The Role of Extracellular Vesicles in the Progression of Human Neuroblastoma

The long-underestimated role of extracellular vesicles in cancer is now reconsidered worldwide by basic and clinical scientists, who recently highlighted novel and crucial activities of these moieties. Extracellular vesicles are now considered as king transporters of specific cargoes, including molecular components of parent cells, thus mediating a wide variety of cellular activities both in normal and neoplastic tissues. Here, we discuss the multifunctional activities and underlying mechanisms of extracellular vesicles in neuroblastoma, the most frequent common extra-cranial tumor in childhood. The ability of extracellular vesicles to cross-talk with different cells in the tumor microenvironment and to modulate an anti-tumor immune response, tumorigenesis, tumor growth, metastasis and drug resistance will be pinpointed in detail. The results obtained on the role of extracellular vesicles may represent a panel of suggestions potentially useful in practice, due to their involvement in the response to chemotherapy, and, moreover, their ability to predict resistance to standard therapies—all issues of clinical relevance.     

Relevance of AIF/CypA Lethal Pathway in SH-SY5Y Cells Treated with Staurosporine

The AIF/CypA complex exerts a lethal activity in several rodent models of acute brain injury. Upon formation, it translocates into the nucleus of cells receiving apoptotic stimuli, inducing chromatin condensation, DNA fragmentation, and cell death by a caspase-independent mechanism. Inhibition of this complex in a model of glutamate-induced cell death in HT-22 neuronal cells by an AIF peptide (AIF(370-394)) mimicking the binding site on CypA, restores cell survival and prevents brain injury in neonatal mice undergoing hypoxia-ischemia without apparent toxicity. Here, we explore the effects of the peptide on SH-SY5Y neuroblastoma cells stimulated with staurosporine (STS), a cellular model widely used to study Parkinson’s disease (PD). This will pave the way to understanding the role of the complex and the potential therapeutic efficacy of inhibitors in PD. We find that AIF(370-394) confers resistance to STS-induced apoptosis in SH-SY5Y cells similar to that observed with CypA silencing and that the peptide works on the AIF/CypA translocation pathway and not on caspases activation. These findings suggest that the AIF/CypA complex is a promising target for developing novel therapeutic strategies against PD.     

Coordination Environment of Cu(II) Ions Bound to N-Terminal Peptide Fragments of Angiogenin Protein

Angiogenin (Ang) is a potent angiogenic factor, strongly overexpressed in patients affected by different types of cancers. The specific Ang cellular receptors have not been identified, but it is known that Ang–actin interaction induces changes both in the cell cytoskeleton and in the extracellular matrix. Most in vitro studies use the recombinant form (r-Ang) instead of the form that is normally present in vivo (“wild-type”, wt-Ang). The first residue of r-Ang is a methionine, with a free amino group, whereas wt-Ang has a glutamic acid, whose amino group spontaneously cyclizes in the pyro-glutamate form. The Ang biological activity is influenced by copper ions. To elucidate the role of such a free amino group on the protein–copper binding, we scrutinized the copper(II) complexes with the peptide fragments Ang(1–17) and AcAng(1–17), which encompass the sequence 1–17 of angiogenin (QDNSRYTHFLTQHYDAK-NH₂), with free amino and acetylated N-terminus, respectively. Potentiometric, ultraviolet-visible (UV-vis), nuclear magnetic resonance (NMR) and circular dichroism (CD) studies demonstrate that the two peptides show a different metal coordination environment. Confocal microscopy imaging of neuroblastoma cells with the actin staining supports the spectroscopic results, with the finding of different responses in the cytoskeleton organization upon the interaction, in the presence or not of copper ions, with the free amino and the acetylated N-terminus peptides.     

Fabrication,characterization and evaluation of the effect of PLGA and PLGA–PEG biomaterials on the proliferation and neurogenesis potential of human neural SH-SY5Y cells

In recent years with regard to the development of nanotechnology and neural stem cell discovery, the combinatorial therapeutic strategies of neural progenitor cells and appropriate biomaterials have raised the hope for brain regeneration following neurological disorders. This study aimed to explore the proliferation and neurogenic effect of PLGA and PLGA–PEG nanofibers on human SH-SY5Y cells in in vitro condition. Nanofibers of PLGA and PLGA–PEG biomaterials were synthesized and fabricated using electrospinning method. Physicochemical features were examined using HNMR, FT-IR, and water contact angle assays. Ultrastructural morphology, the orientation of nanofibers, cell distribution and attachment were visualized by SEM imaging. Cell survival and proliferation rate were measured. Differentiation capacity was monitored by immunofluorescence staining of Map-2. HNMR, FT-IR assays confirmed the integration of PEG to PLGA backbone. Water contact angel assay showed increasing surface hydrophilicity in PLGA–PEG biomaterial compared to the PLGA substrate. SEM analysis revealed the reduction of PLGA–PEG nanofibers' diameter compared to the PLGA group. Cell attachment was observed in both groups while PLGA–PEG had a superior effect in the promotion of survival rate compared to other groups (p < .05). Compared to the PLGA group, PLGA–PEG increased the number of Ki67⁺ cells (p < .01). PLGA–PEG biomaterial induced neural maturation by increasing protein Map-2 compared to the PLGA scaffold in a three-dimensional culture system. According to our data, structural modification of PLGA with PEG could enhance orientated differentiation and the dynamic growth of neural cells.     

