Mesenchymal plasticity of devil facial tumour cells during in vivo vaccine and immunotherapy trials.

Immune evasion is critical to the growth and survival of cancer cells. This is especially pertinent to transmissible cancers, which evade immune detection across genetically diverse hosts. The Tasmanian devil (Sarcophilus harrisii) is threatened by the emergence of Devil Facial Tumour Disease (DFTD), comprising two transmissible cancers (DFT1 and DFT2). The development of effective prophylactic vaccines and therapies against DFTD has been restricted by an incomplete understanding of how allogeneic DFT1 and DFT2 cells maintain immune evasion upon activation of tumour-specific immune responses. In this study, we used RNA sequencing to examine tumours from three experimental DFT1 cases. Two devils received a vaccine prior to inoculation with live DFT1 cells, providing an opportunity to explore changes to DFT1 cancers under immune pressure. Analysis of DFT1 in the non-immunised devil revealed a 'myelinating Schwann cell' phenotype, reflecting both natural DFT1 cancers and the DFT1 cell line used for the experimental challenge. Comparatively, immunised devils exhibited a 'dedifferentiated mesenchymal' DFT1 phenotype. A third 'immune-enriched' phenotype, characterised by increased PDL1 and CTLA-4 expression, was detected in a DFT1 tumour that arose after immunotherapy. In response to immune pressure, mesenchymal plasticity and upregulation of immune checkpoint molecules are used by human cancers to evade immune responses. Similar mechanisms are associated with immune evasion by DFTD cancers, providing novel insights that will inform modification of DFTD vaccines. As DFT1 and DFT2 are clonal cancers transmitted across genetically distinct hosts, the Tasmanian devil provides a 'natural' disease model for more broadly exploring these immune evasion mechanisms in cancer.

Two of a kind: transmissible Schwann cell cancers in the endangered Tasmanian devil (Sarcophilus harrisii).

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.

A second transmissible cancer in Tasmanian devils.

Clonally transmissible cancers are somatic cell lineages that are spread between individuals via the transfer of living cancer cells. There are only three known naturally occurring transmissible cancers, and these affect dogs, soft-shell clams, and Tasmanian devils, respectively. The Tasmanian devil transmissible facial cancer was first observed in 1996, and is threatening its host species with extinction. Until now, this disease has been consistently associated with a single aneuploid cancer cell lineage that we refer to as DFT1. Here we describe a second transmissible cancer, DFT2, in five devils located in southern Tasmania in 2014 and 2015. DFT2 causes facial tumors that are grossly indistinguishable but histologically distinct from those caused by DFT1. DFT2 bears no detectable cytogenetic similarity to DFT1 and carries a Y chromosome, which contrasts with the female origin of DFT1. DFT2 shows different alleles to both its hosts and DFT1 at microsatellite, structural variant, and major histocompatibility complex (MHC) loci, confirming that it is a second cancer that can be transmitted between devils as an allogeneic, MHC-discordant graft. These findings indicate that Tasmanian devils have spawned at least two distinct transmissible cancer lineages and suggest that transmissible cancers may arise more frequently in nature than previously considered. The discovery of DFT2 presents important challenges for the conservation of Tasmanian devils and raises the possibility that this species is particularly prone to the emergence of transmissible cancers. More generally, our findings highlight the potential for cancer cells to depart from their hosts and become dangerous transmissible pathogens.

Immunology of a Transmissible Cancer Spreading among Tasmanian Devils.

Devil facial tumor disease (DFTD) is a transmissible cancer that has killed most of the Tasmanian devil (Sarcophilus harrissii) population. Since the first case appeared in the mid-1990s, it has spread relentlessly across the Tasmanian devil's geographic range. As Tasmanian devils only exist in Tasmania, Australia, DFTD has the potential to cause extinction of this species. The origin of DFTD was a Schwann cell from a female devil. The disease is transmitted when devils bite each other around the facial areas, a behavior synonymous with this species. Every devil that is 'infected' with DFTD dies from the cancer. Once the DFTD cells have been transmitted, they appear to develop into a cancer without inducing an immune response. The DFTD cancer cells avoid allogeneic recognition because they do not express MHC class I molecules on the cell surface. A reduced genetic diversity and the production of immunosuppressive cytokines may also contribute.

