Analysis of Serial Neuroblastoma PDX Passages in Mice Allows the Identification of New Mediators of Neuroblastoma Aggressiveness.

Neuroblastoma is a neural crest cell-derived pediatric tumor characterized by high inter- and intra-tumor heterogeneity, and by a poor outcome in advanced stages. Patient-derived xenografts (PDXs) have been shown to be useful models for preserving and expanding original patient biopsies in vivo, and for studying neuroblastoma biology in a more physiological setting. The maintenance of genetic, histologic, and phenotypic characteristics of the original biopsy along serial PDX passages in mice is a major concern regarding this model. Here we analyze consecutive PDX passages in mice, at both transcriptomic and histological levels, in order to identify potential changes or highlight similarities to the primary sample. We studied temporal changes using mRNA and miRNA expression and correlate those with neuroblastoma aggressiveness using patient-derived databases. We observed a shortening of tumor onset and an increase in proliferative potential in the PDXs along serial passages. This behavior correlates with changes in the expression of genes related to cell proliferation and neuronal differentiation, including signaling pathways described as relevant for neuroblastoma malignancy. We also identified new genes and miRNAs that can be used to stratify patients according to survival, and which could be potential new players in neuroblastoma aggressiveness. Our results highlight the usefulness of the PDX neuroblastoma model and reflect phenotypic changes that might be occurring in the mouse environment. These findings could be useful for understanding the progression of tumor aggressiveness in this pathology.

A protocol to enrich in undifferentiated cells from neuroblastoma tumor tissue samples and cell lines.

The existence of a subpopulation of undifferentiated cells with stem-like properties has been suggested in neuroblastoma tumors, but a definitive biomarker for their successful isolation is missing. Here we describe an in vitro culture system for the enrichment in undifferentiated stem-like tumor cells for subsequent functional assays. We make use of clonal non-adherent cell culture conditions together with cell sorting with specific expression markers. This protocol allows for the differential study of heterogeneous cell population in neuroblastoma tumors. For complete details on the use and execution of this protocol, please refer to Vega et al. (2019).

Identification of VRK1 as a New Neuroblastoma Tumor Progression Marker Regulating Cell Proliferation.

Neuroblastoma (NB) is one of the most common pediatric cancers and presents a poor survival rate in affected children. Current pretreatment risk assessment relies on a few known molecular parameters, like the amplification of the oncogene MYCN. However, a better molecular knowledge about the aggressive progression of the disease is needed to provide new therapeutical targets and prognostic markers and to improve patients' outcomes. The human protein kinase VRK1 phosphorylates various signaling molecules and transcription factors to regulate cell cycle progression and other processes in physiological and pathological situations. Using neuroblastoma tumor expression data, tissue microarrays from fresh human samples and patient-derived xenografts (PDXs), we have determined that VRK1 kinase expression stratifies patients according to tumor aggressiveness and survival, allowing the identification of patients with worse outcome among intermediate risk. VRK1 associates with cell cycle signaling pathways in NB and its downregulation abrogates cell proliferation in vitro and in vivo. Through the analysis of ChIP-seq and methylation data from NB tumors, we show that VRK1 is a MYCN gene target, however VRK1 correlates with NB aggressiveness independently of MYCN gene amplification, synergizing with the oncogene to drive NB progression. Our study also suggests that VRK1 inhibition may constitute a novel cell-cycle-targeted strategy for anticancer therapy in neuroblastoma.

CD44-high neural crest stem-like cells are associated with tumour aggressiveness and poor survival in neuroblastoma tumours.

BACKGROUND: Neuroblastoma is a paediatric tumour originated from sympathoadrenal precursors and characterized by its heterogeneity and poor outcome in advanced stages. Intra-tumoral cellular heterogeneity has emerged as an important feature in neuroblastoma, with a potential major impact on tumour aggressiveness and response to therapy. CD44 is an adhesion protein involved in tumour progression, metastasis and stemness in different cancers; however, there has been controversies about the significance of CD44 expression in neuroblastoma and its relationship with tumour progression. METHODS: We have performed transcriptomic analysis on patient tumour samples studying the outcome of patients with high CD44 expression. Adhesion, invasion and proliferation assays were performed in sorted CD44high neuroblastoma cells. Tumoursphere cultures have been used to enrich in undifferentiated stem-like cells and to asses self-renewal and differentiation potential. We have finally performed in vivo tumorigenic assays on cell line-derived or Patient-derived xenografts. FINDINGS: We show that high CD44 expression is associated with low survival in high-grade human neuroblastoma, independently of MYCN amplification. CD44 is expressed in a cell population with neural crest stem-like features, and with the capacity to generate multipotent, undifferentiated tumourspheres in culture. These cells are more invasive and proliferative in vitro. CD44 positive cells obtained from tumours are more tumorigenic and metastatic, giving rise to aggressive neuroblastic tumours at high frequency upon transplantation. INTERPRETATION: We describe an unexpected intra-tumoural heterogeneity within cellular entities expressing CD44 in neuroblastoma, and propose that CD44 has a role in neural crest stem-like undifferentiated cells, which can contribute to tumorigenesis and malignancy in this type of cancer. FUNDING: Research supported by grants from the "Asociación Española contra el Cáncer" (AECC), the Spanish Ministry of Science and Innovation SAF program (SAF2016-80412-P), and the European Research Council (ERC Starting Grant to RP).

