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Patient-derived xenograft (PDX) models are widely used in cancer research. To investigate the genomic fidelity of non-small cell lung cancer PDX models, we established 48 PDX models from 22 patients enrolled in the TRACERx study. Multi-region tumor sampling increased successful PDX engraftment and most models were histologically similar to their parent tumor. Whole-exome sequencing enabled comparison of tumors and PDX models and we provide an adapted mouse reference genome for improved removal of NOD scid gamma (NSG) mouse-derived reads from sequencing data. PDX model establishment caused a genomic bottleneck, with models often representing a single tumor subclone. While distinct tumor subclones were represented in independent models from the same tumor, individual PDX models did not fully recapitulate intratumor heterogeneity. On-going genomic evolution in mice contributed modestly to the genomic distance between tumors and PDX models. Our study highlights the importance of considering primary tumor heterogeneity when using PDX models and emphasizes the benefit of comprehensive tumor sampling.
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Carcinoma de Pulmón de Células no Pequeñas , Heterogeneidad Genética , Neoplasias Pulmonares , Ratones Endogámicos NOD , Ratones SCID , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Humanos , Animales , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Femenino , Secuenciación del Exoma , Genómica/métodos , Masculino , Ensayos Antitumor por Modelo de Xenoinjerto , Xenoinjertos , Modelos Animales de Enfermedad , Anciano , Persona de Mediana EdadRESUMEN
The airway epithelium is a protective barrier that is maintained by the self-renewal and differentiation of basal stem cells. Increasing age is a principle risk factor for chronic lung diseases, but few studies have explored age-related molecular or functional changes in the airway epithelium. We retrieved epithelial biopsies from histologically normal tracheobronchial sites from pediatric and adult donors and compared their cellular composition and gene expression profile (in laser capture-microdissected whole epithelium, fluorescence-activated cell-sorted basal cells, and basal cells in cell culture). Histologically, pediatric and adult tracheobronchial epithelium was similar in composition. We observed age-associated changes in RNA sequencing studies, including higher interferon-associated gene expression in pediatric epithelium. In cell culture, pediatric cells had higher colony formation ability, sustained in vitro growth, and outcompeted adult cells in a direct competitive proliferation assay. Our results demonstrate cell-intrinsic differences between airway epithelial cells from children and adults in both homeostatic and proliferative states.
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BACKGROUND: Lung squamous cell carcinoma (LUSC) accounts for a significant proportion of cancer deaths worldwide, and is preceded by the appearance of progressively disorganised pre-invasive lesions in the airway epithelium. Yet the biological mechanisms underlying progression of pre-invasive lesions into invasive LUSC are not fully understood. LRIG1 (leucine-rich repeats and immunoglobulin-like domains 1) is downregulated in pre-invasive airway lesions and invasive LUSC tumours and this correlates with decreased lung cancer patient survival. METHODS AND RESULTS: Using an Lrig1 knock-in reporter mouse and human airway epithelial cells collected at bronchoscopy, we show that during homeostasis LRIG1 is heterogeneously expressed in the airway epithelium. In basal airway epithelial cells, the suspected cell of origin of LUSC, LRIG1 identifies a subpopulation of progenitor cells with higher in vitro proliferative and self-renewal potential in both the mouse and human. Using the N-nitroso-tris-chloroethylurea (NTCU)-induced murine model of LUSC, we find that Lrig1 loss-of-function leads to abnormally high cell proliferation during the earliest stages of pre-invasive disease and to the formation of significantly larger invasive tumours, suggesting accelerated disease progression. CONCLUSION: Together, our findings identify LRIG1 as a marker of basal airway progenitor cells with high proliferative potential and as a regulator of pre-invasive lung cancer progression. This work highlights the clinical relevance of LRIG1 and the potential of the NTCU-induced LUSC model for functional assessment of candidate tumour suppressors and oncogenes.
