DHODH is an independent prognostic marker and potent therapeutic target in neuroblastoma.

Despite intensive therapy, children with high-risk neuroblastoma are at risk of treatment failure. We applied a multiomic system approach to evaluate metabolic vulnerabilities in human neuroblastoma. We combined metabolomics, CRISPR screening, and transcriptomic data across more than 700 solid tumor cell lines and identified dihydroorotate dehydrogenase (DHODH), a critical enzyme in pyrimidine synthesis, as a potential treatment target. Of note, DHODH inhibition is currently under clinical investigation in patients with hematologic malignancies. In neuroblastoma, DHODH expression was identified as an independent risk factor for aggressive disease, and high DHODH levels correlated to worse overall and event-free survival. A subset of tumors with the highest DHODH expression was associated with a dismal prognosis, with a 5-year survival of less than 10%. In xenograft and transgenic neuroblastoma mouse models treated with the DHODH inhibitor brequinar, tumor growth was dramatically reduced, and survival was extended. Furthermore, brequinar treatment was shown to reduce the expression of MYC targets in 3 neuroblastoma models in vivo. A combination of brequinar and temozolomide was curative in the majority of transgenic TH-MYCN neuroblastoma mice, indicating a highly active clinical combination therapy. Overall, DHODH inhibition combined with temozolomide has therapeutic potential in neuroblastoma, and we propose this combination for clinical testing.

Single-cell transcriptomics of human embryos identifies multiple sympathoblast lineages with potential implications for neuroblastoma origin.

Characterization of the progression of cellular states during human embryogenesis can provide insights into the origin of pediatric diseases. We examined the transcriptional states of neural crest- and mesoderm-derived lineages differentiating into adrenal glands, kidneys, endothelium and hematopoietic tissue between post-conception weeks 6 and 14 of human development. Our results reveal transitions connecting the intermediate mesoderm and progenitors of organ primordia, the hematopoietic system and endothelial subtypes. Unexpectedly, by using a combination of single-cell transcriptomics and lineage tracing, we found that intra-adrenal sympathoblasts at that stage are directly derived from nerve-associated Schwann cell precursors, similarly to local chromaffin cells, whereas the majority of extra-adrenal sympathoblasts arise from the migratory neural crest. In humans, this process persists during several weeks of development within the large intra-adrenal ganglia-like structures, which may also serve as reservoirs of originating cells in neuroblastoma.

MYCN Function in Neuroblastoma Development.

Dysregulated expression of the transcription factor MYCN is frequently detected in nervous system tumors such as childhood neuroblastoma. Here, gene amplification of MYCN is a single oncogenic driver inducing neoplastic transformation in neural crest-derived cells. This abnormal MYCN expression is one of the strongest predictors of poor prognosis. It is present at diagnosis and is never acquired during later tumorigenesis of MYCN non-amplified neuroblastoma. This suggests that increased MYCN expression is an early event in these cancers leading to a peculiar dysregulation of cells that results in embryonal or cancer stem-like qualities, such as increased self-renewal, apoptotic resistance, and metabolic flexibility.

FGF Signalling in the Self-Renewal of Colon Cancer Organoids.

The progression of colorectal cancer (CRC) is supposedly driven by cancer stem cells (CSC) which are able to self-renew and simultaneously fuel bulk tumour mass with highly proliferative and differentiated tumour cells. However, the CSC-phenotype in CRC is unstable and dependent on environmental cues. Fibroblast growth factor 2 (FGF2) is essential and necessary for the maintenance of self-renewal in adult and embryonic stem cells. Investigating its role in self-renewal in advanced CRC patient-derived organoids, we unveiled that FGF-receptor (FGFR) inhibition prevents organoid formation in very early expanding cells but induces cyst formation when applied to pre-established organoids. Comprehensive transcriptome analyses revealed that the induction of the transcription factor activator-protein-1 (AP-1) together with MAPK activation was most prominent after FGFR-inhibition. These effects resemble mechanisms of an acquired resistance against other described tyrosine kinase inhibitors such as EGF-receptor targeted therapies. Furthermore, we detected elevated expression levels of several self-renewal and stemness-associated genes in organoid cultures with active FGF2 signalling. The combined data assume that CSCs are a heterogeneous population while self-renewal is a common feature regulated by distinct but converging pathways. Finally, we highlight FGF2 signalling as one of numerous components of the complex regulation of stemness in cancer.

