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Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However, for scientists wishing to publish obtained images and image-analysis results, there are currently no unified guidelines for best practices. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here, we present community-developed checklists for preparing light microscopy images and describing image analyses for publications. These checklists offer authors, readers and publishers key recommendations for image formatting and annotation, color selection, data availability and reporting image-analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby to heighten the quality and explanatory power of microscopy data.
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Lista de Verificación , Edición , Reproducibilidad de los Resultados , Procesamiento de Imagen Asistido por Computador , MicroscopíaRESUMEN
Atrial fibrillation (AF) remains challenging to prevent and treat. A key feature of AF is atrial enlargement. However, not all atrial enlargement progresses to AF. Atrial enlargement in response to physiological stimuli such as exercise is typically benign and reversible. Understanding the differences in atrial function and molecular profile underpinning pathological and physiological atrial remodelling will be critical for identifying new strategies for AF. The discovery of molecular mechanisms responsible for pathological and physiological ventricular hypertrophy has uncovered new drug targets for heart failure. Studies in the atria have been limited in comparison. Here, we characterised mouse atria from (1) a pathological model (cardiomyocyte-specific transgenic (Tg) that develops dilated cardiomyopathy [DCM] and AF due to reduced protective signalling [PI3K]; DCM-dnPI3K), and (2) a physiological model (cardiomyocyte-specific Tg with an enlarged heart due to increased insulin-like growth factor 1 receptor; IGF1R). Both models presented with an increase in atrial mass, but displayed distinct functional, cellular, histological and molecular phenotypes. Atrial enlargement in the DCM-dnPI3K Tg, but not IGF1R Tg, was associated with atrial dysfunction, fibrosis and a heart failure gene expression pattern. Atrial proteomics identified protein networks related to cardiac contractility, sarcomere assembly, metabolism, mitochondria, and extracellular matrix which were differentially regulated in the models; many co-identified in atrial proteomics data sets from human AF. In summary, physiological and pathological atrial enlargement are associated with distinct features, and the proteomic dataset provides a resource to study potential new regulators of atrial biology and function, drug targets and biomarkers for AF.
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Fibrilación Atrial , Remodelación Atrial , Atrios Cardíacos , Ratones Transgénicos , Miocitos Cardíacos , Fibrilación Atrial/fisiopatología , Fibrilación Atrial/metabolismo , Fibrilación Atrial/genética , Animales , Atrios Cardíacos/metabolismo , Atrios Cardíacos/fisiopatología , Atrios Cardíacos/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Receptor IGF Tipo 1/metabolismo , Receptor IGF Tipo 1/genética , Cardiomiopatía Dilatada/fisiopatología , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/patología , Modelos Animales de Enfermedad , Fibrosis , Ratones , Humanos , Transducción de Señal , Fosfatidilinositol 3-Quinasas/metabolismo , Insuficiencia Cardíaca/fisiopatología , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patologíaRESUMEN
BACKGROUND: Computational identification of non-coding RNAs (ncRNAs) is a challenging problem. We describe a genome-wide analysis using Bayesian segmentation to identify intronic elements highly conserved between three evolutionarily distant vertebrate species: human, mouse and zebrafish. We investigate the extent to which these elements include ncRNAs (or conserved domains of ncRNAs) and regulatory sequences. RESULTS: We identified 655 deeply conserved intronic sequences in a genome-wide analysis. We also performed a pathway-focussed analysis on genes involved in muscle development, detecting 27 intronic elements, of which 22 were not detected in the genome-wide analysis. At least 87% of the genome-wide and 70% of the pathway-focussed elements have existing annotations indicative of conserved RNA secondary structure. The expression of 26 of the pathway-focused elements was examined using RT-PCR, providing confirmation that they include expressed ncRNAs. Consistent with previous studies, these elements are significantly over-represented in the introns of transcription factors. CONCLUSIONS: This study demonstrates a novel, highly effective, Bayesian approach to identifying conserved non-coding sequences. Our results complement previous findings that these sequences are enriched in transcription factors. However, in contrast to previous studies which suggest the majority of conserved sequences are regulatory factor binding sites, the majority of conserved sequences identified using our approach contain evidence of conserved RNA secondary structures, and our laboratory results suggest most are expressed. Functional roles at DNA and RNA levels are not mutually exclusive, and many of our elements possess evidence of both. Moreover, ncRNAs play roles in transcriptional and post-transcriptional regulation, and this may contribute to the over-representation of these elements in introns of transcription factors. We attribute the higher sensitivity of the pathway-focussed analysis compared to the genome-wide analysis to improved alignment quality, suggesting that enhanced genomic alignments may reveal many more conserved intronic sequences.
