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BACKGROUND & AIMS: High expression of phosphatidylinositol 4-kinase III alpha (PI4KIIIα) correlates with poor survival rates in patients with hepatocellular carcinoma. In addition, hepatitis C virus (HCV) infections activate PI4KIIIα and contribute to hepatocellular carcinoma progression. We aimed at mechanistically understanding the impact of PI4KIIIα on the progression of liver cancer and the potential contribution of HCV in this process. METHODS: Several hepatic cell culture and mouse models were used to study the functional importance of PI4KIIIα on liver pathogenesis. Antibody arrays, gene silencing, and PI4KIIIα-specific inhibitor were applied to identify the involved signaling pathways. The contribution of HCV was examined by using HCV infection or overexpression of its nonstructural protein. RESULTS: High PI4KIIIα expression and/or activity induced cytoskeletal rearrangements via increased phosphorylation of paxillin and cofilin. This led to morphologic alterations and higher migratory and invasive properties of liver cancer cells. We further identified the liver-specific lipid kinase phosphatidylinositol 3-kinase C2 domain-containing subunit gamma (PIK3C2γ) working downstream of PI4KIIIα in regulation of the cytoskeleton. PIK3C2γ generates plasma membrane phosphatidylinositol 3,4-bisphosphate-enriched, invadopodia-like structures that regulate cytoskeletal reorganization by promoting Akt2 phosphorylation. CONCLUSIONS: PI4KIIIα regulates cytoskeleton organization via PIK3C2γ/Akt2/paxillin-cofilin to favor migration and invasion of liver cancer cells. These findings provide mechanistic insight into the contribution of PI4KIIIα and HCV to the progression of liver cancer and identify promising targets for therapeutic intervention.
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Factores Despolimerizantes de la Actina , Carcinoma Hepatocelular , Movimiento Celular , Citoesqueleto , Neoplasias Hepáticas , Invasividad Neoplásica , Paxillin , Transducción de Señal , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Humanos , Animales , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Citoesqueleto/metabolismo , Citoesqueleto/patología , Paxillin/metabolismo , Ratones , Factores Despolimerizantes de la Actina/metabolismo , Factores Despolimerizantes de la Actina/genética , Fosforilación , Hepacivirus , Línea Celular Tumoral , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Células Hep G2 , Hepatitis C/patología , Hepatitis C/metabolismo , Hepatitis C/virología , Interferencia de ARNRESUMEN
Vorticity has recently been suggested to be a property of highly spinning black holes. The connection between vorticity and limiting spin represents a universal feature shared by objects of maximal microstate entropy, so-called saturons. Using Q-ball-like saturons as a laboratory for black holes, we study the collision of two such objects and find that vorticity can have a large impact on the emitted radiation as well as on the charge and angular momentum of the final configuration. As black holes belong to the class of saturons, we expect that the formation of vortices can cause similar effects in black hole mergers, leading to macroscopic deviations in gravitational radiation. This could leave unique signatures detectable with upcoming gravitational-wave searches, which can thereby serve as a portal to macroscopic quantum effects in black holes.
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The stress-associated molecular chaperone system is an actionable target in cancer therapies. It is ubiquitously upregulated in cancer tissues and enables tumorigenicity by stabilizing hundreds of oncoproteins and disturbing the stoichiometry of protein complexes. Most inhibitors target the key component heat-shock protein 90 (HSP90). However, although classical HSP90 inhibitors are highly tumor-selective, they fail in phase 3 clinical oncology trials. These failures are at least partly due to an interference with a negative feedback loop by HSP90 inhibition, known as heat-shock response (HSR): in response to HSP90 inhibition there is compensatory synthesis of stress-inducible chaperones, mediated by the transcription factor heat-shock factor 1 (HSF1). We recently identified that wildtype p53 (p53) actively reduces the HSR by repressing HSF1 via a p21-CDK4/6-MAPK-HSF1 axis. Here we test the hypothesis that in HSP90-based therapies simultaneous p53 activation or direct cell cycle inhibition interrupts the deleterious HSF1-HSR axis and improves the efficiency of HSP90 inhibitors. Indeed, we find that the clinically relevant p53 activator Idasanutlin suppresses the HSF1-HSR activity in HSP90 inhibitor-based therapies. This combination synergistically reduces cell viability and accelerates cell death in p53-proficient colorectal cancer (CRC) cells, murine tumor-derived organoids and patient-derived organoids (PDOs). Mechanistically, upon combination therapy human CRC cells strongly upregulate p53-associated pathways, apoptosis, and inflammatory immune pathways. Likewise, in the chemical AOM/DSS CRC model in mice, dual HSF1-HSP90 inhibition strongly represses tumor growth and remodels immune cell composition, yet displays only minor toxicities in mice and normal mucosa-derived organoids. Importantly, inhibition of the cyclin dependent kinases 4 and 6 (CDK4/6) under HSP90 inhibition phenocopies synergistic repression of the HSR in p53-proficient CRC cells. Even more important, in p53-deficient (mutp53-harboring) CRC cells, an HSP90 inhibition in combination with CDK4/6 inhibitors similarly suppresses the HSF1-HSR system and reduces cancer growth. Likewise, p53-mutated PDOs strongly respond to dual HSF1-HSP90 pathway inhibition and thus, providing a strategy to target CRC independent of the p53 status. In sum, activating p53 (in p53-proficient cancer cells) or inhibiting CDK4/6 (independent of the p53 status) provide new options to improve the clinical outcome of HSP90-based therapies and to enhance colorectal cancer therapy.
