RESUMEN
Missense mutations in the p53 tumor suppressor inactivate its antiproliferative properties but can also promote metastasis through a gain-of-function activity. We show that sustained expression of mutant p53 is required to maintain the prometastatic phenotype of a murine model of pancreatic cancer, a highly metastatic disease that frequently displays p53 mutations. Transcriptional profiling and functional screening identified the platelet-derived growth factor receptor b (PDGFRb) as both necessary and sufficient to mediate these effects. Mutant p53 induced PDGFRb through a cell-autonomous mechanism involving inhibition of a p73/NF-Y complex that represses PDGFRb expression in p53-deficient, noninvasive cells. Blocking PDGFRb signaling by RNA interference or by small molecule inhibitors prevented pancreatic cancer cell invasion in vitro and metastasis formation in vivo. Finally, high PDGFRb expression correlates with poor disease-free survival in pancreatic, colon, and ovarian cancer patients, implicating PDGFRb as a prognostic marker and possible target for attenuating metastasis in p53 mutant tumors.
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Carcinoma Ductal Pancreático/metabolismo , Metástasis de la Neoplasia , Neoplasias Pancreáticas/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Carcinoma Ductal Pancreático/patología , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Humanos , Ratones , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Proteína p53 Supresora de Tumor/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
Genetically engineered mouse models (GEMMs) have greatly expanded our knowledge of pancreatic ductal adenocarcinoma (PDAC) and serve as a critical tool to identify and evaluate new treatment strategies. However, the cost and time required to generate conventional pancreatic cancer GEMMs limits their use for investigating novel genetic interactions in tumor development and maintenance. To address this problem, we developed flexible embryonic stem cell (ESC)-based GEMMs that facilitate the rapid generation of genetically defined multiallelic chimeric mice without further strain intercrossing. The ESCs harbor a latent Kras mutant (a nearly ubiquitous feature of pancreatic cancer), a homing cassette, and other genetic elements needed for rapid insertion and conditional expression of tetracycline-controlled transgenes, including fluorescence-coupled shRNAs capable of efficiently silencing gene function by RNAi. This system produces a disease that recapitulates the progression of pancreatic cancer in human patients and enables the study and visualization of the impact of gene perturbation at any stage of pancreas cancer progression. We describe the use of this approach to dissect temporal roles for the tumor suppressor Pten and the oncogene c-Myc in pancreatic cancer development and maintenance.
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Modelos Animales de Enfermedad , Células Madre Embrionarias , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Animales , Animales Modificados Genéticamente , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Técnicas de Silenciamiento del Gen , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Reproducibilidad de los ResultadosRESUMEN
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
The outstanding clinical success of immune checkpoint blockade has revived the interest in underlying mechanisms of the immune system that are capable of eliminating tumors even in advanced stages. In this scenario, CD4 and CD8 T cell responses are part of the cancer immune cycle and both populations significantly influence the clinical outcome. In general, the immune system has evolved several mechanisms to protect the host against cancer. Each of them has to be undermined or evaded during cancer development to enable tumor outgrowth. In this review, we give an overview of T lymphocyte-driven control of tumor growth and discuss the involved tumor-suppressive mechanisms of the immune system, such as senescence surveillance, cancer immunosurveillance, and cancer immunoediting with respect to recent clinical developments of immunotherapies. The main focus is on the currently existing knowledge about the CD4 and CD8 T lymphocyte interplay that mediates the control of tumor growth.
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Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/fisiología , Comunicación Celular/inmunología , Proliferación Celular , Neoplasias/inmunología , Neoplasias/patología , Animales , Humanos , Inmunoterapia/métodos , Escape del Tumor/inmunologíaRESUMEN
Genome-wide analysis at the level of single cells has recently emerged as a powerful tool to dissect genome heterogeneity in cancer, neurobiology, and development. To be truly transformative, single-cell approaches must affordably accommodate large numbers of single cells. This is feasible in the case of copy number variation (CNV), because CNV determination requires only sparse sequence coverage. We have used a combination of bioinformatic and molecular approaches to optimize single-cell DNA amplification and library preparation for highly multiplexed sequencing, yielding a method that can produce genome-wide CNV profiles of up to a hundred individual cells on a single lane of an Illumina HiSeq instrument. We apply the method to human cancer cell lines and biopsied cancer tissue, thereby illustrating its efficiency, reproducibility, and power to reveal underlying genetic heterogeneity and clonal phylogeny. The capacity of the method to facilitate the rapid profiling of hundreds to thousands of single-cell genomes represents a key step in making single-cell profiling an easily accessible tool for studying cell lineage.
