Jocelyn Darby

A second transmissible cancer in Tasmanian devils

Significance Transmissible cancers are somatic cell lineages that are spread between individuals via the transfer of living cancer cells. Only three transmissible cancers have been reported in nature, suggesting that such diseases emerge rarely. One of the known transmissible cancers affects Tasmanian devils, and is threatening this species with extinction. Here we report the discovery of a second transmissible cancer in Tasmanian devils. This cancer causes facial tumors that are grossly indistinguishable from those caused by the first-described transmissible cancer in this species; however, tumors derived from this second clone are genetically distinct. These findings indicate that Tasmanian devils have spawned at least two different transmissible cancers, and suggest that transmissible cancers may arise more frequently in nature than previously considered. 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.

The La Autoantigen Is a Malignancy-Associated Cell Death Target That Is Induced by DNA-Damaging Drugs

Purpose: To evaluate the La autoantigen as a target for specific monoclonal antibody (mAb) binding in dead cancer cells after use of DNA-damaging chemotherapy. Experimental Design:In vitro studies of La-specific 3B9 mAb binding to malignant and normal primary cells with and without cytotoxic drug treatment were done using immunoblotting and flow cytometry. Chromatin-binding studies and immunofluorescence detection of γH2AX as a marker of DNA double-stranded breaks together with 3B9 binding assays were done to measure DNA damage responses. Incorporation of a transglutaminase 2 (TG2) substrate and TG2 inhibition were studied to measure protein cross-linking in dead cells. Results: La was overexpressed in human cancer cell lines with respect to normal primary cells. Within 3 h of the DNA-damaging stimulus, La became chromatin bound when it colocalized with γH2AX. Later, after the stimulus produced cell death, La-specific 3B9 mAb bound specifically and preferentially in the cytoplasm of dead cancer cells. Moreover, 3B9 binding to dead cancer cells increased with increasing DNA damage. Both La and 3B9 became cross-linked in dead cancer cells via TG2 activity. Conclusion: La autoantigen represents a promising cancer cell death target to determine chemotherapy response because its expression was selectively induced in dead cancer cells after DNA-damaging chemotherapy.

Chemotherapy Synergizes with Radioimmunotherapy Targeting La Autoantigen in Tumors

Background To date, inefficient delivery of therapeutic doses of radionuclides to solid tumors limits the clinical utility of radioimmunotherapy. We aim to test the therapeutic utility of Yttrium-90 (90Y)-radio-conjugates of a monoclonal antibody, which we showed previously to bind specifically to the abundant intracellular La ribonucleoprotein revealed in dead tumor cells after DNA-damaging treatment. Methodology/Principal Findings Immunoconjugates of the DAB4 clone of the La-specific monoclonal antibody, APOMAB®, were prepared using the metal chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and then radiolabeled with 90Y. Mice bearing established subcutaneous tumors were treated with 90Y-DOTA-DAB4 alone or after chemotherapy. Non-radiosensitizing cyclophosphamide/etoposide chemotherapy was used for the syngeneic EL4 lymphoma model. Radiosensitizing cisplatin/gemcitabine chemotherapy was used for the syngeneic Lewis Lung carcinoma (LL2) model, and for the xenograft models of LNCaP prostatic carcinoma and Panc-1 pancreatic carcinoma. We demonstrate the safety, specificity, and efficacy of 90Y-DOTA-DAB4-radioimmunotherapy alone or combined with chemotherapy. EL4 lymphoma-bearing mice either were cured at higher doses of radioimmunotherapy alone or lower doses of radioimmunotherapy in synergy with chemotherapy. Radioimmunotherapy alone was less effective in chemo- and radio-resistant carcinoma models. However, radioimmunotherapy synergized with radiosensitizing chemotherapy to retard significantly tumor regrowth and so prolong the survival of mice bearing LL2, LNCaP, or Panc-1 subcutaneous tumor implants. Conclusions/Significance We report proof-of-concept data supporting a unique form of radioimmunotherapy, which delivers bystander killing to viable cancer cells after targeting the universal cancer antigen, La, created by DNA-damaging treatment in neighboring dead cancer cells. Subsequently we propose that DAB4-targeted ionizing radiation induces additional cycles of tumor cell death, which further augments DAB4 binding to produce a tumor-lethal ‘genotoxic chain reaction’. Clinically, this approach may be useful as consolidation treatment after a drug-induced cell death among (small-volume) metastatic deposits, the commonest cause of cancer death. This article is part II of a two-part series providing proof-of-concept for the diagnostic and therapeutic use of the DAB4 clone of the La-specific monoclonal antibody, APOMAB®.

