Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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ACGH: Human SUM149-Luc triple-negative Inflammatory Breast Cancer cell line versus its metabolically adaptable variants MA1 and MA2

ABSTRACT: CGH array of MA1 and MA2 variant cells as compared to the parental SUM149-Luc breast cancer cell line. The MA1 and MA2 variants were isolated based on the ability of rare cancer cells to survive and grow without adding glutamine in culture medium. To gain insight into the characteristic of metabolically adaptable MA cells that enables them to survive severe metabolic challenge, i.e., prolonged lack of glutamine and other challenges [Singh et al., PLoS ONE 7: e36510, 2012], we analyzed CGH array to compare these cells with the parental SUM149-Luc (luciferase-transfected) cells. We analyzed two independently selected cell populations, one from 0.5 million parental cells (designated MA1) and one from 1 million parental cells (designated MA2). Comparing the MA1 and MA2 variants to the common parental SUM149-Luc cell line. One sample each.

ORGANISM(S): Homo sapiens

SUBMITTER: Anthony Lucci

PROVIDER: E-GEOD-60015 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Establishment and subsequent validation of a aCGH protocol for WGA (whole genome amplification) products originating from single cell or low amount of starting material (i.e. microdissected FFPE tissue samples). The establishment of the protocol involved testing of three DNA labeling protocols. Two labeling protocols were designed specifically for Ampli1(TM) WGA products. Additionally random primed isothermal (Klenow-based) labeling approach was tested (MM-CM-6hlendick et al., PLoS One. 2013 Jun 25;8(6):e67031.). In addition two different types of reference samples were tested and reference based on single-cell WGA products was chosen as most suitable in the end. The validation of the protocol assessed the following aspects: (1) performance of the protocol on primary and reamplified WGA products, (2) accuracy of the protocol in term of sensitivity of the CNA detection, (3) accuracy in terms of recapitulation of complex patterns of CNAs, (4) accuracy in terms of quantitative assessment of the CNAs, (5) ability to detect genomic heterogeneity of single cells (obtained either from in vitro cultures or from clinical patient material), (6) ability to detect minimal regions of aberration within a panel of disseminated cancer cells and corresponding tumor tissues. Establishment and validation of the single-cell aCGH protocol: two condition experiment (i.e. PCR-based labeling technique 1 vs. PCR-based labeling technique 2; PCR-based labeling technique 2 vs. random-primed isothermal (Klenow) labeling approach; reference DNA from cell pool WGA product vs. reference DNA from single-cell WGA products). Validation of the protocol: comparison of the CNA profiles between single-cell WGA products and corresponding bulk DNA. Analysis of the DCC and corresponding FFPE tumor tissue samples: single-condition experiment performed on samples collected at different stages of the disease (DCCs from bone marrow) and/or from different sites (primary tumor-breast; metastasis-lymph nodes; DCCs-bone marrow). Microarray data is corresponding data depicted in the paper manuscript titled: Reliable single cell array CGH for clinical samples

Project description:About 230 clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but no assays enable comprehensive molecular characterization of CTCs with diagnostic precision. We therefore combined a workflow for CTC enrichment and isolation with 100% purity with a non-random whole genome amplifiation method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 breast cancer patients. We defined a genome integrity index (GII) to identify cells suited for molecular chracterization by different molecular assay in more than 90% of single cells, such as diagnostic profiling for point mutations, gene amplifications and whole genomes of single cells. The high reliability on clinical samples enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified therapeutically relevant genomic disparity between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered preexisting cells reistsant to ERBB2 targeted therapies suggesting ongoing microevolution at late stage disease whose exploration may provide essential information for personalized treatment decisions. The analysis aimed to indentify profiles of copy number changes in genomic DNA of single circulating tumor cells (CTCs). For this, CTCs were enrched using the FDA approved CellSearch System and single-cell were isolated using the DEPArray System. Subsequently, single-cell DNA was amplified using the Ampli1 WGA Kits and subjected to single-cell aCGH analysis according to previously published protocol (Czyz ZT et al., PLoS One. 2014 Jan 21;9(1):e85907). The analysis included 38 single CTCs and 10 white blood cells (WBCs) obtained from 16 breast cancer patient. WBCs were used as controls for the analysis. In addition, four CTC cell pools were included in the analysis. This was done to show the discrepancies between the profiles of individual cells and corresponing average genomic profile of CTCs in a patient material (cell pools), thereby demonstrating the importance of the analysis on the cell-by-cell basis. The reference sample used for all aCGH experiments consisted of a pool of four single-cell WGA products generated from WBCs of a healthy female donor.

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ACGH: Human SUM149-Luc triple-negative Inflammatory Breast Cancer cell line versus its metabolically adaptable variants MA1 and MA2

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