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Variaciones en el Número de Copia de ADN , ADN de Neoplasias/genética , Reacción en Cadena de la Polimerasa Multiplex/métodos , Análisis de Secuencia de ADN/métodos , Análisis de la Célula Individual/métodos , Algoritmos , Secuencia de Bases , Línea Celular Tumoral , Genoma Humano , Humanos , Datos de Secuencia MolecularRESUMEN
OBJECTIVES: The clinical relevance of spontaneous portosystemic shunts detected by ultrasound is insufficiently investigated. The aim of this retrospective study was to assess the frequency and clinical relevance of spontaneous portosystemic shunts in patients with liver cirrhosis. METHODS: We evaluated portosystemic shunts, liver cirrhosis and spleen size by ultrasound in 982 patients with liver cirrhosis and correlated these with laboratory results, clinical data and the incidence of clinical endpoint deaths, liver transplantation and the development of HCC during the follow-up period (mean 1.26 ± 1.53 years [range 0-7.2 years]). RESULTS: Portosystemic shunts were detected in 34% of the patients. These patients had a higher rate of alcohol-related cirrhosis (37% vs. 30%, p = .003), a higher MELD score (p < .001) and Child-Pugh grade (p < .001), as well as more frequent hepatic encephalopathy (p < .001) and oesophageal varices (p < .003). The most frequent portosystemic shunt in this cohort was an umbilical vein shunt (69%) followed by splenorenal (16%), mesenteric (7%) and combined/other shunts (8%). Patients with umbilical vein shunts had a higher rate of alcohol-related cirrhosis (p = .041) and suffered more frequently from Child B/C stages (p = .03), hepatorenal syndrome (p = .03), massive ascites (p = .001) and spontaneous bacterial peritonitis (p = .011). CONCLUSIONS: Patients with portosystemic shunts that are detected by ultrasound should be monitored carefully as these patients are associated with advanced liver disease and multiple clinical risk factors.
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Encefalopatía Hepática/complicaciones , Hipertensión Portal/diagnóstico por imagen , Cirrosis Hepática/fisiopatología , Bazo/diagnóstico por imagen , Ultrasonografía , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Ascitis/complicaciones , Niño , Várices Esofágicas y Gástricas/complicaciones , Femenino , Humanos , Hipertensión Portal/etiología , Modelos Lineales , Cirrosis Hepática/complicaciones , Masculino , Persona de Mediana Edad , Vena Porta/diagnóstico por imagen , Estudios Retrospectivos , Centros de Atención Terciaria , Adulto JovenRESUMEN
BACKGROUND & AIMS: Even after potentially curative R0 resection, patients with pancreatic ductal adenocarcinoma (PDAC) have a poor prognosis owing to high rates of local recurrence and metastasis to distant organs. However, we have no suitable transgenic animal models for surgical interventions. METHODS: To induce formation of pancreatic tumor foci, we electroporated oncogenic plasmids into pancreata of LSL-KrasG12D × p53fl/fl mice; mutant Kras was expressed in p53fl/fl mice using a sleeping beauty transposon. We co-delivered a transposon encoding a constitutively active form of Akt2 (myrAkt2). Carcinogenesis and histopathologic features of tumors were examined. Metastasis was monitored by bioluminescence imaging. Tumors were resected and mice were given gemcitabine, and tumor recurrence patterns and survival were determined. Immune cells were collected from resection sites and analyzed by flow cytometry and in depletion experiments. RESULTS: After electroporation of oncogenic plasmids, mice developed a single pancreatic tumor nodule with histopathologic features of human PDAC. Pancreatic tumors that expressed myrAkt2 infiltrated the surrounding pancreatic tissue and neurons and became widely metastatic, reflecting the aggressive clinical features of PDAC in patients. Despite early tumor resection, mice died from locally recurring and distant tumors, but adjuvant administration of gemcitabine after tumor resection prolonged survival. In mice given adjuvant gemcitabine or vehicle, gemcitabine significantly inhibited local recurrence of tumors, but not metastasis to distant organs, similar to observations in clinical trials. Gemcitabine inhibited accumulation of CD11b+Gr1intF4/80int myeloid-derived suppressor cells at the resection margin and increased the number of natural killer (NK) cells at this location. NK cells but not T cells were required for gemcitabine-mediated antitumor responses. CONCLUSIONS: Gemcitabine administration after resection of pancreatic tumors in mice activates NK cell-mediated antitumor responses and inhibits local recurrence of tumors, consistent with observations from patients with PDAC. Transgenic mice with resectable pancreatic tumors might be promising tools to study adjuvant therapy strategies for patients.