Mitogen-activated Tasmanian devil blood mononuclear cells kill devil facial tumour disease cells.

Devil facial tumour disease (DFTD) is a transmissible cancer that has brought the host species, the Tasmanian devil, to the brink of extinction. The cancer cells avoid allogeneic immune recognition by downregulating cell surface major histocompatibility complex (MHC) I expression. This should prevent CD8(+) T cell, but not natural killer (NK) cell, cytotoxicity. The reason why NK cells, normally reactive to MHC-negative cells, are not activated to kill DFTD cells has not been determined. The immune response of wild devils to DFTD, if it occurs, is uncharacterised. To investigate this, we tested 12 wild devils with DFTD, and found suggestive evidence of low levels of antibodies against DFTD cells in one devil. Eight of these devils were also analysed for cytotoxicity, however, none showed evidence for cytotoxicity against cultured DFTD cells. To establish whether mimicking activation of antitumour responses could induce cytotoxic activity against DFTD, Tasmanian devil peripheral blood mononuclear cells (PBMCs) were treated with either the mitogen Concanavalin A, the Toll-like receptor agonist polyinosinic:polycytidylic acid or recombinant Tasmanian devil IL-2. All induced the PBMC cells to kill cultured DFTD cells, suggesting that activation does not occur after encounter with DFTD cells in vivo, but can be induced. The identification of agents that activate cytotoxicity against DFTD target cells is critical for developing strategies to protect against DFTD. Such agents could function as adjuvants to induce functional immune responses capable of targeting DFTD cells and tumours in vivo.

Reversible epigenetic down-regulation of MHC molecules by devil facial tumour disease illustrates immune escape by a contagious cancer.

Contagious cancers that pass between individuals as an infectious cell line are highly unusual pathogens. Devil facial tumor disease (DFTD) is one such contagious cancer that emerged 16 y ago and is driving the Tasmanian devil to extinction. As both a pathogen and an allograft, DFTD cells should be rejected by the host-immune response, yet DFTD causes 100% mortality among infected devils with no apparent rejection of tumor cells. Why DFTD cells are not rejected has been a question of considerable confusion. Here, we show that DFTD cells do not express cell surface MHC molecules in vitro or in vivo, due to down-regulation of genes essential to the antigen-processing pathway, such as ß2-microglobulin and transporters associated with antigen processing. Loss of gene expression is not due to structural mutations, but to regulatory changes including epigenetic deacetylation of histones. Consequently, MHC class I molecules can be restored to the surface of DFTD cells in vitro by using recombinant devil IFN-γ, which is associated with up-regulation of the MHC class II transactivator, a key transcription factor with deacetylase activity. Further, expression of MHC class I molecules by DFTD cells can occur in vivo during lymphocyte infiltration. These results explain why T cells do not target DFTD cells. We propose that MHC-positive or epigenetically modified DFTD cells may provide a vaccine to DFTD. In addition, we suggest that down-regulation of MHC molecules using regulatory mechanisms allows evolvability of transmissible cancers and could affect the evolutionary trajectory of DFTD.

Heat shock proteins expressed in the marsupial Tasmanian devil are potential antigenic candidates in a vaccine against devil facial tumour disease.

