RESUMO
Embryonic malignant transformation is concomitant to organogenesis, often affecting multipotent and migratory progenitors. While lineage relationships between malignant cells and their physiological counterparts are extensively investigated, the contribution of exogenous embryonic signals is not fully known. Neuroblastoma (NB) is a childhood malignancy of the peripheral nervous system arising from the embryonic trunk neural crest (NC) and characterized by heterogeneous and interconvertible tumor cell identities. Here, using experimental models mimicking the embryonic context coupled to proteomic and transcriptomic analyses, we show that signals released by embryonic sympathetic ganglia, including Olfactomedin-1, induce NB cells to shift from a noradrenergic to mesenchymal identity, and to activate a gene program promoting NB metastatic onset and dissemination. From this gene program, we extract a core signature specifically shared by metastatic cancers with NC origin. This reveals non-cell autonomous embryonic contributions regulating the plasticity of NB identities and setting pro-dissemination gene programs common to NC-derived cancers.
Assuntos
Crista Neural , Neuroblastoma , Diferenciação Celular/genética , Criança , Sinais (Psicologia) , Humanos , Neuroblastoma/genética , Neuroblastoma/patologia , ProteômicaRESUMO
Neuroblastoma (NB) is a childhood cancer arising from sympatho-adrenal neural crest cells. Disseminated forms have high frequency of multiple tumoral foci whose etiology remains unknown; NB embryonic origin limits investigations in patients and current models. We developed an avian embryonic model driving human NB tumorigenesis in tissues homologous to patients. We found that aggressive NBs display a metastatic mode, secondary dissemination via peripheral nerves and aorta. Through tumor transcriptional profiling, we found that NB dissemination is induced by the shutdown of a pro-cohesion autocrine signal, SEMA3C, which constrains the tumoral mass. Lowering SEMA3C levels shifts the balance toward detachment, triggering NB cells to collectively evade the tumor. Together with patient cohort analysis, this identifies a microenvironment-driven pro-metastatic switch for NB.
Assuntos
Neuroblastoma/secundário , Microambiente Tumoral , Adolescente , Adulto , Animais , Adesão Celular , Embrião de Galinha , Criança , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Estadiamento de Neoplasias , Proteínas do Tecido Nervoso/fisiologia , Neuroblastoma/etiologia , Neuroblastoma/patologia , Receptores de Superfície Celular/fisiologia , Semaforinas/genética , Semaforinas/fisiologiaRESUMO
Transmission of polarity established early during cell lineage history is emerging as a key process guiding cell differentiation. Highly polarized neurons provide a fascinating model to study inheritance of polarity over cell generations and across morphological transitions. Neural crest cells (NCCs) migrate to the dorsal root ganglia to generate neurons directly or after cell divisions in situ. Using live imaging of vertebrate embryo slices, we found that bipolar NCC progenitors lose their polarity, retracting their processes to round for division, but generate neurons with bipolar morphology by emitting processes from the same locations as the progenitor. Monitoring the dynamics of Septins, which play key roles in yeast polarity, indicates that Septin 7 tags process sites for re-initiation of process growth following mitosis. Interfering with Septins blocks this mechanism. Thus, Septins store polarity features during mitotic rounding so that daughters can reconstitute the initial progenitor polarity.
Assuntos
Polaridade Celular/genética , Forma Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Neurogênese/genética , Neurônios/fisiologia , Septinas/metabolismo , Animais , Ciclo Celular/genética , Diferenciação Celular/genética , Células Cultivadas , Córtex Cerebral/citologia , Embrião de Galinha , Eletroporação , Gânglios Espinais/citologia , Gânglios Espinais/embriologia , Neuritos/fisiologia , Neurônios/citologia , Técnicas de Cultura de Órgãos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Septinas/genética , Medula Espinal/citologia , Medula Espinal/embriologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Leveduras/genéticaRESUMO
Robo-Slit and Plexin-Semaphorin signaling participate in various developmental and pathogenic processes. During commissural axon guidance in the spinal cord, chemorepulsion by Semaphorin3B and Slits controls midline crossing. Slit processing generates an N-terminal fragment (SlitN) that binds to Robo1 and Robo2 receptors and mediates Slit repulsive activity, as well as a C-terminal fragment (SlitC) with an unknown receptor and bioactivity. We identified PlexinA1 as a Slit receptor and found that it binds the C-terminal Slit fragment specifically and transduces a SlitC signal independently of the Robos and the Neuropilins. PlexinA1-SlitC complexes are detected in spinal cord extracts, and ex vivo, SlitC binding to PlexinA1 elicits a repulsive commissural response. Analysis of various ligand and receptor knockout mice shows that PlexinA1-Slit and Robo-Slit signaling have complementary roles during commissural axon guidance. Thus, PlexinA1 mediates both Semaphorin and Slit signaling, and Slit processing generates two active fragments, each exerting distinct effects through specific receptors.