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Background: Checkpoint inhibitors act on exhausted CD8+ T cells and restore their effector function in chronic infections and cancer. The underlying mechanisms of action appear to differ between different types of cancer and are not yet fully understood. Methods: Here, we established a new orthotopic HCC model to study the effects of checkpoint blockade on exhausted CD8+ tumor-infiltrating lymphocytes (TILs). The tumors expressed endogenous levels of HA, which allowed the study of tumor-specific T cells. Results: The induced tumors developed an immune-resistant TME in which few T cells were found. The few recovered CD8+ TILs were mostly terminally exhausted and expressed high levels of PD-1. PD-1/CTLA-4 blockade resulted in a strong increase in the number of CD8+ TILs expressing intermediate amounts of PD-1, also called progenitor-exhausted CD8+ TILs, while terminally exhausted CD8+ TILs were almost absent in the tumors of treated mice. Although transferred naïve tumor-specific T cells did not expand in the tumors of untreated mice, they expanded strongly after treatment and generated progenitor-exhausted but not terminally exhausted CD8+ TILs. Unexpectedly, progenitor-exhausted CD8+ TILs mediated the antitumor response after treatment with minimal changes in their transcriptional profile. Conclusion: In our model, few doses of checkpoint inhibitors during the priming of transferred CD8+ tumor-specific T cells were sufficient to induce tumor remission. Therefore, PD-1/CTLA-4 blockade has an ameliorative effect on the expansion of recently primed CD8+ T cells while preventing their development into terminally exhausted CD8+ TILs in the TME. This finding could have important implications for future T-cell therapies.
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We argue that black holes admit vortex structure. This is based both on a graviton-condensate description of a black hole as well as on a correspondence between black holes and generic objects with maximal entropy compatible with unitarity, so-called saturons. We show that due to vorticity, a Q-ball-type saturon of a calculable renormalizable theory obeys the same extremality bound on the spin as the black hole. Correspondingly, a black hole with extremal spin emerges as a graviton condensate with vorticity. This offers a topological explanation for the stability of extremal black holes against Hawking evaporation. Next, we show that in the presence of mobile charges, the global vortex traps a magnetic flux of the gauge field. This can have macroscopically observable consequences. For instance, the most powerful jets observed in active galactic nuclei can potentially be accounted for. As a signature, such emissions can occur even without a magnetized accretion disk surrounding the black hole. The flux entrapment can provide an observational window to various hidden sectors, such as millicharged dark matter.
