37 research outputs found

    Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

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    Background: Transgenic zebrafish lines with the expression of a fluorescent reporter under the control of a cell-type specific promoter, enable transcriptome analysis of FACS sorted cell populations. RNA quality and yield are key determinant factors for accurate expression profiling. Limited cell number and FACS induced cellular stress make RNA isolation of sorted zebrafish cells a delicate process. We aimed to optimize a workflow to extract sufficient amounts of high-quality RNA from a limited number of FACS sorted cells from Tg(fli1a:GFP) zebrafish embryos, which can be used for accurate gene expression analysis. Results: We evaluated two suitable RNA isolation kits (theRNAqueous micro and the RNeasy plus micro kit) and determined that sorting cells directly into lysis buffer is a critical step for success. For low cell numbers, this ensures direct cell lysis, protects RNA from degradation and results in a higher RNA quality and yield. We showed that this works well up to 0.5x dilution of the lysis buffer with sorted cells. In our sort settings, this corresponded to 30,000 and 75,000 cells for the RNAqueous micro kit and RNeasy plus micro kit respectively. Sorting more cells dilutes the lysis buffer too much and requires the use of a collection buffer. We also demonstrated that an additional genomic DNA removal step after RNA isolation is required to completely clear the RNA from any contaminating genomic DNA. For cDNA synthesis and library preparation, we combined SmartSeq v4 full length cDNA library amplification, Nextera XT tagmentation and sample barcoding. Using this workflow, we were able to generate highly reproducible RNA sequencing results. Conclusions: The presented optimized workflow enables to generate high quality RNA and allows accurate transcriptome profiling of small populations of sorted zebrafish cells

    Integrated and cross-species omics analyses identify the DNA damage response pathway as an important vulnerable pathway in aggressive neuroblastoma tumors

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    Introduction: The past decade several high throughput technologies have been developed that allow to profile cancer cells at global omics level. The big datasets generated on these new platforms can significantly help in generating a comprehensive view and understanding of the tumor biology and subsequently fuels studies on the development and implementation of new molecular targeted therapies. For neuroblastoma, a childhood tumor of the developing sympathetic nervous system, new insights on targetable driver genes are currently limited. Methods: We studied neuroblastoma oncogenesis through analysis of big omics datasets using different bio-informatics tools. Available datasets include transcriptomic and genomic profiles of large sets of human and mouse neuroblastoma tumors and cell lines. Using those data (1) we performed dynamic gene expression analysis during tumor initiation and formation in a MYCN driven mouse model, (2) designed and tested an embryonic stem cell (ESC) signature and (3) integrated DNA copy number and m(i)RNA expression data using the Conexic tool in order to identify new driver genes. Results: Most interestingly, these independent 3 data-mining approaches lead to the identification of a converging theme that can be attributed to tumor aggressiveness and which can serve as an important novel target for therapy, i.e. the DNA damage response pathway. First, master regulator analysis of genes involved in MYCN driven neuroblastoma formation identified the transcription factor FOXM1, an important DNA damage regulator. Second, analysis of a m(i)RNA expression ESC signature in data of more than 200 neuroblastoma tumors allowed us to mark a subgroup of neuroblastoma tumors with increased stem cell capabilities which matched with the most agressive subset of neuroblastoma tumors. Remarkably, the top list of coding genes that correlate with the ESC signature was dominated by genes implicated in DNA damage response, including the FOXM1 gene. Third, driver gene identification in aggressive neuroblastoma tumors using the Conexic tool pointed at 2 DNA repair genes that are both known to be regulated by FOXM1. Conclusions: Based on these data, we hypothesize that FOXM1 plays an important central role in the MYC(N) induced DNA damage response and predict that FOXM1 and its downstream DNA repair genes impact on chemotherapy resistance. Currently, we are testing the effect of treatment with a FOXM1 inhibitor on neuroblastoma chemo-resistance

    An embryonic stem cell activated FOXM1 transcriptional program marks ultra-high-risk primary neuroblastoma patients for FDI-6 small molecule inhibition

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    Introduction: Chemotherapy resistance is responsible for high mortality rates in high-risk neuroblastoma patients. MYCN is a major oncogenic driver in these tumors controlling pluripotency genes including LIN28B. Therefore, we hypothesized that enhanced embryonic stem cell (ESC) gene regulatory programs could mark tumors with increased risk for therapy failure enabling the selection of patients for novel targeted therapies. M&M: A microRNA expression ESC-signature was established based on publically available data. In addition, an mRNA ESC-signature of top 500 protein coding genes with highest positive correlation with the microRNA ESC-signature score was generated. Results: High ESC-signature scores were significantly correlated with worse neuroblastoma patient survival, both in the global patient cohort as well as in the subset of stage 4 tumors without MYCN-amplification. In addition, both in neuroblastoma and other embryonal tumors exhibiting MYCN-activation, the scores were significantly higher. This was confirmed in MYCN cell model systems where the scores altered upon MYCN-overexpression/knock-down. Using GSEA, we identified that genes implicated in DNA damage response and cell cycle control were strongly enriched in the signature. One of the genes in the signature is the transcription factor FOXM1, which is a master regulator driving those pathways. The upstream activator of FOXM1, MELK, was also part of the signature. Inhibition of FOXM1 in neuroblastoma cells using the small molecule FDI-6 significantly reduced cell viability. In addition, MELK inhibitors are currently tested in vitro and both FOXM1 and MELK inhibitors are evaluated in MYCN transgenic zebrafish models. Conclusion: A novel ESC-signature score marks neuroblastomas with poor prognosis enabling the identification of ultra-high-risk neuroblastoma patients that may benefit from targeted therapies using FOXM1 or MELK inhibitors