RESUMEN
BACKGROUND: Cytoreductive surgery (CRS) in combination with hyperthermic intraperitoneal chemotherapy (HIPEC) is an option in advanced peritoneal sarcomatosis. Nevertheless, CRS and HIPEC are not successful in all patients. An enhancement of HIPEC using photodynamic therapy (PDT) might be beneficial. Therefore, a combination of the photosensitizer hypericin (HYP) with HIPEC was evaluated in an animal model. PROCEDURE: An established HIPEC animal model for rhabdomyosarcoma (NOD/LtSz-scid IL2Rγnullmice, n = 80) was used. All groups received HYP (100 µg/200 µl) intraperitoneally with and without cisplatin-based (30 or 60 mg/m2 ) HIPEC (37°C or 42°C, for 60 minutes) (five groups, each n = 16). Peritoneal cancer index (PCI) was documented visually and by HYP-based photodynamic diagnosis (PDD). HYP-based PDT of the tumor was performed. Tissue samples were evaluated regarding proliferation (Ki-67) and apoptosis (TUNEL). RESULTS: HYP uptake was detected even in smallest tumor nodes (<1 mm) with improved tumor detection during PDD (PCI with PDD vs. PCI without PDD: 8.5 vs. 7, p < .001***). Apoptotic effects after PDT without HIPEC were limited to the tumor surface, whereas PDT after HIPEC (60 mg/m2 , 42°C) showed additional reduction of tumor proliferation in the top nine to 11 cell layers (50 µm). CONCLUSION: HYP as fluorescent photosensitizer offers an intraoperative diagnostic advantage detecting intraperitoneal tumor dissemination. The combination of HYP and cisplatin-based HIPEC was feasible in vivo, showing enhanced effects on tumor proliferation and apoptosis induction across the tumor surface. Further studies combining HYP and HIPEC will follow to establish a clinical application.
Asunto(s)
Hipertermia Inducida , Neoplasias Peritoneales , Rabdomiosarcoma Embrionario , Rabdomiosarcoma , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Cisplatino/uso terapéutico , Terapia Combinada , Humanos , Quimioterapia Intraperitoneal Hipertérmica , Antígeno Ki-67 , Modelos Animales , Neoplasias Peritoneales/tratamiento farmacológico , Fármacos Fotosensibilizantes/uso terapéutico , Rabdomiosarcoma/tratamiento farmacológico , Rabdomiosarcoma Embrionario/tratamiento farmacológico , Tasa de SupervivenciaRESUMEN
The identification of epitopes that elicit cytotoxic T-lymphocyte activity is a prerequisite for the development of cancer-specific immunotherapies. However, especially the parallel characterization of several epitopes is limited by the availability of T cells. Microarrays have enabled an unprecedented miniaturization and parallelization in biological assays. Here, we developed peptide microarrays for the detection of CTL activity. MHC class I-binding peptide epitopes were pipetted onto polymer-coated glass slides. Target cells, loaded with the cell-impermeant dye calcein, were incubated on these arrays, followed by incubation with antigen-expanded CTLs. Cytotoxic activity was detected by release of calcein and detachment of target cells. With only 200,000 cells per microarray, CTLs could be detected at a frequency of 0.5% corresponding to 1,000 antigen-specific T cells. Target cells and CTLs only settled on peptide spots enabling a clear separation of individual epitopes. Even though no physical boundaries were present between the individual spots, peptide loading only occurred locally and cytolytic activity was confined to the spots carrying the specific epitope. The peptide microarrays provide a robust platform that implements the whole process from antigen presentation to the detection of CTL activity in a miniaturized format. The method surpasses all established methods in the minimum numbers of cells required. With antigen uptake occurring on the microarray, further applications are foreseen in the testing of antigen precursors that require uptake and processing prior to presentation.