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Genoma , ARN no Traducido/metabolismo , Animales , Teorema de Bayes , Sitios de Unión , Secuencia Conservada , Humanos , Intrones , Ratones , Desarrollo de Músculos/genética , Conformación de Ácido Nucleico , ARN no Traducido/química , ARN no Traducido/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Interfaz Usuario-Computador , Pez Cebra/genéticaRESUMEN
Ribosome biogenesis underpins cell growth and division. Disruptions in ribosome biogenesis and translation initiation are deleterious to development and underlie a spectrum of diseases known collectively as ribosomopathies. Here, we describe a novel zebrafish mutant, titania (tti(s450)), which harbours a recessive lethal mutation in pwp2h, a gene encoding a protein component of the small subunit processome. The biochemical impacts of this lesion are decreased production of mature 18S rRNA molecules, activation of Tp53, and impaired ribosome biogenesis. In tti(s450), the growth of the endodermal organs, eyes, brain, and craniofacial structures is severely arrested and autophagy is up-regulated, allowing intestinal epithelial cells to evade cell death. Inhibiting autophagy in tti(s450) larvae markedly reduces their lifespan. Somewhat surprisingly, autophagy induction in tti(s450) larvae is independent of the state of the Tor pathway and proceeds unabated in Tp53-mutant larvae. These data demonstrate that autophagy is a survival mechanism invoked in response to ribosomal stress. This response may be of relevance to therapeutic strategies aimed at killing cancer cells by targeting ribosome biogenesis. In certain contexts, these treatments may promote autophagy and contribute to cancer cells evading cell death.
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Autofagia/genética , Proteínas de Ciclo Celular , Ribosomas , Serina-Treonina Quinasas TOR , Proteína p53 Supresora de Tumor , Proteínas de Pez Cebra , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Supervivencia Celular , Genes Letales/genética , Mutación , Biosíntesis de Proteínas/genética , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Proteína p53 Supresora de Tumor/genética , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Nemaline myopathy is characterized by muscle weakness and the presence of rod-like (nemaline) bodies. The genetic etiology of nemaline myopathy is becoming increasingly understood with mutations in ten genes now known to cause the disease. Despite this, the mechanism by which skeletal muscle weakness occurs remains elusive, with previous studies showing no correlation between the frequency of nemaline bodies and disease severity. To investigate the formation of nemaline bodies and their role in pathogenesis, we generated overexpression and loss-of-function zebrafish models for skeletal muscle α-actin (ACTA1) and nebulin (NEB). We identify three distinct types of nemaline bodies and visualize their formation in vivo, demonstrating these nemaline bodies not only exhibit different subcellular origins, but also have distinct pathological consequences within the skeletal muscle. One subtype is highly dynamic and upon breakdown leads to the accumulation of cytoplasmic actin contributing to muscle weakness. Examination of a Neb-deficient model suggests this mechanism may be common in nemaline myopathy. Another subtype results from a reduction of actin and forms a more stable cytoplasmic body. In contrast, the final type originates at the Z-disk and is associated with myofibrillar disorganization. Analysis of zebrafish and muscle biopsies from ACTA1 nemaline myopathy patients demonstrates that nemaline bodies also possess a different protein signature. In addition, we show that the ACTA1(D286G) mutation causes impaired actin incorporation and localization in the sarcomere. Together these data provide a novel examination of nemaline body origins and dynamics in vivo and identifies pathological changes that correlate with muscle weakness.
