RESUMEN
Medulloblastoma is the most common high-grade brain tumor in childhood. Medulloblastomas with c-myc amplification, classified as group 3, are the most aggressive among the four disease subtypes resulting in a 5-year overall survival of just above 50%. Despite current intensive therapy regimens, patients suffering from group 3 medulloblastoma urgently require new therapeutic options. Using a recently established c-myc amplified human medulloblastoma cell line, we performed an in-vitro-drug screen with single and combinatorial drugs that are either already clinically approved or agents in the advanced stage of clinical development. Candidate drugs were identified in vitro and then evaluated in vivo. Tumor growth was closely monitored by BLI. Vessel development was assessed by 3D light-sheet-fluorescence-microscopy. We identified the combination of gemcitabine and axitinib to be highly cytotoxic, requiring only low picomolar concentrations when used in combination. In the orthotopic model, gemcitabine and axitinib showed efficacy in terms of tumor control and survival. In both models, gemcitabine and axitinib were better tolerated than the standard regimen comprising of cisplatin and etoposide phosphate. 3D light-sheet-fluorescence-microscopy of intact tumors revealed thinning and rarefication of tumor vessels, providing one explanation for reduced tumor growth. Thus, the combination of the two drugs gemcitabine and axitinib has favorable effects on preventing tumor progression in an orthotopic group 3 medulloblastoma xenograft model while exhibiting a favorable toxicity profile. The combination merits further exploration as a new approach to treat high-risk group 3 medulloblastoma.
Asunto(s)
Antineoplásicos/farmacología , Axitinib/farmacología , Desoxicitidina/análogos & derivados , Resistencia a Antineoplásicos/efectos de los fármacos , Amplificación de Genes , Meduloblastoma/genética , Proteínas Proto-Oncogénicas c-myc/genética , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Desoxicitidina/farmacología , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/patología , Ratones , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
Donor CD4(+)Foxp3(+) regulatory T cells (T reg cells) suppress graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (HCT [allo-HCT]). Current clinical study protocols rely on the ex vivo expansion of donor T reg cells and their infusion in high numbers. In this study, we present a novel strategy for inhibiting GvHD that is based on the in vivo expansion of recipient T reg cells before allo-HCT, exploiting the crucial role of tumor necrosis factor receptor 2 (TNFR2) in T reg cell biology. Expanding radiation-resistant host T reg cells in recipient mice using a mouse TNFR2-selective agonist before allo-HCT significantly prolonged survival and reduced GvHD severity in a TNFR2- and T reg cell-dependent manner. The beneficial effects of transplanted T cells against leukemia cells and infectious pathogens remained unaffected. A corresponding human TNFR2-specific agonist expanded human T reg cells in vitro. These observations indicate the potential of our strategy to protect allo-HCT patients from acute GvHD by expanding T reg cells via selective TNFR2 activation in vivo.
Asunto(s)
Enfermedad Injerto contra Huésped/prevención & control , Receptores Tipo II del Factor de Necrosis Tumoral/fisiología , Linfocitos T Reguladores/inmunología , Enfermedad Aguda , Animales , Femenino , Enfermedad Injerto contra Huésped/inmunología , Trasplante de Células Madre Hematopoyéticas , Interleucina-2/farmacología , Ratones , Ratones Endogámicos , Células Supresoras de Origen Mieloide/fisiologíaRESUMEN
BACKGROUND: Medulloblastoma is the most common malignant brain tumor in children and can be divided in different molecular subgroups. Patients whose tumor is classified as a Group 3 tumor have a dismal prognosis. However only very few tumor models are available for this subgroup. METHODS: We established a robust orthotopic xenograft model with a cell line derived from the malignant pleural effusions of a child suffering from a Group 3 medulloblastoma. RESULTS: Besides classical characteristics of this tumor subgroup, the cells display cancer stem cell characteristics including neurosphere formation, multilineage differentiation, CD133/CD15 expression, high ALDH-activity and high tumorigenicity in immunocompromised mice with xenografts exactly recapitulating the original tumor architecture. CONCLUSIONS: This model using unmanipulated, human medulloblastoma cells will enable translational research, specifically focused on Group 3 medulloblastoma.