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Pruebas Inmunológicas de Citotoxicidad , Mapeo Epitopo/métodos , Inmunoterapia Adoptiva , Neoplasias/terapia , Linfocitos T Citotóxicos/metabolismo , Recuento de Células , Línea Celular Tumoral , Fluoresceínas/metabolismo , Colorantes Fluorescentes/metabolismo , Antígenos HLA-A/metabolismo , Antígeno HLA-A2 , Humanos , Análisis por Micromatrices , Miniaturización , Mucina-1/inmunología , Mucina-1/metabolismo , Neoplasias/inmunología , Neoplasias/patología , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/patologíaRESUMEN
The human DEAD-box helicase DDX3 is a multi-functional protein involved in the regulation of gene expression and additional non-conventional roles as signalling adaptor molecule that are independent of its enzymatic RNA remodeling activity. It is a nucleo-cytoplasmic shuttling protein and it has previously been suggested that dysregulation of its subcellular localization could contribute to tumourigenesis. Indeed, both tumour suppressor and oncogenic functions have been attributed to DDX3. In this study, we investigated the regulation of DDX3's nucleocytoplasmic shuttling. We confirmed that an N-terminal conserved Nuclear Export Signal (NES) is required for export of human DDX3 from the nucleus, and identified three regions within DDX3 that can independently facilitate its nuclear import. We also aimed to identify conditions that alter DDX3's subcellular localisation. Viral infection, cytokine treatment and DNA damage only induced minor changes in DDX3's subcellular distribution as determined by High Content Analysis. However, DDX3's nuclear localization increased in early mitotic cells (during prophase) concomitant with an increase in DDX3 expression levels. Our results are likely to have implications for the proposed use of (nuclear) DDX3 as a prognostic biomarker in cancer.
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Núcleo Celular/metabolismo , ARN Helicasas DEAD-box/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Ciclo Celular , Secuencia Conservada , ARN Helicasas DEAD-box/química , Células HEK293 , Células HeLa , Humanos , Carioferinas/metabolismo , Mutación/genética , Señales de Exportación Nuclear , Señales de Localización Nuclear/metabolismo , Fosforilación , Fosfotreonina/metabolismo , Unión Proteica , Dominios Proteicos , Receptores Citoplasmáticos y Nucleares/metabolismo , Fracciones Subcelulares/metabolismo , Regulación hacia Arriba/genética , Proteína Exportina 1RESUMEN
Screening of RNA interference (RNAi) libraries in primary T cells is labor-intensive and technically challenging because these cells are hard to transfect. Chemically modified, self-delivering small interfering RNAs (siRNAs) offer a solution to this problem, because they enter hard-to-transfect cell types without needing a delivery reagent and are available in library format for RNAi screening. In this study, we have screened a library of chemically modified, self-delivering siRNAs targeting the expression of 72 distinct genes in conjunction with an image-based high-content-analysis platform as a proof-of-principle strategy to identify genes involved in lymphocyte function-associated antigen-1 (LFA-1)-mediated migration in primary human T cells. Our library-screening strategy identified the small GTPase RhoA as being crucial for T cell polarization and migration in response to LFA-1 stimulation and other migratory ligands. We also demonstrate that multiple downstream assays can be performed within an individual RNAi screen and have used the remainder of the cells for additional assays, including cell viability and adhesion to ICAM-1 (the physiological ligand for LFA-1) in the absence or presence of the chemokine SDF-1α. This study therefore demonstrates the ease and benefits of conducting siRNA library screens in primary human T cells using self-delivering, chemically modified siRNAs, and it emphasizes the feasibility and potential of this approach for elucidating the signaling pathways that regulate T cell function.
Asunto(s)
Movimiento Celular/genética , Interferencia de ARN , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Linfocitos T/fisiología , Expresión Génica , Biblioteca de Genes , Silenciador del Gen , Estudios de Asociación Genética , Humanos , Antígeno-1 Asociado a Función de Linfocito/genética , Cultivo Primario de Células , Reproducibilidad de los Resultados , Transfección , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
High-content screening (HCS) technologies are becoming increasingly used in both large-scale drug discovery and basic research programs. These automated imaging and analysis technologies enable the researcher to elucidate the complex biology that underlies the functions of genes, proteins, and other biomolecules at the cellular level. HCS combines the power of automated digital microscopy and advanced software-based image analysis algorithms to detect and quantify biological changes in cells and tissues. This technology is a particularly powerful tool when used to interrogate the cellular effects of exogenously applied agents such as RNAi and/or small molecules. HCS allows for the evaluation of cellular perturbations that occur both at the level of the single cell and within cellular populations. In a multivariate approach, multiple cellular parameters are collected, allowing for more complex analysis. However, in these scenarios, data flow and management still represent substantial bottlenecks in HCS projects. HCS data include a diversity of information from multiple sources such as details pertaining to screening libraries (e.g., siRNA and small molecules), image stacks acquired from automated microscopes (of which there may be up to several million), and the image analysis data. From this, postprocessing algorithms are required to generate statistical, quality control bioinformatic information and ultimately a final hit list. To accomplish these individual tasks, numerous tools can be used to perform each analytical step; however, management of the entire information flow currently requires the use of commercially available proprietary software, the scope of which is often limited, or bespoke customized scripts. In this article, the authors introduce an open-source research tool that allows for the management of the entire data flow of the HCS data chain, by handling and linking information and providing many powerful postprocessing and visualization tools.