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BACKGROUND: In hepatocellular carcinoma (HCC), histone deacetylases (HDACs) are frequently overexpressed. This results in chromatin compaction and silencing of tumor-relevant genes and microRNAs. Modulation of microRNA expression is a potential treatment option for HCC. Therefore, we aimed to characterize the epigenetically regulated miR-129-5p regarding its functional effects and target genes to understand its relevance for HCC tumorigenesis. METHODS: Global miRNA expression of HCC cell lines (HLE, HLF, Huh7, HepG2, Hep3B) and normal liver cell lines (THLE-2, THLE-3) was analyzed after HDAC inhibition by miRNA sequencing. An in vivo xenograft mouse model and in vitro assays were used to investigate tumor-relevant functional effects following miR-129-5p transfection of HCC cells. To validate hepatoma-derived growth factor (HDGF) as a direct target gene of miR-129-5p, luciferase reporter assays were performed. Survival data and HDGF expression were analyzed in public HCC datasets. After siRNA-mediated knockdown of HDGF, its cancer-related functions were examined. RESULTS: HDAC inhibition induced the expression of miR-129-5p. Transfection of miR-129-5p increased the apoptosis of HCC cells, decreased proliferation, migration and ERK signaling in vitro and inhibited tumor growth in vivo. Direct binding of miR-129-5p to the 3'UTR of HDGF via a noncanonical binding site was validated by luciferase reporter assays. HDGF knockdown reduced cell viability and migration and increased apoptosis in Wnt-inactive HCC cells. These in vitro results were in line with the analysis of public HCC datasets showing that HDGF overexpression correlated with a worse survival prognosis, primarily in Wnt-inactive HCCs. CONCLUSIONS: This study provides detailed insights into the regulatory network of the tumor-suppressive, epigenetically regulated miR-129-5p in HCC. Our results reveal for the first time that the therapeutic application of mir-129-5p may have significant implications for the personalized treatment of patients with Wnt-inactive, advanced HCC by directly regulating HDGF. Therefore, miR-129-5p is a promising candidate for a microRNA replacement therapy to prevent HCC progression and tumor metastasis.
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Virotherapy research involves the development, exploration, and application of oncolytic viruses that combine direct killing of cancer cells by viral infection, replication, and spread (oncolysis) with indirect killing by induction of anti-tumor immune responses. Oncolytic viruses can also be engineered to genetically deliver therapeutic proteins for direct or indirect cancer cell killing. In this review-as part of the special edition on "State-of-the-Art Viral Vector Gene Therapy in Germany"-the German community of virotherapists provides an overview of their recent research activities that cover endeavors from screening and engineering viruses as oncolytic cancer therapeutics to their clinical translation in investigator-initiated and sponsored multi-center trials. Preclinical research explores multiple viral platforms, including new isolates, serotypes, or fitness mutants, and pursues unique approaches to engineer them towards increased safety, shielded or targeted delivery, selective or enhanced replication, improved immune activation, delivery of therapeutic proteins or RNA, and redirecting antiviral immunity for cancer cell killing. Moreover, several oncolytic virus-based combination therapies are under investigation. Clinical trials in Germany explore the safety and potency of virotherapeutics based on parvo-, vaccinia, herpes, measles, reo-, adeno-, vesicular stomatitis, and coxsackie viruses, including viruses encoding therapeutic proteins or combinations with immune checkpoint inhibitors. These research advances represent exciting vantage points for future endeavors of the German virotherapy community collectively aimed at the implementation of effective virotherapeutics in clinical oncology.
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Neoplasias/terapia , Viroterapia Oncolítica , Virus Oncolíticos , Animales , Ensayos Clínicos como Asunto , Ingeniería Genética , Alemania , Humanos , Virus Oncolíticos/genéticaRESUMEN
BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer and a highly lethal malignancy. Chemotherapeutic options are limited, but a considerable subset of patients harbors genetic lesions for which targeted agents exist. Fibroblast growth factor receptor 2 (FGFR2) fusions belong to the most frequent and therapeutically relevant alterations in ICC, and the first FGFR inhibitor was recently approved for the treatment of patients with progressed, fusion-positive ICC. Response rates of up to 35% indicate that FGFR-targeted therapies are beneficial in many but not all patients. Thus far, no established biomarkers exist that predict resistance or response to FGFR-targeted therapies in patients with ICC. APPROACH AND RESULTS: In this study, we use an autochthonous murine model of ICC to demonstrate that FGFR2 fusions are potent drivers of malignant transformation. Furthermore, we provide preclinical evidence that the co-mutational spectrum acts not only as an accelerator of tumor development, but also modifies the response to targeted FGFR inhibitors. Using pharmacologic approaches and RNA-interference technology, we delineate that Kirsten rat sarcoma oncogene (KRAS)-activated mitogen-activated protein kinase signaling causes primary resistance to FGFR inhibitors in FGFR2 fusion-positive ICC. The translational relevance is supported by the observation that a subset of human FGFR2 fusion patients exhibits transcriptome profiles reminiscent of KRAS mutant ICC. Moreover, we demonstrate that combination therapy has the potential to overcome primary resistance and to sensitize tumors to FGFR inhibition. CONCLUSIONS: Our work highlights the importance of the co-mutational spectrum as a significant modifier of response in tumors that harbor potent oncogenic drivers. A better understanding of the genetic underpinnings of resistance will be pivotal to improve biomarker-guided patient selection and to design clinically relevant combination strategies.