Neural crest derived progenitor cells contribute to tumor stroma and aggressiveness in stage 4/M neuroblastoma.

Pediatric tumors arise upon oncogenic transformation of stem/progenitor cells during embryonic development. Given this scenario, the existence of non-tumorigenic stem cells included within the aberrant tumoral niche, with a potential role in tumor biology, is an intriguing and unstudied possibility. Here, we describe the presence and function of non-tumorigenic neural crest-derived progenitor cells in aggressive neuroblastoma (NB) tumors. These cells differentiate into neural crest typical mesectodermal derivatives, giving rise to tumor stroma and promoting proliferation and tumor aggressiveness. Furthermore, an analysis of gene expression profiles in stage 4/M NB revealed a neural crest stem cell (NCSC) gene signature that was associated to stromal phenotype and high probability of relapse. Thus, this NCSC gene expression signature could be used in prognosis to improve stratification of stage 4/M NB tumors. Our results might facilitate the design of new therapies by targeting NCSCs and their contribution to tumor stroma.

Physiological Plasticity of Neural-Crest-Derived Stem Cells in the Adult Mammalian Carotid Body.

Adult stem cell plasticity, or the ability of somatic stem cells to cross boundaries and differentiate into unrelated cell types, has been a matter of debate in the last decade. Neural-crest-derived stem cells (NCSCs) display a remarkable plasticity during development. Whether adult populations of NCSCs retain this plasticity is largely unknown. Herein, we describe that neural-crest-derived adult carotid body stem cells (CBSCs) are able to undergo endothelial differentiation in addition to their reported role in neurogenesis, contributing to both neurogenic and angiogenic processes taking place in the organ during acclimatization to hypoxia. Moreover, CBSC conversion into vascular cell types is hypoxia inducible factor (HIF) dependent and sensitive to hypoxia-released vascular cytokines such as erythropoietin. Our data highlight a remarkable physiological plasticity in an adult population of tissue-specific stem cells and could have impact on the use of these cells for cell therapy.

Evaluation of the immunomodulatory effect of melatonin on the T-cell response in peripheral blood from systemic lupus erythematosus patients.

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by the production of antinuclear autoantibodies. In addition, the involvement of CD4+ T-helper (Th) cells in SLE has become increasingly evident. Although the role of melatonin has been tested in some experimental models of lupus with inconclusive results, there are no studies evaluating the melatonin effect on cells from patients with SLE. Therefore, the aim of this study was to analyse the role of in vitro administered melatonin in the immune response of peripheral leukocytes from treated patients with SLE (n = 20) and age- and sex-matched healthy controls. Melatonin was tested for its effect on the production of key Th1, Th2, Th9, Th17 and innate cytokines. The frequency of T regulatory (Treg) cells and the expression of FOXP3 and BAFF were also explored. Our results are the first to show that melatonin decreased the production of IL-5 and to describe the novel role of melatonin in IL-9 production by human circulating cells. Additionally, we highlighted a two-faceted melatonin effect. Although it acted as a prototypical anti-inflammatory compound, reducing exacerbated Th1 and innate responses in PHA-stimulated cells from healthy subjects, it caused the opposite actions in immune-depressed cells from patients with SLE. Melatonin also increased the number of Treg cells expressing FOXP3 and offset BAFF overexpression in SLE patient cells. These findings open a new field of research in lupus that could lead to the use of melatonin as treatment or cotreatment for SLE.

Melatonin-synthesizing enzymes and melatonin receptor in rat thyroid cells.

Melatonin is an indoleamine with a wide spectrum of biological activities other than transmitting photoperiod information, including antioxidant, oncostatic, anti-aging and immunomodulatory properties. Although melatonin is synthesized mainly in the pineal gland, other tissues have the same capacity. In the present study, we examined whether two key enzymes in melatonin biosynthesis, arylalkylamine Nacetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT) and its receptor MT1 are expressed in the two endocrine thyroid cells of the rat, follicular cells and C cells. Reverse transcriptase polymerase chain reaction analyses demonstrated that both AANAT and HIOMT mRNAs are expressed in the rat thyroid C-cells, and MT1 expression has been detected in C cells and follicular cells. Immunofluorescence revealed that AANAT protein is localized in C-cell cytoplasm, and MT1 protein in both cell populations. These findings demonstrate that the rat thyroid expresses AANAT, HIOMT, and its receptor MT1, showing that C cells are the main melatonin-synthesizing sites in the thyroid. This local C-cell-secreted melatonin may protect follicular cells from the oxidative stress inherent to the thyroid gland, and could also have paracrine and autocrine functions.