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Carcinoma de Pulmón de Células no Pequeñas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Animales , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Humanos , Pulmón/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Glicoproteínas de Membrana/efectos adversos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Proteínas del Tejido Nervioso/metabolismo , OncogenesRESUMEN
Before squamous cell lung cancer develops, precancerous lesions can be found in the airways. From longitudinal monitoring, we know that only half of such lesions become cancer, whereas a third spontaneously regress. Although recent studies have described the presence of an active immune response in high-grade lesions, the mechanisms underpinning clinical regression of precancerous lesions remain unknown. Here, we show that host immune surveillance is strongly implicated in lesion regression. Using bronchoscopic biopsies from human subjects, we find that regressive carcinoma in situ lesions harbor more infiltrating immune cells than those that progress to cancer. Moreover, molecular profiling of these lesions identifies potential immune escape mechanisms specifically in those that progress to cancer: antigen presentation is impaired by genomic and epigenetic changes, CCL27-CCR10 signaling is upregulated, and the immunomodulator TNFSF9 is downregulated. Changes appear intrinsic to the carcinoma in situ lesions, as the adjacent stroma of progressive and regressive lesions are transcriptomically similar. SIGNIFICANCE: Immune evasion is a hallmark of cancer. For the first time, this study identifies mechanisms by which precancerous lesions evade immune detection during the earliest stages of carcinogenesis and forms a basis for new therapeutic strategies that treat or prevent early-stage lung cancer.See related commentary by Krysan et al., p. 1442.This article is highlighted in the In This Issue feature, p. 1426.
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Carcinoma de Células Escamosas/inmunología , Vigilancia Inmunológica/inmunología , Neoplasias Pulmonares/inmunología , HumanosRESUMEN
Current methods to replace damaged upper airway epithelium with exogenous cells are limited. Existing strategies use grafts that lack mucociliary function, leading to infection and the retention of secretions and keratin debris. Strategies that regenerate airway epithelium with mucociliary function are clearly desirable and would enable new treatments for complex airway disease.Here, we investigated the influence of the extracellular matrix (ECM) on airway epithelial cell adherence, proliferation and mucociliary function in the context of bioengineered mucosal grafts. In vitro, primary human bronchial epithelial cells (HBECs) adhered most readily to collagen IV. Biological, biomimetic and synthetic scaffolds were compared in terms of their ECM protein content and airway epithelial cell adherence.Collagen IV and laminin were preserved on the surface of decellularised dermis and epithelial cell attachment to decellularised dermis was greater than to the biomimetic or synthetic alternatives tested. Blocking epithelial integrin α2 led to decreased adherence to collagen IV and to decellularised dermis scaffolds. At air-liquid interface (ALI), bronchial epithelial cells cultured on decellularised dermis scaffolds formed a differentiated respiratory epithelium with mucociliary function. Using in vivo chick chorioallantoic membrane (CAM), rabbit airway and immunocompromised mouse models, we showed short-term preservation of the cell layer following transplantation.Our results demonstrate the feasibility of generating HBEC grafts on clinically applicable decellularised dermis scaffolds and identify matrix proteins and integrins important for this process. The long-term survivability of pre-differentiated epithelia and the relative merits of this approach against transplanting basal cells should be assessed further in pre-clinical airway transplantation models.
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Colágeno , Matriz Extracelular , Laminina , Mucosa Respiratoria , Andamios del Tejido , Animales , Bronquios , Células Cultivadas , Células Epiteliales , Humanos , ConejosRESUMEN
Therapeutic approaches requiring the intravenous injection of autologous or allogeneic mesenchymal stromal cells (MSCs) are currently being evaluated for treatment of a range of diseases, including orthopaedic injuries. An alternative approach would be to mobilise endogenous MSCs into the blood, thereby reducing costs and obviating regulatory and technical hurdles associated with development of cell therapies. However, pharmacological tools for MSC mobilisation are currently lacking. Here we show that ß3 adrenergic agonists (ß3AR) in combination with a CXCR4 antagonist, AMD3100/Plerixafor, can mobilise MSCs into the blood in mice and rats. Mechanistically we show that reversal of the CXCL12 gradient across the bone marrow endothelium and local generation of endocannabinoids may both play a role in this process. Using a spine fusion model we provide evidence that this pharmacological strategy for MSC mobilisation enhances bone formation.
