RESUMEN
The wild Tasmanian devil (Sarcophilus harrisii) population has suffered a devastating decline due to two clonal transmissible cancers. The first devil facial tumor 1 (DFT1) was observed in 1996, followed by a second genetically distinct transmissible tumor, the devil facial tumor 2 (DFT2), in 2014. DFT1/2 frequently metastasize, with lymph nodes being common metastatic sites. MHC-I downregulation by DFT1 cells is a primary means of evading allograft immunity aimed at polymorphic MHC-I proteins. DFT2 cells constitutively express MHC-I, and MHC-I is upregulated on DFT1/2 cells by interferon gamma, suggesting other immune evasion mechanisms may contribute to overcoming allograft and anti-tumor immunity. Human clinical trials have demonstrated PD1/PDL1 blockade effectively treats patients showing increased expression of PD1 in tumor draining lymph nodes, and PDL1 on peritumoral immune cells and tumor cells. The effects of DFT1/2 on systemic immunity remain largely uncharacterized. This study applied the open-access software QuPath to develop a semiautomated pipeline for whole slide analysis of stained tissue sections to quantify PD1/PDL1 expression in devil lymph nodes. The QuPath protocol provided strong correlations to manual counting. PD-1 expression was approximately 10-fold higher than PD-L1 expression in lymph nodes and was primarily expressed in germinal centers, whereas PD-L1 expression was more widely distributed throughout the lymph nodes. The density of PD1 positive cells was increased in lymph nodes containing DFT2 metastases, compared to DFT1. This suggests PD1/PDL1 exploitation may contribute to the poorly immunogenic nature of transmissible tumors in some devils and could be targeted in therapeutic or prophylactic treatments.Abbreviations: PD1: programmed cell death protein 1; PDL1: programmed death ligand 1; DFT1: devil facial tumor 1; DFT2: devil facial tumor 2; DFTD: devil facial tumor disease; MCC: Matthew's correlation coefficient; DAB: diaminobenzidine; ROI: region of interest.
Asunto(s)
Antígeno B7-H1 , Neoplasias Faciales , Humanos , Antígeno B7-H1/genética , Receptor de Muerte Celular Programada 1/genética , Ganglios Linfáticos/patología , Microambiente TumoralRESUMEN
The devil facial tumour disease (DFTD) has led to a massive decline in the wild Tasmanian devil (Sarcophilus harrisii) population. The disease is caused by two independent devil facial tumours (DFT1 and DFT2). These transmissible cancers have a mortality rate of nearly 100â%. An adenoviral vector-based vaccine has been proposed as a conservation strategy for the Tasmanian devil. This study aimed to determine if a human adenovirus serotype 5 could express functional transgenes in devil cells. As DFT1 cells do not constitutively express major histocompatibility complex class I (MHC-I), we developed a replication-deficient adenoviral vector that encodes devil interferon gamma (IFN-γ) fused to a fluorescent protein reporter. Our results show that adenoviral-expressed IFN-γ was able to stimulate upregulation of beta-2 microglobulin, a component of MHC-I, on DFT1, DFT2 and devil fibroblast cell lines. This work suggests that human adenoviruses can serve as a vaccine platform for devils and potentially other marsupials.
Asunto(s)
Infecciones por Adenoviridae , Adenovirus Humanos , Neoplasias Faciales , Marsupiales , Animales , Humanos , Adenovirus Humanos/genética , Interferón gamma , Adenoviridae/genética , Neoplasias Faciales/genética , Neoplasias Faciales/veterinaria , Antígenos de Histocompatibilidad Clase I/genéticaRESUMEN
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.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias Faciales/veterinaria , Marsupiales/fisiología , Proteoma/análisis , Células de Schwann/patología , Transcriptoma , Animales , Biomarcadores de Tumor/genética , Neoplasias Faciales/genética , Neoplasias Faciales/metabolismo , Neoplasias Faciales/patología , Humanos , Células de Schwann/metabolismoRESUMEN
Introduction: Macrophage phagocytosis of pathogens and tumour cells is an important early event in protection against infectious disease and cancer. As tumour necrosis factor α (TNF) is an important cytokine in macrophage activation, we investigated the involvement of TNF in macrophage phagocytosis of tumour cells. Methods: We used Devil Facial Tumour Disease (DFTD) cancer cells as the target tumour cells. The Tasmanian devil (Sarcophilus harrisii) population is threatened by the transmissible DFTD. Using DFTD cells provided the opportunity to determine if these cells can be phagocytosed and investigate requirement for TNF. As effector cells, bone marrow derived macrophages (BMDMs), generated from C57BL/6 wild type (B6.WT) and C57BL/6 TNF-/- (B6.TNF-/-) mice were used. Phagocytosis of DFTD cells was investigated by confocal microscopy and flow cytometry. Results: DFTD cells were consistently phagocytosed by B6.WT and B6.TNF-/- BMDMs with similar efficiency in vitro. Consequently the DFTD cells are not resistant to phagocytosis. Following activation by exposure to IFNγ and LPS or LPS alone, B6.TNF-/- BMDMs had higher phagocytic efficiency and lower nitric oxide (NO) production compared to wild-type controls. In addition, NO seems to be unlikely to be the involved in phagocytosis efficiency in IFNγ and LPS activated B6.TNF-/- macrophages and consequences thereof. Conclusion: Our results indicate that TNF is not required for IFNγ and LPS or LPS alone activation of macrophage phagocytosis. TNF may negatively regulate macrophage phagocytosis of tumour cells.
