Regulation of exosome heterogeneity and function

Lead Research Organisation: University of Oxford
Department Name: Physiology Anatomy and Genetics

Abstract

In all animals, cells communicate with each other by releasing signals that affect nearby and distant target cells. These signals are vital to ensure all tissues develop in a co-ordinated way, and respond appropriately to the environment. Diseases including cancer, diabetes and neurodegenerative disorders can involve defects in these processes. We have known for decades that many signals are proteins that bind to receptors and activate a cascade of events that changes a cell's behaviour. More recently, secreted membrane-bound vesicles called exosomes have been identified as an alternative and more complex mode of communication. They carry signals and their receptors, as well as intracellular signalling proteins and RNAs. Their multifaceted signalling activity allows them to completely reprogramme cell behaviours. Because of this, they have attracted much attention as potential markers and messengers of disease, and as possible vehicles to deliver bioactive molecules to defective cells in patients.

Exosomes are proposed to form inside intracellular membrane-bound 'multivesicular' compartments that are thought to originate from organelles called late endosomes. Multiple exosome subtypes seem to exist, but it has proved difficult to experimentally separate them from each other and other secreted vesicles. The regulation and functions of different classes of exosome have therefore remained poorly understood.

We have investigated this problem in the fruit fly, Drosophila melanogaster. The functions of different genes can be much more readily tested in flies than in mammals. Our groups and many other researchers have found that basic cellular mechanisms are remarkably similar in humans and flies, allowing us to use flies to answer fundamental questions in biology and then apply the findings to investigate problems relating to animal and human health. We identified a specific cell in flies that has huge intracellular endosomal and secretory compartments, and have demonstrated that contrary to current dogma, exosomes are formed in several different compartments in addition to late endosomes. We have discovered proteins that selectively mark each type of exosome and already have evidence that secretion of these subtypes can be independently controlled. Most notably, one of these new classes of exosome is also made in human cancer cells. These exosomes are secreted when cells are subjected to adverse conditions and they have specialised properties that may help tumours to adapt to their environment.

Here we propose to fully characterise the different exosomes made by cells in our fly system. We will block the function of multiple genes that we think may control these exosomes to work out how specific exosome subtypes are formed and secreted. We will then extend our studies into human cells, blocking formation of selected exosome subtypes to identify their cargos and functions, and determining which of the control mechanisms we have identified is conserved from flies to humans. This will allow us to work out what the different types of exosome do and how exosome signalling can be changed to influence the behaviour of surrounding cells.

With huge interest in analysing exosome function in health and disease, and in engineering exosomes as new delivery systems for therapeutics, there is an urgent need to determine what different types of exosome exist and how they are made. Findings from this proposal will immediately establish a new framework for many researchers worldwide to define different exosome subtypes in isolates and then potentially isolate them selectively or block their secretion. They may also provide insights into novel ways in which exosomes and their biogenesis mechanisms could be studied or exploited in other areas, such as reproductive biology, infectious disease and pest control, where cell-cell and inter-organism communication play critical roles.

Technical Summary

We have developed the adult prostate-like secondary cell (SC) in flies as a new in vivo model to study exosome biogenesis. By using the temperature-dependent GAL4/UAS/GAL80ts system, we can inducibly knock down exosome regulators while expressing fluorescently tagged exosome markers. The remarkably large size of exosome-forming compartments in these cells allows us to resolve intraluminal vesicles (ILVs) in situ in living cells for the first time with super-resolution and real-time microscopy techniques. Employing a range of new Rab gene traps, we have shown that distinct compartments marked with five different Rabs produce exosomes, and these Rabs provide a signature for the subcellular origin of ILVs. Four of these compartments were not previously associated with ILV formation. We have already shown for one of them, marked by Rab11a, this function is conserved in human cancer cell lines and generates a new class of exosome with distinct cargos (verified by Western analysis and proteomics) and functions.

Here, our key objectives are now to employ the established methodologies described above to:

1. Fully characterise the different ILV-forming compartments in SCs and identify their specific cargos and functions in flies;

2. Genetically dissect the mechanisms that control biogenesis of each exosome subtype in SCs;

3. Using human cancer cell lines, which produce exosomes carrying the homologues of all five ILV-associated Rabs identified in flies, screen for evolutionarily conserved mechanisms of exosome subtype-specific control. For at least one new exosome subtype, we will determine its protein and miRNA cargos, and its functions.

