RESUMEN
Neuroblastoma is a paediatric cancer of the sympathetic nervous system and the most common solid tumour of infancy, contributing to 15% of paediatric oncology deaths. Current therapies are not effective in the long-term treatment of almost 80% of patients with this clinically aggressive disease. The primary challenge in the identification and validation of new agents for paediatric drug development is the accurate representation of tumour biology and diversity. In addition to this limitation, the low incidence of neuroblastoma makes the recruitment of eligible patients for early phase clinical trials highly challenging and highlights the need for robust preclinical testing to ensure that the best treatments are selected. The research field requires new preclinical models, technologies, and concepts to tackle these problems. Tissue engineering offers attractive tools to assist in the development of three-dimensional (3D) cell models using various biomaterials and manufacturing approaches that recreate the geometry, mechanics, heterogeneity, metabolic gradients, and cell communication of the native tumour microenvironment. In this review, we discuss current experimental models and assess their abilities to reflect the structural organisation and physiological conditions of the human body, in addition to current and new techniques to recapitulate the tumour niche using tissue-engineered platforms. Finally, we will discuss the possible use of novel 3D in vitro culture systems to address open questions in neuroblastoma biology.
Asunto(s)
Modelos Animales de Enfermedad , Neuroblastoma/inmunología , Neuroblastoma/patología , Microambiente Tumoral/inmunología , Animales , HumanosRESUMEN
3D scaffold-based in vitro cell culturing is a recent technological advancement in cancer research bridging the gap between conventional 2D culture and in vivo tumours. The main challenge in treating neuroblastoma, a paediatric cancer of the sympathetic nervous system, is to combat tumour metastasis and resistance to multiple chemotherapeutic drugs. The aim of this study was to establish a physiologically relevant 3D neuroblastoma tissue-engineered system and explore its therapeutic relevance. Two neuroblastoma cell lines, chemotherapeutic sensitive Kelly and chemotherapeutic resistant KellyCis83 were cultured in a 3D in vitro model on two collagen-based scaffolds containing either glycosaminoglycan (Coll-GAG) or nanohydroxyapatite (Coll-nHA) and compared to 2D cell culture and an orthotopic murine model. Both neuroblastoma cell lines actively infiltrated the scaffolds and proliferated displaying >100-fold increased resistance to cisplatin treatment when compared to 2D cultures, exhibiting chemosensitivity similar to orthotopic xenograft in vivo models. This model demonstrated its applicability to validate miRNA-based gene delivery. The efficacy of liposomes bearing miRNA mimics uptake and gene knockdown was similar in both 2D and 3D in vitro culturing models highlighting the proof-of-principle for the applicability of 3D collagen-based scaffolds cell system for validation of miRNA function. Collectively, this data shows the successful development and characterisation of a physiologically relevant, scaffold-based 3D tissue-engineered neuroblastoma cell model, strongly supporting its value in the evaluation of chemotherapeutics, targeted therapies and investigation of neuroblastoma pathogenesis. While neuroblastoma is the specific disease being focused upon, the platform may have multi-functionality beyond this tumour type. STATEMENT OF SIGNIFICANCE: Traditional 2D cell cultures do not completely capture the 3D architecture of cells and extracellular matrix contributing to a gap in our understanding of mammalian biology at the tissue level and may explain some of the discrepancies between in vitro and in vivo results. Here, we demonstrated the successful development and characterisation of a physiologically relevant, scaffold-based 3D tissue-engineered neuroblastoma cell model, strongly supporting its value in the evaluation of chemotherapeutics, targeted therapies and investigation of neuroblastoma pathogenesis. The ability to test drugs in this reproducible and controllable tissue-engineered model system will help reduce the attrition rate of the drug development process and lead to more effective and tailored therapies. Importantly, such 3D cell models help to reduce and replace animals for pre-clinical research addressing the principles of the 3Rs.