Human iPSC-derived MSCs (iMSCs) from aged individuals acquire a rejuvenation signature.

BACKGROUND: Primary mesenchymal stem cells (MSCs) are fraught with aging-related shortfalls. Human-induced pluripotent stem cell (iPSC)-derived MSCs (iMSCs) have been shown to be a useful clinically relevant source of MSCs that circumvent these aging-associated drawbacks. To date, the extent of the retention of aging-hallmarks in iMSCs differentiated from iPSCs derived from elderly donors remains unclear. METHODS: Fetal femur-derived MSCs (fMSCs) and adult bone marrow MSCs (aMSCs) were isolated, corresponding iPSCs were generated, and iMSCs were differentiated from fMSC-iPSCs, from aMSC-iPSCs, and from human embryonic stem cells (ESCs) H1. In addition, typical MSC characterization such as cell surface marker expression, differentiation capacity, secretome profile, and trancriptome analysis were conducted for the three distinct iMSC preparations-fMSC-iMSCs, aMSC-iMSCs, and ESC-iMSCs. To verify these results, previously published data sets were used, and also, additional aMSCs and iMSCs were analyzed. RESULTS: fMSCs and aMSCs both express the typical MSC cell surface markers and can be differentiated into osteogenic, adipogenic, and chondrogenic lineages in vitro. However, the transcriptome analysis revealed overlapping and distinct gene expression patterns and showed that fMSCs express more genes in common with ESCs than with aMSCs. fMSC-iMSCs, aMSC-iMSCs, and ESC-iMSCs met the criteria set out for MSCs. Dendrogram analyses confirmed that the transcriptomes of all iMSCs clustered together with the parental MSCs and separated from the MSC-iPSCs and ESCs. iMSCs irrespective of donor age and cell type acquired a rejuvenation-associated gene signature, specifically, the expression of INHBE, DNMT3B, POU5F1P1, CDKN1C, and GCNT2 which are also expressed in pluripotent stem cells (iPSCs and ESC) but not in the parental aMSCs. iMSCs expressed more genes in common with fMSCs than with aMSCs. Independent real-time PCR comparing aMSCs, fMSCs, and iMSCs confirmed the differential expression of the rejuvenation (COX7A, EZA2, and TMEM119) and aging (CXADR and IGSF3) signatures. Importantly, in terms of regenerative medicine, iMSCs acquired a secretome (e.g., angiogenin, DKK-1, IL-8, PDGF-AA, osteopontin, SERPINE1, and VEGF) similar to that of fMSCs and aMSCs, thus highlighting their ability to act via paracrine signaling. CONCLUSIONS: iMSCs irrespective of donor age and cell source acquire a rejuvenation gene signature. The iMSC concept could allow circumventing the drawbacks associated with the use of adult MSCs und thus provide a promising tool for use in various clinical settings in the future.

New insights into human primordial germ cells and early embryonic development from single-cell analysis.

Human preimplantation developmental studies are difficult to accomplish due to associated ethical and moral issues. Preimplantation cells are rare and exist only in transient cell states. From a single cell, it is very challenging to analyse the origination of the heterogeneity and complexity inherent to the human body. However, recent advances in single-cell technology and data analysis have provided new insights into the process of early human development and germ cell specification. In this Review, we examine the latest single-cell datasets of human preimplantation embryos and germ cell development, compare them to bulk cell analyses, and interpret their biological implications.

Combined ultra-low input mRNA and whole-genome sequencing of human embryonic stem cells.

BACKGROUND: Next Generation Sequencing has proven to be an exceptionally powerful tool in the field of genomics and transcriptomics. With recent development it is nowadays possible to analyze ultra-low input sample material down to single cells. Nevertheless, investigating such sample material often limits the analysis to either the genome or transcriptome. We describe here a combined analysis of both types of nucleic acids from the same sample material. METHODS: The method described enables the combined preparation of amplified cDNA as well as amplified whole-genome DNA from an ultra-low input sample material derived from a sub-colony of in-vitro cultivated human embryonic stem cells. cDNA is prepared by the application of oligo-dT coupled magnetic beads for mRNA capture, first strand synthesis and 3'-tailing followed by PCR. Whole-genome amplified DNA is prepared by Phi29 mediated amplification. Illumina sequencing is applied to short fragment libraries prepared from the amplified samples. RESULTS: We developed a protocol which enables the combined analysis of the genome as well as the transcriptome by Next Generation Sequencing from ultra-low input samples. The protocol was evaluated by sequencing sub-colony structures from human embryonic stem cells containing 150 to 200 cells. The method can be adapted to any available sequencing system. CONCLUSIONS: To our knowledge, this is the first report where sub-colonies of human embryonic stem cells have been analyzed both at the genomic as well as transcriptome level. The method of this proof of concept study may find useful practical applications for cases where only a limited number of cells are available, e.g. for tissues samples from biopsies, tumor spheres, circulating tumor cells and cells from early embryonic development. The results we present demonstrate that a combined analysis of genomic DNA and messenger RNA from ultra-low input samples is feasible and can readily be applied to other cellular systems with limited material available.

