Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq analysis of several in vitro models of hepatocyte function: Project 4 of Open Targets - Epigenomes of Cell Lines

ABSTRACT: The aim of this experiment was to characterize the gene expression of several in vitro cellular models of hepatocyte function: primary hepatocytes and common immortalized liver cell lines (HepG2, Hep3B and Huh7), cultured in parallel in both 2D and 3D configurations. Two independent cultures were carried out for each of the cell lines. For primary cultures, cells were obtained from three independent donors and cultured in parallel in both conditions. For the 2D culture, cell lines were grown using standard culture methods and harvested sub-confluence in exponential growth phase. For the 2D culture of primary hepatocytes, cells were grown in sandwich configuration between two layers of extracellular matrix (collagen coated plates and medium containing 0.25mg/ml Matrigel) and harvested at day 9 post seeding. For 3D cultures, cells were seeded at 1500 cells per well in ultra-low adherence multi-well plates to facilitate cellular aggregation and spheroid formation. Cell line spheroids aggregated rapidly and were harvested 4-5 days post seeding. Primary cell spheroids formed more slowly and were harvested 14-17 days post seeding. This RNA-seq experiment is being carried out as part of the Open Targets project to identify a gene expression signature of common immortalized cell lines/models. This signature will be used in combination with data from ChIP-seq experiments from the same cell lines against primary cells and tissues. The overall aim of the Open Targets Cell Line Epigenome project is to establish a systematic approach for the determination of human biological and disease relevance through the generation of transcriptomic and epigenomic data in cell lines of interest. Comparison of cell line mRNA expression and epigenome data with existing and newly generated reference data from human tissue and cell types will identify assay systems that will provide greater confidence in translating target biology and compound pharmacology to patients.

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Homo sapiens

SUBMITTER: Aidan MacNamara

PROVIDER: E-MTAB-6870 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Summary: Melanoma spheroids grown under neural crest cell conditions are highly plastic migratory/invasive tumor cells endowed with immunomodulator function Background: The aggressiveness of melanoma tumors is likely to rely on their well-recognized heterogeneity and plasticity. Melanoma comprises multi subpopulations of cancer cells some of which may possess stem cell-like properties supporting the notion of plasticity. Although useful for certain tumors, the use of the sphere-formation assay to identify stem cell-like or tumor initiating cells subpopulations in human melanoma has been recently challenged. Our study reveals that this assay predicts a functional phenotype associated with aggressive behavior of tumor cells. Methodology/Principal Findings: We analyzed the molecular and functional phenotypes of melanoma spheroids formed in neural crest cell medium. Whether from metastatic (SLM8) or advanced primary (Mela1) tumors, spheroid cells expressed melanoma-associated markers. They displayed higher capacity to differentiate along mesenchymal lineages, and showed enhanced expression of SOX2, NANOG, KLF4, and/or OCT4 transcription factors, but not extensive self-renewal or enhanced tumorigenicity when compared to their adherent counterparts. To determine whether melanoma spheroids in our model could predict a molecular or functional phenotype, we performed gene expression profiling experiments using Affymetrix microarrays. Gene expression profiling attributed a neural crest cell signature to these spheroids and indicated that a migratory/invasive and immune-function modulating program could be associated with these cells. In vitro assays confirmed that these spheroids are endowed with enhanced migratory/invasive capacities. In immune activation assays, spheroid cells elicited a poorer allogenic response from immune cells and inhibited mitogen-dependent T cells activation and proliferation more efficiently than their adherent counterparts. Thus, our findings reveal novel immune-modulator function of melanoma spheroid cells and suggest specific roles for these spheroids in invasion and in evasion of antitumor immunity. Conclusion/Significance: The association of a more plastic, invasive and evasive, thus a more aggressive tumor phenotype with melanoma spheroid cells reveals a previously unrecognized aspect of tumor cells expanded as spheroid cultures. While of limited efficiency for melanoma initiating cell identification, our melanoma spheroid model predicted aggressive phenotype and could therefore, be constructive to investigate melanoma aggressiveness, relevant to patients and clinical transferability. 12 Total samples were analyzed: SLM8 adherent (SLMA) and spheroids (SLMS) cells, and Mela1 adherent (MelaA) and spheroid (MelaS) cells, all performed in triplicates. Paired statistical analyses were performed using Student's paired t-test on the gene signal intensities (gene level) and results were considered statistically significant at p-values <=0.05 and fold-change >=1.5.

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RNA-seq analysis of several in vitro models of hepatocyte function: Project 4 of Open Targets - Epigenomes of Cell Lines

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