, the work will uncover a wide range of information which will be of benefit to a wide range of research and could profoundly influence our understanding of EV-mediated communication.
In addition to measuring uptake, our experimental design will allow us to simultaneously identify proteins involved in the release of EVs and their cargo from endosomal compartments following internalisation as well uncovering whether EVs with different functional effects on cells are taken up by different pathways. We will also be able to assess whether EVs released by stressed cells, which we have shown are qualitatively different and are involved in coordinating a homeostatic intercellular stress response, are taken up by different mechanisms to regular EVs. We will also be able to identify and test the role of different genes in regulating this intercellular stress response mediated by EVs.
Thus, the work will uncover a wide range of information which will be of benefit to a wide range of research and could profoundly influence our understanding of EV-mediated communication.
Planned Impact
This project will be the first comprehensive analysis of EV uptake and processing mechanisms in cells. It will also help us to identify genes involved in the mechanisms of intercellular stress response, as well as give insight into how EVs and their cargo can escape the endosome. The work would therefore have a wide impact on the research community. It would lead to collaborations with our group and stimulate a wide variety of further work in the EV, intracellular trafficking and stress response fields. This impact would be further felt by increased competitiveness in these fields within the UK and further funding from research councils and elsewhere. The work could also have impact by improving delivery of biological tools or therapeutic drugs, which could increase the efficiency of research or improve the outlook for patients. Commercialisation of potential applications would also impact on the local and UK economy. Thus there will be a high potential impact to the research community, industry and patients within the UK and beyond.
People |
ORCID iD |
David Carter (Principal Investigator) |
Publications
Couch Y
(2021)
A brief history of nearly EV-erything - The rise and rise of extracellular vesicles.
in Journal of extracellular vesicles
Russell AE
(2019)
Biological membranes in EV biogenesis, stability, uptake, and cargo transfer: an ISEV position paper arising from the ISEV membranes and EVs workshop.
in Journal of extracellular vesicles
Van Niel G
(2022)
Challenges and directions in studying cell-cell communication by extracellular vesicles.
in Nature reviews. Molecular cell biology
Samuel P
(2018)
Cisplatin induces the release of extracellular vesicles from ovarian cancer cells that can induce invasiveness and drug resistance in bystander cells.
in Philosophical transactions of the Royal Society of London. Series B, Biological sciences
Melling GE
(2022)
Confocal microscopy analysis reveals that only a small proportion of extracellular vesicles are successfully labelled with commonly utilised staining methods.
in Scientific reports
Nieuwland R
(2018)
Essentials of extracellular vesicles: posters on basic and clinical aspects of extracellular vesicles
in Journal of Extracellular Vesicles
Bewicke-Copley F
(2017)
Extracellular vesicles released following heat stress induce bystander effect in unstressed populations.
in Journal of extracellular vesicles
Fontana F
(2021)
Extracellular Vesicles: Emerging Modulators of Cancer Drug Resistance.
in Cancers
Théry C
(2018)
Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines
in Journal of Extracellular Vesicles
Akbar N
(2023)
Rapid neutrophil mobilization by VCAM-1+ endothelial cell-derived extracellular vesicles.