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Neoplasias de los Conductos Biliares/genética , Conductos Biliares Intrahepáticos , Transformación Celular Neoplásica/genética , Colangiocarcinoma/genética , Fusión Génica/genética , Neoplasias Hepáticas Experimentales/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Adenosilhomocisteinasa/genética , Animales , Antígenos de Neoplasias/genética , Antimetabolitos Antineoplásicos/farmacología , Neoplasias de los Conductos Biliares/patología , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Colangiocarcinoma/patología , Proteínas Co-Represoras/genética , Proteína de Unión al Elemento de Respuesta al AMP Cíclico/genética , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Proteínas Fetales/genética , Ratones , Proteínas Asociadas a Microtúbulos/genética , Mutación , Compuestos de Fenilurea/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Proteínas de Transporte Vesicular/genética , GemcitabinaRESUMEN
BACKGROUND AND AIMS: Programmed death 1 (PD-1) checkpoint inhibition has shown promising results in patients with hepatocellular carcinoma, inducing objective responses in approximately 20% of treated patients. The roles of other coinhibitory molecules and their individual contributions to T-cell dysfunction in liver cancer, however, remain largely elusive. APPROACH AND RESULTS: We performed a comprehensive mRNA profiling of cluster of differentiation 8 (CD8) T cells in a murine model of autochthonous liver cancer by comparing the transcriptome of naive, functional effector, and exhausted, tumor-specific CD8 T cells. Subsequently, we functionally validated the role of identified genes in T-cell exhaustion. Our results reveal a unique transcriptome signature of exhausted T cells and demonstrate that up-regulation of the inhibitory immune receptor T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitor motif domains (TIGIT) represents a hallmark in the process of T-cell exhaustion in liver cancer. Compared to PD-1, expression of TIGIT more reliably identified exhausted CD8 T cells at different stages of their differentiation. In combination with PD-1 inhibition, targeting of TIGIT with antagonistic antibodies resulted in synergistic inhibition of liver cancer growth in immunocompetent mice. Finally, we demonstrate expression of TIGIT on tumor-infiltrating CD8 T cells in tissue samples of patients with hepatocellular carcinoma and intrahepatic cholangiocarcinoma and identify two subsets of patients based on differential expression of TIGIT on tumor-specific T cells. CONCLUSIONS: Our transcriptome analysis provides a valuable resource for the identification of key pathways involved in T-cell exhaustion in patients with liver cancer and identifies TIGIT as a potential target in checkpoint combination therapies.
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Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/inmunología , Linfocitos T CD8-positivos/inmunología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/inmunología , Colangiocarcinoma/genética , Colangiocarcinoma/inmunología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/inmunología , Receptores Inmunológicos/genética , Transcriptoma , Anciano , Animales , Neoplasias de los Conductos Biliares/patología , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Colangiocarcinoma/patología , Modelos Animales de Enfermedad , Quimioterapia Combinada , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptores Inmunológicos/antagonistas & inhibidores , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacosRESUMEN
BACKGROUND: Nerve-cancer interactions are increasingly recognized to be of paramount importance for the emergence and progression of pancreatic cancer (PCa). Here, we investigated the role of indirect cholinergic activation on PCa progression through inhibition of acetylcholinesterase (AChE) via clinically available AChE-inhibitors, i.e. physostigmine and pyridostigmine. METHODS: We applied immunohistochemistry, immunoblotting, MTT-viability, invasion, flow-cytometric-cell-cycle-assays, phospho-kinase arrays, multiplex ELISA and xenografted mice to assess the impact of AChE inhibition on PCa cell growth and invasiveness, and tumor-associated inflammation. Survival analyses were performed in a novel genetically-induced, surgically-resectable mouse model of PCa under adjuvant treatment with gemcitabine+/-physostigmine/pyridostigmine (n = 30 mice). Human PCa specimens (n = 39) were analyzed for the impact of cancer AChE expression on tumor stage and survival. RESULTS: We discovered a strong expression of AChE in cancer cells of human PCa specimens. Inhibition of this cancer-cell-intrinsic AChE via pyridostigmine and physostigmine, or administration of acetylcholine (ACh), diminished PCa cell viability and invasion in vitro and in vivo via suppression of pERK signaling, and reduced tumor-associated macrophage (TAM) infiltration and serum pro-inflammatory cytokine levels. In the novel genetically-induced, surgically-resectable PCa mouse model, adjuvant co-therapy with AChE blockers had no impact on survival. Accordingly, survival of resected PCa patients did not differ based on tumor AChE expression levels. Patients with higher-stage PCa also exhibited loss of the ACh-synthesizing enzyme, choline-acetyltransferase (ChAT), in their nerves. CONCLUSION: For future clinical trials of PCa, direct cholinergic stimulation of the muscarinic signaling, rather than indirect activation via AChE blockade, may be a more effective strategy.