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Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Miopatías Nemalínicas/patología , Miopatías Nemalínicas/fisiopatología , Actinina/genética , Actinina/metabolismo , Actinas/metabolismo , Animales , Animales Modificados Genéticamente , Citoplasma/metabolismo , Citoplasma/patología , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Morfolinos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Debilidad Muscular/patología , Debilidad Muscular/fisiopatología , Mutación , Fenotipo , Sarcómeros/metabolismo , Sarcómeros/patología , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key regulators of blood vessel formation, including in tumors, where their deregulated function can promote the production of aberrant, leaky blood vessels, supporting tumor development. Here we investigated the VEGFR1 ligand VEGF-B, which we demonstrate to be expressed in tumor cells and in tumor stroma and vasculature across a range of tumor types. We examined the anti-VEGF-B-specific monoclonal antibody 2H10 in preclinical xenograft models of breast and colorectal cancer, in comparison with the anti-VEGF-A antibody bevacizumab. Similar to bevacizumab, 2H10 therapy was associated with changes in tumor blood vessels and intra-tumoral diffusion consistent with normalization of the tumor vasculature. Accordingly, treatment resulted in partial inhibition of tumor growth, and significantly improved the response to chemotherapy. Our studies indicate the importance of VEGF-B in tumor growth, and the potential of specific anti-VEGF-B treatment to inhibit tumor development, alone or in combination with established chemotherapies.
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Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However for scientists wishing to publish the obtained images and image analyses results, there are to date no unified guidelines. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here we present community-developed checklists for preparing light microscopy images and image analysis for publications. These checklists offer authors, readers, and publishers key recommendations for image formatting and annotation, color selection, data availability, and for reporting image analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby heighten the quality and explanatory power of microscopy data is in publications.
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This protocol features parallel isolation of myocytes and non-myocytes from murine hearts. It was designed with considerations for (1) time required to extract cardiac cells, (2) cell viability, and (3) protocol scalability. Here, a peristaltic pump and 3D-printed elements are combined to perfuse the heart with enzymes to dissociate cells. Myocytes and non-myocytes extracted using this protocol are separated by centrifugation and/or fluorescence-activated cell sorting for use in downstream applications including single-cell omics or other bio-molecular analyses. For complete details on the use and execution of this protocol, please refer to McLellan et al. (2020).
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Separación Celular/métodos , Miocardio/citología , Miocitos Cardíacos , Análisis de la Célula Individual/métodos , Animales , Técnicas de Cultivo de Célula , Células Cultivadas , Genómica , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/clasificación , Miocitos Cardíacos/citologíaRESUMEN
IL11 is a member of the IL6 family of cytokines and signals through its cognate receptor subunits, IL11RA and glycoprotein 130 (GP130), to elicit biological responses via the JAK/STAT signaling pathway. IL11 contributes to cancer progression by promoting the survival and proliferation of cancer cells, but the potential immunomodulatory properties of IL11 signaling during tumor development have thus far remained unexplored. Here, we have characterized a role for IL11 in regulating CD4+ T cell-mediated antitumor responses. Absence of IL11 signaling impaired tumor growth in a sporadic mouse model of colon cancer and syngeneic allograft models of colon cancer. Adoptive bone marrow transfer experiments and in vivo depletion studies demonstrated that the tumor-promoting activity of IL11 was mediated through its suppressive effect on host CD4+ T cells in the tumor microenvironment. Indeed, when compared with Il11ra-proficient CD4+ T cells associated with MC38 tumors, their Il11ra-deficient counterparts displayed elevated expression of mRNA encoding the antitumor mediators IFNγ and TNFα. Likewise, IL11 potently suppressed the production of proinflammatory cytokines (IFNγ, TNFα, IL6, and IL12p70) by CD4+ T cells in vitro, which we corroborated by RNAscope analysis of human colorectal cancers, where IL11RAhigh tumors showed less IFNG and CD4 expression than IL11RAlow tumors. Therefore, our results ascribe a tumor cell-extrinsic immunomodulatory role to IL11 during colon cancer development that could be amenable to an anticytokine-based therapy.See related Spotlight by van der Burg, p. 724.
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Linfocitos T CD4-Positivos/inmunología , Neoplasias del Colon/inmunología , Subunidad alfa del Receptor de Interleucina-11/metabolismo , Interleucina-11/metabolismo , Animales , Antígenos CD4/análisis , Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Línea Celular Tumoral , Colon/inmunología , Colon/patología , Neoplasias del Colon/patología , Conjuntos de Datos como Asunto , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Humanos , Interferón gamma/análisis , Interferón gamma/metabolismo , Subunidad alfa del Receptor de Interleucina-11/análisis , Subunidad alfa del Receptor de Interleucina-11/genética , Ratones , Ratones Noqueados , Neoplasias de Tejido Óseo , Receptores de Interleucina-11/metabolismo , Microambiente Tumoral/inmunologíaRESUMEN
Intratumoral heterogeneity is a driver of breast cancer progression, but the nature of the clonal interactive network involved in this process remains unclear. Here, we optimized the use of optical barcoding to visualize and characterize 31 cancer subclones in vivo. By mapping the clonal composition of thousands of metastases in two clinically relevant sites, the lungs and liver, we found that metastases were highly polyclonal in lungs but not in the liver. Furthermore, the transcriptome of the subclones varied according to their metastatic niche. We also identified a reversible niche-driven signature that was conserved in lung and liver metastases collected during patient autopsies. Among this signature, we found that the tumor necrosis factor-α pathway was up-regulated in lung compared to liver metastases, and inhibition of this pathway affected metastasis diversity. These results highlight that the cellular and molecular heterogeneity observed in metastases is largely dictated by the tumor microenvironment.