Water infusion versus air insufflation for colonoscopy.

BACKGROUND: Colonoscopy is a widely used diagnostic and therapeutic modality. A large proportion of the population is likely to undergo colonoscopy for diagnosis and treatment of colorectal diseases, or when participating in colorectal cancer screening programs. To reduce pain, water infusion instead of traditional air insufflation during the insertion phase of the colonoscopy has been proposed, thereby improving patients' acceptance of the procedure. Moreover, the water infusion method may improve early detection of precancerous neoplasms. OBJECTIVES: To compare water infusion techniques with standard air insufflation, specifically evaluating technical quality and screening efficacy, as well as patients' acceptance of the water infusion procedure. SEARCH METHODS: We searched the Cochrane Colorectal Cancer Group Specialized Register (February 2014), the Cochrane Central Register of Controlled Trials (CENTRAL; 2014, Issue 1), Ovid MEDLINE (1950 to February 2014), Ovid EMBASE (1974 to February 2014), and ClinicalTrials.gov (1999 to February 2014) for eligible randomised controlled trials. SELECTION CRITERIA: We included randomised controlled trials comparing water infusion (water exchange or water immersion methods) against standard air insufflation during the insertion phase of the colonoscopy. DATA COLLECTION AND ANALYSIS: Two review authors independently assessed the studies for inclusion and extracted data from eligible studies. We performed analysis using Review Manager software (RevMan 5). MAIN RESULTS: We included 16 randomised controlled trials consisting of 2933 colonoscopies. Primary outcome measures were cecal intubation rate and adenoma detection; secondary outcomes were time needed to reach the cecum, pain experienced by participants during the procedure, completion of cecal intubation without sedation/analgesia, and adverse events. Completeness of colonoscopy, that is cecal intubation rate, was similar between water infusion and standard air insufflation (risk ratio 1.00, 95% confidence interval (CI) 0.97 to 1.03, P = 0.93). Adenoma detection rate, that is number of participants with at least one detected adenoma, was slightly improved with water infusion (risk ratio 1.16, 95% CI 1.04 to 1.30, P = 0.007). Assuming the fraction of patients undergoing screening colonoscopy who had one or more adenomas detected was 20 per 100 with standard colonoscopy, the use of water colonoscopy may increase the fraction to 23 per 100 individuals. From our findings, it is possible that up to 68,000 more of the 1.7 million outpatient screening colonoscopies performed annually in the United States, could detect adenomas if water infusion colonoscopy was used. In addition, with water infusion participants experienced significantly less pain (mean difference in pain score on a 0 to 10 scale: -1.57, 95% CI -2.00 to -1.14, P < 0.00001) and a significantly lower proportion of participants requested on-demand sedation or analgesia, or both (risk ratio 1.20, 95% CI 1.14 to 1.27, P < 0.00001). Qualitative analysis suggests that water infusion colonoscopy was not associated with a markedly increased rate of adverse events compared with the standard procedure. AUTHORS' CONCLUSIONS: Completeness of colonoscopy, that is cecal intubation rate, was not improved by water infusion compared with standard air insufflation colonoscopy. However, adenoma detection, assessed with two different measures (that is adenoma detection rate and number of detected adenomas per procedure), was slightly augmented by the water infusion colonoscopy. Improved adenoma detection might be due to the cleansing effects of water infusions on the mucosa. Detection of premalignant lesions during standard colonoscopy is suboptimal, and so improvements in adenoma detection by water infusion colonoscopy, although small, may help to reduce the risk of interval colorectal carcinoma. The most obvious benefit of water infusion colonoscopy was reduction of procedure-related abdominal pain, which may enhance the acceptance of screening/surveillance colonoscopy.