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

ABSTRACT: 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 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 from 17 of the 30 patients.

ORGANISM(S): Homo sapiens

PROVIDER: GSE112757 | GEO | 2019/04/04

REPOSITORIES: GEO

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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

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

MicroRNA expression signatures of CD14-CD45-EpCAM+ cells in human bone marrow for colorectal cancer liver metastasis

Project description:To further development of our gene expression approach to biodosimetry, we have employed microRNA microarray expression profiling to identify genes with the potential to distinguish liver metastasis related microRNA. Colorectal cancer patients were administered anesthesia and 20 mL BM was taken from the right and left anterior iliac crests before surgery. Mononucleated cells were collected using a standard Ficoll-Hypaque gradient technique. To enrich for EpCAM+ cells, CD14+ cells were removed from the whole bone marrow using auto MACSTM pro (Milteny Biotec, Bergisch Gladbach, Germany) with anti-CD14 immunomagnetic beads (clone; TÜK4, Milteny Biotec). Next, CD45+ cells were removed by treatment with anti-CD45 immunomagnetic beads (clone; 5B1; Milteny Biotec). The residual CD14?CD45? cells were then incubated with FcR blocking reagent (Milteny Biotec), followed by incubation with anti-EpCAM immunomagnetic beads (clone; HEA-125, Milteny Biotec), and the CD14?CD45?EpCAM+ cells were taken up. Total RNA of these cells we analyzed the microRNA levels of CD14?CD45?EpCAM+ cells obtained from non-metastasis patients (n = 12) and liver metastasis patients (n = 7). Ten-microRNA consensus signature was identified that distinguished between CD14?CD45?EpCAM+ cells from liver metastasis patients and CD14?CD45?EpCAM+ cells from non-liver metastasis patients. MicroRNA expression of CD14-CD45-EpCAM+ cells in human bone marrow was measured. RNA of these cells we analyzed the microRNA levels of CD14?CD45?EpCAM+ cells obtained from non-metastasis patients (n = 12) and liver metastasis patients (n = 7).

2013-11-11 | E-GEOD-51716 | biostudies-arrayexpress

MicroRNA expression signatures of CD14-CD45-EpCAM+ cells in human bone marrow for colorectal cancer liver metastasis

Project description:To further development of our gene expression approach to biodosimetry, we have employed microRNA microarray expression profiling to identify genes with the potential to distinguish liver metastasis related microRNA. Colorectal cancer patients were administered anesthesia and 20 mL BM was taken from the right and left anterior iliac crests before surgery. Mononucleated cells were collected using a standard Ficoll-Hypaque gradient technique. To enrich for EpCAM+ cells, CD14+ cells were removed from the whole bone marrow using auto MACSTM pro (Milteny Biotec, Bergisch Gladbach, Germany) with anti-CD14 immunomagnetic beads (clone; TÜK4, Milteny Biotec). Next, CD45+ cells were removed by treatment with anti-CD45 immunomagnetic beads (clone; 5B1; Milteny Biotec). The residual CD14−CD45− cells were then incubated with FcR blocking reagent (Milteny Biotec), followed by incubation with anti-EpCAM immunomagnetic beads (clone; HEA-125, Milteny Biotec), and the CD14−CD45−EpCAM+ cells were taken up. Total RNA of these cells we analyzed the microRNA levels of CD14−CD45−EpCAM+ cells obtained from non-metastasis patients (n = 12) and liver metastasis patients (n = 7). Ten-microRNA consensus signature was identified that distinguished between CD14−CD45−EpCAM+ cells from liver metastasis patients and CD14−CD45−EpCAM+ cells from non-liver metastasis patients.

2013-11-11 | GSE51716 | GEO

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

2012-12-31 | GSE27511 | GEO

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

microRNA expression profiles in bone marrow fractions from gastric cancer patients

Project description:Both disseminated tumor cells and noncancerous host cells contributed to cancer progression cooperatively in bone marrow. Bone marrow samples were obtained from 4 gastric cancer patients, and were separated into 3 fractions (CD45 positive, CD45 negative/EpCAM positive, and CD14 positive fractions) by the automagnetic-activated cell separation (AutoMACS) system using CD45, EpCAM, and CD14 microbeads (Miltenyi Biotec, Germany). microRNA expression profiles in each fractions were evaluated in order to identify candidate prognostic markers for gastric cancer patients. In 4 patients with gastric cancer, bone marrow samples (40 mL) were obtained from iliac bones. Nucleated cells were collected by gradient centrifugation using Ficoll-Paque PREMIUM (GE Healthcare Life Science, USA) and Leucosep (Greiner Bio-One, Germany) according to the manufacturer’s instructions. Next, we separated bone marrow cells into 3 fractions using MACS: CD45 positive (CD45+), CD45 negative/EpCAM positive (CD45-/EpCAM+), and CD14 positive (CD14+). microRNA expression levels of whole bone marrow cells and each fractions were measured by the miRCURY™ LNA array microarray (6th gen-hsa, mmu & rno#208402, Exiqon, Vedbaek, Denmark). The miRCURY™ LNA array microarray slides were scanned using the Agilent G2505C Microarray Scanner System (Agilent Technologies, Inc., USA) and the data analysis was carried out using the Feature Extraction 10.7.3.1 (Agilent Technologies, Inc., USA).

2013-08-23 | E-GEOD-40325 | biostudies-arrayexpress

microRNA expression profiles in bone marrow fractions from gastric cancer patients

2013-08-23 | GSE40325 | GEO

Single-cell RNA-seq of lung cells following bacterial pneumonia with or without LIF blockade

Project description:C57BL/6 mice were intratracheally challenged for 24 hours with saline or E. coli (1 x 10^6 CFU) with or without LIF neutralization. Left lobes were collected and digested into single-cell suspensions for FACS. Equal ratios of CD45+ (leukocytes), CD45-EpCAM+ (epithelium), and CD45-EpCAM- (other) cells were pooled from a mouse undergoing each condition.

2022-02-17 | GSE179855 | GEO

Single cell analysis of Villin-Cre;Apc min;Lef1 fl/fl (VApcMinL) and ApcMin Epcam- cells and CD45-Epcam- stromal cells from lamina propria at the age of 11 wks.

Project description:Comparison of Villin-Cre;Apc min;Lef1 fl/fl (VApcMinL) and ApcMin Epcam- cells and CD45-Epcam- stromal cells from lamina propria at the age of 11 wks. Two male mice per group was used in the experiment.

2021-12-10 | GSE173565 | GEO

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