Dataset Information

Semiautomated isolation and molecular characterisation of single or highly purified tumour cells from CellSearch enriched blood samples using dielectrophoretic cell sorting.

ABSTRACT:

Background

Molecular characterisation of single circulating tumour cells (CTCs) holds considerable promise for predictive biomarker assessment and to explore CTC heterogeneity. We evaluate a new method, the DEPArray system, that allows the dielectrophoretic manipulation and isolation of single and 100% purified groups of CTCs from pre-enriched blood samples and explore the feasibility of their molecular characterisation.

Methods

Samples containing known numbers of two cell populations were used to assess cell loss during sample loading. Cultured breast cancer cells were isolated from spiked blood samples using CellSearch CTC and Profile kits. Single tumour cells and groups of up to 10 tumour cells were recovered with the DEPArray system and subjected to transcriptional and mutation analysis.

Results

On average, 40% cell loss was observed when loading samples to the DEPArray system. Expected mutations in clinically relevant markers could be obtained for 60% of single recovered tumour cells and all groups of tumour cells. Reliable gene expression profiles were obtained from single cells and groups of up to 10 cells for 2 out of 3 spiked breast cancer cell lines.

Conclusion

We describe a semiautomated workflow for the isolation of small groups of 1 to 10 tumour cells from whole blood samples and provide proof of principle for the feasibility of their comprehensive molecular characterisation.

SUBMITTER: Peeters DJ

PROVIDER: S-EPMC3619252 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Publications

Semiautomated isolation and molecular characterisation of single or highly purified tumour cells from CellSearch enriched blood samples using dielectrophoretic cell sorting.

Peeters D J E DJ De Laere B B Van den Eynden G G GG Van Laere S J SJ Rothé F F Ignatiadis M M Sieuwerts A M AM Lambrechts D D Rutten A A van Dam P A PA Pauwels P P Peeters M M Vermeulen P B PB Dirix L Y LY

British journal of cancer 20130307 6

<h4>Background</h4>Molecular characterisation of single circulating tumour cells (CTCs) holds considerable promise for predictive biomarker assessment and to explore CTC heterogeneity. We evaluate a new method, the DEPArray system, that allows the dielectrophoretic manipulation and isolation of single and 100% purified groups of CTCs from pre-enriched blood samples and explore the feasibility of their molecular characterisation.<h4>Methods</h4>Samples containing known numbers of two cell populat ...[more]

PMID: 23470469

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Project description:Enumeration of circulating tumor cells (CTCs) obtained from minimally invasive blood samples has been well established as a valuable monitoring tool in metastatic and early breast cancer, as well as in several other cancer types. The gold standard technology for detecting CTCs in blood against a backdrop of millions of leukocytes is the FDA-approved CellSearch system (Janssen Diagnostics), which relies on EpCAM-based immunomagnetic separation. Secondary characterization of these cells could enable treatment selection based on specific targets in these cells, as well as providing a real time window into the metastatic process and offering unique insights into tumor heterogeneity. The objective of this study was to develop a method for downstream characterization of CTCs following isolation with the CellSearch system.An in vitro CTC model system focusing on clinically useful treatment predictive biomarkers in breast cancer, specifically the estrogen receptor ? (ER?) and the human epidermal growth factor receptor 2 (HER2), was established using healthy donor blood spiked with breast cancer cell lines MCF7 (ER?(+)/HER2(-)) and SKBr3 (ER?(-)/HER2(+)). Following CTC isolation by CellSearch, the captured CTCs were further enriched and fixed on a microscope slide using the in-house-developed CTC-DropMount technique.The recovery rate of CTCs after CellSearch Profile analysis and CTC-DropMount was 87%. A selective and consistent triple-immunostaining protocol was optimized. Cells positive for DAPI, cytokeratin (CK) 8, 18 and 19, but negative for the leukocyte-specific marker CD45, were classified as CTCs and subsequently analyzed for ER? and HER2 expression. The method was verified in breast cancer patient samples, thus demonstrating its clinical relevance.Our results show that it is possible to ascertain the status of important predictive biomarkers expressed in breast cancer CTCs using the newly developed CTC-DropMount technique. Downstream characterization of multiple biomarkers using a standard fluorescence microscope demonstrates that important clinical and biological information may be obtained from a single patient blood sample following either CellSearch epithelial or profile analyses.Clinical Trials NCT01322893.

Project description:BACKGROUND: Tumor cells in the blood of patients with metastatic carcinomas are associated with poor survival. Knowledge of the cells' genetic make-up can help to guide targeted therapy. We evaluated the efficiency and quality of isolation and amplification of DNA from single circulating tumor cells (CTC). METHODS: The efficiency of the procedure was determined by spiking blood with SKBR-3 cells, enrichment with the CellSearch system, followed by single cell sorting by fluorescence-activated cell sorting (FACS) and whole genome amplification. A selection of single cell DNA from fixed and unfixed SKBR-3 cells was exome sequenced and the DNA quality analyzed. Single CTC from patients with lung cancer were used to demonstrate the potential of single CTC molecular characterization. RESULTS: The overall efficiency of the procedure from spiked cell to amplified DNA was approximately 20%. Losses attributed to the CellSearch system were around 20%, transfer to FACS around 25%, sorting around 5% and DNA amplification around 25%. Exome sequencing revealed that the quality of the DNA was affected by the fixation of the cells, amplification, and the low starting quantity of DNA. A single fixed cell had an average coverage at 20× depth of 30% when sequencing to an average of 40× depth, whereas a single unfixed cell had 45% coverage. GenomiPhi-amplified genomic DNA had a coverage of 72% versus a coverage of 87% of genomic DNA. Twenty-one percent of the CTC from patients with lung cancer identified by the CellSearch system could be isolated individually and amplified. CONCLUSIONS: CTC enriched by the CellSearch system were sorted by FACS, and DNA retrieved and amplified with an overall efficiency of 20%. Analysis of the sequencing data showed that this DNA could be used for variant calling, but not for quantitative measurements such as copy number detection. Close to 55% of the exome of single SKBR-3 cells were successfully sequenced to 20× depth making it possible to call 72% of the variants. The overall coverage was reduced to 30% at 20× depth, making it possible to call 56% of the variants in CellSave-fixed cells.

Dataset Information

Semiautomated isolation and molecular characterisation of single or highly purified tumour cells from CellSearch enriched blood samples using dielectrophoretic cell sorting.

Background

Methods

Results

Conclusion

Publications

Semiautomated isolation and molecular characterisation of single or highly purified tumour cells from CellSearch enriched blood samples using dielectrophoretic cell sorting.

Similar Datasets

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