Devil facial tumour disease (DFTD) comprises two genetically distinct transmissible cancers (DFT1 and DFT2) endangering the survival of the Tasmanian devil (Sarcophilus harrisii) in the wild. DFT1 first arose from a cell of the Schwann cell lineage; however, the tissue-of-origin of the recently discovered DFT2 cancer is unknown. In this study, we compared the transcriptome and proteome of DFT2 tumours to DFT1 and normal Tasmanian devil tissues to determine the tissue-of-origin of the DFT2 cancer. Our findings demonstrate that DFT2 expresses a range of Schwann cell markers and exhibits expression patterns consistent with a similar origin to the DFT1 cancer. Furthermore, DFT2 cells express genes associated with the repair response to peripheral nerve damage. These findings suggest that devils may be predisposed to transmissible cancers of Schwann cell origin. The combined effect of factors such as frequent nerve damage from biting, Schwann cell plasticity and low genetic diversity may allow these cancers to develop on rare occasions. The emergence of two independent transmissible cancers from the same tissue in the Tasmanian devil presents an unprecedented opportunity to gain insight into cancer development, evolution and immune evasion in mammalian species.

Devil facial tumor disease (DFTD) is renowned for its successful evasion of the host immune system. Down regulation of the major histocompatabilty complex class I molecule (MHC-I) on the DFTD cells is a primary mechanism of immune escape. Immunization trials on captive Tasmanian devils have previously demonstrated that an immune response against DFTD can be induced, and that immune-mediated tumor regression can occur. However, these trials were limited by their small sample sizes. Here, we describe the results of two DFTD immunization trials on cohorts of devils prior to their wild release as part of the Tasmanian Government’s Wild Devil Recovery project. 95% of the devils developed anti-DFTD antibody responses. Given the relatively large sample sizes of the trials (N = 19 and N = 33), these responses are likely to reflect those of the general devil population. DFTD cells manipulated to express MHC-I were used as the antigenic basis of the immunizations in both trials. Although the adjuvant composition and number of immunizations differed between trials, similar anti-DFTD antibody levels were obtained. The first trial comprised DFTD cells and the adjuvant combination of ISCOMATRIX™, polyIC, and CpG with up to four immunizations given at monthly intervals. This compared to the second trial whereby two immunizations comprising DFTD cells and the adjuvant combination ISCOMATRIX™, polyICLC (Hiltonol®) and imiquimod were given a month apart, providing a shorter and, therefore, more practical protocol. Both trials incorporated a booster immunization given up to 5 months after the primary course. A key finding was that devils in the second trial responded more quickly and maintained their antibody levels for longer compared to devils in the first trial. The different adjuvant combination incorporating the RNAase resistant polyICLC and imiquimod used in the second trial is likely to be responsible. The seroconversion in the majority of devils in these anti-DFTD immunization trials was remarkable, especially as DFTD is hallmarked by its immune evasion mechanisms. Microsatellite analyzes of MHC revealed that some MHC-I microsatellites correlated to stronger immune responses. These trials signify the first step in the long-term objective of releasing devils with immunity to DFTD into the wild.

Around 40% of humans and Tasmanian devils (Sarcophilus harrisii) develop cancer in their lifetime, compared to less than 10% for most species. In addition, devils are affected by two of the three known transmissible cancers in mammals. Immune checkpoint immunotherapy has transformed human medicine, but a lack of species-specific reagents has limited checkpoint immunology in most species. We developed a cut-and-paste reagent development system and used the fluorescent fusion protein system to show that immune checkpoint interactions are conserved across 160,000,000 years of evolution, CD200 is highly expressed on transmissible tumor cells, and coexpression of CD200R1 can block CD200 surface expression. The system’s versatility across species was demonstrated by fusing a fluorescent reporter to a camelid-derived nanobody that binds human programmed death ligand 1. The evolutionarily conserved pathways suggest that naturally occurring cancers in devils and other species can be used to advance our understanding of cancer and immunological tolerance.