Evaluation of seafood toxicity in the Australes archipelago (French Polynesia) using the neuroblastoma cell-based assay

Ciguatera fish poisoning (CFP), a disease caused by consuming fish that have accumulated ciguatoxins (CTXs) in their tissue, is regarded as the most prevalent form of intoxication in French Polynesia. Recently, the Australes, one of the least affected archipelago until the early 1980s, has shown a dramatic increase in its incidence rates in 2009 with unusual CFP cases. In the present work, potential health hazards associated with the proliferation of various marine phytoplankton species and the consumption of fish and marine invertebrates highly popular among local population were assessed in three Australes islands: Raivavae, Rurutu and Rapa. Extracts from the marine dinoflagellates Gambierdiscus, Ostreospis and mat-forming cyanobacteria as well as fish, giant clams and sea urchin samples were examined for the presence of CTXs and palytoxin (PLTX) by using the neuroblastoma cell-based assay (CBA-N2a).

Cytotoxic responses observed with both standards (Pacific CTX-3C and PLTX) and targeted marine products indicate that CBA-N2a is a robust screening tool, with high sensitivity and good repeatability and reproducibility. In Rurutu and Raivavae islands, our main findings concern the presence of CTX-like compounds in giant clams and sea urchins, suggesting a second bio-accumulation route for CFP toxins in the ciguatera food chain. In Rapa, the potential CFP risk from Gambierdiscus bloom and fish was confirmed for the first time, with levels of CTXs found above the consumer advisory level of 0.01?ng Pacific CTX-1B?g^?1 of flesh in three fish samples. However, despite the presence of trace level of PLTX in Ostreopsis natural assemblages of Rapa, no sign of PLTX accumulation is yet observed in tested fish samples.

Because this multi-toxinic context is likely to emerge in most French Polynesian islands, CBA-N2a shows great potential for future applications in the algal- and toxin-based field monitoring programmes currently on hand locally.     

吗啡对 SH-SY5Y 细胞中 HSP70蛋白的调控作用

目的：观察吗啡对人神经母细胞瘤（SH-SY5Y）细胞中热休克蛋白70（HSP70）的调控作用。方法将维甲酸诱导分化的 SH-SY5Y 细胞随机分为4组：正常对照组（Con 组）,给予 PBS 作用48 h;吗啡组（Mor 组）,给予吗啡10μmol 作用48 h;吗啡＋纳络酮组（Mor＋Nal 组）,给予纳洛酮100μmol 作用30 min,之后给予吗啡10μmol 作用48 h;纳络酮组（Nal 组）,给予纳洛酮100μmol 作用48 h。采用免疫印迹方法检测各组 HSP70蛋白表达水平。结果与 Con 组比较,Mor 组 SH-SY5Y 细胞中 HSP70蛋白表达水平显著升高（P ＜0．001）;与 Mor 组比较,Mor＋Nal 组、Nal 组 SH-SY5Y 细胞中 HSP70蛋白表达水平均明显抑制（P ＜0．01或 P ＜0．001）。结论吗啡对 HSP70蛋白表达具有促进作用。     

Defining Pathological Activities of ALK in Neuroblastoma,a Neural Crest-Derived Cancer

Neuroblastoma is a common extracranial solid tumour of childhood, responsible for 15% of cancer-related deaths in children. Prognoses vary from spontaneous remission to aggressive disease with extensive metastases, where treatment is challenging. Tumours are thought to arise from sympathoadrenal progenitor cells, which derive from an embryonic cell population called neural crest cells that give rise to diverse cell types, such as facial bone and cartilage, pigmented cells, and neurons. Tumours are found associated with mature derivatives of neural crest, such as the adrenal medulla or paraspinal ganglia. Sympathoadrenal progenitor cells express anaplastic lymphoma kinase (ALK), which encodes a tyrosine kinase receptor that is the most frequently mutated gene in neuroblastoma. Activating mutations in the kinase domain are common in both sporadic and familial cases. The oncogenic role of ALK has been extensively studied, but little is known about its physiological role. Recent studies have implicated ALK in neural crest migration and sympathetic neurogenesis. However, very few downstream targets of ALK have been identified. Here, we describe pathological activation of ALK in the neural crest, which promotes proliferation and migration, while preventing differentiation, thus inducing the onset of neuroblastoma. Understanding the effects of ALK activity on neural crest cells will help find new targets for neuroblastoma treatment.     

Quantification of the Immune Content in Neuroblastoma: Deep Learning and Topological Data Analysis in Digital Pathology

We introduce here a novel machine learning (ML) framework to address the issue of the quantitative assessment of the immune content in neuroblastoma (NB) specimens. First, the EUNet, a U-Net with an EfficientNet encoder, is trained to detect lymphocytes on tissue digital slides stained with the CD3 T-cell marker. The training set consists of 3782 images extracted from an original collection of 54 whole slide images (WSIs), manually annotated for a total of 73,751 lymphocytes. Resampling strategies, data augmentation, and transfer learning approaches are adopted to warrant reproducibility and to reduce the risk of overfitting and selection bias. Topological data analysis (TDA) is then used to define activation maps from different layers of the neural network at different stages of the training process, described by persistence diagrams (PD) and Betti curves. TDA is further integrated with the uniform manifold approximation and projection (UMAP) dimensionality reduction and the hierarchical density-based spatial clustering of applications with noise (HDBSCAN) algorithm for clustering, by the deep features, the relevant subgroups and structures, across different levels of the neural network. Finally, the recent TwoNN approach is leveraged to study the variation of the intrinsic dimensionality of the U-Net model. As the main task, the proposed pipeline is employed to evaluate the density of lymphocytes over the whole tissue area of the WSIs. The model achieves good results with mean absolute error 3.1 on test set, showing significant agreement between densities estimated by our EUNet model and by trained pathologists, thus indicating the potentialities of a promising new strategy in the quantification of the immune content in NB specimens. Moreover, the UMAP algorithm unveiled interesting patterns compatible with pathological characteristics, also highlighting novel insights into the dynamics of the intrinsic dataset dimensionality at different stages of the training process. All the experiments were run on the Microsoft Azure cloud platform.