in Cardiovascular research
Title | 3D EV animation |
Description | I coordinated the production of a 3D video animation explaining what extracellular vesicles are and what they can do. We made two versions of the video, one for the general audience (https://www.youtube.com/watch?v=sx6M8UAkUSM) and one for a more scientific audience (https://www.youtube.com/watch?v=OQeRfaQkZCk). The videos can be viewed on a 2D screen but also in 3D using either a virtual reality headset (such as a vive or occulus rift) or cheaper headset. We have used this at several outreach and engagement events and have had a lot of positive feedback. |
Type Of Art | Film/Video/Animation |
Year Produced | 2018 |
Impact | The general public and scientific videos have been viewed more than 4,500 times on YouTube alone, and have been featured on blogs and websites (e.g. https://www.exosome-rna.com/extracellular-vesicles-the-cells-secret-messengers/). |
URL | https://www.youtube.com/watch?v=sx6M8UAkUSM |
Description | We have investigated EV uptake technologies and have made a couple of key findings which we are currently writing up for submission to journals: 1. EV transfer systems based on CRISPR/Cas9 need to be based on gain of function rather than loss of function. 2. EV labeling dyes, used by many in the field, do not label EVs effectively. This is a key finding that will have a big impact on the field. |
Exploitation Route | This will change the way many in the field do their EV uptake experiments. It's also going to upset some people as it would suggest many of the experiments published in the field are based on incorrect assumptions and are potentially factually incorrect. |
Sectors | Healthcare Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology |
Description | Advising DEFRA (Veterinary Medicines Directorate) |
Geographic Reach | National |
Policy Influence Type | Participation in a guidance/advisory committee |
Description | MOOC |
Geographic Reach | Multiple continents/international |
Policy Influence Type | Influenced training of practitioners or researchers |
URL | https://www.isev.org/page/MOOC |
Description | MOOC II |
Geographic Reach | Multiple continents/international |
Policy Influence Type | Influenced training of practitioners or researchers |
URL | https://www.coursera.org/learn/extracellular-vesicles-health-disease |
Description | ExoPop: Extracellular Vesicle Profiling for early detection of leukaemic progression |
Amount | £100,000 (GBP) |
Funding ID | C64210/A28052 |
Organisation | Cancer Research UK |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 04/2019 |
End | 05/2019 |
Description | Collaboration with Dr Alberto Baena |
Organisation | University of Oxford |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We have provided our expertise in EVs to establish a new collaboration to investigate EV transfer and stress response using the model organism Drosophila Melanogaster. |
Collaborator Contribution | We provided intellectual input and help to supervise two PhD students working directly on this collaboration. |
Impact | The students are in their second year and have produced excellent preliminary data which are being prepared for a manuscript |
Start Year | 2017 |
Description | Collaboration with Dr Bhavik Patel |
Organisation | University of Brighton |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Dr Patel is an electrochemist with whom we're working on a novel approach to detect and characterise EVs. My group is providing expertise in EVs. |
Collaborator Contribution | Dr Patel is providing expertise in impact electrochemistry. |
Impact | We have won a grant from CRUK to explore the application of impact electrochemistry. The project is at an early stage and is multidisciplinary, bringing together expertise in EVs and electrochemistry. Through the rest of the research team (which includes Dr Nick Turner - a materials scientist, Dr Beth Psalia - a clinical researcher, and Dr Tingting Zhu - an AI and machine learning expert) we have a truly cross-disciplinary approach. |
Start Year | 2017 |
Description | Collaboration with Prof Venu Nair |
Organisation | The Pirbright Institute |
Department | Avian viral Diseases |
Country | United Kingdom |
Sector | Private |
PI Contribution | We have offered our expertise in EV uptake to investigate whether vesicles released by virally-infected chicken cells can transfer miRNAs to other recipient cells. |
Collaborator Contribution | Prof Nair contributes his expertise in virology that relates to the poultry industry. |
Impact | Manuscript in preparation. |
Start Year | 2017 |
Description | DTP studentship - Becker |
Organisation | University of Oxford |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | This is a collaboration with Dr Esther Becker at the Department of Physiology and Genetics at the University of Oxford. An Oxford BBSRC DTP student registered at Oxford Brookes University undertook two rotations for her PhD, one in my lab and one in Dr Becker's lab. As a result she is doing a joint project between myself and Dr Becker to elucidate the role of TRPC3, a Calcium ion channel, in the biogenesis of extracellular vesicles. This project is using expertise gained during the current project to test the role of this channel in regulating EV release. |
Collaborator Contribution | Dr Esther Becker provides expertise in TRPC3, which is a key protein in regulating normal neurobiological processes. |