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Tobacco smoking causes lung cancer1-3, a process that is driven by more than 60 carcinogens in cigarette smoke that directly damage and mutate DNA4,5. The profound effects of tobacco on the genome of lung cancer cells are well-documented6-10, but equivalent data for normal bronchial cells are lacking. Here we sequenced whole genomes of 632 colonies derived from single bronchial epithelial cells across 16 subjects. Tobacco smoking was the major influence on mutational burden, typically adding from 1,000 to 10,000 mutations per cell; massively increasing the variance both within and between subjects; and generating several distinct mutational signatures of substitutions and of insertions and deletions. A population of cells in individuals with a history of smoking had mutational burdens that were equivalent to those expected for people who had never smoked: these cells had less damage from tobacco-specific mutational processes, were fourfold more frequent in ex-smokers than current smokers and had considerably longer telomeres than their more-mutated counterparts. Driver mutations increased in frequency with age, affecting 4-14% of cells in middle-aged subjects who had never smoked. In current smokers, at least 25% of cells carried driver mutations and 0-6% of cells had two or even three drivers. Thus, tobacco smoking increases mutational burden, cell-to-cell heterogeneity and driver mutations, but quitting promotes replenishment of the bronchial epithelium from mitotically quiescent cells that have avoided tobacco mutagenesis.
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Bronquios/metabolismo , Mutagénesis , Mutación/genética , Mucosa Respiratoria/metabolismo , Fumar Tabaco/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Bronquios/citología , Bronquios/patología , Niño , Células Clonales/citología , Células Clonales/metabolismo , Análisis Mutacional de ADN , Femenino , Humanos , Neoplasias Pulmonares/etiología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Mucosa Respiratoria/citología , Mucosa Respiratoria/patología , Fumadores , Telómero/genética , Telómero/metabolismo , Fumar Tabaco/efectos adversos , Fumar Tabaco/patología , Adulto JovenRESUMEN
IMPACT STATEMENT: This article describes a method for engrafting epithelial progenitor cells to a revascularized scaffold in a protective and supportive collagen-rich environment. This method has the potential to overcome two key limitations of existing grafting techniques as epithelial cells are protected from mechanical shear and the relatively hypoxic phase that occurs while grafts revascularize, offering the opportunity to provide epithelial cells to decellularized allografts at the point of implantation. Advances in this area will improve the safety and efficacy of bioengineered organ transplantation.
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Colágeno/metabolismo , Fibroblastos/citología , Pulmón/citología , Trasplante de Células Madre , Células Madre/citología , Ingeniería de Tejidos , Tráquea/fisiología , Animales , Supervivencia Celular , Pollos , Membrana Corioalantoides/metabolismo , Células Epiteliales/citología , Masculino , Conejos , Andamios del TejidoRESUMEN
There is considerable interest in the ex vivo propagation of primary human basal epithelial stem/progenitor cells with a view to their use in drug development, toxicity testing and regenerative medicine. These cells can be expanded in co-culture with mitotically inactivated 3T3-J2 murine embryonic feeder cells but, similar to other epithelial cell culture systems employing 3T3-J2 cells, the aspects of cross-talk between 3T3-J2 cells and human airway basal cells that are critical for their expansion remain largely unknown. In this study, we investigated secreted growth factors that are produced by 3T3-J2 cells and act upon primary human airway basal cells. We found robust production of hepatocyte growth factor (HGF) from fibroblast feeder cells following mitotic inactivation. Consistent with the limited cross-species reactivity of murine HGF on the human HGF receptor (MET; HGFR), MET inhibition did not affect proliferative responses in human airway basal cells and HGF could not replace feeder cells in this culture system. However, we found that murine HGF is not completely inactive on human airway epithelial cells or cancer cell lines but stimulates the phosphorylation of GRB2-associated-binding protein 2 (GAB2) and signal transducer and activator of transcription 6 (STAT6). Although HGF induces phosphorylation of STAT6 tyrosine 641 (Y641), there is no subsequent STAT6 nuclear translocation or STAT6-driven transcriptional response. Overall, these findings highlight the relevance of cross-species protein interactions between murine feeder cells and human epithelial cells in 3T3-J2 co-culture and demonstrate that STAT6 phosphorylation occurs in response to MET activation in epithelial cells. However, STAT6 nuclear translocation does not occur in response to HGF, precluding the transcriptional activity of STAT6.
