Project description:Many research organizations are exploring the use of high-throughput profiling (HTP) assays such as transcriptomics and image-based phenotypic profiling for in vitro bioactivity screening and hazard evaluation of chemicals. HTP assays can be used to characterize the response of many human-derived cell types to chemicals. However, no cell-based in vitro model will express all the potential molecular targets and pathways that are present in the human body that could be perturbed, resulting in toxicity. Therefore, an envisioned approach for the use of HTP assays in chemical bioactivity screening involves panels of cell lines that express complementary (partially non-overlapping) sets of molecular targets. In this study, baseline gene expression in 53 different human-derived cell lines was characterized using the TempO-Seq human whole transcriptome assay. The cell line set included 36 human cancer-derived cell lines and 17 engineered or spontanously immortalized human cell lines. The baseline gene expression data can inform what set(s) of molecular targets are expressed across the cell line collection and inform researchers regarding the choice of cell lines for chemical screening studies.
Project description:Introduction: DU145 and LNCaP are classic prostate cancer cell lines. Characterizing their baseline transcriptomics profiles (without any intervention) can offer insights into baseline genetic features and oncogenic pathways that should be considered while interpreting findings after various experimental interventions such as exogenous gene transfection or drug treatment. Methods: LNCaP and DU145 cell lines were cultured under normal conditions, followed by RNA extraction, cDNA conversion, library preparation, and RNA sequencing using the Illumina NovaSeq platform. The sequences were analyzed to identify differentially expressed genes (DEGs) and for gene ontology (GO) and pathway enrichment. Results: A total of 3916 and 2301 genes were found to be differentially upregulated and downregulated between LNCaP and DU145 cell lines, respectively. The GO and pathway analysis of up-regulated DEGs indicated significant enrichment of genes involved in extracellular matrix organization and cell-substrate adhesion, while down-regulated genes are involved in epithelial cell migration, cell death regulation, and cell proliferation. Conclusion: The results showed significant differences in baseline gene expression and cellular pathways that may account for the varying metastatic potentials between LNCaP and DU145 cell lines, which should be considered when interpreting findings after experimental interventions.
Project description:There is a need for robust phosphopeptide enrichment methods to allow signaling network analysis in cancer cell lines and tissues with minimal fractionation. With recent instrument developments thousands of unique phosphopeptides can be detected by single-shot LC-MS/MS. However, successful phosphoproteomics experiments still rely on efficient phosphopeptide enrichment from a tryptic digest prior to LC-MS/MS analysis. Here we describe a performance assessment of HAMMOC (hydroxyl acid modified metal affinity chromatography) (Sugiyama MCP2007, Kyono, JPR 2008) combined with single shot label-free quantitation at 500 µg peptide input level. We apply the method to profile the baseline phosphorylation landscape of a panel of 8 colorectal cancer (CRC) cell lines. These CRC cell lines represent the 3 CRC subtypes (CCS1, CCS2 and CCS3) as reported by large-scale transcriptome analysis. We report an analysis of the phosphoprotein network and processes enriched in the cell lines representing the poor prognosis CCS3 subtype.
Project description:Baseline gene expression for two primary and two recurrent tumor cell lines derived from MTB;TAN transgenic mice. Microarrays were performed in biological duplicate to determine differential gene expression between primary and recurrent tumor cell cohorts.
Project description:There is a need for robust phosphopeptide enrichment methods to allow signaling network analysis in cancer cell lines and tissues with minimal fractionation. With recent instrument developments thousands of unique phosphopeptides can be detected by single-shot LC-MS/MS. However, successful phosphoproteomics experiments still rely on efficient phosphopeptide enrichment from a tryptic digest prior to LC-MS/MS analysis. Here we describe a performance assessment of HAMMOC (hydroxyl acid modified metal affinity chromatography) (Sugiyama MCP2007, Kyono, JPR 2008) combined with single shot label-free quantitation at 500 µg peptide input level. We apply the method to profile the baseline phosphorylation landscape of a panel of 8 colorectal cancer (CRC) cell lines. These CRC cell lines represent the 3 CRC subtypes (CCS1, CCS2 and CCS3) as reported by large-scale transcriptome analysis. We report an analysis of the phosphoprotein network and processes enriched in the cell lines representing the poor prognosis CCS3 subtype.
Project description:Malignant pleural mesothelioma (MPM) is an asbestos-related lethal malignancy refractory to conventional therapies. Since its symptoms are not specific, MPM can be easily confused with other chest diseases, especially metastatic lung adenocarcinomas (ADCA), and diagnosis is often established late when the disease is at an advanced stage. Classically, a reliable diagnosis requires histological analysis of multiple pleural biopsies. However, there is still no absolute marker for MPM. Thus, with the aim of identifying novel markers with higher specificity and sensitivity, gene expression profiling studies have been conducted using tumor specimens. Because of the cell heterogeneity of tumor samples, we decided to apply counterflow centrifugal elutriation to isolate cancer cells from pleural effusions, which are a common feature of MPM and ADCA. We profiled a total of 54 biological samples corresponding to triplicates of 13 MPM and 4 ADCA as well as the SV40-immortalized cell line MeT-5A. Our microarray results confirmed some of the existing markers which are currently used to distinguish MPM from ADCA by immunostaining of pleural biopsies and which have also been proposed for use in a PCR-based assay. Of particular interest, we also identified novel cellular markers (including predominantly COL3A1, OSAP, OCIAD2, XAGE1), which we validated by real time RT-PCR, and novel soluble markers, such as osteonectin and galectin-3, whose clinical utility as molecular targets remains to be determined. Keywords: cell type comparison three-condition experiment: ADCA vs MPM vs Met5A cells 13 MPM, 4 ADCA and Met5A were independantly grown and harvested 3 biological replicates per cell line, one replicate per array