These studies will not only reveal how cells use different ILV-forming compartments to generate exosomes and how regulating these processes can alter exosome signalling, but also inform many other studies of exosome biology in which the inability to distinguish different classes of exosome has hampered progress.

Planned Impact

We outline the potential academic impact of our proposal above. Other possible impact areas are:

1. Clinical Medicine

Because of the gaps in our basic understanding of exosome biogenesis and the increasing realisation that exosomes have a range of important biological functions, there are several areas of medicine that might benefit from our studies. The most immediate impact might come from developing exosomes as biomarkers for health and disease. Such assays are already emerging for cancer screening. As techniques like microfluidics advance, this could become a routine procedure. A better understanding of the regulation of exosome signalling and cargos should highlight candidate markers to screen for specific clinical conditions. Much of this translational work will happen elsewhere. However, Goberdhan's CRUK-funded links to clinicians, Harris (Oncology, Oxford) and Hamdy (Chair of Surgery, Oxford), Wilson's broad contacts as a medical tutor, and our associations with clinicians in multiple relevant research- and disease-focus groups, means that we can participate in and inform developments through further jointly funded work. Importantly, Goberdhan and Wilson both have clinicians working with them (DPhil student and Clinical Lecturer respectively), who can provide important input in their biweekly joint meetings. They also have monthly research meetings both with Harris and Hamdy.

Another longer-term outcome would be to identify ways to block secretion of specific exosome subtypes, potentially, for example, allowing clinicians to inhibit tumour adaptation mechanisms that drive progression and metastasis. Furthermore, as the roles of exosomes in normal physiology and diseases, such as neurodegenerative disorders, become better established, our work will inform the interpretation and development of this work. For example, in Oxford and elsewhere, clinical researchers are testing seminal fluid EVs as enhancers of IVF and embryo implantation. The potency of such treatments might be improved by prior isolation of specific exosome subtypes.

2. Pharmaceutical Industry and Biotechnology

As discussed above, some of our findings could inform development of biomarkers or potential therapies. Regarding the former, Goberdhan's clinical collaborations already give us access to patient samples, so we can test our ideas in a clinical context. There are opportunities in Oxford to develop monoclonal antibodies for biomarker detection. Indeed, Goberdhan and Harris are commercialising an antibody from their work on transporters with Novus, Ximbio and Cancer Research Technology (CRT), the commercial arm of CRUK. Through the collaborations described above, therapy development over the long term could also involve Goberdhan, as would biodelivery development by extending collaborations with Wood. It is possible that the role of exosomes in insect reproduction could be exploited in pest control strategies, though it would be critical to show advantages over current male-sterile techniques, etc. We are in annual contact with OU Innovation, the University's Technology Transfer Company, if our findings could be commercially exploited in these ways.

3. General Public and Schools

Our work exemplifies how flies can be used to undertake in vivo studies, which target problems in basic biology that are relevant to human health. During the project, Goberdhan and her group will undertake activities that she has already developed with the CRUK Engagement Manager to enthuse members of the public with an interest in health science through meetings and lab visits. Wilson's group will also take part in these activities and continue to be involved in University- and College-based schools events that showcase links between academic science, healthcare and biotechnology. Involvement of the PDRA and Gandy will help them develop multiple skills relevant to many employment sectors (see Justification for Resources).
 
Description Since 2016, we have presented aspects of our work in several engagement activities, particularly with schoolchildren. They are particularly interested and surprised to hear how using flies can impact our understanding of cancer and basic biological processes involved in human disease. Feedback is routinely positive and some of the individuals who come from schools that have previously very limited links to Oxford have applied here, a key objective of some of the events that we hold. We are now also involved in the UNIQ and UNIQ+ schemes in Oxford, where schoolchildren and undergraduate students from disadvantaged backgrounds come to Oxford to experience the academic opportunities here: schoolchildren stay for a week and receive a range of academic sessions, including a cell biology tutorial that I participate in, and undergraduates work in my lab on accessory gland projects inspired by our funded studies. These students often successfully apply to Oxford (and other world-class Universities) subsequently.
First Year Of Impact 2016
Sector Education
Impact Types Cultural,Societal