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Comunicación Celular , Células Epiteliales/metabolismo , Células Nutrientes/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Mucosa Respiratoria/metabolismo , Animales , Línea Celular , Técnicas de Cocultivo , Activación Enzimática , Células Epiteliales/citología , Células Nutrientes/citología , Humanos , Ratones , Mucosa Respiratoria/citología , Factor de Transcripción STAT6/metabolismoRESUMEN
Pre-clinical non-small cell lung cancer (NSCLC) models are poorly representative of the considerable inter- and intra-tumor heterogeneity of the disease in patients. Primary cell-based in vitro models of NSCLC are therefore desirable for novel therapy development and personalized cancer medicine. Methods have been described to generate rapidly proliferating epithelial cell cultures from multiple human epithelia using 3T3-J2 feeder cell culture in the presence of Y-27632, a RHO-associated protein kinase (ROCK) inhibitor, in what are known as "conditional reprograming conditions" (CRC) or 3T3 + Y. In some cancer studies, variations of this methodology have allowed primary tumor cell expansion across a number of cancer types but other studies have demonstrated the preferential expansion of normal epithelial cells from tumors in such conditions. Here, we report our experience regarding the derivation of primary NSCLC cell cultures from 12 lung adenocarcinoma patients enrolled in the Tracking Cancer Evolution through Therapy (TRACERx) clinical study and discuss these in the context of improving the success rate for in vitro cultivation of cells from NSCLC tumors.
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Carcinoma de Pulmón de Células no Pequeñas/patología , Técnicas de Cocultivo/métodos , Células Epiteliales/citología , Neoplasias Pulmonares/patología , Células 3T3 , Anciano , Anciano de 80 o más Años , Amidas/farmacología , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Proliferación Celular , Células Epiteliales/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Pulmonares/genética , Masculino , Ratones , Persona de Mediana Edad , Trasplante de Neoplasias , Piridinas/farmacología , Mucosa Respiratoria/citología , Análisis de Secuencia de ADN , Células Tumorales CultivadasRESUMEN
Autologous airway epithelial cells have been used in clinical tissue-engineered airway transplantation procedures with a view to assisting mucosal regeneration and restoring mucociliary escalator function. However, limited time is available for epithelial cell expansion due to the urgent nature of these interventions and slow epithelial regeneration has been observed in patients. Human airway epithelial cells can be expanded from small biopsies or brushings taken during bronchoscopy procedures, but the optimal mode of tissue acquisition from patients has not been investigated. Here, we compared endobronchial brushing and endobronchial biopsy samples in terms of their cell number and their ability to initiate basal epithelial stem cell cultures. We found that direct co-culture of samples with 3T3-J2 feeder cells in culture medium containing a Rho-associated protein kinase inhibitor, Y-27632, led to the selective expansion of greater numbers of basal epithelial stem cells during the critical early stages of culture than traditional techniques. Additionally, we established the benefit of initiating cell cultures from cell suspensions, either using brushing samples or through enzymatic digestion of biopsies, over explant culture. Primary epithelial cell cultures were initiated from endobronchial biopsy samples that had been cryopreserved before the initiation of cell cultures, suggesting that cryopreservation could eliminate the requirement for close proximity between the clinical facility in which biopsy samples are taken and the specialist laboratory in which epithelial cells are cultured. Overall, our results suggest ways to expedite epithelial cell preparation in future airway cell therapy or bioengineered airway transplantation procedures.