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Bioensayo/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Programas Informáticos , Flujo de Trabajo , Estadística como AsuntoRESUMEN
Cellular signal transduction proceeds through a complex network of molecular interactions and enzymatic activities. The timing of these molecular events is critical for the propagation of a signal and the generation of a specific cellular response. To define the timing of signalling events, we introduce the combination of high-resolution confocal microscopy with the application of small-molecule inhibitors at various stages of signal transduction in T cells. Inhibitors of Src-family tyrosine kinases and actin dynamics were employed to dissect the role of the lymphocyte-specific tyrosine kinase Lck in the formation and maintenance of T cell receptor/CD3-dependent contacts. Anti-CD3epsilon-coated coverslips served as a highly defined stimulus. The kinetics of the recruitment of the yellow fluorescent protein-tagged signalling protein ZAP-70 were detected by high-resolution confocal microscopy. The analysis revealed that at 5 min after receptor engagement, Lck activity was required for maintenance of contacts. In contrast, after 20 min of receptor engagement, the contacts were Lck-independent. The relevance of the timing of inhibitor application provides a pharmacological concept for the maturation of T cell-substrate contacts.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/enzimología , Animales , Complejo CD3/fisiología , Humanos , Activación de Linfocitos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/fisiología , Microscopía Confocal/métodos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/fisiología , Proteínas Recombinantes de Fusión/farmacología , Linfocitos T/inmunología , Factores de Tiempo , Proteína Tirosina Quinasa ZAP-70RESUMEN
The generation of microarrays by functionalization of hydrophobic glass surfaces with conjugates of triacylated lipophilic end-groups and with a peptide or hapten as a test substance is presented. Immobilization on the hydrophobic surfaces through the triacylated anchor group is fully orthogonal to the reactivity of functional groups within the test substances. The technique is therefore free of risk that reactions of these functional groups may influence the biological activity of the test compounds in screening applications. In addition, no preactivation of either the surface or the compounds is required. Reagents and substrates may be stored at ambient conditions for long periods of time. The lipoconjugates are administered from aqueous solution enabling automated nanopipetting down to spot dimensions of 100 microm across. The microstructures are stable with respect to the conditions of biochemical assays and applications in cell biology. Due to the hydrophobicity of the nonfunctionalized surfaces, standard blocking protocols used in microtiter-plate testing can be employed, thereby inhibiting nonspecific binding of assay reagents. Generation of these microstructures on hydrophobic glass slides or coverslips enables highly sensitive multichannel read-outs with high-resolution fluorescence microscopy.
Asunto(s)
Antígenos de Superficie/metabolismo , Inmunoensayo/métodos , Lípidos , Microquímica/métodos , Proteínas/química , Biotinilación , Señalización del Calcio , Técnicas Químicas Combinatorias , Técnicas Citológicas , Humanos , Inmunoensayo/instrumentación , Células Jurkat , Microquímica/instrumentación , Unión ProteicaRESUMEN
Microarrays that mediate the uptake of small molecules into living cells are described. Tissue culture cells were seeded onto glass substrates functionalized locally with fluorescently labelled test substances. In order to enable a localized transfer of substances after contact of cells with the substrate, substances were immobilized on the surface either by non-covalent interactions or chemolabile linker groups. These chemolabile linker groups were incorporated into covalently immobilized compounds. Different ester linkages were evaluated as chemolabile linker groups. As model compounds, esters of the carboxy group of a cysteine with the hydroxy groups of carboxyfluorescein-labelled serine amide and tyrosine amide residues or the thiol group of another fluorescein-labelled cysteine amide were generated. Covalent immobilization occurred on maleimide-functionalized glass cover slips. The surface functionalization and release kinetics were assessed by confocal laser scanning microscopy. The fastest release was obtained for the phenolic tyrosine ester. Alternatively, fluorescently labelled peptides were immobilized by non-covalent interactions on glass and on a hydrogel matrix. In order to increase the efficiency of cellular uptake, peptides were N-terminally extended with a cell-penetrating peptide. Uptake of these peptides into cells was confined to the functionalized spots, and was specific for peptides extended with the cell-penetrating peptide.