A novel five-gene signature predicts overall and recurrence-free survival in NSCLC
ABSTRACT: Earlier studies had shown that side population cells isolated from established non-small cell lung cancer (NSCLC) cell lines exhibit cancer stem cell properties. Microarray data from side population (SP) and main population (MP) cells isolated from 4 NSCLC lines (A549, H1650, H460, H1975) were used to examine gene expression profiles associated with stemness. Total RNA extracted from SP and MP samples were used to generate cRNA targets, which were hybridized to Human Genome U133 Plus 2.0 probe arrays. Raw data was processed and the mean center expression level for each gene was determined. Four cell lines (A549, H1650, H460, H1975), each having 1 SP and 1 MP sample.
Project description:Side population (SP) cells are highly enriched in stem and progenitor cells. CD45+and CD45- SP cells were found at all developmental lung stages with the highest frequency of cells present at embryonic day 17.5 (E17.5), In order to clarify the role of these cells in lung development, we used oligonucleotide microarrays to evaluate their gene expression profiles. To do this, oligonucleotide gene arrays were performed in triplicate on RNA derived from CD45+ and CD45-SP cells, and CD45+ and CD45- non-SP populations (main population; MP) isolated from the E17.5 lung.
Project description:Gene expression profiling has been used to characterize prognosis in various cancers. Earlier studies had shown that side population cells isolated from Non-Small Cell Lung Cancer (NSCLC) cell lines exhibit cancer stem cell properties. In this study we apply a systems biology approach to gene expression profiling data from cancer stem like cells isolated from lung cancer cell lines to identify novel gene signatures that could predict prognosis. Microarray data from side population (SP) and main population (MP) cells isolated from 4 NSCLC lines (A549, H1650, H460, H1975) were used to examine gene expression profiles associated with stem like properties. Differentially expressed genes that were over or under-expressed at least two fold commonly in all 4 cell lines were identified. We found 354 were upregulated and 126 were downregulated in SP cells compared to MP cells; of these, 89 up and 62 downregulated genes (average 2 fold changes) were used for Principle Component Analysis (PCA) and MetaCore pathway analysis. The pathway analysis demonstrated representation of 4 up regulated genes (TOP2A, AURKB, BRRN1, CDK1) in chromosome condensation pathway and 1 down regulated gene FUS in chromosomal translocation. Microarray data was validated using qRT-PCR on the 5 selected genes and all showed robust correlation between microarray and qRT-PCR. Further, we analyzed two independent gene expression datasets that included 360 lung adenocarcinoma patients from NCI Director's Challenge Set for overall survival and 63 samples from Sungkyunkwan University (SKKU) for recurrence free survival. Kaplan-Meier and log-rank test analysis predicted poor survival of patients in both data sets. Our results suggest that genes involved in chromosome condensation are likely related with stem-like properties and might predict survival in lung adenocarcinoma. Our findings highlight a gene signature for effective identification of lung adenocarcinoma patients with poor prognosis and designing more aggressive therapies for such patients.
Project description:This SuperSeries is composed of the following subset Series: GSE31246: Expression data from SP and non-SP sorted anti-EpCAM treated A2C12 cells GSE31313: Expression data from anti-EpCAM treated and untreated SP cells compared to lung tissue GSE31315: Expression data from SP and non-SP sorted anti-EpCAM treated A549 cells Refer to individual Series
Project description:Targeted therapies against cancer stem cells, which are enriched in side populations (SP), involves interruption of Wnt-signalling. Furthermore, EpCAM is a SP marker and modulator of Wnt-signalling. Therefore, the effects of an anti-EpCAM treatment on SP-cells and WNT/β-catenin signalling was studied. SP of the human lung adenocarcinoma cell line A549 was obtained by fluorescence activated cell sorting and whole genome scans helped to define their molecular phenotype after anti-EpCAM antibody treatment. Anti-EpCAM treated and untreated A549 cells were subjected to Hoechst 33342 dye exclusion assay and sorted to SP and non-SP fractions by FACS. Three biological replicates.
Project description:Targeted therapies against cancer stem cells which are enriched in side populations (SP) involves interruption of Wnt-signalling. Furthermore, EpCAM is a SP marker and modulator of Wnt-signalling. Therefore, the effects of an anti-EpCAM treatment on SP-cells and WNT/β-catenin signalling was studied. SP of the murine lung adenocarcinoma cell line A2C12 was obtained by fluorescence activated cell sorting and whole genome scans helped to define their molecular phenotype after anti-EpCAM antibody treatment. Anti-EpCAM treated and untreated A2C12 cells were subjected to Hoechst 33342 dye exclusion assay and sorted to SP and non-SP fractions by FACS. Three biological replicates.