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Antimetabolitos Antineoplásicos/farmacología , Desoxicitidina/análogos & derivados , Células Asesinas Naturales/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológico , Animales , Terapia Combinada , Desoxicitidina/farmacología , Modelos Animales de Enfermedad , Ratones , Invasividad Neoplásica , Recurrencia Local de Neoplasia/prevención & control , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/cirugía , Proteínas Proto-Oncogénicas c-akt/metabolismo , GemcitabinaRESUMEN
There is evidence that viral oncolysis is synergistic with immune checkpoint inhibition in cancer therapy but the underlying mechanisms are unclear. Here, we investigated whether local viral infection of malignant tumors is capable of overcoming systemic resistance to PD-1-immunotherapy by modulating the spectrum of tumor-directed CD8 T-cells. To focus on neoantigen-specific CD8 T-cell responses, we performed transcriptomic sequencing of PD-1-resistant CMT64 lung adenocarcinoma cells followed by algorithm-based neoepitope prediction. Investigations on neoepitope-specific T-cell responses in tumor-bearing mice demonstrated that PD-1 immunotherapy was insufficient whereas viral oncolysis elicited cytotoxic T-cell responses to a conserved panel of neoepitopes. After combined treatment, we observed that PD-1-blockade did not affect the magnitude of oncolysis-mediated antitumoral immune responses but a broader spectrum of T-cell responses including additional neoepitopes was observed. Oncolysis of the primary tumor significantly abrogated systemic resistance to PD-1-immunotherapy leading to improved elimination of disseminated lung tumors. Our observations were confirmed in a transgenic murine model of liver cancer where viral oncolysis strongly induced PD-L1 expression in primary liver tumors and lung metastasis. Furthermore, we demonstrated that combined treatment completely inhibited dissemination in a CD8 T-cell-dependent manner. Therefore, our results strongly recommend further evaluation of virotherapy and concomitant PD-1 immunotherapy in clinical studies.
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Neoplasias/etiología , Neoplasias/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Infecciones Tumorales por Virus , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Antineoplásicos , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Modelos Animales de Enfermedad , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Expresión Génica , Inmunoterapia , Isoinjertos , Ligandos , Ratones , Ratones Transgénicos , Mutación , Neoplasias/patología , Neoplasias/terapia , Viroterapia Oncolítica , Virus Oncolíticos/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptores Toll-Like/metabolismoRESUMEN
Cholangiocarcinoma is the second most common primary liver cancer and responds poorly to existing therapies. Intrahepatic cholangiocarcinoma (ICC) likely originates from the biliary tree and develops within the hepatic parenchyma. We have generated a flexible orthotopic allograft mouse model of ICC that incorporates common genetic alterations identified in human ICC and histologically resembles the human disease. We examined the utility of this model to validate driver alterations in ICC and tested their suitability as therapeutic targets. Specifically, we showed that the fused-in-glioblastoma-c-ros-oncogene1 (FIG-ROS1(S); FIG-ROS) fusion gene dramatically accelerates ICC development and that its inactivation in established tumors has a potent antitumor effect. Our studies establish a versatile model of ICC that will be a useful preclinical tool and validate ROS1 fusions as potent oncoproteins and therapeutic targets in ICC and potentially other tumor types.
