RESUMEN
Neuroblastoma is a paediatric malignancy of the developing sympathetic nervous system. About half of the patients have metastatic disease at the time of diagnosis and a survival rate of less than 50%. Our understanding of the cellular processes promoting neuroblastoma metastases will be facilitated by the development of appropriate experimental models. In this study, we aimed to explore the invasion of neuroblastoma cells and organoids from patient-derived xenografts (PDXs) grown embedded in 3D extracellular matrix (ECM) hydrogels by time-lapse microscopy and quantitative image analysis. We found that the ECM composition influenced the growth, viability and local invasion of organoids. The ECM compositions induced distinct cell behaviours, with Matrigel being the preferred substratum for local organoid invasion. Organoid invasion was cell line- and PDX-dependent. We identified six distinct phenotypes in PDX-derived organoids. In contrast, NB cell lines were more phenotypically restricted in their invasion strategies, as organoids isolated from cell line-derived xenografts displayed a broader range of phenotypes compared to clonal cell line clusters. The addition of FBS and bFGF induced more aggressive cell behaviour and a broader range of phenotypes. In contrast, the repression of the prognostic neuroblastoma marker, MYCN, resulted in less aggressive cell behaviour. The combination of PDX organoids, real-time imaging and the novel 3D culture assays developed herein will enable rapid progress in elucidating the molecular mechanisms that control neuroblastoma invasion.
RESUMEN
We investigated the effects of cigarette smoke extract (CSE) on lung fibroblasts and found that the invasiveness of lung cancer cells was facilitated by the conditioned medium from CSE-treated fibroblasts. CSE induced autophagy in fibroblasts and increased the expression of autophagy-related proteins, including optineurin and Ras-related protein Rab1B. Afterward, the fibroblasts produced high levels of interleukin-8 (IL-8), which promoted cancer cell invasion. The inhibition of either optineurin or Rab1B abrogated a rise in microtubule-associated protein 1 light chain 3 ß and a decrease in p62 protein, as well as the production of IL-8, in CSE-treated fibroblasts. A three-dimensional invasion assay using cancer cell spheroids revealed that the invasion of cancer cells alone and the fibroblast-led cancer cell invasion were both enhanced by the conditioned media from CSE-treated fibroblasts. These results suggest that cigarette smoke may induce autophagy and IL-8 secretion in lung fibroblasts and modify the microenvironment to favor invasion of lung cancer cells.
Asunto(s)
Autofagia/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , Nicotiana/efectos adversos , Humo/efectos adversos , Fumar/efectos adversos , Autofagia/inmunología , Fibroblastos Asociados al Cáncer/efectos de los fármacos , Fibroblastos Asociados al Cáncer/inmunología , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Medios de Cultivo Condicionados/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Interleucina-8/metabolismo , Pulmón/citología , Pulmón/patología , Neoplasias Pulmonares/inmunología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Invasividad Neoplásica/patología , Fumar/inmunología , Esferoides Celulares , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Proteínas de Unión al GTP rab1/genética , Proteínas de Unión al GTP rab1/metabolismoRESUMEN
Widespread tumor cell invasion is a fundamental property of diffuse gliomas and is ultimately responsible for their poor prognosis. A greater understanding of basic mechanisms underlying glioma invasion is needed to provide insights into therapies that could potentially counteract them. While none of the currently available in vitro models can fully recapitulate the complex interactions of glioma cells within the brain tumor microenvironment, if chosen and developed appropriately, these models can provide controlled experimental settings to study molecular and cellular phenomena that are challenging or impossible to model in vivo. Therefore, selecting the most appropriate in vitro model, together with its inherent advantages and limitations, for specific hypotheses and experimental questions achieves primary significance. In this review, we describe and discuss commonly used methods for modeling and studying glioma invasion in vitro, including platforms, matrices, cell culture, and visualization techniques, so that choices for experimental approach are informed and optimal.