Introduction: The Tasmanian devil (Sarcophilus harrisii) is the largest extant carnivorous marsupial. Since 1996, its population has declined by 77% primarily due to a clonal transmissible tumor, known as devil facial tumor (DFT1) disease. In 2014, a second transmissible devil facial tumor (DFT2) was discovered. DFT1 and DFT2 are nearly 100% fatal.Areas covered: We review DFT control approaches and propose a rabies-style oral bait vaccine (OBV) platform for DFTs. This approach has an extensive safety record and was a primary tool in large-scale rabies virus elimination from wild carnivores across diverse landscapes. Like rabies virus, DFTs are transmitted by oral contact, so immunizing the oral cavity and stimulating resident memory cells could be advantageous. Additionally, exposing infected devils that already have tumors to OBVs could serve as an oncolytic virus immunotherapy.The primary challenges may be identifying appropriate DFT-specific antigens and optimization of field delivery methods.Expert commentary: DFT2 is currently found on a peninsula in southern Tasmania, so an OBV that could eliminate DFT2 should be the priority for this vaccine approach. Translation of an OBV approach to control DFTs will be challenging, but the approach is feasible for combatting ongoing and future disease threats.

The Tasmanian devil ( Sarcophilus harrisii ) is the only mammalian species known to be affected by multiple transmissible cancers. Devil facial tumour 1 and 2 (DFT1 and DFT2) are independent neoplastic cell lineages that produce large, disfiguring cancers known as devil facial tumour disease (DFTD). The long-term persistence of wild Tasmanian devils is threatened due to the ability of DFTD cells to propagate as contagious allografts and the high mortality rate of DFTD. Recent studies have demonstrated that both DFT1 and DFT2 cancers originated from founder cells of the Schwann cell lineage, an uncommon origin of malignant cancer in humans. This unprecedented finding has revealed a potential predisposition of Tasmanian devils to transmissible cancers of the Schwann cell lineage. In this review, we compare the molecular nature of human Schwann cells and nerve sheath tumours with DFT1 and DFT2 to gain insights into the emergence of transmissible cancers in the Tasmanian devil. We discuss a potential mechanism whereby Schwann cell plasticity and frequent wounding in Tasmanian devils combine with an inherent cancer predisposition and low genetic diversity to give rise to transmissible Schwann cell cancers in devils on rare occasions. Response to Reviewers:We appreciate the suggestions and have incorporated all into the revised version. Thank you.

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Downregulation of major histocompatibility complex I (MHC-I) on tumor cells is a primary means of immune evasion by many types of cancer. Additionally, MHC-I proteins are a primary target of humoral and cellular mechanisms associated with transplant rejection. Transmissible tumors that overcome allograft rejection mechanisms and evade anti-tumor immunity have killed thousands of wild Tasmanian devils (Sarcophilus harrisii). Interferon-gamma (IFNG) upregulates surface MHC-I expression on devil facial tumor (DFT) cells but is not sufficient to induce tumor regressions. Transcriptome analysis of IFNG-treated DFT cells revealed strong upregulation of NLRC5, a master regulator of MHC-I in humans and mice. To explore the role of NLRC5 in transmissible cancers, we developed DFT cells lines that constitutively overexpress NLRC5. Transcriptomic results suggest that the role of NLRC5 as a master regulator of MHC-I is conserved in devils. Furthermore, NLRC5 was shown to drive the expression of many components of the antigen presentation pathway. To determine if MHC-I is a target of allogeneic immune responses, we tested serum from devils with natural DFT regressions against DFT cells. Antibody binding occurred with cells treated with IFNG and overexpressed NLRC5. However, CRISPR/Cas9-mediated knockout of MHC-I subunit beta-2-microglobulin (B2M) eliminated antibody binding to DFT cells. Consequently, MHC-I could be identified as a target for anti-tumor and allogeneic immunity and provides mechanistic insight into MHC-I expression and antigen presentation in marsupials. NLRC5 could be a promising target for immunotherapy and vaccines to protect devils from transmissible cancers and inform development of transplant and cancer therapies for humans.

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