Asunto(s)
Colágeno/química , Técnicas de Transferencia de Gen , Neuroblastoma , Andamios del Tejido/química , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Línea Celular Tumoral , Femenino , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patología , Neuroblastoma/terapiaRESUMEN
MicroRNAs (miRNAs) contribute to the pathogenesis of many forms of cancer, including the pediatric cancer neuroblastoma, but the underlying mechanisms leading to altered miRNA expression are often unknown. Here, a novel integrated approach for analyzing DNA methylation coupled with miRNA and mRNA expression data sets identified 67 epigenetically regulated miRNA in neuroblastoma. A large proportion (42%) of these miRNAs was associated with poor patient survival when underexpressed in tumors. Moreover, we demonstrate that this panel of epigenetically silenced miRNAs targets a large set of genes that are overexpressed in tumors from patients with poor survival in a highly redundant manner. The genes targeted by the epigenetically regulated miRNAs are enriched for a number of biological processes, including regulation of cell differentiation. Functional studies involving ectopic overexpression of several of the epigenetically silenced miRNAs had a negative impact on neuroblastoma cell viability, providing further support to the concept that inactivation of these miRNAs is important for neuroblastoma disease pathogenesis. One locus, miR-340, induced either differentiation or apoptosis in a cell context dependent manner, indicating a tumor suppressive function for this miRNA. Intriguingly, it was determined that miR-340 is upregulated by demethylation of an upstream genomic region that occurs during the process of neuroblastoma cell differentiation induced by all-trans retinoic acid (ATRA). Further biological studies of miR-340 revealed that it directly represses the SOX2 transcription factor by targeting of its 3'-untranslated region, explaining the mechanism by which SOX2 is downregulated by ATRA. Although SOX2 contributes to the maintenance of stem cells in an undifferentiated state, we demonstrate that miR-340-mediated downregulation of SOX2 is not required for ATRA induced differentiation to occur. In summary, our results exemplify the dynamic nature of the miRNA epigenome and identify a remarkable network of miRNA/mRNA interactions that significantly contribute to neuroblastoma disease pathogenesis.
Asunto(s)
Epigénesis Genética/genética , Redes Reguladoras de Genes/genética , MicroARNs/genética , Neuroblastoma/etiología , Neuroblastoma/genética , Regiones no Traducidas 3'/genética , Línea Celular Tumoral , Biología Computacional , Metilación de ADN/efectos de los fármacos , Metilación de ADN/genética , Epigénesis Genética/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Genómica , Humanos , Neuroblastoma/patología , Factores de Transcripción SOXB1/genética , Análisis de Supervivencia , Tretinoina/farmacologíaRESUMEN
The discovery of new biomarkers is a rapidly advancing area in cancer biology. The challenge of biomarker development for broad clinical use requires the translation of lab-based knowledge into clinical practice. The Long Interspersed Nuclear Elements-1 (LINE-1s or L1 elements) are active members of an autonomous family of non-LTR retrotransposons and occupy nearly 17% of the human genome. There is strong experimental evidence that the global hypomethylation of genomic DNA in cancer cells results in the activation of L1s and their expression is detectable at genome, transcriptome and proteome levels in human cancer cells. Thus, human L1s constitute a potential marker for cancer cells. In this review we have attempted to scrutinize L1 expression profiles in clinical cancer studies by undertaking a comprehensive systematic analysis of papers published in the field so far with a view to providing a more complete picture of the detection methods used, improvements achieved and potential future directions. Ultimately, we will try to evaluate the potential of L1s as a molecular marker in cancer detection.
Asunto(s)
Biomarcadores de Tumor/análisis , Transformación Celular Neoplásica/genética , Metilación de ADN , Genoma Humano , Elementos de Nucleótido Esparcido Largo/genética , Neoplasias/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Transformación Celular Neoplásica/metabolismo , ADN/metabolismo , Predicción , Perfilación de la Expresión Génica , Genómica/métodos , Humanos , Neoplasias/diagnóstico , Neoplasias/metabolismo , Neoplasias/patología , PronósticoAsunto(s)
Elementos de Nucleótido Esparcido Largo , Proteínas de Unión al ARN/análisis , Animales , Células CHO , Cricetinae , Cricetulus , Citoplasma/química , Genoma Humano , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Humanos , Microscopía Confocal , Sistemas de Lectura AbiertaRESUMEN
The effect of a support composed of polymers based on poly-N-isopropyl acrylamide and poly-t-butyl acrylamide and collagen on human fibroblasts was studied. As the temperature was decreased to 4 degrees C, the polymeric support is converted to a diluted state and cells spontaneously detached from it. The presence of collagen in the support prevented the detachment of cells and increased cell growth. It was shown by microcalorimetry, that in a copolymer-collagen mixture, a microstratification takes place.