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Neoplasias de la Mama , Neoplasias Hepáticas , Neoplasias Pulmonares , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Pulmonares/patología , Metástasis de la Neoplasia , Transcriptoma , Microambiente Tumoral/genéticaRESUMEN
BACKGROUND & AIMS: Zebrafish mutants generated by ethylnitrosourea-mutagenesis provide a powerful tool for dissecting the genetic regulation of developmental processes, including organogenesis. One zebrafish mutant, "flotte lotte" (flo), displays striking defects in intestinal, liver, pancreas, and eye formation at 78 hours postfertilization (hpf). In this study, we sought to identify the underlying mutated gene in flo and link the genetic lesion to its phenotype. METHODS: Positional cloning was employed to map the flo mutation. Subcellular characterization of flo embryos was achieved using histology, immunocytochemistry, bromodeoxyuridine incorporation analysis, and confocal and electron microscopy. RESULTS: The molecular lesion in flo is a nonsense mutation in the elys (embryonic large molecule derived from yolk sac) gene, which encodes a severely truncated protein lacking the Elys C-terminal AT-hook DNA binding domain. Recently, the human ELYS protein has been shown to play a critical, and hitherto unsuspected, role in nuclear pore assembly. Although elys messenger RNA (mRNA) is expressed broadly during early zebrafish development, widespread early defects in flo are circumvented by the persistence of maternally expressed elys mRNA until 24 hpf. From 72 hpf, elys mRNA expression is restricted to proliferating tissues, including the intestinal epithelium, pancreas, liver, and eye. Cells in these tissues display disrupted nuclear pore formation; ultimately, intestinal epithelial cells undergo apoptosis. CONCLUSIONS: Our results demonstrate that Elys regulates digestive organ formation.
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Apoptosis/fisiología , Mucosa Intestinal/anomalías , Mucosa Intestinal/fisiología , Proteínas de Complejo Poro Nuclear/genética , Poro Nuclear/patología , Proteínas de Pez Cebra/genética , Animales , Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Sistema Nervioso Entérico/anomalías , Sistema Nervioso Entérico/patología , Sistema Nervioso Entérico/fisiología , Anomalías del Ojo/patología , Anomalías del Ojo/fisiopatología , Regulación del Desarrollo de la Expresión Génica , Mucosa Intestinal/patología , Intestinos/anomalías , Intestinos/patología , Intestinos/fisiología , Hígado/anomalías , Hígado/patología , Hígado/fisiología , Microscopía Electrónica , Poro Nuclear/fisiología , Poro Nuclear/ultraestructura , Proteínas de Complejo Poro Nuclear/metabolismo , Páncreas/anomalías , Páncreas/patología , Páncreas/fisiología , Fenotipo , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
INTRODUCTION: The importance of ErbB3 receptor tyrosine kinase in cancer progression, primary and acquired drug resistance, has become steadily evident since its discovery in 1989. ErbB3 overexpression in various solid organ malignancies is associated with shorter survival of patients. However, initial strategies to therapeutically target ErbB3 have not been rewarding. AREAS COVERED: Here, we provide an overview of ErbB3 biology in carcinogenesis. We outline the role of ErbB3 as a critical pathway for resistance to other anti-cancer drugs. We focus on emerging clinical data, which will steer the potential future development of ErbB3 directed therapies. EXPERT OPINION: Initial approaches to ErbB3 targeting have been challenging. However, the lack of success of anti-ErbB3 therapies in ongoing clinical trials may relate more to the complex biology of the receptor and challenges with the biomarkers used to date. Furthermore, it seems certain that the expression of the receptor per se is necessary but not sufficient for the response to ErbB3 therapies. Emerging data suggest that more sophisticated biomarkers are needed. Nonetheless, it is also likely that ErbB3 therapies may have the most efficacy in combination therapy, and their favorable toxicity profile makes this feasible.
