Gene expression analysis in NB adherent primary cell lines after endothelin-1 treatment
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ABSTRACT: We cultured adherent primary cell lines from NB tumor samples. Adherent primary cell lines from NB tumor samples have a subpopulation of neural crest progenitors that grow as spheres when cultured in low-binding conditions. We tested the changes in gene expression induced by endothelin-1 (ET1), a cytokine that regulates proliferation, migration, differentiation and survival of NC cells .
Project description:We cultured adherent primary cell lines from NB tumor samples. Adherent primary cell lines from NB tumor samples have a subpopulation of neural crest progenitors that grow as spheres when cultured in low-binding conditions.
Project description:Transcriptional profile of PCSC spheres in SCM-1% KO (stem-like cells) vs adherent cultures in PCSC-Celprogen medium (differentiated-like cells) Two-condition experiment: Sphere vs. Parental/adherent cells. Biological replicates: 2 sphere replicates , 2 adherent replicates. Early passaged tumor cells were expanded in adherent conditions and then plated in sphere forming conditions (SCM-1KO% medium at low density) to isolate sphere growing cells.
Project description:High-risk neuroblastoma (NB) often involves amplification of the neural MYC (MYCN) oncogene as well as mutations in ALK. Currently, high-risk NB presents significant clinical challenges, and additional therapeutic options are needed. Oncogenes such as MYCN and ALK result in increased replication stress in cancer cells, offering one such therapeutically exploitable option. Here, we followed up on earlier phosphoproteomic analyses that identified ATR activity in ALK-driven NB cell lines. We tested several ATR inhibitors, identifying BAY 1895344 as the most potent inhibitor of NB cell growth and proliferation. Using RNA-Seq, proteomics and phosphoproteomics we characterized the response of NB cells and tumours to ATR inhibition, identifying key components of the DNA damage response (DDR) as well as ATRX, MYCN, E2F and DCK among other ATR targets in NB cells. ATR inhibition with BAY 1895344 also produced robust responses in mouse NB models. Remarkably, a 2 week protocol combining ATR and ALK inhibition led to complete regression of NB tumours in two independent NB genetically modified mouse tumour models. These results suggest that NB patients, particularly in high-risk groups with oncogene induced replication stress, may benefit from inhibition of ATR as therapeutic intervention.
Project description:PTM cells can be induced to contract by endothelin 1 (ET1). We performed miRNA-Seq of untreated testicular peritubular myoid cells (PTM_1, PTM_2, PTM_3) and ET1-treated peritubular myoid cells (ET1_1, ET1_2, ET1_3) using BGISEQ-500 platform (BGI, China).
Project description:PTM cells can be induced to contract by endothelin 1 (ET1). We performed RNA-Seq of untreated testicular peritubular myoid cells (PTM_1, PTM_2, PTM_3) and ET1-treated peritubular myoid cells (ET1_1, ET1_2, ET1_3) using BGISEQ-500 platform (BGI, China).
Project description:To determine if aberrant activation of endothelin-1 (Et1) could lead to the dysregulation of many downstream genes, we exposed fibroblasts to exogenous ET1 peptide and assayed for transcriptional changes by microarray. Mouse dermal fibroblasts were treated with exogenous Et1 peptide for 24 hours. ET1 treatment resulted in significant expression changes - primarily downregulation - of a number of genes. In particular, Tgf-beta-2 and Tgf-beta-3 were among the downregulated genes, which in turn alter the expression status of their many target genes. These data suggest that the stable silencing of Et1 is important for the phenotypic stability of dermal fibroblasts, and perhaps many other cell types as well.<br><br>Three separate biological replicates were derived for both control and treated samples. The primary dermal fibroblasts were derived by explant procedure from the skin of mouse pups aged 0-3 days. By passage 5, cells were split to two separate cultures-- one with 100nM synthetic Et1 peptide added to the medium (treated) and the other with nothing added (control). Cells were exposed to Et1 for 24 hrs, then treated and control populations were harvested for total RNA.
Project description:We have recently demonstrated that human paediatric mesenchymal stem cells can be reprogrammed toward a Ewing’s sarcoma family tumor (ESFT) cancer stem cell (CSC) phenotype by mechanisms that implicate microRNAs (miRNAs). Here, we show that the miRNA profile of ESFT CSC is shared by embryonic stem cells and CSC from divergent tumor types. We also provide evidence that the miRNA profile of ESFT CSC is the result of reversible disruption of TARBP2-dependent miRNA maturation. Restoration of TARBP2 activity and systemic delivery of synthetic forms of either of two of its targets, miRNA-143 or miRNA-145, inhibited ESFT CSC clonogenicity and tumor growth in vivo. Our observations suggest that CSC self-renewal and tumor initiation may depend on deregulation of TARBP2-dependent miRNA expression. 2 Ewing sarcoma primary samples: comparison between spheres and derived adherent cells.
Project description:Mouse primary dermal fibroblasts were treated with 100 nM endothelin-1 (ET1) synthetic peptide for 24 hours. Control samples received no ET1 peptide. The experiment compared treated to untreated to identify gene expression changes due to ET1 exposure. There are three biological replicates for both control and treated samples. These biological replicates represent separate derivations of primary dermal fibroblasts from genetically identical mouse litters aged 0-3 days.
Project description:Metastases in the bone marrow (BM) are grim prognostic factors in patients with neuroblastoma (NB). In spite of extensive analysis of primary tumor cells from high- and low-risk NB patients, a characterization of freshly isolated BM-infiltrating metastatic NB cells is still lacking. Our aim was to identify proteins specifically expressed by metastatic NB cells, that may be relevant for prognostic and therapeutic purposes. Metastatic NB cells were freshly isolated from patients’ BM by positive immunomagnetic bead manipulation using anti-GD2 monoclonal antibody. Unselected BM samples from patients with metastatic NB were also included. Gene expression profiles were compared with those obtained from archived NB primary tumors from patients with 5y-follow-up. After validation by RT-qPCR, expression/secretion of the proteins encoded by the up-regulated genes in the BM-infiltrating NB cells was evaluated by flow cytometry and ELISA. Compared to primary tumor cells, BM-infiltrating NB cells down-modulated the expression of CX3CL1, AGT, ATP1A2 mRNAs, whereas they up-regulated several genes commonly expressed by various lineages of BM resident cells. BM-infiltrating NB cells expressed indeed the proteins encoded by the top-ranked genes, S100A8 and A9 (calprotectin), CD177 and CD3, and secreted the CXCL7 chemokine. BM-infiltrating NB cells also expressed CD271 and HLA-G. We have identified proteins specifically expressed by BM-infiltrating NB cells. Among them, calprotectin, a potent inflammatory protein, and HLA-G, endowed with tolerogenic properties facilitating tumor escape from host immune response, may represent novel biomarkers and/or targets for therapeutic intervention in high-risk NB patients.