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Immunomagnetic enrichment and flow cytometric isolation of breast cancer circulating tumor cells (CTCs) for genomic characterization

ABSTRACT: We developed a novel approach to isolate tumor cells with high purity from blood via immunomagnetic enrichment followed by fluorescence activated cell sorting (IE/FACS) and examined copy number alterations in these cells. Magnetic beads coated with EpCAM mAb were added to blood to enrich for tumor cells. Enriched samples were then subjected to FACS analysis using differentially labeled mAbs to distinguish tumor cells (EpCAM+) from leukocytes (CD45+) during sorting. DNA from isolated tumor cells was subjected to whole genome amplification (WGA) and copy number analysis via array comparative genomic hybridization (CGH). The assay was evaluated using BT474 and MCF7 breast cancer cell lines and in CTCs from 5 metastatic breast cancer (MBC) patients with matched archival primary tumors and later extended to an additional 97 MBC patients.

ORGANISM(S): Homo sapiens

PROVIDER: GSE27511 | GEO | 2012/12/31

SECONDARY ACCESSION(S): PRJNA138579

REPOSITORIES: GEO

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Immunomagnetic enrichment and flow cytometric isolation of breast cancer circulating tumor cells (CTCs) for genomic characterization

Project description:We developed a novel approach to isolate tumor cells with high purity from blood via immunomagnetic enrichment followed by fluorescence activated cell sorting (IE/FACS) and examined copy number alterations in these cells. Magnetic beads coated with EpCAM mAb were added to blood to enrich for tumor cells. Enriched samples were then subjected to FACS analysis using differentially labeled mAbs to distinguish tumor cells (EpCAM+) from leukocytes (CD45+) during sorting. DNA from isolated tumor cells was subjected to whole genome amplification (WGA) and copy number analysis via array comparative genomic hybridization (CGH). The assay was evaluated using BT474 and MCF7 breast cancer cell lines and in CTCs from 5 metastatic breast cancer (MBC) patients with matched archival primary tumors and later extended to an additional 97 MBC patients. Evaluation of the assay on isolated breast cancer cell lines spiked into blood correctly identified the known genomic alterations with high reproducibility. In clinical studies, comparison of CTCs with matched archival primary tumors confirmed shared lineage with notable divergence. In addition, serial testing of CTCs confirmed reproducibility, and indicated genomic change over time. Analysis of genomic profiles of CTCs from 102 MBC patients revealed common copy number alterations including gains in 1q and 8q and losses in 8p and 11q. Comparison with a published CGH dataset of primary breast tumors revealed similar frequencies of recurrent genomic copy number aberrations.

2012-12-31 | E-GEOD-27511 | biostudies-arrayexpress

Expanded Genomic Profiling of Circulating Tumor Cells in Metastatic Breast Cancer Patients to Assess Biomarker Status and Biology Over Time (CALGB 40502 and CALGB 40503, Alliance).

Project description:We developed a novel approach to isolate tumor cells with high purity from blood which was subjected to immunomagnetic enrichment using EpCAM beads followed by fluorescence activated cell sorting (IE/FACS) to isolate EpCAM-positive cells away from leukocytes (CD45+). Duplicate samples of 20 cells were isolated from the same enriched blood from MBC patients and then subjected to DNA and RNA profiling in parallel. For DNA profiling, sorted cells were subjected to BAC array comparative genomic hybridization analysis following whole genome amplification. For RNA profiling, QPCR analysis was performed on sixty four (64) cancer-related genes using Taqman® low density arrays.