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Colina O-Acetiltransferasa/metabolismo , Colinérgicos/farmacología , Inflamación/prevención & control , Neoplasias Pancreáticas/tratamiento farmacológico , Acetilcolina/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Apoptosis , Ciclo Celular , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Persona de Mediana Edad , Invasividad Neoplásica , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Pronóstico , Tasa de Supervivencia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tumor-selective replicating "oncolytic" viruses are novel and promising tools for immunotherapy of cancer. However, despite their first success in clinical trials, previous experience suggests that currently used oncolytic virus monotherapies will not be effective enough to achieve complete tumor responses and long-term cure in a broad spectrum of cancers. Nevertheless, there are reasonable arguments that suggest advanced oncolytic viruses will play an essential role as enablers of multi-stage immunotherapies including established systemic immunotherapies. Oncolytic adenoviruses (oAds) display several features to meet this therapeutic need. oAds potently lyse infected tumor cells and induce a strong immunogenic cell death associated with tumor inflammation and induction of antitumor immune responses. Furthermore, established and versatile platforms of oAds exist, which are well suited for the incorporation of heterologous genes to optimally exploit and amplify the immunostimulatory effect of viral oncolysis. A considerable spectrum of functional genes has already been integrated in oAds to optimize particular aspects of immune stimulation including antigen presentation, T cell priming, engagement of additional effector functions, and interference with immunosuppression. These advanced concepts have the potential to play a promising future role as enablers of multi-stage immunotherapies involving adoptive cell transfer and systemic immunotherapies.
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Beyond the nearly uniform presence of KRAS mutations, pancreatic cancer is increasingly recognized as a heterogeneous disease. Preclinical in vivo model systems exist, but with the advent of precision oncology, murine models with enhanced genetic flexibility are needed to functionally annotate genetic alterations found in the human malignancy. Here, we describe the generation of focal gene disruptions and large chromosomal deletions via inducible and pancreas-specific expression of Cas9 in adult mice. Experimental mice are derived on demand directly from genetically engineered embryonic stem cells, without the need for further intercrossing. To provide initial validation of our approach, we show that disruption of the E3 ubiquitin ligase Rnf43 accelerates KrasG12D-dependent tumourigenesis. Moreover, we demonstrate that this system can be used to rapidly interrogate the impact of complex cancer-associated alleles through the generation of a previously unstudied 1.2 megabase deletion surrounding the CDKN2A and CDKN2B tumour suppressors. Thus, our approach is capable of reproducibly generating biallelic and precise loss of large chromosomal fragments that, in conjunction with mutant Kras, leads to development of pancreatic ductal adenocarcinoma with full penetrance.
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Carcinogénesis/genética , Carcinoma Ductal Pancreático/genética , Edición Génica , Neoplasias Pancreáticas/genética , Animales , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Genoma Humano/genética , Humanos , Ratones , Mutación/genética , Páncreas/patología , Neoplasias Pancreáticas/patología , Medicina de Precisión , Eliminación de Secuencia/genética , Neoplasias PancreáticasRESUMEN
Tumor-selectively replicating "oncolytic" adenoviruses based on serotype 5 are promising tools for the treatment of solid tumors. However, their effective delivery to the tumor by systemic administration remains challenging. Several strategies of molecular retargeting have been pursued to equip adenoviruses with molecular features that facilitate their efficient uptake by tumors and to protect healthy tissue from damage. Transductional retargeting can be conveniently achieved using bispecific molecular adapter proteins based on the ectodomain of the coxsackievirus and adenovirus receptor linked to tumor ligands of choice. In this chapter, we describe methods for their design, purification, and application.