The Tasmanian devil (Sarcophilus harrisii), the largest extant carnivorous marsupial and endemic to Tasmania, is at the verge of extinction due to the emergence of a transmissible cancer known as devil facial tumour disease (DFTD). DFTD has spread over the distribution range of the species and has been responsible for a severe decline in the global devil population. To protect the Tasmanian devil from extinction in the wild, our group has focused on the development of a prophylactic vaccine. Although this work has shown that vaccine preparations using whole DFTD tumour cells supplemented with adjuvants can induce anti-DFTD immune responses, alternative strategies that induce stronger and more specific immune responses are required. In humans, heat shock proteins (HSPs) derived from tumour cells have been used instead of whole-tumour cell preparations as a source of antigens for cancer immunotherapy. As HSPs have not been studied in the Tasmanian devil, this study presents the first characterisation of HSPs in this marsupial and evaluates the suitability of these proteins as antigenic components for the enhancement of a DFTD vaccine. We show that tissues and cancer cells from the Tasmanian devil express constitutive and inducible HSP. Additionally, this study suggests that HSP derived from DFTD cancer cells are immunogenic supporting the future development of a HSP-based vaccine against DFTD.

Transcriptome and proteome profiling reveals stress-induced expression signatures of imiquimod-treated Tasmanian devil facial tumor disease (DFTD) cells.

As a topical cancer immunotherapy, the toll-like receptor 7 ligand imiquimod activates tumor regression via stimulation of immune cell infiltration and cytotoxic responses. Imiquimod also exerts direct pro-apoptotic effects on tumor cells in vitro, but a role for these effects in imiquimod-induced tumor regression remains undefined. We previously demonstrated that cell lines derived from devil facial tumor disease (DFTD), a transmissible cancer threatening the survival of the Tasmanian devil (Sarcophilus harrisii), are sensitive to imiquimod-induced apoptosis. In this study, the pro-apoptotic effects of imiquimod in DFTD have been investigated using RNA-sequencing and label-free quantitative proteomics. This analysis revealed that changes to gene and protein expression in imiquimod treated DFTD cells are consistent with the onset of oxidative and endoplasmic reticulum stress responses, and subsequent activation of the unfolded protein response, autophagy, cell cycle arrest and apoptosis. Imiquimod also regulates the expression of oncogenic pathways, providing a direct mechanism by which this drug may increase tumor susceptibility to immune cytotoxicity in vivo. Our study has provided the first global analysis of imiquimod-induced effects in any tumor cell line. These findings have highlighted the potential of cell stress pathways as therapeutic targets in DFTD, and will allow for improved mechanistic use of imiquimod as a therapy in both the Tasmanian devil and human cancers.

Two Decades of the Impact of Tasmanian Devil Facial Tumor Disease.

The Tasmanian devil, a marsupial carnivore, has been restricted to the island state of Tasmania since its extinction on the Australian mainland about 3000 years ago. In the past two decades, this species has experienced severe population decline due to the emergence of devil facial tumor disease (DFTD), a transmissible cancer. During these 20 years, scientists have puzzled over the immunological and evolutionary responses by the Tasmanian devil to this transmissible cancer. Targeted strategies in population management and disease control have been developed as well as comparative processes to identify variation in tumor and host genetics. A multi-disciplinary approach with multi-institutional teams has produced considerable advances over the last decade. This has led to a greater understanding of the molecular pathogenesis and genomic classification of this cancer. New and promising developments in the Tasmanian devil's story include evidence that most immunized, and some wild devils, can produce an immune response to DFTD. Furthermore, epidemiology combined with genomic studies suggest a rapid evolution to the disease and that DFTD will become an endemic disease. Since 1998 there have been more than 350 publications, distributed over 37 Web of Science categories. A unique endemic island species has become an international curiosity that is in the spotlight of integrative and comparative biology research.

The newly-arisen Devil facial tumour disease 2 (DFT2) reveals a mechanism for the emergence of a contagious cancer.

Devil Facial Tumour 2 (DFT2) is a recently discovered contagious cancer circulating in the Tasmanian devil (Sarcophilus harrisii), a species which already harbours a more widespread contagious cancer, Devil Facial Tumour 1 (DFT1). Here we show that in contrast to DFT1, DFT2 cells express major histocompatibility complex (MHC) class I molecules, demonstrating that loss of MHC is not necessary for the emergence of a contagious cancer. However, the most highly expressed MHC class I alleles in DFT2 cells are common among host devils or non-polymorphic, reducing immunogenicity in a population sharing these alleles. In parallel, MHC class I loss is emerging in vivo, thus DFT2 may be mimicking the evolutionary trajectory of DFT1. Based on these results we propose that contagious cancers may exploit partial histocompatibility between the tumour and host, but that loss of allogeneic antigens could facilitate widespread transmission of DFT2.