Asunto(s)
Meduloblastoma/patología , Neoplasias Experimentales/patología , Animales , Biomarcadores de Tumor , Línea Celular Tumoral , Femenino , Humanos , Lactante , Masculino , Ratones , Ratones SCID , Células Madre Neoplásicas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Inhibition of the tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible 14 (Fn14) system reduces intestinal cell death and disease development in several models of colitis. In view of the crucial role of TNF and intestinal cell death in graft-versus-host disease (GVHD) and the ability of TWEAK to enhance TNF-induced cell death, we tested here the therapeutic potential of Fn14 blockade on allogeneic hematopoietic cell transplantation (allo-HCT)-induced intestinal GVHD. An Fn14-specific blocking human immunoglobulin G1 antibody variant with compromised antibody-dependent cellular cytotoxicity (ADCC) activity strongly inhibited the severity of murine allo-HCT-induced GVHD. Treatment of the allo-HCT recipients with this monoclonal antibody reduced cell death of gastrointestinal cells but neither affected organ infiltration by donor T cells nor cytokine production. Fn14 blockade also inhibited intestinal cell death in mice challenged with TNF. This suggests that the protective effect of Fn14 blockade in allo-HCT is based on the protection of intestinal cells from TNF-induced apoptosis and not due to immune suppression. Importantly, Fn14 blockade showed no negative effect on graft-versus-leukemia/lymphoma (GVL) activity. Thus, ADCC-defective Fn14-blocking antibodies are not only possible novel GVL effect-sparing therapeutics for the treatment of GVHD but might also be useful for the treatment of other inflammatory bowel diseases where TNF-induced cell death is of relevance.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Apoptosis , Enfermedad Injerto contra Huésped/prevención & control , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Intestinos/patología , Receptores del Factor de Necrosis Tumoral/antagonistas & inhibidores , Inhibidores del Factor de Necrosis Tumoral , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , Western Blotting , Células Cultivadas , Citocina TWEAK , Modelos Animales de Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/metabolismo , Enfermedad Injerto contra Huésped/patología , Humanos , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/inmunología , Mucosa Intestinal/metabolismo , Intestinos/inmunología , Mediciones Luminiscentes , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/inmunología , Receptores del Factor de Necrosis Tumoral/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rituximab , Receptor de TWEAK , Factor de Necrosis Tumoral alfa/farmacología , Factores de Necrosis Tumoral/inmunología , Factores de Necrosis Tumoral/metabolismoRESUMEN
Cytokine secretion and degranulation represent key components of CD8(+) T-cell cytotoxicity. While transcriptional blockade of IFN-γ and inhibition of degranulation by TGF-ß are well established, we wondered whether TGF-ß could also induce immune-regulatory miRNAs in human CD8(+) T cells. We used miRNA microarrays and high-throughput sequencing in combination with qRT-PCR and found that TGF-ß promotes expression of the miR-23a cluster in human CD8(+) T cells. Likewise, TGF-ß up-regulated expression of the cluster in CD8(+) T cells from wild-type mice, but not in cells from mice with tissue-specific expression of a dominant-negative TGF-ß type II receptor. Reporter gene assays including site mutations confirmed that miR-23a specifically targets the 3'UTR of CD107a/LAMP1 mRNA, whereas the further miRNAs expressed in this cluster-namely, miR-27a and -24-target the 3'UTR of IFN-γ mRNA. Upon modulation of the miR-23a cluster by the respective miRNA antagomirs and mimics, we observed significant changes in IFN-γ expression, but only slight effects on CD107a/LAMP1 expression. Still, overexpression of the cluster attenuated the cytotoxic activity of antigen-specific CD8(+) T cells. These functional data thus reveal that the miR-23a cluster not only is induced by TGF-ß, but also exerts a suppressive effect on CD8(+) T-cell effector functions, even in the absence of TGF-ß signaling.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Citotoxicidad Inmunológica , Epítopos de Linfocito T/inmunología , Interferón gamma/metabolismo , MicroARNs/genética , Factor de Crecimiento Transformador beta/metabolismo , Regiones no Traducidas 3' , Secuencia de Bases , Sitios de Unión , Linfocitos T CD8-positivos/efectos de los fármacos , Línea Celular , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interferón gamma/química , Interferón gamma/genética , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Proteína 1 de la Membrana Asociada a los Lisosomas/química , Proteína 1 de la Membrana Asociada a los Lisosomas/genética , Antígeno MART-1/inmunología , Familia de Multigenes , Interferencia de ARN , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
To promote cancer research and to develop innovative therapies, refined pre-clinical mouse tumor models that mimic the actual disease in humans are of dire need. A number of neoplasms along the B cell lineage are commonly initiated by a translocation recombining c-myc with the immunoglobulin heavy-chain gene locus. The translocation is modeled in the C.129S1-Igha(tm1(Myc)Janz)/J mouse which has been previously engineered to express c-myc under the control of the endogenous IgH promoter. This transgenic mouse exhibits B cell hyperplasia and develops diverse B cell tumors. We have isolated tumor cells from the spleen of a C.129S1-Igha(tm1(Myc)Janz)/J mouse that spontaneously developed a plasmablastic lymphoma-like disease. These cells were cultured, transduced to express eGFP and firefly luciferase, and gave rise to a highly aggressive, transplantable B cell lymphoma cell line, termed IM380. This model bears several advantages over other models as it is genetically induced and mimics the translocation that is detectable in a number of human B cell lymphomas. The growth of the tumor cells, their dissemination, and response to treatment within immunocompetent hosts can be imaged non-invasively in vivo due to their expression of firefly luciferase. IM380 cells are radioresistant in vivo and mice with established tumors can be allogeneically transplanted to analyze graft-versus-tumor effects of transplanted T cells. Allogeneic hematopoietic stem cell transplantation of tumor-bearing mice results in prolonged survival. These traits make the IM380 model very valuable for the study of B cell lymphoma pathophysiology and for the development of innovative cancer therapies.