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Bronquios/patología , Separación Celular/métodos , Células Epiteliales/patología , Células 3T3 , Animales , Biopsia , Proliferación Celular , Criopreservación , Humanos , RatonesRESUMEN
Extracellular vesicles (EVs) are lipid membrane-enclosed nanoparticles released by cells. They mediate intercellular communication by transferring biological molecules and therefore have potential as innovative drug delivery vehicles. TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis of cancer cells. Unfortunately, the clinical application of recombinant rTRAIL has been hampered by its low bioavailability and resistance of cancer cells. EV-mediated TRAIL delivery may circumvent these problems. Mesenchymal stromal cells (MSCs) produce EVs and could be a good source for therapeutic EV production. We investigated if TRAIL could be expressed in MSC-derived EVs and examined their cancer cell-killing efficacy. EVs were isolated by ultracentrifugation and were membranous particles of 50-70 nm in diameter. Both MSC- and TRAIL-expressing MSC (MSCT)-derived EVs express CD63, CD9 and CD81, but only MSCT-EVs express surface TRAIL. MSCT-EVs induced apoptosis in 11 cancer cell lines in a dose-dependent manner but showed no cytotoxicity in primary human bronchial epithelial cells. Caspase activity inhibition or TRAIL neutralisation blocked the cytotoxicity of TRAIL-positive EVs. MSCT-EVs induced pronounced apoptosis in TRAIL-resistant cancer cells and this effect could be further enhanced using a CDK9 inhibitor. These data indicate that TRAIL delivery by MSC-derived EVs is an effective anticancer therapy.
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Patients with large tracheal lesions unsuitable for conventional endoscopic or open operations may require a tracheal replacement but there is no present consensus of how this may be achieved. Tissue engineering using decellularized or synthetic tracheal scaffolds offers a new avenue for airway reconstruction. Decellularized human donor tracheal scaffolds have been applied in compassionate-use clinical cases but naturally derived extracellular matrix (ECM) scaffolds demand lengthy preparation times. Here, we compare a clinically applied detergent-enzymatic method (DEM) with an accelerated vacuum-assisted decellularization (VAD) protocol. We examined the histological appearance, DNA content and extracellular matrix composition of human donor tracheae decellularized using these techniques. Further, we performed scanning electron microscopy (SEM) and biomechanical testing to analyze decellularization performance. To assess the biocompatibility of scaffolds generated using VAD, we seeded scaffolds with primary human airway epithelial cells in vitro and performed in vivo chick chorioallantoic membrane (CAM) and subcutaneous implantation assays. Both DEM and VAD protocols produced well-decellularized tracheal scaffolds with no adverse mechanical effects and scaffolds retained the capacity for in vitro and in vivo cellular integration. We conclude that the substantial reduction in time required to produce scaffolds using VAD compared to DEM (approximately 9 days vs. 3-8 weeks) does not compromise the quality of human tracheal scaffold generated. These findings might inform clinical decellularization techniques as VAD offers accelerated scaffold production and reduces the associated costs.
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Sistema Libre de Células/química , Matriz Extracelular/química , Matriz Extracelular/ultraestructura , Ingeniería de Tejidos/instrumentación , Andamios del Tejido , Tráquea/citología , Tráquea/crecimiento & desarrollo , Fraccionamiento Celular/instrumentación , Fraccionamiento Celular/métodos , Células Cultivadas , Diseño de Equipo , Análisis de Falla de Equipo , Humanos , Ingeniería de Tejidos/métodos , Tráquea/ultraestructura , VacioRESUMEN
RATIONALE: Stem cell-based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell-seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts. OBJECTIVES: To define a scalable cell culture system to deliver airway epithelium to clinical grafts. METHODS: Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T3+Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air-liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air-liquid interface cultures. MEASUREMENTS AND MAIN RESULTS: 3T3-J2 feeder cells and ROCK inhibition allowed rapid expansion of airway basal cells. These cells were capable of multipotent differentiation in vitro, generating both ciliated and goblet cell lineages. Cilia were functional with normal beat frequency and pattern. Cultured cells repopulated tracheal scaffolds in a heterotopic transplantation xenograft model. CONCLUSIONS: Our method generates large numbers of functional airway basal epithelial cells with the efficiency demanded by clinical transplantation, suggesting its suitability for use in tracheal reconstruction.