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Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Receptor ErbB-3/metabolismo , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Antineoplásicos/farmacología , Biomarcadores de Tumor/metabolismo , Progresión de la Enfermedad , Desarrollo de Medicamentos , Resistencia a Antineoplásicos , Humanos , Neoplasias/patologíaRESUMEN
BACKGROUND: Targeted therapy of HER2 positive breast cancer has led to clinical success in some cases with primary and secondary resistance being major obstacles. Due to the substantial involvement of mTOR kinase in cell growth and proliferation pathways it is now targeted in combination treatments to counteract HER2 targeted therapy resistance. However, the selection of receptive patient populations for a specific drug combination is crucial. This work aims to develop a molecular probe capable of identifying patients with tumour populations which are receptive to RAD001 combination therapy. Based on the structure of a mTOR inhibitor specific for mTORC1, we designed, synthesised and characterised a novel benzofuran based molecular probe which suits late stage fluorination via Click chemistry. RESULTS: Synthesis of the alkyne precursor 5 proceeded in 27.5% yield over 7 linear steps. Click derivatisation gave the non-radioactive standard in 25% yield. Radiosynthesis of [18F]1-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-yl) methyl)-4-((5-methoxy-2-phenylbenzofuran-4-yl) methyl) piperazine ([18F]mBPET-1) proceeded over two steps which were automated on an iPhase FlexLab synthesis module. In the first step, 2-[18F]fluoroethylazide ([18F]6) was produced, purified by automated distillation in 60% non-decay-corrected yield and subjected to Click conditions with 5. Semi-preparative RP-HPLC purification and reformulation gave [18F]mBPET-1 in 40% ± 5% (n = 6) overall RCY with a process time of 90 min. Radiochemical purity was ≥99% at end of synthesis (EOS) and ≥ 98% after 4 h at room temperature. Molar activities ranged from typically 24.8 GBq/µmol (EOS) to a maximum of 78.6 GBq/µmol (EOS). Lipophilicity of [18F]mBPET-1 was determined at pH 7.4 (logD7.4 = 0.89). [18F]mBPET-1 showed high metabolic stability when incubated with mouse S9 liver fractions which resulted in a 0.8% drop in radiochemical purity after 3 h. Cell uptake assays showed 1.3-1.9-fold increased uptake of the [18F]mBPET-1 in RAD001 sensitive compared to insensitive cells across a panel of 4 breast cancer cell lines. CONCLUSION: Molecular targeting of mTOR with [18F]mBPET-1 distinguishes mTOR inhibitor sensitive and insensitive cell lines. Future studies will explore the ability of [18F]mBPET-1 to predict response to mTOR inhibitor treatment in in vivo models.
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The zebrafish provides an ideal model for the study of vertebrate organogenesis, including the formation of the digestive tract and its associated organs. Despite optical transparency of embryos, the internal position of the developing digestive system and its close juxtaposition with the yolk initially made morphological analysis relatively challenging, particularly during the first 3 d of development. However, methodologies have been successfully developed to address these problems and comprehensive morphologic analysis of the developing digestive system has now been achieved using a combination of light and fluorescence microscope approaches-including confocal analysis-to visualize wholemount and histological preparations of zebrafish embryos. Furthermore, the expanding number of antibodies that cross-react with zebrafish proteins and the generation of tissue-specific transgenic green fluorescent protein reporter lines that mark specific cell and tissue compartments have greatly enhanced our ability to successfully image the developing zebrafish digestive system.