Impact | Outputs pending. |
Start Year | 2018 |
Description | EV cargo delivery screen - Dr Pieter Vader |
Organisation | Utrecht University |
Country | Netherlands |
Sector | Academic/University |
PI Contribution | We have provided access to siRNA libraries and support with screening bulk EV uptake. |
Collaborator Contribution | Dr Vader's team will use their recently developed Crispr system for measuring the uptake and delivery of EV cargo. |
Impact | In progress |
Start Year | 2020 |
Description | Prof Jeremy Simpson |
Organisation | University College Dublin |
Department | UCD Conway Institute of Biomedical annd Biomolecular Research |
Country | Ireland |
Sector | Academic/University |
PI Contribution | The project is a BBSRC-SFI funded award to both labs. |
Collaborator Contribution | My lab is looking at the delivery of extracellular vesicle (EV) cargo and the function of stress EVs, and Prof Simpson is looking at bulk uptake of EVs into cells. In both cases we are running screens to identify genes involved and the mechanisms underlying these processes. |
Impact | The collaboration is still at an early stage as the project has only been going for 7 months. Thus far we have skyped several times and I have given a seminar at University College Dublin. There aren't any publications at this stage. |
Start Year | 2017 |
Description | EV 3D Video |
Form Of Engagement Activity | A broadcast e.g. TV/radio/film/podcast (other than news/press) |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | I coordinated the production of a 3D video animation explaining what extracellular vesicles are and what they can do. We made two versions of the video, one for the general audience (https://www.youtube.com/watch?v=sx6M8UAkUSM) and one for a more scientific audience (https://www.youtube.com/watch?v=OQeRfaQkZCk). The videos can be viewed on a 2D screen but also in 3D using either a virtual reality headset (such as a vive or occulus rift) or cheaper headset. We have used this at several outreach and engagement events and have had a lot of positive feedback. The general public and scientific videos have been viewed more than 4,500 times on YouTube alone. |
Year(s) Of Engagement Activity | 2018 |
URL | https://www.youtube.com/watch?v=sx6M8UAkUSM |
Description | EV Landscape meeting - Nature editorial group |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other audiences |
Results and Impact | The Spinger Nature editorial team are becoming increasingly aware of extracellular vesicles (EVs) as an important topic area. They invited three international experts to a 'landscape meeting' where we each presented some information about our work and the EV field. Their goal was to better understand the challenges of the EV field. |
Year(s) Of Engagement Activity | 2019 |
Description | Interview for Nature Biotechnology |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | I gave an interview about the therapeutic potential of basic research on extracellular vesicles. My quotes appeared in an article in Nature Biotechnology (impact factor 35.7) published in december 2019. As of 9th march 2020 this article has been accessed more than 2,000 times and has been cited in one scientific article. So far, Altmetric has seen 36 tweets from 31 users, with an upper bound of 162,472 followers. |
Year(s) Of Engagement Activity | 2019 |
URL | https://www.nature.com/articles/s41587-019-0326-5 |
Description | Ni Science Festival |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | I was invited to give a talk about my research at the Northern Ireland Science Festival 2019. It was intended for a general audience and, from speaking to audience members afterwards, it had a positive impact on the knowledge and perceptions of people who were present. |
Year(s) Of Engagement Activity | 2019 |
URL | https://www.nisciencefestival.com/ |
Description | Participation in Oxford Brookes Science Bazaar 2019 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | We were once again involved in running an EV-themed stand at the science bazaar at Oxford Brookes University (which attracted more than 2,000 people). |
Year(s) Of Engagement Activity | 2019 |
Description | Participation in Oxford Brookes Science Bazaar 2020 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | We were once again involved in running an EV-themed stand at the science bazaar at Oxford Brookes University (which attracted more than 2,000 people). |
Year(s) Of Engagement Activity | 2020 |
Description | Science Bazaar and Oxford Science Week |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Other audiences |
Results and Impact | We were involved in two public outreach events, the science bazaar at Oxford Brookes University (which attracted more than 2,000 people) and the Oxford Science Week at the Oxford Town Hall which attracted over 1,000 members of the public. |
Year(s) Of Engagement Activity | 2017 |
URL | https://www.oxscifest.com/ |
Description | UK Society for Extracellular Vesicles Summer School |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | I co-organised the first UK Summer School for EVs. We had approximately 52 attendees, mainly from the UK but also coming from other countries including Spain, Italy, Romania and Singapore. |
Year(s) Of Engagement Activity | 2019 |
URL | https://www.ukev.org.uk/ukev-summer-school/ |