Asunto(s)
Diagnóstico por Imagen/métodos , Mediciones Luminiscentes/métodos , Linfoma de Células B/patología , Animales , Linfocitos B , Línea Celular Tumoral , Femenino , Citometría de Flujo , Trasplante de Células Madre Hematopoyéticas , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Trasplante HomólogoRESUMEN
Multiple activities are ascribed to the cytokine tumor necrosis factor (TNF) in health and disease. In particular, TNF was shown to affect carcinogenesis in multiple ways. This cytokine acts via the activation of two cell surface receptors, TNFR1, which is associated with inflammation, and TNFR2, which was shown to cause anti-inflammatory signaling. We assessed the effects of TNF and its two receptors on the progression of pancreatic cancer by in vivo bioluminescence imaging in a syngeneic orthotopic tumor mouse model with Panc02 cells. Mice deficient for TNFR1 were unable to spontaneously reject Panc02 tumors and furthermore displayed enhanced tumor progression. In contrast, a fraction of wild type (37.5%), TNF deficient (12.5%), and TNFR2 deficient mice (22.2%) were able to fully reject the tumor within two weeks. Pancreatic tumors in TNFR1 deficient mice displayed increased vascular density, enhanced infiltration of CD4(+) T cells and CD4(+) forkhead box P3 (FoxP3)(+) regulatory T cells (Treg) but reduced numbers of CD8(+) T cells. These alterations were further accompanied by transcriptional upregulation of IL4. Thus, TNF and TNFR1 are required in pancreatic ductal carcinoma to ensure optimal CD8(+) T cell-mediated immunosurveillance and tumor rejection. Exogenous systemic administration of human TNF, however, which only interacts with murine TNFR1, accelerated tumor progression. This suggests that TNFR1 has basically the capability in the Panc02 model to trigger pro-and anti-tumoral effects but the spatiotemporal availability of TNF seems to determine finally the overall outcome.
Asunto(s)
Carcinoma Ductal/fisiopatología , Regulación de la Expresión Génica/inmunología , Neoplasias Pancreáticas/fisiopatología , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Linfocitos T CD4-Positivos/inmunología , Carcinoma Ductal/inmunología , Carcinoma Ductal/metabolismo , Línea Celular Tumoral , Cartilla de ADN/genética , Citometría de Flujo , Interleucina-4/metabolismo , Mediciones Luminiscentes , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Receptores Tipo II del Factor de Necrosis Tumoral/deficiencia , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Src-kinase inhibitors hold great potential as targeted therapy against malignant cells. However, such inhibitors may also affect nonmalignant cells and cause pronounced off-target effects. We investigated the role of the dual kinase inhibitor dasatinib on human myeloid cells. Dasatinib is clinically used for the treatment of bcr/abl⺠leukemias because it blocks the mutated tyrosine kinase abl. To understand its effect on the development of antigen-specific T-cell responses, we assessed antigen-specific priming of human, naïve T cells. In surprising contrast to the direct inhibition of T-cell activation by dasatinib, pretreatment of maturing dendritic cells (DCs) with dasatinib strongly enhanced their stimulatory activity. This effect strictly depended on the activating DC stimulus and led to enhanced interleukin 12 (IL-12) production and T-cell responses of higher functional avidity. Src-kinase inhibitors, and not conventional tyrosine kinase inhibitors, increased IL-12 production in several cell types of myeloid origin, such as monocytes and classical or nonclassical DCs. Interestingly, only human cells, but not mouse or macaques DCs, were affected. These data highlight the potential immunostimulatory capacity of a group of novel drugs, src-kinase inhibitors, thereby opening new opportunities for chemoimmunotherapy. These data also provide evidence for a regulatory role of src kinases in the activation of myeloid cells.