Asunto(s)
Neoplasias Faciales/inmunología , Neoplasias Faciales/veterinaria , Macrófagos/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Línea Celular Tumoral , Células Cultivadas , Neoplasias Faciales/patología , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Marsupiales , Ratones Endogámicos C57BL , Ratones Mutantes , Óxido Nítrico/metabolismo , Fagocitosis , Factor de Necrosis Tumoral alfa/deficienciaRESUMEN
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.
Asunto(s)
Marsupiales/fisiología , Neoplasias/veterinaria , Alelos , Animales , Rotura Cromosómica , Análisis Citogenético , Exones/genética , Genoma , Geografía , Haplotipos/genética , Cariotipificación , Repeticiones de Microsatélite/genética , Datos de Secuencia Molecular , Neoplasias/genética , Neoplasias/patología , Polimorfismo de Nucleótido Simple/genética , Tasmania , Cromosoma X/genéticaRESUMEN
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.
Asunto(s)
Neoplasias Faciales/patología , Leucocitos Mononucleares/citología , Marsupiales/metabolismo , Mitógenos/farmacología , Animales , Formación de Anticuerpos/efectos de los fármacos , Formación de Anticuerpos/inmunología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Concanavalina A/farmacología , Medios de Cultivo Condicionados/farmacología , Citotoxicidad Inmunológica/efectos de los fármacos , Neoplasias Faciales/inmunología , Interleucina-2/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Poli I-C/farmacología , Receptor Toll-Like 3/agonistasRESUMEN
The hybridoma method for production of monoclonal antibodies has been a cornerstone of biomedical research for several decades. Here we convert the monoclonal antibody sequence from mouse-derived hybridomas into a "devilized" recombinant antibody with devil IgG heavy chain and IgK light chain. The chimeric recombinant antibody can be used in functional assays, immunotherapy, and to improve understanding of antibodies and Fc receptors in Tasmanian devils. The process can be readily modified for other species.
Asunto(s)
Hibridomas , Inmunoglobulina G , Marsupiales , Animales , Ratones , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Hibridomas/inmunología , Marsupiales/inmunología , Marsupiales/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Immune checkpoint immunotherapy is a pillar of human oncology treatment with potential for non-human species. The first checkpoint immunotherapy approved for human cancers targeted the CTLA4 protein. CTLA4 can inhibit T cell activation by capturing and internalizing CD80 and CD86 from antigen presenting cells, a process called trans-endocytosis. Similarly, CD28 can capture CD80 and CD86 via trogocytosis and retain the captured ligands on the surface of the CD28-expressing cells. The wild Tasmanian devil (Sarcophilus harrisii) population has declined by 77% due to transmissible cancers that evade immune defenses despite genetic mismatches between the host and tumors. We used a live cell-based assay to demonstrate that devil CTLA4 and CD28 can capture CD80 and CD86. Mutation of evolutionarily conserved motifs in CTLA4 altered functional interactions with CD80 and CD86 in accordance with patterns observed in other species. These results suggest that checkpoint immunotherapies can be translated to evolutionarily divergent species.