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Colangiocarcinoma/genética , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica/genética , Terapia Genética/métodos , Neoplasias Hepáticas/genética , Proteínas de Fusión Oncogénica/genética , Animales , Neoplasias de los Conductos Biliares , Conductos Biliares Intrahepáticos , Línea Celular Tumoral , Colangiocarcinoma/terapia , Técnicas de Transferencia de Gen , Genes p53/genética , Neoplasias Hepáticas/terapia , Ratones , Mutación/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Interferencia de ARN , RetroviridaeRESUMEN
PURPOSE: Several targeted agents demonstrated efficacy in early clinical trials for gastrointestinal (GI) cancers, but in many cases, phase-III trials and/or approval by the European Medicines Agency (EMA) are lacking. The primary focus of this study was to assess the regulatory processes associated with use and reimbursement of off-label treatment in precision oncology and to evaluate the benefit of targeted therapy in a real-world population in Germany. METHODS: Our cohort comprises 137 patients with GI cancers and is biased towards cancer entities with a high frequency of known targetable alterations, such as cholangiocarcinoma. Genetic testing was used to identify molecular targets, and therapy response was evaluated based on CT scans. RESULTS: A molecular target for precision oncology was identified in 53 patients and 43 requests for cost coverage were submitted to health insurance companies. 60% of the requests received approval after initial application and another 7% after appeal. Half of the rejected requests were denied despite ESCAT IA level evidence. The median time between initiation of molecular testing and start of therapy was 75 days. 35 patients received matched targeted therapies (n = 28) or, in the case of MSI, immunotherapy (IO) (n = 7). We observed a trend in favor of molecular therapy when compared to the immediate prior treatment. CONCLUSION: Relevant treatment options were identified by molecular testing in a significant subset of patients. When targeted therapies that lack EMA approval are considered, treatment initiation may be delayed by the duration of the molecular analysis and the regulatory processes.
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Antineoplásicos , Neoplasias Gastrointestinales , Antineoplásicos/uso terapéutico , Estudios de Cohortes , Neoplasias Gastrointestinales/diagnóstico , Neoplasias Gastrointestinales/genética , Neoplasias Gastrointestinales/terapia , Humanos , Terapia Molecular Dirigida , Patología Molecular , Medicina de PrecisiónRESUMEN
BACKGROUND & AIMS: A coordinated stress and regenerative response is important after hepatocyte damage. Here, we investigate the phenotypes that result from genetic abrogation of individual components of the checkpoint kinase 2/transformation-related protein 53 (p53)/cyclin-dependent kinase inhibitor 1A (p21) pathway in a murine model of metabolic liver injury. METHODS: Nitisinone was reduced or withdrawn in Fah-/- mice lacking Chk2, p53, or p21, and survival, tumor development, liver injury, and regeneration were analyzed. Partial hepatectomies were performed and mice were challenged with the Fas antibody Jo2. RESULTS: In a model of metabolic liver injury, loss of p53, but not Chk2, impairs the oxidative stress response and aggravates liver damage, indicative of a direct p53-dependent protective effect on hepatocytes. Cell-cycle control during chronic liver injury critically depends on the presence of both p53 and its downstream effector p21. In p53-deficient hepatocytes, unchecked proliferation occurs despite a strong induction of p21, showing a complex interdependency between p21 and p53. The increased regenerative potential in the absence of p53 cannot fully compensate the surplus injury and is not sufficient to promote survival. Despite the distinct phenotypes associated with the loss of individual components of the DNA damage response, gene expression patterns are dominated by the severity of liver injury, but reflect distinct effects of p53 on proliferation and the anti-oxidative stress response. CONCLUSIONS: Characteristic phenotypes result from the genetic abrogation of individual components of the DNA damage-response cascade in a liver injury model. The extent to which loss of gene function can be compensated, or affects injury and proliferation, is related to the level at which the cascade is interrupted. Accession numbers of repository for expression microarray data: GSE156983, GSE156263, GSE156852, and GSE156252.
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Lesión Pulmonar Aguda/complicaciones , Carcinogénesis/patología , Quinasa de Punto de Control 2/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Neoplasias Hepáticas/patología , Regeneración Hepática , Proteína p53 Supresora de Tumor/fisiología , Animales , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Cholangiocarcinoma (CCA) is a genetically and histologically complex disease with a highly dismal prognosis. A deeper understanding of the underlying cellular and molecular mechanisms of human CCA will increase our current knowledge of the disease and expedite the eventual development of novel therapeutic strategies for this fatal cancer. This endeavor is effectively supported by genetic mouse models, which serve as sophisticated tools to systematically investigate CCA pathobiology and treatment response. These in vivo models feature many of the genetic alterations found in humans, recapitulate multiple hallmarks of cholangiocarcinogenesis (encompassing cell transformation, preneoplastic lesions, established tumors and metastatic disease) and provide an ideal experimental setting to study the interplay between tumor cells and the surrounding stroma. This review is intended to serve as a compendium of CCA mouse models, including traditional transgenic models but also genetically flexible approaches based on either the direct introduction of DNA into liver cells or transplantation of pre-malignant cells, and is meant as a resource for CCA researchers to aid in the selection of the most appropriate in vivo model system.