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Sistema Digestivo/embriología , Pez Cebra/embriología , Animales , Sistema Digestivo/citología , Sistema Digestivo/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes , Procesamiento de Imagen Asistido por Computador/instrumentación , Procesamiento de Imagen Asistido por Computador/métodos , Inmunohistoquímica/instrumentación , Inmunohistoquímica/métodos , Microscopía/instrumentación , Microscopía/métodos , Organogénesis , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Excessive signaling through gp130, the shared receptor for the interleukin (IL)6 family of cytokines, is a common hallmark in solid malignancies and promotes their progression. Here, we established the in vivo utility of bazedoxifene, a steroid analog clinically approved for the treatment of osteoporosis, to suppress gp130-dependent tumor growth of the gastrointestinal epithelium. Bazedoxifene administration reduced gastric tumor burden in gp130Y757F mice, where tumors arise exclusively through excessive gp130/STAT3 signaling in response to the IL6 family cytokine IL11. Likewise, in mouse models of sporadic colon and intestinal cancers, which arise from oncogenic mutations in the tumor suppressor gene Apc and the associated ß-catenin/canonical WNT pathway, bazedoxifene treatment reduces tumor burden. Consistent with the proposed orthogonal tumor-promoting activity of IL11-dependent gp130/STAT3 signaling, tumors of bazedoxifene-treated Apc-mutant mice retain excessive nuclear accumulation of ß-catenin and aberrant WNT pathway activation. Likewise, bazedoxifene treatment of human colon cancer cells harboring mutant APC did not reduce aberrant canonical WNT signaling, but suppressed IL11-dependent STAT3 signaling. Our findings provide compelling proof of concept to support the repurposing of bazedoxifene for the treatment of gastrointestinal cancers in which IL11 plays a tumor-promoting role.
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Reposicionamiento de Medicamentos , Neoplasias Gastrointestinales/tratamiento farmacológico , Indoles/uso terapéutico , Moduladores Selectivos de los Receptores de Estrógeno/uso terapéutico , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Proliferación Celular/efectos de los fármacos , Receptor gp130 de Citocinas/química , Receptor gp130 de Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Neoplasias Gastrointestinales/patología , Humanos , Indoles/metabolismo , Indoles/farmacología , Interleucina-11/química , Interleucina-11/metabolismo , Interleucina-11/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor de Transcripción STAT3/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/metabolismoRESUMEN
Optically transparent zebrafish embryos provide an excellent vertebrate model system in which to reveal specific mRNA and protein expression patterns during development. Whole-mount preparations can be used to generate three-dimensional color or fluorescent readouts of the expression pattern of a given gene (or genes), matched with a bright-field image of all the tissues in the developing embryo. Whole-mount mRNA in situhybridization (WISH) has long been the method of choice for revealing gene expression patterns in zebrafish because this method depends only on being able to identify a relatively short region of nucleotide sequence unique for the gene of interest. In contrast, the scarcity of antibodies that are specific to or cross-react with zebrafish proteins has limited the widespread use of immunocytochemical applications, though this situation will improve in the future. The elucidation of the specific expression patterns of Wnt pathway genes in zebrafish has made a major contribution to our current understanding of their roles in vertebrate development.
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Expresión Génica , Pez Cebra/embriología , Pez Cebra/genética , Animales , Microinyecciones/instrumentación , Microinyecciones/métodos , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Transducción de Señal/fisiología , Proteínas Wnt/antagonistas & inhibidores , Proteínas Wnt/metabolismo , Pez Cebra/anatomía & histología , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.
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Microscopía Confocal/métodos , Receptores de Superficie Celular/metabolismo , Análisis Espectral/métodos , HumanosRESUMEN
The selection of therapeutic dose for the most effective treatment of tumours is an intricate interplay of factors. Molecular imaging with positron emission tomography (PET) or single-photon emission computed tomography (SPECT) can address questions central to this selection: Does the drug reach its target? Does the drug engage with the target of interest? Is the drug dose sufficient to elicit the desired pharmacological effect? Does the dose saturate available target sites? Combining functional PET and SPECT imaging with anatomical imaging technologies such as magnetic resonance imaging (MRI) or computed tomography (CT) allows drug occupancy at the target to be related directly to anatomical or physiological changes in a tissue resulting from therapy. In vivo competition studies, using a tracer amount of radioligand that binds to the tumour receptor with high specificity, enable direct assessment of the relationship between drug plasma concentration and target occupancy. Including imaging studies in early drug development can aid with dose selection and suggest improvements for patient stratification to obtain higher effective utility from a drug after approval. In this review, the potential value of including translational receptor occupancy studies and molecular imaging strategies early on in drug development is addressed.
