Neuroblastoma is an embryonal tumor with a heterogeneous clinical course. The tumor is presumed to be derived from the neural crest, but the cells of origin remain to be determined. To date, few recurrent genetic changes contributing to neuroblastoma formation, such as amplification of the MYCN oncogene and activating mutations of the ALK oncogene, have been identified. The possibility to model neuroblastoma in mice allows investigation of the cell of origin hypothesis in further detail. Here we present the evidence that murine neural crest progenitor cells can give rise to neuroblastoma upon transformation with MYCN or ALK(F1174L). For this purpose we used JoMa1, a multipotent neural crest progenitor cell line, which is kept in a viable and undifferentiated state by a tamoxifen-activated c-Myc transgene (c-MycER(T)). Expression of MYCN or ALK(F1174L), one of the oncogenic ALK variants identified in primary neuroblastomas, enabled these cells to grow independently of c-MycER(T) activity in vitro and caused formation of neuroblastoma-like tumors in vivo in contrast to parental JoMa1 cells and JoMa1 cells-expressing TrkA or GFP. Tumorigenicity was enhanced upon serial transplantation of tumor-derived cells, and tumor cells remained susceptible to the MYC-inhibitor, NBT-272, indicating that cell growth depended on functional MYCN. Our findings support neural crest progenitor cells as the precursor cells of neuroblastoma, and indicate that neuroblastomas arise as their malignant progeny.

The CHESS may become a valuable diagnostic tool for early estimation of shunt dependency after SAH. Further evaluation and external validation will be required in prospective studies.

MicroRNAs (miRNAs) are deregulated in a variety of human cancers, including neuroblastoma, the most common extracranial tumor of childhood. We previously reported a signature of 42 miRNAs to be highly predictive of neuroblastoma outcome. One miRNA in this signature, miR-542, was downregulated in tumors from patients with adverse outcome. Reanalysis of quantitative PCR and next-generation sequencing transcript data revealed that miR-542-5p as well as miR-542-3p expression is inversely correlated with poor prognosis in neuroblastoma patients. We, therefore, analyzed the function of miR-542 in neuroblastoma tumor biology. Ectopic expression of miR-542-3p in neuroblastoma cell lines reduced cell viability and proliferation, induced apoptosis and downregulated Survivin. Survivin expression was also inversely correlated with miR-542-3p expression in primary neuroblastomas. Reporter assays confirmed that miR-542-3p directly targeted Survivin. Downregulating Survivin using siRNA copied the phenotype of miR-542-3p expression in neuroblastoma cell lines, while cDNA-mediated ectopic expression of Survivin partially rescued the phenotype induced by miR-542-3p expression. Treating nude mice bearing neuroblastoma xenografts with miR-542-3p-loaded nanoparticles repressed Survivin expression, decreased cell proliferation and induced apoptosis in the respective xenograft tumors. We conclude that miR-542-3p exerts its tumor suppressive function in neuroblastoma, at least in part, by targeting Survivin. Expression of miR-542-3p could be a promising therapeutic strategy for treating aggressive neuroblastoma.

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest 1 . Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest 2,3,4,5 . These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone 6,7,8,9 . To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required 10 . The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE. ProtocolThe authors state that experiments on animals were performed in accordance with the European Communities Council Directive (86/609/EEC), following the Guidelines of the NIH regarding the care and use of animals for experimental procedures and the regulations set forth by the Institutional Animal Care and Use Committee (IACUC) at the University of Duisburg-Essen (Germany). • Eggs have to be incubated in a humidified incubator for 11 days at 37°C (100°F).• 70% ethanol spray, clean plates, precise clean forceps, 4°C (40°F) cold sterile DMEM on ice and 60 mL sterile syringes • 50 mL Sterile corning tubes have to be half-filled with 4°C cold DMEM to mix the macerated embryos at a ratio of 1:1 (macerated mass: DMEM). As soon as the eggs are taken out of the incubator dissection of the chick embryos has to be started. • Sterilize dissection tools in the autoclave or on the day of dissection, immerse the tools in 70% ethanol for 20 minutes.1. Wet the incubated eggs with a 70% ethanol spray for at least 30 seconds and dry them carefully with clean soft paper towels while not shaking the eggs excessively. Note: We recommend not dissecting more than 60 chick embryos simultaneously. 2. Open the eggs carefully by dashing against a sharp edge and take out the embryo with caution not destroying the yolk sack or the chick embryo itself. 3. Search for the beginning of the umbilical cord quickly and open the clear wrapping without destroying the yolk sack or any little vessels. Then dissect the umbilical cord with precise clean forceps. 4. Take the embryo out of the yolk sack.Note: Embryos should be decapitated quickly (not shown in the video). 5. Collect the embryos in minimal essential medium at 4°C.1. Take the plunger out of a 60 mL sterile syringe. 2. Transfer approximately 10 embryos into one 60 mL syringe. 3...