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Immunological tools and early diagnostic approaches to monitor the effects of devil facial tumour disease on wild Tasmanian devils

Espejo Benavides, CE ORCID: 0000-0002-9031-6935 2021 , 'Immunological tools and early diagnostic approaches to monitor the effects of devil facial tumour disease on wild Tasmanian devils', PhD thesis, University of Tasmania.

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The Tasmanian devil (Sarcophilus harrissi) has been threatened with extinction because of two independently arising, genetically distinct transmissible cancers (DFT1 and DFT2) that cause devil facial tumour disease (DFTD). These cancers are both caused by clonal cells of Schwann cell origin, transmitted among devils through biting during social interactions like an allograft. Fatal in almost 100% of cases, DFTD usually kills its host within 6 to 12 months after the presentation of tumour masses on facial, oral and neck regions. Since DFT1 was discovered in 1996, multiple conservation interventions have been implemented to protect the species from extinction. However, these efforts have been hindered by the lack of species-specific reagents and diagnostic tools, which is a common challenge in the study of diseases affecting non-human or non-model species.
Immunological reagents are required to identify and quantify immune cells of defined phenotypes to understand how the animal immune system is affected by pathogens. For example, devils infected with DFTD show a reduction of lymphocytes, however the T lymphocyte subset that is affected by the disease is unknown due to the lack of a subset-specific antibody. Additionally, a cornerstone of disease management, including cancers, is effective diagnosis. This is especially critical in the case of DFTD due to the epidemic nature of these transmissible cancers. In this thesis, I have 1) developed a needed anti-devil CD8α monoclonal antibody (mAb) to characterise CD8+ T cells in devils; 2) characterised the proteome of extracellular vesicles derived from cultured DFTD cells to better understand DFTD cell signalling and to create a baseline for diagnostic tool development; 3) discovered a differential diagnostic protein biomarker for DFTD and 4) discovered and validated an early DFT1 biomarker using an extracellular vesicle approach.
First, I developed and validated a mAb for devil CD8+ T cells. The extracellular domain of the Tasmanian devil CD8-alpha was identified and used to develop recombinant proteins for mAb production. The anti-devil CD8α mAb was validated using a transfected Chinese hamster ovary cell line expressing devil CD8 alpha protein, and peripheral blood mononuclear cells (PBMC) from captive devils by flow cytometry. I also stimulated captive devil PBMCs to analyse the production of IFNG as a surrogate measure of T cell activity and found that the majority of INFG production was by CD4-/CD8-/B- cells. Finally, I developed a sterile and portable laboratory to isolate PBMCs from wild devils in the field to immunophenotype their CD4 and CD8 T cells as proof of concept that this antibody can be utilized to assess immune competence in wild populations.
Next, I employed extracellular vesicle (EV) approaches to evaluate how DFTD cancer cells may be manipulating devil physiology to aid tumorigenesis and cancer progression. EVs are nano-sized bilipid structures secreted by most cells, including cancer cells, that deliver bioactive cargo, such as a proteins, genetic material, and lipids to recipient cells. EVs play a crucial role in intercellular communication and are active participants in cancer progression. I optimised a methodology to isolate EVs derived from DFTD cultured cells to be characterised by mass spectrometry techniques. This first proteomic characterisation of EVs derived from cultured DFTD cells lead to the discovery that EVs from both DFT1 and DFT2 cell lines expressed higher levels of proteins associated with focal adhesion functions known to be linked to metastasis. Additionally, hallmark proteins of epithelial mesenchymal transition (EMT) were enriched in DFT2 EVs relative to DFT1 EVs, demonstrating the potential of EVs to detect functional differences between two clinically indistinguishable tumours, even more so than the proteome changes within the parent cancer cells. I next investigated whether the protein markers found in EVs derived from cultured cells could be used as potential differential DFTD biomarkers. I then characterised the EV proteome from 27 devil serum samples. The protein tenascin (TNC), one of the EMT hallmark proteins detected in DFT2 EVs in vitro, displayed a high predictive power to distinguish devils infected with DFT2 compared to those infected with DFT1 and healthy controls, demonstrating potential for a differential diagnosis biomarker.
Finally, I analysed the EV proteome from independent discovery and validation cohorts of captive and wild Tasmanian devil serum samples (n = 87) to identify potential DFT1 biomarkers. The protein cathelicidin-3 (CATH3) was enriched in serum EVs of both devils with clinical DFTD infection and latent devils 3 to 6 months before DFTD diagnosis. CATH3 enrichment identified overt DFT1 with 87.9% sensitivity and 94.1% specificity and predicted latent DFT1 with 93.8% and 94.1% specificity, presenting a critically needed biomarker for the early detection of DFT1.
This thesis has expanded the management toolbox for the conservation of the iconic Tasmanian devil in the wild. I developed the first monoclonal antibody against devil CD8α, which will be crucial to adequately analyse the cell mediated immune response in healthy, diseased, or vaccinated devils. These findings are among the very first to demonstrate clinical relevance of EVs in a wild disease system, providing a considerable step towards development of a clinical tool needed for conservation of an iconic, endangered species. The discovery and validation of an early DFT1 biomarker provides a major advance for DFTD diagnosis. The use of the preclinical biomarker I discovered will greatly enhance the capabilities of ongoing management efforts critical to ensure the viability of the remaining devil populations, such as epidemiological monitoring, maintenance of insurance populations, and deployment of vaccine and preventative measures. Finally, these results demonstrated that EV-approaches have considerable potential for other species (including humans) to provide much needed preclinical diagnostic tools that have defied previous development efforts.

Item Type: Thesis - PhD
Authors/Creators:Espejo Benavides, CE
Keywords: cancer diagnostics, exosomes, marsupials, microvesicles, proteomics, T cells, animal models, early cancer detection.
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Copyright 2021 the author

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Chapter 4 appears to be the equivalent of a pre-print version of an article published as: Espejo, C., Wilson, R., Willms, E. Ruiz‑Aravena, M., Pye, R. J., Jones, M. E., Hill, A. F., Woods, G. M., Lyons, A. B., 2021. Extracellular vesicle proteomes of two transmissible cancers of Tasmanian devils reveal tenascin-C as a serum-based differential diagnostic biomarker, Cellular and molecular life sciences, 78(23), 7537-7555.

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