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Antineoplásicos/farmacología , Evaluación Preclínica de Medicamentos/métodos , Imagen Molecular/métodos , Neoplasias/tratamiento farmacológico , Receptores de Superficie Celular/metabolismo , Animales , Antineoplásicos/uso terapéutico , Modelos Animales de Enfermedad , Humanos , Imagen por Resonancia Magnética , Neoplasias/diagnóstico por imagen , Tomografía de Emisión de Positrones , Tomografía Computarizada de Emisión de Fotón ÚnicoRESUMEN
The development of HER2-directed monoclonal antibodies and tyrosine kinase inhibitors have provided benefits to cancer patients, as well as produced many insights into the biology of the ErbB receptor family. Current therapies based on ErbB family members have resulted in improved overall survival with associated improvements in quality of life for the cancer patients that respond to treatment. Compared to monotherapy using either two antibodies to block the HER2 receptor blockade or combinatorial approaches with HER2 antibodies and standard therapies has provided additional benefits. Despite the therapeutic success of existing HER2 therapies, personalising treatment and overcoming resistance to these therapies remains a significant challenge. The heterogeneous intra-tumoural HER2 expression and lack of fully predictive and prognostic biomarkers remain significant barriers to improving the use of HER2 antibodies. Imaging modalities using radiolabelled pertuzumab and trastuzumab allow quantitative assessment of intra-tumoural HER2 expression, HER2 antibody saturation and the success of different drug delivery systems to be assessed. Molecular imaging with HER2 antibodies has the potential to be a non-invasive, predictive and prognostic technique capable of influencing therapeutic decisions, predicting response and failure of treatments as well as providing insights into receptor recycling and signalling. Similarly, conjugating HER2 antibodies with novel toxic payloads or combining HER2 antibodies with cellular immunotherapy provide exciting new opportunities for the management of tumours overexpressing HER2. Future research will lead to higher therapeutic responses, lower toxicities and providing insight into the mechanisms of resistance to HER2-targeted treatments.
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Anticuerpos Monoclonales Humanizados/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Terapia Molecular Dirigida/métodos , Receptor ErbB-2/genética , Adulto , Anciano , Neoplasias de la Mama/mortalidad , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Femenino , Humanos , Persona de Mediana Edad , Pronóstico , Calidad de Vida , Receptor ErbB-2/efectos de los fármacos , Análisis de Supervivencia , Trastuzumab/administración & dosificación , Resultado del TratamientoRESUMEN
Drugs that target the Renin-Angiotensin System (RAS) have recently come into focus for their potential utility as cancer treatments. The use of Angiotensin Receptor Blockers (ARBs) and Angiotensin-Converting Enzyme (ACE) Inhibitors (ACEIs) to manage hypertension in cancer patients is correlated with improved survival outcomes for renal, prostate, breast and small cell lung cancer. Previous studies demonstrate that the Angiotensin Receptor Type I (AT1R) is linked to breast cancer pathogenesis, with unbiased analysis of gene-expression studies identifying significant up-regulation of AGTR1, the gene encoding AT1R in ER+ve/HER2-ve tumors correlating with poor prognosis. However, there is no evidence, so far, of the functional contribution of AT1R to breast tumorigenesis. We explored the potential therapeutic benefit of ARB in a carcinogen-induced mouse model of breast cancer and clarified the mechanisms associated with its success.Mammary tumors were induced with 7,12-dimethylbenz[α]antracene (DMBA) and medroxyprogesterone acetate (MPA) in female wild type mice and the effects of the ARB, Losartan treatment assessed in a preventative setting (n = 15 per group). Tumor histopathology was characterised by immunohistochemistry, real-time qPCR to detect gene expression signatures, and tumor cytokine levels measured with quantitative bioplex assays. AT1R was detected with radiolabelled ligand binding assays in fresh frozen tumor samples.We showed that therapeutic inhibition of AT1R, with Losartan, resulted in a significant reduction in tumor burden; and no mammary tumor incidence in 20% of animals. We observed a significant reduction in tumor progression from DCIS to invasive cancer with Losartan treatment. This was associated with reduced tumor cell proliferation and a significant reduction in IL-6, pSTAT3 and TNFα levels. Analysis of tumor immune cell infiltrates, however, demonstrated no significant differences in the recruitment of lymphocytes or tumour-associated macrophages in Losartan or vehicle-treated mammary tumors.Analysis of AT1R expression with radiolabelled ligand binding assays in human breast cancer biopsies showed high AT1R levels in 30% of invasive ductal carcinomas analysed. Furthermore, analysis of the TCGA database identified that high AT1R expression to be associated with luminal breast cancer subtype.Our in vivo data and analysis of human invasive ductal carcinoma samples identify the AT1R is a potential therapeutic target in breast cancer, with the availability of a range of well-tolerated inhibitors currently used in clinics. We describe a novel signalling pathway critical in breast tumorigenesis, that may provide new therapeutic avenues to complement current treatments.