Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiling of circulating tumor cells in breast cancer

ABSTRACT: CTCs are the purpoted intermediates of metastatic dissemination and are likely to contain cellular clones responsible for disease progression representing a preferred source for identification of druggable targets. Unfortunately a molecular characterization of CTCs is seriously hampered by their low numbers even in metastatic patients and by the elevated contamination of isolated CTCs with leukocytes. With the final goal of providing a reliable assay allowing to obtain valuable information on CTC features in the clinical setting, we have developed a method based on capture with beads linked to EpCAM and to MUC1 exploiting a commercially available kit and performing an extensive gene expression profiling with the DASL platform. For spike-in experiments, low pre-defined numbers of cells derived from established breast cancer cell lines (MCF7 and MDA-MB-468) were spiked into 5 mL of whole blood of healthy donor and captured using AdnaTest EMT-1/StemCell Select kit. Total RNA isolated from the captured cells together with total RNA isolated from the same in vitro cultered cells was processed in duplicate or triplicate onto Illumina Whole-Genome DASL HT platform. Finally, gene expression profiling was carried out on CTCs isolated from blood of 7 patients with advanced breast cancer. According with the characteristics of the study, data normalization was not applicable

ORGANISM(S): Homo sapiens

SUBMITTER: Maurizio Callari

PROVIDER: E-GEOD-55470 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Gene expression profiling of circulating tumor cells in breast cancer.

Fina Emanuela E Callari Maurizio M Reduzzi Carolina C D'Aiuto Francesca F Mariani Gabriella G Generali Daniele D Pierotti Marco A MA Daidone Maria G MG Cappelletti Vera V

Clinical chemistry 20141119 1

<h4>Background</h4>Determining the transcriptional profile of circulating tumor cells (CTCs) may allow the acquisition of clinically relevant information while overcoming tumor heterogeneity-related biases associated with use of tissue samples for biomarker assessment. However, such molecular characterization is challenging because CTCs are rare and outnumbered by blood cells.<h4>Methods</h4>Here, we describe a technical protocol to measure the expression of >29 000 genes in CTCs captured from w ...[more]

PMID: 25411184

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Project description:Metastasis is responsible for the vast majority of breast cancer related deaths. Metastatic breast cancer (MBC) is inherently different than primary breast cancer (BC), evolving under selection pressure at different organ sites or during systemic therapy. The current ASCO guidelines call for biopsy of a metastatic site to guide decision making for systemic therapy. Yet, biopsies of macro metastasis are oftentimes not feasible in the clinical setting. Circulating tumor cells (CTCs) have been shown to be prognostic in MBC, but their use as clinical biomarker beyond CTC enumeration has been limited. A better understanding of CTC-biology compared to metastasis may shed light on treatment opportunities and help advance the application of CTCs as liquid biopsies in clinical practice. The ANGLE Parsortix system is a microfluidics device that separates CTCs based on size and deformability, without the need for cell-surface marker selection. Our lab has previously demonstrated the feasibility of gene expression profiling of rare CTCs. Here, we evaluated whether whole transcriptome sequencing (RNA Seq) gene expression profiling of ANGLE Parsortix isolated CTCs may serve as a surrogate for biopsies of macro metastases. CTCs from 21 MBC patients were enumerated and captured from 10mL peripheral blood (PB) via the ANGLE Parsortix system. RNA Seq was performed on fresh metastatic tumor biopsies (mets), CTCs and peripheral blood from all patients. Biopsy sites included: skin (n=1), lung (n=1), pleural effusion (n=5), pericardial effusion (n=1), breast (n=3), lymph node (n=2), brain (n=4), liver (n=1), ascites (n=3), cerebrospinal fluid (n=2) and bone (n=1). 19/21 patients were included in subsequent data analysis. We present the whole transcriptomic landscape of CTCs with comparison to metastases and peripheral blood all acquired prior to treatment of Stage IV breast cancer. Multiple genes, including oncogenes, breast epithelial, mesenchymal genes and cancer stem cell genes were found with higher expression in CTCs versus metastases. When focusing on 66 known potentially clinically actionable genes in breast cancer, represented by 7 molecular signaling pathways, CTCs did not show significantly different patterns of expression versus mets compared to PB. RNA Seq of CTCs may be utilized to identify alterations in MBC patients that are potentially clinically actionable.

Project description:Melanoma is an invasive malignancy with a high frequency of blood-borne metastases, but circulating tumor cells (CTCs) have not been readily isolated. We adapted microfluidic CTC capture to a tamoxifen-driven B-RAF/PTEN mouse melanoma model. CTCs were detected in all tumor-bearing mice, rapidly declining after B-RAF inhibitor treatment. CTCs were shed early from localized tumors and a short course of B-RAF inhibition following surgical resection was sufficient to dramatically suppress distant metastases. The large number of CTCs in melanoma-bearing mice enabled comparison of RNA sequencing profiles with the matched primary tumor. A mouse melanoma CTC-derived signature correlated with invasiveness and cellular motility in human melanoma. In patients with metastatic melanoma, CTCs were detected in smaller numbers in patients with metastatic melanoma and declined with successful B-RAF targeted therapy. Together, the capture of CTCs and their molecular characterization provide insight into the hematogenous spread of melanoma. We adapted a microfluidic platform, the HbCTC-Chip (Stott et al., 2010, Pubmed ID: 20930119), to capture melanoma CTCs derived from mouse tumors, using panels of antibodies against melanoma-specific cell surface markers, followed by staining for melanoma antigens and optimized on-chip fluorescent imaging. We used a tamoxifen inducible BRAF(CA/+)/PTEN(flox/flox) melanoma mouse model (Dankort et al., 2009, Pubmed ID: 19282848) derived from a C57BL/6 background. Such mice received focal subcutaneous injection of tamoxifen (Sigma) (50ul at 5mg/ml in 50% EtOH suspension) at the left flank at 6-7 weeks after birth. Blood samples were collected from five mice with high tumor burden following tamoxifen injection. Blood from each mouse was split and processed through the CTC-chips functionalized with anti-CSPG4/MCAM antibody and control IgGs, respectively. Matched primary (from the tamoxifen injection site) and metastatic (from upper or lower back) tumors were harvested from the same mouse and immediately flash-frozen in liquid nitrogen. RNA extraction, single molecular sequencing and determination of Digital Gene Expression was as in Yu et al., 2012 (Pubmed ID: 22763454). One of the five mice yielded no CTC or IgG data. In addition, skin was taken from a sixth mouse, which was a complete wild type C57BL/6 mouse (without the BRAF/PTEN transgenes).The skin was taken after euthanasia of the animal and was processed as the tissue from the matched primary and metastatic tumors.

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Gene expression profiling of circulating tumor cells in breast cancer

Publications

Gene expression profiling of circulating tumor cells in breast cancer.

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