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Adenoviridae/genética , Terapia Genética , Vectores Genéticos/genética , Neoplasias/terapia , Viroterapia Oncolítica , Virus Oncolíticos/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Modelos Animales de Enfermedad , Terapia Genética/métodos , Humanos , Neoplasias/etiología , Neoplasias/patología , Viroterapia Oncolítica/métodosRESUMEN
Tumour immunotherapies have been a breakthrough in clinical oncology but only a few patients benefit from this progress. Additional interventions that sensitize immunologically cold tumours for the administration of checkpoint modifiers are urgently needed. In this issue of EMBO Molecular Medicine, Aznar et al present the already approved yellow fever vaccine 17D as an oncolytic agent for tumour immunoactivation. In tumour-bearing mice, they demonstrated a convincing synergy of the vaccine with CD137 agonistic antibodies resulting in significantly improved survival.
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Neoplasias , Vacuna contra la Fiebre Amarilla , Animales , Humanos , Inmunoterapia , Ratones , Vacunas AtenuadasRESUMEN
Gallbladder cancer is associated with a dismal prognosis, and accurate in vivo models will be elemental to improve our understanding of this deadly disease and develop better treatment options. We have generated a transplantation-based murine model for gallbladder cancer that histologically mimics the human disease, including the development of distant metastasis. Murine gallbladder-derived organoids are genetically modified by either retroviral transduction or transfection with CRISPR/Cas9 encoding plasmids, thereby allowing the rapid generation of complex cancer genotypes. We characterize the model in the presence of two of the most frequent oncogenic drivers-Kras and ERBB2-and provide evidence that the tumor histology is highly dependent on the driver oncogene. Further, we demonstrate the utility of the model for the preclinical assessment of novel therapeutic approaches by showing that liposomal Irinotecan (Nal-IRI) is retained in tumor cells and significantly prolongs the survival of gallbladder cancer-bearing mice compared to conventional irinotecan.
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Virus-neutralizing antibodies are a severe obstacle in oncolytic virotherapy. Here, we present a strategy to convert this unfavorable immune response into an anticancer immunotherapy via molecular retargeting. Application of a bifunctional adapter harboring a tumor-specific ligand and the adenovirus hexon domain DE1 for engaging antiadenoviral antibodies, attenuates tumor growth and prolongs survival in adenovirus-immunized mice. The therapeutic benefit achieved by tumor retargeting of antiviral antibodies is largely due to NK cell-mediated triggering of tumor-directed CD8 T-cells. We further demonstrate that antibody-retargeting (Ab-retargeting) is a feasible method to sensitize tumors to PD-1 immune checkpoint blockade. In therapeutic settings, Ab-retargeting greatly improves the outcome of intratumor application of an oncolytic adenovirus and facilitates long-term survival in treated animals when combined with PD-1 checkpoint inhibition. Tumor-directed retargeting of preexisting or virotherapy-induced antiviral antibodies therefore represents a promising strategy to fully exploit the immunotherapeutic potential of oncolytic virotherapy and checkpoint inhibition.