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Células Epiteliales/metabolismo , Enfermedades Respiratorias/terapia , Células Madre/metabolismo , Ingeniería de Tejidos/métodos , Diferenciación Celular/fisiología , Células Cultivadas , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Depuración Mucociliar/fisiología , Reacción en Cadena de la Polimerasa , Mucosa Respiratoria/fisiologíaRESUMEN
BACKGROUND AIMS: Mesenchymal stromal cell (MSC) delivery of pro-apoptotic tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an attractive strategy for anticancer therapy. MSCs expressing full-length human TRAIL (flT) or its soluble form (sT) have previously been shown to be effective for cancer killing. However, a comparison between the two forms has never been performed, leaving it unclear which approach is most effective. This study addresses the issue for the possible clinical application of TRAIL-expressing MSCs in the future. METHODS: MSCs were transduced with lentiviruses expressing flT or an isoleucine zipper-fused sT. TRAIL expression was examined and cancer cell apoptosis was measured after treatment with transduced MSCs or with MSC-derived soluble TRAIL. RESULTS: The transduction does not adversely affect cell phenotype. The sT-transduced MSCs (MSC-sT) secrete abundant levels of soluble TRAIL but do not present the protein on the cell surface. Interestingly, the flT-transduced MSCs (MSC-flT) not only express cell-surface TRAIL but also release flT into medium. These cells were examined for inducing apoptosis in 20 cancer cell lines. MSC-sT cells showed very limited effects. By contrast, MSC-flT cells demonstrated high cancer cell-killing efficiency. More importantly, MSC-flT cells can overcome some cancer cell resistance to recombinant TRAIL. In addition, both cell surface flT and secreted flT are functional for inducing apoptosis. The secreted flT was found to have higher cancer cell-killing capacity than either recombinant TRAIL or MSC-secreted sT. CONCLUSIONS: These observations demonstrate that MSC delivery of flT is superior to MSC delivery of sT for cancer therapy.
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Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Células Madre Mesenquimatosas/metabolismo , Neoplasias/terapia , Ligando Inductor de Apoptosis Relacionado con TNF/uso terapéutico , Apoptosis/genética , Apoptosis/fisiología , Línea Celular Tumoral , Proliferación Celular/genética , Humanos , Células Madre Mesenquimatosas/citología , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Transducción Genética/métodosRESUMEN
The mechanism by which trauma initiates healing remains unclear. Precise understanding of these events may define interventions for accelerating healing that could be translated to the clinical arena. We previously reported that addition of low-dose recombinant human TNF (rhTNF) at the fracture site augmented fracture repair in a murine tibial fracture model. Here, we show that local rhTNF treatment is only effective when administered within 24 h of injury, when neutrophils are the major inflammatory cell infiltrate. Systemic administration of anti-TNF impaired fracture healing. Addition of rhTNF enhanced neutrophil recruitment and promoted recruitment of monocytes through CCL2 production. Conversely, depletion of neutrophils or inhibition of the chemokine receptor CCR2 resulted in significantly impaired fracture healing. Fragility, or osteoporotic, fractures represent a major medical problem as they are associated with permanent disability and premature death. Using a murine model of fragility fractures, we found that local rhTNF treatment improved fracture healing during the early phase of repair. If translated clinically, this promotion of fracture healing would reduce the morbidity and mortality associated with delayed patient mobilization.
