Insights into the nature and immune escape of a transmissible cancer in Tasmanian devils

Devil facial tumour disease (DFTD) is a transmissible cancer that threatens the survival of the Tasmanian devil (Sarcophilus harrisii), the world's largest living carnivorous marsupial. Despite devils having a competent immune system, there is no evidence of natural immunity to the tumours. Affected animals die within months of tumour appearance. The species could face extinction within 25 to 35 years. Cytogenetic and molecular studies have confirmed that the infectious agent is the cancer cell itself. However, our knowledge about the nature and biology of this tumour is limited. Tumour transcriptome analyses of DFTD tumours and devil tissues revealed that DFTD expresses a set of genes related to the myelination pathway in the peripheral nervous system. This thesis examined the protein expression of peripheral nerve and other neuronal markers in DFTD to further elucidate the nature of the tumour. It also evaluated the utility of neuronal and peripheral nerve proteins as diagnostic markers for the disease. Immunohistochemistry and flow cytometry confirmed that DFTD tumour cells express a number of proteins found throughout the neuronal system. Notably, DFTD tumour cells expressed structural proteins found in myelinating Schwann cells in the peripheral nervous system. These included peripheral myelin protein 22 (PMP22), myelin protein zero (MPZ), myelin basic protein (MBP), and the recently described periaxin (PRX). Nerve growth factor receptor (NGFR), which is involved in the differentiation of Schwann cells, was also detected in DFTD tumour cells. These results coupled with the available genetic data confirm that DFTD cells are of Schwann cell origin. Furthermore, this study showed that periaxin, a specific marker of Schwann cells, is consistently expressed in DFTD primary tumours, DFTD metastases, DFTD cell lines and murine xenografted DFTD tumours. Therefore, this thesis identified periaxin as a sensitive and specific marker for DFTD. This will greatly facilitate the diagnosis of the disease. How DFTD can be transplanted across major histocompatibility (MHC) barriers is an aspect of DFTD that is not understood. Initially this was attributed to the reduced diversity at the major histocompatibility complex (MHC) loci. However, recent studies showed that the limited MHC diversity in devils is sufficient to produce measurable mixed lymphocyte reactions and the rejection of skin allografts. Thus, this thesis investigated the expression of MHC to determine if this may explain the lack of immune response to DFTD cells. Analysis of the expression of MHC proteins in devils has been hindered by the lack of cross-reacting reagents. During the course of this study collaborators from the University of Cambridge (UK) developed the first anti-devil MHC class I (MHC-I) antibody. This antibody was used to examine the expression of MHC-I in devil tissues and DFTD. MHC-I protein expression in primary tumours and metastases was lower than normal tissues. Its expression was also variable within a tumour and among different tumours. These results suggest that alteration of MHC-I expression could contribute to the immune escape of DFTD. Immunisation trials with non-viable DFTD cell lines revealed that some Tasmanian devils can produce antibodies against DFTD cells. This thesis utilised an immunoproteomic approach to identify immunogenic proteins in DFTD. Several candidates were identified as DFTD tumour associated antigens. These include heat shock proteins, tubulin, histone H2B, stathmin protein, and proliferating cell nuclear antigen (PCNA). These proteins are excellent targets for the development of early diagnostic tools as well as therapeutic approaches. In summary, this thesis provided strong supporting evidence for a Schwann cell origin of DFTD and generated a specific diagnostic marker for the disease. Additionally, this thesis opened new opportunities for the understanding of the mechanisms that allow immune evasion and the interaction between the tumour cells and the devil's immune system. These results will have direct implications for the future development of a vaccine approach.