 
Description Citation in the Position Statement of the International Socieity for Extracellular Vesicles on the minimum information needed for the study of extracellular vesicles
Geographic Reach Multiple continents/international 
Policy Influence Type Citation in other policy documents
Impact The extracellular vesicle field is technically challenging. The guidelines in this document are having an impact on improving the quality of research in the extracellular field. This will have the long term effect of moving the research in this are topical and clinically important area along more rapidly.
URL https://www.ncbi.nlm.nih.gov/pubmed/?term=misev+goberdhan
 
Description Serum extracellular vesicle signatures as biomarkers for non-invasive early detection of oesophageal adenocarcinoma
Amount £98,243 (GBP)
Funding ID C15991/A28123 
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2019 
End 09/2020
 
Description Cell-cell communication via lncRNA transfer 
Organisation University of Toronto
Department Donnelly Centre for Cellular and Biomolecular Research
Country Canada 
Sector Academic/University 
PI Contribution Expertise in analysing exosome- and microcarrier-mediated signalling between cells.
Collaborator Contribution Expertise in lncRNA expression and function in Drosophila.
Impact Canadian Institutes of Health Research grant 2019 - 2024. We will provide training, tools, experimental input and expertise as collaborators in analysing accessory gland cell biology.
Start Year 2019
 
Description Prof Freddie C Hamdy 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Monthly meetings with Prof Hamdy, Prof Wilson and members of our research teams. We have recently instigated plans to help develop the collaboration further by extending these meetings to include other group leaders and members of their teams, eg Mr Richard Bryant, Prof Claire Edwards. I bring expertise, intellectual input and a research programme focussed on trying to tackle challenges in cancer research through a better understanding of the fundamental biology mechanisms relevant to prostate cancer.
Collaborator Contribution Prof Hamdy brings a wealth of expertise in clinical research and surgery to the collaboration. through Prof Hamdy we have been able to establish a number of collaborative partnerships. These have enabled us to extend our collaborative research in to key areas. For example: 1. Analysis of the PAT4 amino acids transporter expression in prostate cancer and correlation with patient outcome with Dan Stevens, Mr Richard Bryant (NDS), Dr Claire Verrill (NDS), Prof Claire Edwards (NDS/NDORMS, Oxford). 2. Development of a fly model of prostate cancer leading to Corrigan et al., 2014; Redhai et al., 2016, Wilson et al., 2017 (detailed below)
Impact Grants: Cancer Research UK Oxford Centre Translational Collaboration Scheme, 'Dissecting the regulation and function of tumour cell exosomes'. (£30,000; one and a half years; Oct 2012-Mar 2014) Goberdhan DCI (Prinicipal Investigator); Edwards C, Edwards J, Harris AL, Sargent IL, Wilson C, Hamdy FC (Co-Is) Publications: Corrigan, L, Redhai S, Leiblich A, Fan SJ, Perera SM, Patel R, Gandy C, Wainwright SM, Morris JF, Hamdy FC, Goberdhan, DC§, Wilson C§ (2014) BMP-regulated exosomes from Drosophila male reproductive glands reprogram female behavior. J Cell Biol 206, 671-688. Redhai S, Hellberg JEEU, Wainwright M, Perera SW, Castellanos F, Kroeger B, Gandy C, Leiblich A, Corrigan L, Hilton T, Patel B, Fan S-J, Hamdy F, Goberdhan DC, Wilson C (2016) Regulation of dense-core granule replenishment by autocrine BMP signalling in Drosophila secondary cells. PLoS Genetics, 12, e1006366. Wilson, C, Leiblich, A, Goberdhan, DC and Hamdy, FC (2017) The Drosophila accessory gland as a model for prostate cancer and other pathologies. Curr Top Dev Biol, 121, 339-375.
Start Year 2012
 