Asunto(s)
Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Antígenos CD28/metabolismo , Antígeno CTLA-4/metabolismo , Marsupiales/inmunología , Secuencias de Aminoácidos/genética , Animales , Antígenos CD28/antagonistas & inhibidores , Células CHO , Antígeno CTLA-4/antagonistas & inhibidores , Antígeno CTLA-4/genética , Células Cultivadas , Clonación Molecular , Cricetulus , Especies en Peligro de Extinción , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Microscopía Intravital , Marsupiales/metabolismo , Mutación , TrogocitosisRESUMEN
PURPOSE: Downregulation of MHC class I (MHC-I) is a common immune evasion strategy of many cancers. Similarly, two allogeneic clonal transmissible cancers have killed thousands of wild Tasmanian devils (Sarcophilus harrisii) and also modulate MHC-I expression to evade anti-cancer and allograft responses. IFNG treatment restores MHC-I expression on devil facial tumor (DFT) cells but is insufficient to control tumor growth. Transcriptional co-activator NLRC5 is a master regulator of MHC-I in humans and mice but its role in transmissible cancers remains unknown. In this study, we explored the regulation and role of MHC-I in these unique genetically mis-matched tumors. METHODS: We used transcriptome and flow cytometric analyses to determine how MHC-I shapes allogeneic and anti-tumor responses. Cell lines that overexpress NLRC5 to drive antigen presentation, and B2M-knockout cell lines incapable of presenting antigen on MHC-I were used to probe the role of MHC-I in rare cases of tumor regressions. RESULTS: Transcriptomic results suggest that NLRC5 plays a major role in MHC-I regulation in devils. NLRC5 was shown to drive the expression of many components of the antigen presentation pathway but did not upregulate PDL1. Serum from devils with tumor regressions showed strong binding to IFNG-treated and NLRC5 cell lines; antibody binding to IFNG-treated and NRLC5 transgenic tumor cells was diminished or absent following B2M knockout. CONCLUSION: MHC-I could be identified as a target for anti-tumor and allogeneic immunity. Consequently, 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.
Asunto(s)
Presentación de Antígeno/inmunología , Biomarcadores de Tumor/metabolismo , Neoplasias Faciales/inmunología , Regulación Neoplásica de la Expresión Génica , Antígenos de Histocompatibilidad Clase I/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Animales , Biomarcadores de Tumor/genética , Neoplasias Faciales/genética , Neoplasias Faciales/metabolismo , Neoplasias Faciales/patología , Antígenos de Histocompatibilidad Clase I/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Marsupiales , Transcriptoma , Células Tumorales CultivadasRESUMEN
This protocol provides a step-by-step method to create recombinant fluorescent fusion proteins that can be secreted from mammalian cell lines. This builds on many other recombinant protein and fluorescent protein techniques, but is among the first to harness fluorescent fusion proteins secreted directly into cell culture supernatant. This opens new possibilities that are not achievable with proteins produced in bacteria or yeast, such as direct use of the fluorescent protein-secreting cells in live co-culture assays. The Fluorescent Adaptable Simple Theranostic (FAST) protein system includes a histidine purification tag and a tobacco etch virus (TEV) cleavage site, allowing the purification tag and fluorescent protein to be removed for therapeutic use. This protocol is split into five parts: (A) In silico characterization of the gene-of-interest (GOI) and protein-of-interest (POI); (B) design of the expression vector; (C) assembly of the expression vector; (D) transfection of a eukaryotic cell line with the expression vector; (E) testing of the recombinant protein. This extensive protocol can be completed with only polymerase chain reaction (PCR) and cell culture training. Additionally, each part of the protocol can be used independently.
RESUMEN
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.
Asunto(s)
Neoplasias Faciales , Marsupiales , Animales , Neoplasias Faciales/metabolismo , Neoplasias Faciales/patología , Evasión Inmune , InmunoterapiaRESUMEN
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 gammaH2AX 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 gammaH2AX. 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.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Autoantígenos/metabolismo , Cisplatino/toxicidad , Roturas del ADN de Doble Cadena/efectos de los fármacos , ADN de Neoplasias/efectos de los fármacos , Neoplasias/patología , Ribonucleoproteínas/metabolismo , Apoptosis/fisiología , Autoantígenos/inmunología , Cromatina/metabolismo , Reactivos de Enlaces Cruzados/farmacología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Proteínas de Unión al GTP/metabolismo , Histonas/metabolismo , Humanos , Immunoblotting , Linfocitos/efectos de los fármacos , Monocitos/efectos de los fármacos , Necrosis , Neoplasias/genética , Neoplasias/metabolismo , Proteína Glutamina Gamma Glutamiltransferasa 2 , Ribonucleoproteínas/antagonistas & inhibidores , Ribonucleoproteínas/inmunología , Transglutaminasas/metabolismo , Células Tumorales Cultivadas , Antígeno SS-BRESUMEN
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'',N''''-tetraacetic acid-indium-111, respectively, and biodistribution of the radiotracers was investigated in EL4 tumor-bearing 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 approximately 50% 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.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Apoptosis/efectos de los fármacos , Autoantígenos/inmunología , Linfoma de Células T/terapia , Ribonucleoproteínas/inmunología , Animales , Antineoplásicos Alquilantes/farmacología , Antineoplásicos Fitogénicos/farmacología , Apoptosis/fisiología , Terapia Combinada , Ciclofosfamida/farmacología , Daño del ADN/efectos de los fármacos , Etopósido/farmacología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Immunoblotting , Inmunoconjugados , Radioisótopos de Indio/farmacocinética , Linfoma de Células T/inmunología , Linfoma de Células T/patología , Ratones , Ratones Endogámicos C57BL , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Radioinmunoterapia , Timo/citología , Timo/efectos de los fármacos , Timo/metabolismo , Distribución Tisular , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Antígeno SS-BRESUMEN
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.
