Regulation of exosome heterogeneity and function

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.

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.

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).