The toll-like receptor ligands Hiltonol® (polyICLC) and imiquimod effectively activate antigen-specific immune responses in Tasmanian devils (Sarcophilus harrisii).

Devil facial tumour disease (DFTD) describes two genetically distinct transmissible tumours that pose a significant threat to the survival of the Tasmanian devil. A prophylactic vaccine could protect devils from DFTD transmission. For this vaccine to be effective, potent immune adjuvants will be required. Toll-like receptors (TLRs) promote robust immune responses in human cancer studies and are highly conserved across mammalian species. In this study, we investigated the proficiency of TLR ligands for immune activation in the Tasmanian devil using in vitro mononuclear cell stimulations and in vivo immunisation trials with a model antigen. We identified two such TLR ligands, polyICLC (Hiltonol®) (TLR3) and imiquimod (TLR7), that in combination induced significant IFNγ production from Tasmanian devil lymphocytes in vitro. Immunisation with these ligands and the model antigen keyhole limpet haemocyanin activated robust antigen-specific primary, secondary and long-term memory IgG responses. Our results support the conserved nature of TLR signaling across mammalian species. PolyICLC and imiquimod will be trialed as immune adjuvants in future DFTD vaccine formulations.

Regression of devil facial tumour disease following immunotherapy in immunised Tasmanian devils.

Devil facial tumour disease (DFTD) is a transmissible cancer devastating the Tasmanian devil (Sarcophilus harrisii) population. The cancer cell is the 'infectious' agent transmitted as an allograft by biting. Animals usually die within a few months with no evidence of antibody or immune cell responses against the DFTD allograft. This lack of anti-tumour immunity is attributed to an absence of cell surface major histocompatibility complex (MHC)-I molecule expression. While the endangerment of the devil population precludes experimentation on large experimental groups, those examined in our study indicated that immunisation and immunotherapy with DFTD cells expressing surface MHC-I corresponded with effective anti-tumour responses. Tumour engraftment did not occur in one of the five immunised Tasmanian devils, and regression followed therapy of experimentally induced DFTD tumours in three Tasmanian devils. Regression correlated with immune cell infiltration and antibody responses against DFTD cells. These data support the concept that immunisation of devils with DFTD cancer cells can successfully induce humoral responses against DFTD and trigger immune-mediated regression of established tumours. Our findings support the feasibility of a protective DFTD vaccine and ultimately the preservation of the species.

The Immunomodulatory Small Molecule Imiquimod Induces Apoptosis in Devil Facial Tumour Cell Lines.

The survival of the Tasmanian devil (Sarcophilus harrisii) is threatened by devil facial tumour disease (DFTD). This transmissible cancer is usually fatal, and no successful treatments have been developed. In human studies, the small immunomodulatory molecule imiquimod is a successful immunotherapy, activating anti-tumour immunity via stimulation of toll-like receptor-7 (TLR7) signaling pathways. In addition, imiquimod is a potent inducer of apoptosis in human tumour cell lines via TLR7 independent mechanisms. Here we investigate the potential of imiquimod as a DFTD therapy through analysis of treated DFTD cell lines and Tasmanian devil fibroblasts. WST-8 proliferation assays and annexin V apoptosis assays were performed to monitor apoptosis, and changes to the expression of pro- and anti-apoptotic genes were analysed using qRT-PCR. Our results show that DFTD cell lines, but not Tasmanian devil fibroblasts, are sensitive to imiquimod-induced apoptosis in a time and concentration dependent manner. Induction of apoptosis was accompanied by down-regulation of the anti-apoptotic BCL2 and BCLXL genes, and up-regulation of the pro-apoptotic BIM gene. Continuous imiquimod treatment was required for these effects to occur. These results demonstrate that imiquimod can deregulate DFTD cell growth and survival in direct and targeted manner. In vivo, this may increase DFTD vulnerability to imiquimod-induced TLR7-mediated immune responses. Our findings have improved the current knowledge of imiquimod action in tumour cells for application to both DFTD and human cancer therapy.