2018-04-01 | GSE40622 | GEO

DNA profiling of EpCAM-positive cells in bone marrow of early breast cancer (EBC) patients

Project description:Parallel DNA and RNA profiling of EpCAM-positive cells in bone marrow and primary tumor tissue with positive disseminated tumor cell (DTC) count via immunomagnetic Enrichment/Flow Cytometry (IE/FC) of metastatic breast cancer (MBC) patients confirm their malignant nature We developed a novel approach to isolate tumor cells with high purity from bone marrow which was subjected to immunomagnetic enrichment using EpCAM beads followed by fluorescence activated cell sorting (IE/FACS) to isolate EpCAM-positive cells away from leukocytes (CD45+). For DNA profiling, sorted cells were subjected to BAC array comparative genomic hybridization analysis following whole genome amplification. For RNA profiling, QPCR analysis was performed on sixty four (64) cancer-related genes using Taqman® low density arrays. For non-tumor controls, RNA profiling was performed on matched leukocytes (CD45+) isolated from the same enriched bone marrow samples.

2019-04-04 | GSE112756 | GEO

Molecular characterization of tumor cells from the cerebrospinal fluid and matched primary tumors from metastatic breast cancer patients with leptomeningeal disease

Project description:We purified tumor cells in the CSF (“CSFTC”) from 15 metastatic breast cancer patients diagnosed with leptomeningeal disease using a two-step method involving immunomagnetic enrichment and fluorescence-activated cell sorting (IE/FACS). Magnetic beads coated with mAb to the epithelial cell adhesion marker (EPCAM) were used to enrich for tumor cells and were further purified by FACS analysis. For DNA profiling, isolated CSFTC were subjected to molecular characterization through genome-wide copy number analyses. Genomic analyses were then compared with those performed on the corresponding archival primary tumors. For RNA profiling, isolated CSFTC were then subjected to molecular characterization through gene expression profiling via QPCR analysis of 64 cancer-related genes

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IGF1R signaling primes epithelial cancer cells to induce epithelial-mesenchymal plasticity by upregulating integrin αV in cutaneous squamous cell carcinoma

Project description:Asses the global (phospho) protein profile of cancer cell lysates of three cell biological replicates of each stage of our model of skin SCC progression (full epithelial, plastic EpCAM-positive, plastic EpCAM-negative, and full mesenchymal) isolated from mouse skin SCCs by FACS sorting. The main research question in this experiment using pTyrIP, total protein lysate, and TiOx profiling is to check for differences in full epithelial vs plastic EpCAM(+) cancer cells, and plastic EpCAM(+) vs plastic EpCAM(-) cancer cells. With this information we would like to identify kinase signalling pathways that are induced in each of these stages and that may promote SCC progression, in order to design strategies to block tumor progression.

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RNA profiling of EpCAM-positive cells in bone marrow of early breast cancer (EBC) patients

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2019-04-04 | GSE112757 | GEO

Molecular characterization of tumor cells from the cerebrospinal fluid and matched primary tumors from metastatic breast cancer patients with leptomeningeal disease

Project description:We purified tumor cells in the CSF (M-bM-^@M-^\CSFTCM-bM-^@M-^]) from 15 metastatic breast cancer patients diagnosed with leptomeningeal disease using a two-step method involving immunomagnetic enrichment and fluorescence-activated cell sorting (IE/FACS). Magnetic beads coated with mAb to the epithelial cell adhesion marker (EPCAM) were used to enrich for tumor cells and were further purified by FACS analysis. For DNA profiling, isolated CSFTC were subjected to molecular characterization through genome-wide copy number analyses. Genomic analyses were then compared with those performed on the corresponding archival primary tumors. For RNA profiling, isolated CSFTC were then subjected to molecular characterization through gene expression profiling via QPCR analysis of 64 cancer-related genes CGH: 17 CSFTC samples from 13 patients were successfully profiled ,1 patient had 5 timepoints, 6 of 13 patients had matched to copy number data archival tumors RNA: 18 samples from 5 patients had successful gene expression data of the 64 genes measured in triplicates. For non-tumor controls, 9 of the samples had matching gene expression data from sorted leuckocytes (+CD45 cells) obtained from the same draw.

2014-01-09 | E-GEOD-46068 | biostudies-arrayexpress

Investigating the transcriptional regulation of intratumoral hypoxia in CTCs

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High throughput sequencing enables detailed analysis of S. cerevisiae DNA replication

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Expression data from SP and non-SP sorted anti-EpCAM treated A549 cells

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