Description Prof John Morris 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution My research team and I provided intellectual input, expertise and researcher group members to help carry out this work.
Collaborator Contribution Prof John Morris provided intellectual input, expertise relating to both EM and immuno-EM analysis. He trained and worked closely with several members of mine and collaborating labs, eg Sumeth Perera, a DPhil student in my research group to produce the data for a manuscript in preparation ( details below),
Impact Publications: Corrigan, L, Redhai S, Leiblich A, Fan SJ, Perera SM, Patel R, Gandy C, Wainwright SM, Morris JF, Hamdy FC, Goberdhan, DC§, Wilson C§ (2014) BMP-regulated exosomes from Drosophila male reproductive glands reprogram female behavior. J Cell Biol 206, 671-688. §denotes equal author contribution Perera, SMW§, Fan, S-J§, Kroeger, B§, Alves, C, Stefana, I, Redhai, S, Wainwright, M, Morris, JF, Harris, AL, Wilson, C, Goberdhan, DC. A novel conserved exosome biogenesis pathway mediates responses to mTORC1-inhibitory microenviromental stresses in cancer cells, in prep
Start Year 2014
 
Description Prof Sarah Blagden (Department of Oncology, Universtty of Oxford) 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution My lab has hosted visits from Prof Sarah Blagden's DPhil student, Fenella Gross, to train her in isolating and analysing extracellular vesicles from human cells grown in culture, under different stress conditions. In my research team Dr Shih-Jung Fan and Dr John Mason have been involved in this collaboration.
Collaborator Contribution Prof Sarah Blagden's research group (Dept of Oncology, University of Oxford) specialises in mRNA dysregulation in cancer and has initiated the collaboration with my lab to investigate whether the mRNA binding protein La-Related Protein 1 (LARP1), which is implicated in cancer progression, may be conveyed on extracellular vesicles. This collaboration has been facilitated by Sarah's DPhil student, Fenella Gross.
Impact Data supporting the idea that LARP1 is transferred in extracellular vesicles has been generated. Meetings involving member of the Goberdhan, Blagden and other collaborators are ensuing. Prof Blagden has continued to hold these meetings every few months, to disccuss data to support a CRUK Programme grant application that Sarah will lead, focussing on the role of extracellular vesicles and the immune system in ovarian cancer.
Start Year 2018
 
Description Qiong Zhang and Ros Rickaby 
Organisation University of Oxford
Department Department of Earth Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution My research team and I will provide advice and training in isolating and characterising extracellular vesicles. his might include, for example, your expertise, intellectual input or the training of staff. It might also include access to data, equipment or facilities. You are asked in the next question to describe the contribution that your partners brought to this collaboration.
Collaborator Contribution My collaborators will provide expertise in isotopic analysis of metaaomes and metalloproteins. This collaboration was led to a successful grant application, entitled, 'The stable isotope signature of exosomes and other extracellular vesicles (EVs)', from the University John Fell OUP Research Fund, submitted by Dr Qiong Zhang, a Postdoctoral Researcher in Prof Ros Rickaby's lab in the Department of Earth Scences at the University of Oxford. The aim of this application is to provide pilot data for a ull application to develop this collaboration further.
Impact There are no specific outcomes to report yet as the grant has only recently started.
Start Year 2020
 
Description Participation in an activity, workshop or similar - UNIQ+ Summer School 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Undergraduate students
Results and Impact UNIQ+ is a new scheme where talented undergraduate students from disadvantaged backgrounds, studying at other Universities, undertake a research project in labs at Oxford, as well as being given the opportunity to experience other aspects of the academic environment here. We hosted an undergraduate from Nottingham this year, who undertook some excellent research and subsequently reported gaining significantly from the experience.
Year(s) Of Engagement Activity 2019
 
Description School Visit (Kent) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Presentation and Q&A session to post-GSCE schoolchildren from a region around St. Olave's Grammar School in Orpington, Kent. Discussing medicine and biomedicine as a degree and career option, admissions to Oxford, etc. After the Q&A session, I answered individual questions from students, two of whom indicated that their views on the most appropriate career choices had changed as a result of the meeting. I also received e-mails of thanks from the co-ordinating teacher at the meeting and some parents.
Year(s) Of Engagement Activity 2018,2019
 
Description UNIQ Summer School 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Education week giving students from disadvantaged backgrounds the opportunity to experience University and Oxford environment and to encourage them to aspire to applying to the top national Universities. I am involved in a cell biology tutorial session with a group of ~ 6 students, where they can personally interact with academics and i can discuss our work, as well as basic aspects of cell biology. This session is highlighted by most students as the most useful event of the week. We are aware that many student on this course decide to apply to Oxford and they have a success rate that is typically higher than other students.
Year(s) Of Engagement Activity 2019