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Target cell pre-enrichment and whole genome amplification for single cell downstream characterization

: Chen, Shukun; El-Heliebi, Amin; Schmid, Julia; Kashofer, Karl; Czyz, Zbigniew T.; Polzer, Bernhard Michael; Pantel, Klaus; Kroneis, Thomas; Sedlmayr, Peter

Fulltext urn:nbn:de:0011-n-5099255 (1009 KByte PDF)
MD5 Fingerprint: f2db238e0e19475573d3889065da0c8a
Created on: 18.9.2018

Journal of visualized experiments : JoVE. Online resource 135 (2018), Art. e56394, 10 pp.
ISSN: 1940-087X
Journal Article, Electronic Publication
Fraunhofer ITEM ()
rare cell analysis; single cell analysis; whole-genome amplification; aarray comparative genome hybridization; In vivo isolation device; targeted next generation sequencing; next generation sequencing immunofluorescence labelling; cancer - research

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)(1). A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell's amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (<10 - >50%) and needs some further attention. This device is not cleared for the use in patients.