Asunto(s)
Interleucina-6/inmunología , Hígado/inmunología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Factor de Necrosis Tumoral alfa/genética , Animales , Arginasa/biosíntesis , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Receptor gp130 de Citocinas/metabolismo , Inflamación/inmunología , Interleucina-4/metabolismo , Interleucina-6/biosíntesis , Lectinas Tipo C/biosíntesis , Leishmania major/inmunología , Leishmaniasis Cutánea/inmunología , Leishmaniasis Cutánea/parasitología , Hígado/parasitología , Hígado/patología , Activación de Macrófagos/genética , Macrófagos/citología , Receptor de Manosa , Lectinas de Unión a Manosa/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/citología , Carga de Parásitos , Receptor de Factor Estimulante de Colonias de Macrófagos/biosíntesis , Receptores de Superficie Celular/biosíntesis , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT6/metabolismoRESUMEN
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.
Asunto(s)
Antígenos de Neoplasias/inmunología , Neoplasias Faciales/prevención & control , Proteínas de Choque Térmico/metabolismo , Marsupiales/inmunología , Adyuvantes Inmunológicos , Animales , Anticuerpos/inmunología , Formación de Anticuerpos , Vacunas contra el Cáncer/inmunología , Línea Celular , Extinción Biológica , Neoplasias Faciales/tratamiento farmacológico , Neoplasias Faciales/inmunología , Inmunoterapia , Espectrometría de Masas , Proteoma , TasmaniaRESUMEN
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.
Asunto(s)
Neoplasias Faciales/inmunología , Inmunización/métodos , Inmunoterapia/métodos , Marsupiales/inmunología , Animales , Formación de Anticuerpos/inmunología , Neoplasias Faciales/terapia , Neoplasias Faciales/veterinaria , Femenino , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunidad Humoral/inmunología , Masculino , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Aminoquinolinas/farmacología , Apoptosis/efectos de los fármacos , Neoplasias Faciales/patología , Neoplasias Cutáneas/patología , Animales , Apoptosis/genética , Línea Celular Tumoral , Humanos , Imiquimod , MarsupialesRESUMEN
A sixth locus (vsp417-6) belonging to the vsp417 gene subfamily, a subset of the family of genes that encodes 'variant-specific' surface proteins (VSP) in Giardia, is described. The sequence of vsp417-6(A-I), the ortholog representing the vsp417-6 locus in isolates of the type A-I (Assemblage A, Group I) genotype of Giardia intestinalis, was determined from a cloned 5.5-kb Hind III fragment of genomic DNA derived from isolate Ad-1/C1. The gene encodes a 704 residue polypeptide (VSP417-6(A-I), Mr 71,674) that has 75% identity (92% similarity) over a 718 residue overlap with the prototype of the VSP417 subfamily, VSP417-1(A-I)-encoded by the vsp417-1 (syn. tsa417) locus in type A-I isolates. Alignment of VSP417-6(A-I) with the deduced sequences of other known members of this subfamily identified one polypeptide, encoded by a gene found in type A-II (Assemblage A, Group II) isolates, whose homology with VSP417-6(A-I) (91% identity, 98% similarity over 713-residues) indicated that it was VSP417-6(A-II), the VSP417-6 ortholog in type A-II isolates. Sequence-based phylogenetic analyses of known VSP417 subfamily members defined several loci that predate the emergence of the A-I and A-II sublineages of G. intestinalis. Related sequences that may correspond to additional, uncharacterised vsp417 subfamily genes were identified in genomic DNA by Southern hybridisation using subfamily- and locus-specific probes. Variant-specific expression of vsp417-1 and vsp417-6 within axenic cultures of G. intestinalis was detected by in situ mRNA hybridization, indicating that these genes are functional and that they are expressed in an alternative fashion with other vsp genes in these organisms.
