Extracellular vesicles (EVs) originate from membranes of all cell types and can be found in all tissues and biological fluids.^{1 – 4} They are $\sim 30$ to 1000 nm in diameter and play a crucial role in intercellular communication by delivering a variety of cargo, including nucleic acids, proteins, glycans, and lipids to local and distant cells where they influence cellular phenotype.^2,3,5,6 EV-mediated intercellular signaling contributes to the regulation of a wide range of biological functions in target cells including immunological processes (e.g., antigen presentation), communication between components of the neurovascular unit, glial and neuronal function, development and differentiation of stem cells, and tissue regeneration. Additionally, in aberrant conditions, EVs have been associated with the pathophysiology of stroke, Alzheimer’s disease, Parkinson’s disease, cancer, obesity, cardiovascular disease, and rheumatoid arthritis.^{2,5,7 – 11} Therefore, there is a rapidly increasing scientific interest in EVs including their detection and imaging in tissue or biological fluids.

Trematodes cause disease in millions of people worldwide, but the absence of commercial vaccines has led to an over-reliance on a handful of monotherapies to control infections. Since drug-resistant fluke populations are emerging, a deeper understanding of parasite biology and host interactions is required to identify new drug targets and immunogenic vaccine candidates. Mass spectrometry-based proteomics represents a key tool to that end. Recent studies have capitalised on the wider availability of annotated helminth genomes to achieve greater coverage of trematode proteomes and discover new aspects of the host–parasite relationship. This review focusses on these latest advances. These include how the protein components of fluke extracellular vesicles have given insight into their biogenesis and cellular interactions. In addition, how the integration of transcriptome/proteome datasets has revealed that the expression and secretion of selected families of liver fluke virulence factors and immunomodulators are regulated in accordance with parasite development and migration within the mammalian host. Furthermore, we discuss the use of immunoproteomics as a tool to identify vaccine candidates associated with protective antibody responses. Finally, we highlight how established and emerging technologies, such as laser microdissection and single-cell proteomics, could be exploited to resolve the protein profiles of discrete trematode tissues or cell types which, in combination with functional tools, could pinpoint optimal targets for fluke control.

The liver fluke, Fasciola hepatica, is an obligate blood-feeder, and the gastrodermal cells of the parasite form the interface with the host’s blood. Despite their importance in the host–parasite interaction, in-depth proteomic analysis of the gastrodermal cells is lacking. Here, we used laser microdissection of F. hepatica tissue sections to generate unique and biologically exclusive tissue fractions of the gastrodermal cells and tegument for analysis by mass spectrometry. A total of 226 gastrodermal cell proteins were identified, with proteases that degrade haemoglobin being the most abundant. Other detected proteins included those such as proton pumps and anticoagulants which maintain a microenvironment that facilitates digestion. By comparing the gastrodermal cell proteome and the 102 proteins identified in the laser microdissected tegument with previously published tegument proteomic datasets, we showed that one-quarter of proteins (removed by freeze–thaw extraction) or one-third of proteins (removed by detergent extraction) previously identified as tegumental were instead derived from the gastrodermal cells. Comparative analysis of the laser microdissected gastrodermal cells, tegument, and F. hepatica secretome revealed that the gastrodermal cells are the principal source of secreted proteins, as well as showed that both the gastrodermal cells and the tegument are likely to release subpopulations of extracellular vesicles (EVs). Microscopical examination of the gut caeca from flukes fixed immediately after their removal from the host bile ducts showed that selected gastrodermal cells underwent a progressive thinning of the apical plasma membrane which ruptured to release secretory vesicles en masse into the gut lumen. Our findings suggest that gut-derived EVs are released via a novel atypical secretory route and highlight the importance of the gastrodermal cells in nutrient acquisition and possible immunomodulation by the parasite.

Extracellular vesicles (EVs) reflect their spatiotemporal cellular origins and, when released into the peripheral bloodstream, represent sources of biomarkers for processes occurring in inaccessible tissues, such as the brain. Positive selection by immunoaffinity isolation targeting a specific EV surface marker can be used to preferentially enrich for EVs from a particular cell type over other EVs in circulation. However, the case of L1CAM immunocapture for enrichment of brain-derived neuronal EVs has exemplified the biological and technical influences affecting the outcomes of positive selection, which are discussed here. These include findings from the wider (non-EV) literature showing that soluble L1CAM is a binding partner of the common EV marker CD9. Additional studies show that CD9 is not expressed by the vast majority of neurons in the brain, but it is highly expressed by neurons of the peripheral nervous system, which could affect the interpretation of studies demonstrating the co-localisation of L1CAM and CD9 on EVs to assert isolation of EVs derived from neurons in the brain. Next, emerging negative selection strategies are discussed which, when combined with positive selection, could overcome some of the challenges associated with enriching for EV-based biomarkers. These strategies include depleting EVs on a cell-by-cell type basis, as well as targeting common EV markers to simultaneously deplete diverse, undesired EV populations whilst selecting for the EVs of interest. An example is given of how CD9 depletion could enrich and select for brain-derived neuronal EVs. Additionally, studies that have used negative selection alone to select for EVs, and the advantages of this approach for biomarker detection, are highlighted. Finally, based on these insights, considerations for the choice of future positive and negative selection targets are discussed, including how understanding the nuances of EV heterogeneity could facilitate the process of EV-based biomarker discovery and their translation to the clinic.

Extracellular vesicles (EVs) reflect their spatiotemporal cellular origins and, when released into the peripheral bloodstream, represent sources of biomarkers for processes occurring in inaccessible tissues, such as the brain. Positive selection by immunoaffinity isolation targeting a specific EV surface marker can be used to preferentially enrich for EVs from a particular cell type over other EVs in circulation. However, the case of L1CAM immunocapture for enrichment of brain-derived neuronal EVs has exemplified the biological and technical influences affecting the outcomes of positive selection, which are discussed here. These include findings from the wider (non-EV) literature showing that soluble L1CAM is a binding partner of the common EV marker CD9. Additional studies show that CD9 is not expressed by the vast majority of neurons in the brain, but it is highly expressed by neurons of the peripheral nervous system, which could affect the interpretation of studies demonstrating the co-localisation of L1CAM and CD9 on EVs to assert isolation of EVs derived from neurons in the brain. Next, emerging negative selection strategies are discussed which, when combined with positive selection, could overcome some of the challenges associated with enriching for EV-based biomarkers. These strategies include depleting EVs on a cell-by-cell type basis, as well as targeting common EV markers to simultaneously deplete diverse, undesired EV populations whilst selecting for the EVs of interest. An example is given of how CD9 depletion could enrich and select for brain-derived neuronal EVs. Additionally, studies that have used negative selection alone to select for EVs, and the advantages of this approach for biomarker detection, are highlighted. Finally, based on these insights, considerations for the choice of future positive and negative selection targets are discussed, including how understanding the nuances of EV heterogeneity could facilitate the process of EV-based biomarker discovery and their translation to the clinic.