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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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The rising incidence of cholangiocarcinoma (CCA) coupled with a low 5-year survival rate that remains below 10% delineates the urgent need for more effective treatment strategies. Although several recent studies provided detailed information on the genetic landscape of this fatal malignancy, versatile model systems to functionally dissect the immediate clinical relevance of the identified genetic alterations are still missing. To enhance our understanding of CCA pathophysiology and facilitate rapid functional annotation of putative CCA driver and tumor maintenance genes, we developed a tractable murine CCA model by combining the cyclization recombination (Cre)-lox system, RNA interference, and clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) technology with liver organoids, followed by subsequent transplantation into immunocompetent, syngeneic mice. Histologically, resulting tumors displayed cytokeratin 19-positive ductal structures surrounded by a desmoplastic stroma-hallmark features of human CCAs. Despite their initial biliary phenotype in vitro, organoids retained the plasticity to induce a broader differentiation spectrum of primary liver cancers following transplantation into recipient mice, depending on their genetic context. Thus, the organoid system combines the advantage of using nontransformed, premalignant cells to recapitulate liver tumorigenesis as a multistep process, with the advantage of a reproducible and expandable cell culture system that abrogates the need for recurrent isolations of primary cells. Conclusion: Genetically modified liver organoids are able to transform into histologically accurate CCAs. Depending on the oncogenic context, they are also able to give rise to liver cancers that show features of hepatocellular carcinomas. The model can be used to functionally explore candidate cancer genes of primary liver cancers in immunocompetent animals and evaluate novel treatment regimens.
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The secreted Frizzled receptor related proteins (Sfrp's) belong to a protein family that comprises antagonists and modifiers of Wnt and BMP signalling. Here we report the isolation and expression pattern of the Sfrp gene "Sizzled" in the chick. Sizzled genes, as well as the closely related crescent genes, exist in the genomes of fishes, frogs and chicks, but not of mammals. The chicken Sizzled gene (Szl) is initially expressed in the anterior endoderm of gastrulating and early head fold embryos. An additional, separate expression domain develops at the posterior end of the embryo from the head process stage onwards. Szl transcripts are then detected in precardial mesodermal cells, are transiently transcribed in the straight heart tube, and later prominently in the splanchnic mesoderm surrounding the arterial pole of the developing heart, the anterior heart field. These cells are subsequently recruited to form the cardiac outflow tract. cSzl expression is downregulated when the septation of the outflow tract by neural crest derived cells begins.
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Proteínas Aviares/metabolismo , Corazón/embriología , Secuencia de Aminoácidos , Animales , Proteínas Aviares/clasificación , Proteínas Aviares/genética , Embrión de Pollo , Expresión Génica , Datos de Secuencia Molecular , Miocardio/citología , Miocardio/metabolismo , Filogenia , ARN Mensajero/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9)-inhibitors have shown great po-tential in efficient lipid lowering to achieve low-density lipoprotein-cholesterol (LDL-C) treatment goals. The aim of the study was too describe the clinical use of PCSK9-inhibitors and to investigate therapy adherence and safety outside of clinical trials. METHODS: Thirty-eight patients were treated with PSCK9-inhibitors. Patients were eligible for this therapy based on their individual cardiovascular risk and when all other available lipid-lowering regi-men had failed. Every patient answered a questionnaire concerning medical history and relevant side effects and therapy adherence. RESULTS: Conventional therapy reduced patient LDL-C levels by about 38%. However, in 26 of the 38 patients, LDL-C treatment goals were not fulfilled because patients did not tolerate further dose es-calation due to side effects. Using a PCSK9 inhibitor, LDL-C levels were reduced by another 54% and 42% of patients reaching treatment goals. The results show that most patients still require concomitant therapy to reach LDL-C target levels. Three patients required dose reduction or change of the PCSK9 inhibitor. 16% did not inject the PCSK9 inhibitor regularly. CONCLUSIONS: Only a minority of patients reached the recommended LDL-C goals. PCSK9-inhibitors were generally well tolerated. Despite low rates of reported side effects, therapy adherence was incom-plete, with 6 patients not injecting PCSK9-inhibitors on a regular basis. In-depth information about the medication and close supervision is advisable. PCSK9 inhibitors have shown great potential in aggressive lipid lowering therapy, but basic therapy is still required in most cases. Close supervision is recommended to improve therapy adherence. (Cardiol J 2018; 25, 1: 32-41).