Asunto(s)
Antígenos de Protozoos/genética , Antígenos de Superficie/genética , Genes Protozoarios , Giardia lamblia/genética , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Animales , Variación Antigénica , Antígenos de Protozoos/metabolismo , Antígenos de Superficie/metabolismo , Evolución Molecular , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas Protozoarias/metabolismo , ARN Mensajero/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: The lupus-associated (La)-specific murine monoclonal antibody DAB4 (APOMAB®) specifically binds dead cancer cells. Using DAB4, we examined La expression in human lung cancer samples to assess its suitability as a cancer-selective therapeutic target. We evaluated the safety and effectiveness of radioimmunotherapy (RIT) using DAB4 radiolabeled with Lutetium-177 (177Lu) in the murine Lewis Lung (LL2) carcinoma model, and determined whether combining RIT with DNA-damaging cisplatin-based chemotherapy, a PARP inhibitor (PARPi), or both alters treatment responses. METHODS: The expression of La mRNA in human lung cancer samples was analysed using the online database Oncomine, and the protein expression of La was examined using a TissueFocus Cancer Survey Tissue Microarray. The binding of DAB4 to cisplatin-treated LL2 cells was assessed in vitro. LL2 tumour-bearing mice were administered escalating doses of 177Lu-DAB4 alone or in combination with chemotherapy, and tumour growth and survival measured. Biodistribution analysis was used to determine tissue uptake of 177Lu-DAB4 or its isotype control (177Lu-Sal5), when delivered alone or after chemotherapy. PARPi (rucaparib; AG-014699) was combined with chemotherapy and the effects of combined treatment on tumour growth, tumour cell DNA damage and death, and intratumoural DAB4 binding were also analysed. The effect of the triple combination of PARPi, chemotherapy and 177Lu-DAB4 on tumour growth and survival of LL2 tumour-bearing mice was tested. RESULTS: La was over-expressed at both mRNA and protein levels in surgical specimens of human lung cancer and the over-expression of La mRNA conferred a poorer prognosis. DAB4 bound specifically to cisplatin-induced dead LL2 cells in vitro. An anti-tumour dose response was observed when escalating doses of 177Lu-DAB4 were delivered in vivo, with supra-additive responses observed when chemotherapy was combined with 177Lu-DAB4. Combining PARPi with chemotherapy was more effective than chemotherapy alone with increased tumour cell DNA damage and death, and intratumoural DAB4 binding. The combination of PARPi, chemotherapy and 177Lu-DAB4 was well-tolerated and maximised tumour growth delay. CONCLUSIONS: The La antigen represents a dead cancer cell-specific target in lung cancer, and DAB4 specifically targeted tumour tissue in vivo, particularly after chemotherapy. Tumour uptake of DAB4 increased further after the combination of PARPi and chemotherapy, which generated new dead tumour cell-binding targets. Consequently, combining 177Lu-DAB4 with PARPi and chemotherapy produced the greatest anti-tumour response. Therefore, the triple combination of PARPi, chemotherapy and RIT may have broad clinical utility.
RESUMEN
UNLABELLED: Early identification of tumor responses to treatment is crucial for devising more effective and safer cancer treatments. No widely applicable, noninvasive method currently exists for specifically detecting tumor cell death after cytotoxic treatment and thus for predicting treatment outcomes. METHODS: We have further characterized the targeting of the murine monoclonal antibody DAB4 specifically to dead tumor cells in vitro, in vivo, and in clinical samples. We found that sustained DAB4 binding to treated cells was closely associated with markers of intrinsic apoptosis and DNA double-strand break formation. In a competition binding assay, DAB4 bound EL4 murine thymic lymphoma cells in preference to the normal counterpart of murine thymocytes. Defective in vivo clearance of apoptotic cells augmented in vivo accumulation of DAB4 in tumors particularly after chemotherapy but was unchanged in normal tissues. Tumor targeting of DAB4 was selective for syngeneic murine tumors and for human tumor xenografts of prostate cancer (PC-3) and pancreatic cancer (Panc-1) before and more so after chemotherapy. Furthermore, DAB4 was shown to bind to dead primary acute lymphoblastic leukemic blasts cultured with cytotoxic drugs and dead epithelial cancer cells isolated from peripheral blood of small cell lung carcinoma patients given chemotherapy. CONCLUSION: Collectively, these results further demonstrate the selectivity of DAB4 for chemotherapy-induced dead tumor cells. This postchemotherapy selectivity is related to a relative increase in the availability of DAB4-binding targets in tumor tissue rather than in normal tissues. The in vitro findings were translated in vivo to human xenograft models and to ex vivo analyses of clinical samples, providing further evidence of the potential of DAB4 as a marker of tumor cell death after DNA-damaging cytotoxic treatment that could be harnessed as a predictive marker of treatment responses.