APOMAB®, a La-Specific Monoclonal Antibody, Detects the Apoptotic Tumor Response to Life-Prolonging and DNA-Damaging Chemotherapy

Background Antineoplastic therapy may impair the survival of malignant cells to produce cell death. Consequently, direct measurement of tumor cell death in vivo is a highly desirable component of therapy response monitoring. We have previously shown that APOMAB® representing the DAB4 clone of a La/SSB-specific murine monoclonal autoantibody is a malignant cell-death ligand, which accumulates preferentially in tumors in an antigen-specific and dose-dependent manner after DNA-damaging chemotherapy. Here, we aim to image tumor uptake of APOMAB® (DAB4) and to define its biological correlates. Methodology/Principal Findings Brisk tumor cell apoptosis is induced in the syngeneic EL4 lymphoma model after treatment of tumor-bearing mice with DNA-damaging cyclophosphamide/etoposide chemotherapy. Tumor and normal organ accumulation of Indium 111 (111In)-labeled La-specific DAB4 mAb as whole IgG or IgG fragments was quantified by whole-body static imaging and organ assay in tumor-bearing mice. Immunohistochemical measurements of tumor caspase-3 activation and PARP-1 cleavage, which are indicators of early and late apoptosis, respectively, were correlated with tumor accumulation of DAB4. Increased tumor accumulation of DAB4 was associated directly with both the extent of chemotherapy-induced tumor cell death and DAB4 binding per dead tumor cell. Tumor DAB4 accumulation correlated with cumulative caspase-3 activation and PARP-1 cleavage as tumor biomarkers of apoptosis and was directly related to the extended median survival time of tumor-bearing mice. Conclusions/Significance Radiolabeled La-specific monoclonal antibody, DAB4, detected dead tumor cells after chemotherapy, rather than chemosensitive normal tissues of gut and bone marrow. DAB4 identified late apoptotic tumor cells in vivo. Hence, radiolabeled DAB4 may usefully image responses to human carcinoma therapy because DAB4 would capture the protracted cell death of carcinoma. We believe that the ability of radiolabeled DAB4 to rapidly assess the apoptotic tumor response and, consequently, to potentially predict extended survival justifies its future clinical development as a radioimmunoscintigraphic agent. This article is part I of a two-part series providing proof-of-concept for the the diagnostic and therapeutic use of a La-specific monoclonal antibody, the DAB4 clone of which is represented by the registered trademark, APOMAB®.

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.

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.

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.

CCR7 facilitates the pro-inflammatory function of dendritic cells in experimental leishmaniasis.

Cutaneous leishmaniasis, caused by the parasite Leishmania major, results in lesions at the site of infection, which are self‐healing in resistant hosts. However, in the absence of the chemokine receptor CCR7, mice are unable to heal the lesion and develop chronic disease. These B6.CCR7−/− mice display an increased number of Th2 cells and immunosuppressive cytokine levels, as well as more regulatory T cells. As CCR7 is expressed on activated dendritic cells (DCs), and these cells require CCR7 to migrate to the draining lymph node, we expected decreased migration of DCs into the lymph node in the absence of CCR7 during cutaneous leishmaniasis. Consequently, in an attempt to initiate a self‐healing response, we adoptively transferred CCR7+ (B6.WT) DCs into the site of infection of B6.CCR7−/− mice. Surprisingly, instead of healing the lesion, B6.CCR7−/− mice inoculated with B6.WT DCs developed augmented lesions and showed increased immunosuppression compared to control B6.CCR7−/− mice transferred with B6.CCR7−/− DCs or B6.WT mice with B6.WT DCs. Finally, B6.WT mice injected with B6.CCR7−/− DCs also presented delayed healing of the lesion. These results indicate that CCR7 must be expressed on DCs, as well as peripheral cells, to allow an efficient immune response to L. major.

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.

Absence of Tumor Necrosis Factor Supports Alternative Activation of Macrophages in the Liver after Infection with Leishmania major

The absence of tumor necrosis factor (TNF) causes lethal infection by Leishmania major in normally resistant C57BL/6J (B6.WT) mice. The underlying pathogenic mechanism of this fatal disease has so far remained elusive. We found that B6.WT mice deficient for the tnf gene (B6.TNF−/−) displayed not only a non-healing cutaneous lesion but also a serious infection of the liver upon L. major inoculation. Infected B6.TNF−/− mice developed an enlarged liver that showed increased inflammation. Furthermore, we detected an accumulating monocyte-derived macrophage population (CD45+F4/80+CD11bhiLy6Clow) that displayed a M2 macrophage phenotype with high expression of CD206, arginase-1, and IL-6, supporting the notion that IL-6 could be involved in M2 differentiation. In in vitro experiments, we demonstrated that IL-6 upregulated M-CSF receptor expression and skewed monocyte differentiation from dendritic cells to macrophages. This was countered by the addition of TNF. Furthermore, TNF interfered with the activation of IL-6-induced gp130-signal transducer and activator of transcription (STAT) 3 and IL-4-STAT6 signaling, thereby abrogating IL-6-facilitated M2 macrophage polarization. Therefore, our results support the notion of a general role of TNF in the inflammatory activation of macrophages and define a new role of IL-6 signaling in macrophage polarization downstream of TNF.