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Adenoviridae/inmunología , Anticuerpos/inmunología , Inmunoterapia/métodos , Neoplasias/terapia , Viroterapia Oncolítica/métodos , Virus Oncolíticos/inmunología , Adenoviridae/genética , Animales , Anticuerpos Neutralizantes/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Células HEK293 , Humanos , Células Asesinas Naturales/inmunología , Ratones , Terapia Molecular Dirigida/métodos , Neoplasias/inmunología , Virus Oncolíticos/genéticaRESUMEN
INTRODUCTION: Cholangiocarcinoma is characterized by aggressive tumor growth, high recurrence rates, and resistance against common chemotherapeutical regimes. The polyether-antibiotic Salinomycin is a promising drug in cancer therapy because of its ability to overcome apoptosis resistance of cancer cells and its selectivity against cancer stem cells. Here, we investigated the effectiveness of Salinomycin against cholangiocarcinoma in vivo, and analyzed interference of Salinomycin with autophagic flux in human cholangiocarcinoma cells. RESULTS: Salinomycin reduces tumor cell viability, proliferation, migration, invasion, and induced apoptosis in vitro. Subcutaneous and intrahepatic cholangiocarcinoma growth in vivo was inhibited upon Salinomycin treatment. Analysis of autophagy reveals inhibition of autophagic activity. This was accompanied by accumulation of mitochondrial mass and increased generation of reactive oxygen species. CONCLUSIONS: This study demonstrates the effectiveness of Salinomycin against cholangiocarcinoma in vivo. Inhibition of autophagic flux represents an underlying molecular mechanism of Salinomycin against cholangiocarcinoma. METHODS: The two murine cholangiocarcinoma cell lines p246 and p254 were used to analyze tumor cell proliferation, viability, migration, invasion, and apoptosis in vitro. For in vivo studies, murine cholangiocarcinoma cells were injected into syngeneic C57-BL/6-mice to initiate subcutaneous cholangiocarcinoma growth. Intrahepatic tumor growth was induced by electroporation of oncogenic transposon-plasmids into the left liver lobe. For mechanistic studies in human cells, TFK-1 and EGI-1 were used, and activation of autophagy was analyzed after exposure to Salinomycin.
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Genetic replacement of adenoviral fiber knobs by ligands that enable tumor specific targeting of oncolytic adenoviruses is challenging because the fiber knob contributes to virus assembly. Here, we present a novel concept by describing stable recombinant adenoviruses with tumor specific infection mode. The fiber knob was replaced by endosialidaseNF (endoNF), the tailspike protein of bacteriophage K1F. EndoNF recognizes polysialic acid, an oncofetal antigen characteristic for high malignant tumors of neuroendocrine origin. An intramolecular chaperone contained in endoNF warrants folding and compensates for the knob function in virus assembly. Obtained recombinant viruses demonstrated polysialic acid dependent infection modes, strong oncolytic capacity with polysialic acid positive cells in culture and a high potential to inhibit tumor growth in a therapeutic mouse model of subcutaneous neuroblastoma. With a single genetic manipulation we achieved ablation of the fiber knob, introduction of a tumor specific ligand, and folding control over the chimeric fiber construct.
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Adenoviridae , Neoplasias/terapia , Neuraminidasa , Viroterapia Oncolítica , Virus Oncolíticos , Ácidos Siálicos , Animales , Bacteriófagos/enzimología , Células HEK293 , Humanos , Ratones , Neoplasias/metabolismo , Neuraminidasa/metabolismo , Neuraminidasa/uso terapéutico , Neuroblastoma/terapia , Proteínas Virales/metabolismo , Proteínas Virales/uso terapéuticoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and disseminating cancer resistant to therapy, including checkpoint immunotherapies, and early tumor resection and (neo)adjuvant chemotherapy fails to improve a poor prognosis. In a transgenic mouse model of resectable PDAC, we investigated the coordinated activation of T and natural killier (NK) cells in addition to gemcitabine chemotherapy to prevent tumor recurrence. Only neoadjuvant, but not adjuvant treatment with a PD-1 antagonist effectively supported chemotherapy and suppressed local tumor recurrence and improved survival involving both NK and T cells. Local T-cell activation was confirmed by increased tumor infiltration with CD103+CD8+ T cells and neoantigen-specific CD8 T lymphocytes against the marker neoepitope LAMA4-G1254V. To achieve effective prevention of distant metastases in a complementary approach, we blocked the NK-cell checkpoint CD96, an inhibitory NK-cell receptor that binds CD155, which was abundantly expressed in primary PDAC and metastases of human patients. In gemcitabine-treated mice, neoadjuvant PD-1 blockade followed by adjuvant inhibition of CD96 significantly prevented relapse of PDAC, allowing for long-term survival. In summary, our results show in an aggressively growing transgenic mouse model of PDAC that the coordinated activation of both innate and adaptive immunity can effectively reduce the risk of tumor recurrence after surgery, facilitating long-term remission of this lethal disease.Significance: Coordinated neoadjuvant and adjuvant immunotherapies reduce the risk of disease relapse after resection of murine PDAC, suggesting this concept for future clinical trials. Cancer Res; 78(2); 475-88. ©2017 AACR.