Assuntos
Axônios/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Fragmentos de Peptídeos/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Células Cultivadas , Embrião de Galinha , Genótipo , Cones de Crescimento , Camundongos , RNA Interferente Pequeno/genética , Medula Espinal/anatomia & histologia , Medula Espinal/citologiaRESUMO
During development, progenitors and post-mitotic neurons receive signals from adjacent territories that regulate their fate. The floor-plate is a group of glial cells lining the ependymal canal at ventral position. The floor-plate expresses key morphogens contributing to the patterning of cell lineages in the spinal cord. At later developmental stages, the floor-plate regulates the navigation of axons in the spinal cord, acting as a barrier to prevent the crossing of ipsilateral axons and controlling midline crossing by commissural axons(1). These functions are achieved through the secretion of various guidance cues. Some of these cues act as attractants and repellents for the growing axons while others regulate guidance receptors and downstream signaling to modulate the sensitivity of the axons to the local guidance cues(2,3). Here we describe a method that allows investigating the properties of floor-plate derived signals in a variety of developmental contexts, based on the production of Floor-Plate conditioned medium (FP(cm))(4-6). We then exemplify the use of this FP(cm) in the context of axon guidance. First, the spinal cord is isolated from mouse embryo at E12.5 and the floor-plate is dissected out and cultivated in a plasma-thrombin matrix (Figure 1). Second two days later, commissural tissue are dissected out from E12.5 embryos, triturated and exposed to the FP(cm). Third, the tissue are processed for Western blot analysis of commissural markers.
Assuntos
Medula Espinal/fisiologia , Técnicas de Cultura de Tecidos/métodos , Animais , Western Blotting , Meios de Cultivo Condicionados , Camundongos , Células-Tronco Neurais/citologia , Medula Espinal/citologia , Medula Espinal/embriologia , Medula Espinal/crescimento & desenvolvimentoRESUMO
Amplicons are helper-dependent herpes simplex virus type 1 (HSV-1)-based vectors that can deliver very large, foreign DNA sequences and, as such, are good candidates for both gene delivery and vaccine development. However, many studies have shown that innate immune responses induced by virus vectors can play a significant role in the control of transgenic expression and in the induction of inflammatory responses. Furthermore, amplicons are very interesting tools to study innate cellular responses elicited by entry of HSV-1 particles in the absence of any virus gene expression. For these reasons, in this study we characterized the innate antiviral response established in human fibroblasts of limited passage (HFFF-2) infected by amplicons. Our results indicate that infection with amplicons triggered an interferon (IFN)-regulatory factors 3 and 7 (IRF3/7)-dependent antiviral response, rendered the cells resistant to vesicular stomatitis virus infection and induced significant changes in the pattern of cellular gene expression, including the upregulation of Toll-like receptor 3 (TLR3), IRF7 and IFN-stimulated genes (ISGs). In contrast, we observed only a mild and contained type I IFN response in infected cells. Amplicon infection induced nuclear translocation and subsequent degradation of IRF3, without hyperphosphorylation of the protein. Inhibition of endosome-resident TLR signalling by blocking lysosome maturation or the knockdown of TLR3 and 4 did not abolish the cellular response to amplicons, whereas knockdown of IRF3 and 7 inhibited ISG and IFN-beta expression severely. Therefore, our results confirm the existence of TLR-independent, IRF3/7-dependent activation pathways triggered by HSV-1 particles in human fibroblasts.