Toll-like receptor signaling is functional in immune cells of the endangered Tasmanian devil.

Devil facial tumour disease (DFTD) is a fatally transmissible cancer that threatens the Tasmanian devil population. As Tasmanian devils do not produce an immune response against DFTD cells, an effective vaccine will require a strong adjuvant. Activation of innate immune system cells through toll-like receptors (TLRs) could provide this stimulation. It is unknown whether marsupials, including Tasmanian devils, express functional TLRs. We isolated RNA from peripheral blood mononuclear cells and, with PCR, detected transcripts for TLRs 2, 3, 4, 5, 6, 7, 8, 9, 10 and 13. Stimulation of the mononuclear cells with agonists to these TLRs increased the expression of downstream TLR signaling products (IL1α, IL6, IL12A and IFNß). Our data provide the first evidence that TLR signaling is functional in the mononuclear cells of the Tasmanian devil. Future DFTD vaccination trials will incorporate TLR agonists to enhance the immune response against DFTD.

Identification of dendritic cells, B cell and T cell subsets in Tasmanian devil lymphoid tissue; evidence for poor immune cell infiltration into devil facial tumors.

The Tasmanian devil is under threat of extinction due to the transmissible devil facial tumor disease (DFTD). This fatal tumor is an allograft that does not induce an immune response, raising questions about the activity of Tasmanian devil immune cells. T and B cell analysis has been limited by a lack of antibodies, hence the need to produce such reagents. Amino acid sequence analysis revealed that CD4, CD8, IgM, and IgG were closely related to other marsupials. Monoclonal antibodies were produced against CD4, CD8, IgM, and IgG by generating bacterial fusion proteins. These, and commercial antibodies against CD1a and CD83, identified T cells, B cells and dendritic cells by immunohistochemistry. CD4(+) and CD8(+) T cells were identified in pouch young thymus, adult lymph nodes, spleen, bronchus- and gut-associated lymphoid tissue. Their anatomical distribution was characteristic of mammalian lymphoid tissues with more CD4(+) than CD8(+) cells in lymph nodes and splenic white pulp. IgM(+) and IgG(+) B cells were identified in adult lymph nodes, spleen, bronchus-associated lymphoid tissue and gut-associated lymphoid tissue, with more IgM(+) than IgG(+) cells. Dendritic cells were identified in lymph node, spleen and skin. This distribution is consistent with eutherian mammals and other marsupials, indicating they have the immune cell subsets for an anti-tumor immunity. Devil facial tumor disease tumors contained more CD8(+) than CD4(+) cells, but in low numbers. There were also low numbers of CD1a(+) and MHC class II(+) cells, but no CD83(+) IgM(+) or IgG(+) B cells, consistent with poor immune cell infiltration.

The Tasmanian devil transcriptome reveals Schwann cell origins of a clonally transmissible cancer.

The Tasmanian devil, a marsupial carnivore, is endangered because of the emergence of a transmissible cancer known as devil facial tumor disease (DFTD). This fatal cancer is clonally derived and is an allograft transmitted between devils by biting. We performed a large-scale genetic analysis of DFTD with microsatellite genotyping, a mitochondrial genome analysis, and deep sequencing of the DFTD transcriptome and microRNAs. These studies confirm that DFTD is a monophyletic clonally transmissible tumor and suggest that the disease is of Schwann cell origin. On the basis of these results, we have generated a diagnostic marker for DFTD and identify a suite of genes relevant to DFTD pathology and transmission. We provide a genomic data set for the Tasmanian devil that is applicable to cancer diagnosis, disease evolution, and conservation biology.