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.

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.

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.

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.

BackgroundMucositis is a toxic side effect of anti-cancer treatments and is a major focus in cancer research. Pro-inflammatory cytokines have previously been implicated in the pathophysiology of chemotherapy-induced gastrointestinal mucositis. However, whether they play a key role in the development of radiotherapy-induced gastrointestinal mucositis is still unknown. Therefore, the aim of the present study was to characterise the expression of pro-inflammatory cytokines in the gastrointestinal tract using a rat model of fractionated radiotherapy-induced toxicity.MethodsThirty six female Dark Agouti rats were randomly assigned into groups and received 2.5 Gys abdominal radiotherapy three times a week over six weeks. Real time PCR was conducted to determine the relative change in mRNA expression of pro-inflammatory cytokines IL-1β, IL-6 and TNF in the jejunum and colon. Protein expression of IL-1β, IL-6 and TNF in the intestinal epithelium was investigated using qualitative immunohistochemistry.ResultsRadiotherapy-induced sub-acute damage was associated with significantly upregulated IL-1β, IL-6 and TNF mRNA levels in the jejunum and colon. The majority of pro-inflammatory cytokine protein expression in the jejunum and colon exhibited minimal change following fractionated radiotherapy.ConclusionsPro-inflammatory cytokines play a key role in radiotherapy-induced gastrointestinal mucositis in the sub-acute onset setting.

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 gH2AX 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 gH2AX. 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 viaTG2 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 DNAdamaging chemotherapy.

Purpose: To investigate the potential of the La-specific monoclonal antibody (mAb) 3B9 as an in vivo tumor-targeting agent. Experimental Design: The murine EL4 lymphoma cell line was used for in vitro studies and the EL4 model in which apoptosis was induced with cyclophosphamide and etoposide was used for in vivo studies. In vitro studies compared 3B9 binding in the EL4 cell with that in its counterpart primary cell type of the thymocyte. For in vivo studies, 3B9 was intrinsically or extrinsically labeled with carbon-14 or 1,4,7,10-tetra-azacylododecane-N,N ¶,N 00 ,N 0000 -tetraacetic acidî ndium-111, respectively, and biodistribution of the radiotracers was investigated in EL4 tumorbearing mice, which were treated or not with chemotherapy. Results: La-specific 3B9 mAb bound EL4 cells rather than thymocytes, and binding was detergent resistant. 3B9 binding to dead EL4 cells in vitro was specific, rapid, and saturable. Significantly, more 3B9 bound dead EL4 tumor explant cells after host mice were treated with chemotherapy, which suggested that DNA damage induced 3B9 binding. Tumor binding of 3B9 in vivo was antigen specific and increased significantly after chemotherapy. Tumor accumulation of 3B9 peaked at f50% of the injected dose per gram of tumor 72 h after chemotherapy and correlated with increased tumor cell death. Tumor/organ ratios of 3B9 biodistribution, which included the tumor/blood ratio, exceeded unity 48 or more hours after chemotherapy. Conclusions: La-specific mAb selectively targeted dead tumor cells in vivo, and targeting was augmented by cytotoxic chemotherapy. This novel cell death radioligand may be useful both for radioimmunoscintigraphy and radioimmunotherapy.

BackgroundAntineoplastic 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 FindingsBrisk 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/SignificanceRadiolabeled 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®.