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Huesos/efectos de los fármacos , Huesos/fisiología , Curación de Fractura/efectos de los fármacos , Fracturas Óseas/patología , Inmunidad Innata/efectos de los fármacos , Factor de Necrosis Tumoral alfa/administración & dosificación , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Huesos/inmunología , Quimiocina CCL2/metabolismo , Modelos Animales de Enfermedad , Curación de Fractura/inmunología , Fracturas Óseas/tratamiento farmacológico , Humanos , Ratones , Monocitos/inmunología , Neutrófilos/inmunología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
UNLABELLED: HIF-1α is a transcription factor that is activated during hypoxia and inflammation and is a key regulator of angiogenesis in vivo. During the development of asthma, peribronchial angiogenesis is induced in response to aeroallergens and is thought to be an important feature of sustained chronic allergic inflammation. Recently, elevated HIF-1α levels have been demonstrated in both the lung tissue and bronchoalveolar lavage of allergic patients, respectively. Therefore, we investigated the role of HIF-1α on the development of angiogenesis and inflammation following acute and chronic allergen exposure. Our data shows that intranasal exposure to house dust mite (HDM) increases the expression of HIF-1α in the lung, whilst reducing the expression of the HIF-1α negative regulators, PHD1 and PHD3. Blockade of HIF-1α in vivo, significantly decreased allergic inflammation and eosinophilia induced by allergen, due to a reduction in the levels of IL-5 and Eotaxin-2. Importantly, HIF-1α blockade significantly decreased levels of VEGF-A and CXCL1 in the lungs, which in turn led to a profound decrease in the recruitment of endothelial progenitor cells and a reduction of peribronchial angiogenesis. Furthermore, HDM or IL-4 treatment of primary lung macrophages resulted in significant production of both VEGF-A and CXCL1; inhibition of HIF-1α activity abrogated the production of these factors via an up-regulation of PHD1 and PHD3. These findings suggest that novel strategies to reduce the expression and activation of HIF-1α in lung macrophages may be used to attenuate allergen-induced airway inflammation and angiogenesis through the modulation of VEGF-A and CXCL1 expression. CLINICAL RELEVANCE: This study provides new insights into the role of HIF-1α in the development of peribronchial angiogenesis and inflammation in a murine model of allergic airway disease. These findings indicate that strategies to reduce activation of macrophage derived HIF-1α may be used as a target to improve asthma pathology.
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Remodelación de las Vías Aéreas (Respiratorias) , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Pulmón/patología , Pulmón/fisiopatología , Macrófagos/metabolismo , Macrófagos/parasitología , Animales , Quimiocina CCL24/metabolismo , Quimiocina CXCL1/metabolismo , Células Endoteliales/metabolismo , Eosinofilia/complicaciones , Eosinofilia/patología , Eosinofilia/fisiopatología , Femenino , Hipersensibilidad/complicaciones , Hipersensibilidad/parasitología , Hipersensibilidad/patología , Hipersensibilidad/fisiopatología , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Inflamación/complicaciones , Inflamación/patología , Inflamación/fisiopatología , Pulmón/parasitología , Ratones , Ratones Endogámicos BALB C , Neovascularización Fisiológica , Pyroglyphidae , Células Madre/metabolismo , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Overexpression of the transforming growth factor ß family signalling molecule smad2 in the airway epithelium provokes enhanced allergen-induced airway remodelling in mice, concomitant with elevated levels of interleukin (IL)-25. OBJECTIVE: We investigated whether IL-25 plays an active role in driving this airway remodelling. METHODS: Anti-IL-25 antibody was given to mice exposed to either inhaled house dust mite (HDM) alone, or in conjunction with an adenoviral smad2 vector which promotes an enhanced remodelling phenotype. RESULTS: Blocking IL-25 in allergen-exposed mice resulted in a moderate reduction in pulmonary eosinophilia and levels of T helper type 2 associated cytokines, IL-5 and IL-13. In addition, IL-25 neutralisation abrogated peribronchial collagen deposition, airway smooth muscle hyperplasia and airway hyperreactivity in control mice exposed to HDM and smad2-overexpressing mice. IL-25 was shown to act directly on human fibroblasts to induce collagen secretion. Recruitment of endothelial progenitor cells to the lung and subsequent neovascularisation was also IL-25 dependent, demonstrating a direct role for IL-25 during angiogenesis in vivo. Moreover, the secretion of innate epithelial derived cytokines IL-33 and thymic stromal lymphopoietin (TSLP) was completely ablated. CONCLUSIONS: In addition to modulating acute inflammation, we now demonstrate a role for IL-25 in orchestrating airway remodelling. IL-25 also drives IL-33 and TSLP production in the lung. These data delineate a wider role for IL-25 in mediating structural changes to the lung following allergen exposure and implicate IL-25 as a novel therapeutic target for the treatment of airway remodelling in asthma.
Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias)/inmunología , Hiperreactividad Bronquial/inmunología , Interleucinas/inmunología , Pyroglyphidae/inmunología , Remodelación de las Vías Aéreas (Respiratorias)/genética , Animales , Asma/inmunología , Asma/metabolismo , Biopsia con Aguja , Hiperreactividad Bronquial/metabolismo , Líquido del Lavado Bronquioalveolar/inmunología , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Fibroblastos/inmunología , Fibroblastos/metabolismo , Regulación de la Expresión Génica/inmunología , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Distribución Aleatoria , Sensibilidad y Especificidad , Proteína Smad2/inmunologíaRESUMEN
The development of genetically marked animal tumour xenografts is an area of ongoing research to enable easier and more reliable testing of cancer therapies. Genetically marked tumour models have a number of advantages over conventional tumour models, including the easy longitudinal monitoring of therapies and the reduced number of animals needed for trials. Several different methods have been used in previous studies to mark tumours genetically, however all have limitations, such as genotoxicity and other artifacts related to the usage of integrating viral vectors. Recently, we have generated an episomally maintained plasmid DNA (pDNA) expression system based on Scaffold/Matrix Attachment Region (S/MAR), which permits long-term luciferase transgene expression in the mouse liver. Here we describe a further usage of this pDNA vector with the human Ubiquitin C promoter to create stably transfected human hepatoma (Huh7) and human Pancreatic Carcinoma (MIA-PaCa2) cell lines, which were delivered into "immune deficient" mice and monitored longitudinally over time using a bioluminometer. Both cell lines revealed sustained episomal long-term luciferase expression and formation of a tumour showing the pathological characteristics of hepatocellular carcinoma (HCC) and pancreatic carcinoma (PaCa), respectively. This is the first demonstration that a pDNA vector can confer sustained episomal luciferase transgene expression in various mouse tumour models and can thus be readily utilised to follow tumour formation without interfering with the cellular genome.
Asunto(s)
Línea Celular Tumoral , Regiones de Fijación a la Matriz/genética , Plásmidos/metabolismo , Trasplante Heterólogo/métodos , Células Tumorales Cultivadas/metabolismo , Animales , Southern Blotting , Cartilla de ADN/genética , Vectores Genéticos/genética , Humanos , Inmunohistoquímica , Estudios Longitudinales , Luciferasas , Ratones , Ratones SCID , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Ubiquitina C/genéticaRESUMEN
Synaptic terminals are known to expand and contract throughout an animal's life. The physiological constraints and demands that regulate appropriate synaptic growth and connectivity are currently poorly understood. In previous work, we identified a Drosophila model of lysosomal storage disease (LSD), spinster (spin), with larval neuromuscular synapse overgrowth. Here we identify a reactive oxygen species (ROS) burden in spin that may be attributable to previously identified lipofuscin deposition and lysosomal dysfunction, a cellular hallmark of LSD. Reducing ROS in spin mutants rescues synaptic overgrowth and electrophysiological deficits. Synapse overgrowth was also observed in mutants defective for protection from ROS and animals subjected to excessive ROS. ROS are known to stimulate JNK and fos signaling. Furthermore, JNK and fos in turn are known potent activators of synapse growth and function. Inhibiting JNK and fos activity in spin rescues synapse overgrowth and electrophysiological deficits. Similarly, inhibiting JNK, fos, and jun activity in animals with excessive oxidative stress rescues the overgrowth phenotype. These data suggest that ROS, via activation of the JNK signaling pathway, are a major regulator of synapse overgrowth. In LSD, increased autophagy contributes to lysosomal storage and, presumably, elevated levels of oxidative stress. In support of this suggestion, we report here that impaired autophagy function reverses synaptic overgrowth in spin. Our data describe a previously unexplored link between oxidative stress and synapse overgrowth via the JNK signaling pathway.