Project description:Prediction of treatment outcome of non-small cell lung cancer (NSCLC) with EGFR inhibitors on the basis of the genetic analysis of the tumor can be incorrect in case of rare or complex mutations, bypass molecular activation pathways, or pharmacodynamic variations. The aim of this study was to develop an ex vivo and in vivo real-time quantitative imaging test for EGFR inhibitors sensitivity assessment.Erlotinib resistant (A549, H460, H1975), insensitive (H1650) and hypersensitive (HCC827) cell lines were injected subcutaneously in Nude mice. Tumor xenografts from mice treated with Erlotinib were imaged ex vivo and in vivo using probe-based confocal laser endomicroscopy (pCLE) and NucView 488 Caspase 3 substrate, a fluorescent probe specific for the activated caspase 3.Assessment of apoptosis at 24h post treatment, both ex vivo in explanted tumor xenografts and in vivo, showed a significant difference between resistant cell lines (A549, H460 and H1975) and insensitive (H1650) or hypersensitive (HCC827) ones (p<0.05 for ex vivo imaging, p?0.02 for in vivo imaging). There was also a significant difference between insensitive and hypersensitive cell lines, both ex vivo (p<0.05) and in vivo (p = 0.01).Real-time in vivo and ex vivo assessment of apoptosis using pCLE differentiates resistant from sensitive NSCLC xenografts to Erlotinib.
Project description:Analysis of Hoechst 33342 dye-effluxing side population cells (SP cells defined as glioma stem cells, GSCs) and dye-retaining main population cells (MP cells defined as non-GSCs) that were FACS-sorted from the C6 glioma cell line stably expressing EGFP (C6-eGFP). ECM-related genes, such as Col4a1 and Col4a2, and the iron carrier gene Tf are upregulated in MP cells. Results provide the insight into molecular basis underlying the maintenance of GSCs by non-GSCs. Gene expression profiles were compared between SP and MP cells just after FACS-sorting from the whole C6-eGFP cells based on their Hoechst-effluxing abilities.
Project description:Abnormal lipid metabolisms are closely associated with cancers. In this study, mass spectrometry was employed to in situ investigate the associations of membrane lipid phenotypes of six human lung cancer cell lines (i.e., A549, H1650, H1975 from adenocarcinoma, H157 and H1703 from squamous cell carcinomas, and H460 from a large cell carcinoma) with cancer cell types and finally total 230 lipids were detected. Based these 230 lipids, partial least-square discriminant analysis indicated that fifteen lipids (i.e., PE 18:0_18:1, PI 18:0_20:4, SM 42:2, PE 16:0_20:4, PE 36:2, PC 36:2, SM 34:1, PA 38:3,C18:0, C22:4, PA 34:2, C20:5, C20:2, C18:2, and CerP 36:2) with variable importance in the projection (VIP) value of > 1.0 could be used to differentiate six cancer cell lines with the Predicted Residual Sum of Square (PRESS) score of 0.1974. Positive correlation between polyunsaturated fatty acids (i.e., C20:4, C22:4, C22:5, and C22:6) and polyunsaturated phospholipids (PE 16:0_20:4, PE 38:4, and PI 18:0_20:4) was observed in lung adenocarcinoma cells, especially for H1975 cells. Three adenocarcinoma cell lines (i.e., A549, H1650, and H1975) could be differentiated from other lung cancer cell lines based on the expression of C18:1, C20:1, C20:2, C20:5, and C22:6.
Project description:Anaplastic Large Cell Lymphoma (ALCL) is propagated by cancer stem cells which are identified by their ability to efflux dye and hence reside in the side population (SP) on FACS analysis. In order to understand the origins of these ALCL SP cells we purified SP and main population (MP; SP-depleted) cell from the ALCL cell line SUDHL-1. On comparison of the gene expression signatures derived from these 2 cellular populations distinct branches dividing the SP and MP populations present indicating that they are distinct subsets within the bulk tumour. We sought to obtain pure populations of SP and MP cells from human SUDHL-1 Anaplastic Large Cell Lymphoma (ALCL) cell line. SUDHL-1 cells were stained with Hoechst 33342 dye, analysed using MoFlo Cell Sorter with UV lasers. The cells were then sorted in triplicates and subjected to RNA extraction. The extracted RNA was subjected to gene expression microarray using the Affymetrix Human Gene 1.0 ST Array.
Project description:Purpose: To explore the side population (SP) in pancreatic ductal adenocarcinoma (PDAC) for its gene expression profile and its association to cancer stem cells (CSC) and to evaluate the value of genes from its gene signature on patient survival. Experimental design: Side and main population (MP) cells were isolated from 11 human PDAC resection specimens using fluorescence-activated cell sorting (FACS) after Hoechst incubation. Total RNA was extracted for whole-genome analysis. A gene signature for the purified SP (pSP; depleted from immune/endothelial CD45+/CD31+ cells) was developed and validated with the nCounter system on expression data of 78 primary PDAC using Cox regression analyses for disease-free and overall survival. Results: An SP was identified in all PDAC samples. Whole-genome expression profiling of pSP revealed upregulation of genes related to therapy-resistance and of CSC-associated genes and pathways. A pSP signature of 32 up- or downregulated genes was capable of discriminating SP from MP in an independent set of 10 PDAC samples, and some contributing genes had a prognostic value in a separate series of 78 patients who underwent surgery for PDAC. Conclusion: Pancreatic cancer contains an SP with chemo-resistant and CSC-associated molecular characteristics. Genes from a newly defined pSP gene signature are related to survival of patients who undergo surgical resection for pancreatic cancer, and might therefore represent potential therapeutic targets. Microarray analysis was performed on SP and MP samples from 11 different human PDAC samples.