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Anticuerpos Monoclonales/uso terapéutico , Hipercolesterolemia/tratamiento farmacológico , Inhibidores de PCSK9 , Cumplimiento y Adherencia al Tratamiento , Anticuerpos Monoclonales Humanizados , Anticolesterolemiantes/uso terapéutico , Apoptosis , Estudios de Cohortes , Femenino , Humanos , Hipercolesterolemia/sangre , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Riesgo , Encuestas y Cuestionarios , Resultado del TratamientoRESUMEN
Mutations in members of the SWI/SNF chromatin remodeling family are common events in cancer, but the mechanisms whereby disruption of SWI/SNF components alters tumorigenesis remain poorly understood. To model the effect of loss of function mutations in the SWI/SNF subunit Arid1a in pancreatic ductal adenocarcinoma (PDAC) initiation, we directed shRNA triggered, inducible and reversible suppression of Arid1a to the mouse pancreas in the setting of oncogenic KrasG12D. Arid1a cooperates with Kras in the adult pancreas as postnatal silencing of Arid1a following sustained KrasG12D expression induces rapid and irreversible reprogramming of acinar cells into mucinous PDAC precursor lesions. In contrast, Arid1a silencing during embryogenesis, concurrent with KrasG12D activation, leads to retention of acinar cell fate. Together, our results demonstrate Arid1a as a critical modulator of Kras-dependent changes in acinar cell identity, and underscore an unanticipated influence of timing and genetic context on the effects of SWI/SNF complex alterations in epithelial tumorigenesis.
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Células Acinares/fisiología , Carcinogénesis , Diferenciación Celular , Transformación Celular Neoplásica , Proteínas de Unión al ADN/metabolismo , Morfogénesis , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Animales , Carcinoma Ductal Pancreático/fisiopatología , Regulación de la Expresión Génica , Ratones , Factores de TranscripciónRESUMEN
CD4 and CD8 T cells play a pivotal role in controlling tumor growth. However, the interplay of both cell types and their role in tumor suppression still remain elusive. In this study, we investigated the regulation of CD4 and CD8 T cell responses to different classes of tumor-specific antigens in liver cancer mouse models. Tumors were induced in p19Arf-deficient mice by hydrodynamic injection of transposon plasmids encoding NrasG12V and pre-defined tumor antigens. This allowed for assessing the regulation of tumor-specific CD4 and CD8 T cell responses. We showed that MHC class I tumor immunogenicity was essential to trigger tumor-directed CD4 T cells. Tumor-specific CD8 T cell responses arose independently of CD4 T cells, but they required Th1-polarized CD4 T cells for efficient tumor suppression. Our results further indicate that the immune system is incapable of eliciting sufficient numbers of T cells directed against antigens derived from immunoedited tumors, which consequently leads to a lack of T-cell-mediated tumor suppression in untreated hosts.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Neoplasias/inmunología , Animales , Antígenos de Neoplasias/inmunología , Carcinogénesis/inmunología , Carcinogénesis/patología , Citotoxicidad Inmunológica , Genes MHC Clase I , Genes ras , Vigilancia Inmunológica , Ratones Endogámicos C57BL , Neoplasias/patología , Análisis de Supervivencia , Linfocitos T Citotóxicos/inmunología , Células TH1/inmunologíaRESUMEN
The inability to visualize the true extent of cancers represents a significant challenge in many areas of oncology. The margins of most cancer types are not well demarcated because the cancer diffusely infiltrates the surrounding tissues. Furthermore, cancers may be multifocal and characterized by the presence of microscopic satellite lesions. Such microscopic foci represent a major reason for persistence of cancer, local recurrences, and metastatic spread, and are usually impossible to visualize with currently available imaging technologies. An imaging method to reveal the true extent of tumors is desired clinically and surgically. We show the precise visualization of tumor margins, microscopic tumor invasion, and multifocal locoregional tumor spread using a new generation of surface-enhanced resonance Raman scattering (SERRS) nanoparticles, which are termed SERRS nanostars. The SERRS nanostars feature a star-shaped gold core, a Raman reporter resonant in the near-infrared spectrum, and a primer-free silication method. In genetically engineered mouse models of pancreatic cancer, breast cancer, prostate cancer, and sarcoma, and in one human sarcoma xenograft model, SERRS nanostars enabled accurate detection of macroscopic malignant lesions, as well as microscopic disease, without the need for a targeting moiety. Moreover, the sensitivity (1.5 fM limit of detection) of SERRS nanostars allowed imaging of premalignant lesions of pancreatic and prostatic neoplasias. High sensitivity and broad applicability, in conjunction with their inert gold-silica composition, render SERRS nanostars a promising imaging agent for more precise cancer imaging and resection.