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Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

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 ...

Medical University of Graz

Whole genome amplification (WGA) is a widely used technique allowing multiplying picogram amounts of target DNA by several orders of magnitude. The technique described here is based on heat-induced random fragmentation yielding DNA strands mainly ranging from 0.1 to 1 kb in length. The fragmented DNA is then subjected to library generation by annealing of adaptor sequences to both ends of the DNA fragments. Using primers hybridizing to the adapter sequences, the DNA is amplified by thermal cycling. This amplification typically yields > 2 mg DNA from a single cell, is suited for amplifying DNA isolated from (partly) degraded samples [e.g. formalin-fixed paraffin-embedded (FFPE) material] and works well when used for array-comparative genome hybridization (array-CGH). 

Heat-Induced Fragmentation and Adapter-Assisted Whole Genome Amplification Using GenomePlex® Single-Cell Whole Genome Amplification Kit (WGA4).

This chapter describes a simple and inexpensive multiplex PCR-based method to assess the quality of whole genome amplification (WGA) products generated from heat-induced random fragmented DNA. A set of four primer pairs is used to amplify DNA sequences of WGA products in and downstream of GAPDH gene in yielding 100, 200, 300, and 400 bp fragments. PCR products are analyzed by agarose gel electrophoresis and the respective WGA quality is classified according to the number of obtained PCR bands. WGA products that yield three or four PCR bands are considered to be of high quality and yield good results when analyzed by means of array comparative genome hybridization (CGH). 

Using Multiplex PCR for Assessing the Quality of Whole Genome Amplified DNA.

Multiple analyses such as DNA profiling, sequencing, or comparative genome hybridization (CGH) done on the single-cell level long for pre-amplification due to the diploid human genome. Isothermal whole genome amplification allows amplification of long DNA templates from single cells. When analysis needs to be performed under rare cell conditions additional care needs to be taken due to the fact that, even after pre-enrichment, few candidate target cells are still dispersed among an overwhelming number of non-target background cells. Here, we describe a protocol where we define a population of candidate target cells based on specific staining. Candidate cells are then isolated by laser microdissection and pressure catapulting (LMPC) and transferred onto a microliter reaction slide. This slide allows monitoring the single-cell isolation process and isothermal whole genome amplification in less than 2 μL. The amplification products obtained from single cells can be forwarded to multiple analyses. 

Low-Volume On-Chip Single-Cell Whole Genome Amplification for Multiple Subsequent Analyses.

Enumeration and especially molecular characterization of circulating tumour cells (CTCs) holds great promise for cancer management. We tested a modified type of an in vivo enrichment device (Catch&Release) for its ability to bind and detach cancer cells for the purpose of single-cell molecular downstream analysis in vitro. The evaluation showed that single-cell analysis using array comparative genome hybridization (array-CGH) and next generation sequencing (NGS) is feasible. We found array-CGH to be less noisy when whole genome amplification (WGA) was performed with Ampli1 as compared to GenomePlex (DLRS values 0.65 vs. 1.39). Moreover, Ampli1-processed cells allowed detection of smaller aberrations (median 14.0 vs. 49.9 Mb). Single-cell NGS data obtained from Ampli1-processed samples showed the expected non-synonymous mutations (deletion/SNP) according to bulk DNA. We conclude that clinical application of this refined in vivo enrichment device allows CTC enumeration and characterization, thus, representing a promising tool for personalized medicine.

Catch and Release: rare cell analysis from a functionalised medical wire.

Circulating tumor cells (CTCs) are a group of rare cells disseminated from either primary or metastatic tumors into the blood stream. CTCs are considered to be the precursor of cancer metastasis. As a critical component of liquid biopsies, CTCs are a unique tool to understand the formation of metastasis and a valuable source of information on intratumor heterogeneity. Much effort has been invested in technologies for the detection of CTCs because they are rare cells among the vast number of blood cells. Studies in various cancers have repeatedly demonstrated that increased CTC counts prior to or during treatment are significantly associated with poor outcomes. In the new era of precision medicine, the study of CTCs reaches far beyond detection and counting. The rapidly growing field of analytical platforms for rare-cell analysis allows in-depth characterization of CTCs at the bulk cell and single-cell level. Genetic profiling of CTCs may provide an insight into the real-time tumor status, may allow the monitoring and evaluation of treatment response in clinical routine, and may lead to the development of novel therapeutic targets as well.

Biological and Molecular Characterization of Circulating Tumor Cells: A Creative Strategy for Precision Medicine?

Molecular characterization of circulating tumor cells (CTCs) is important for selecting patients for targeted treatments. We present, for the first time, results on gene expression profiling of CTCs isolated in vivo from high-risk prostate cancer (PCa) patients compared with CTC detected by 3 protein-based assays-CellSearch, PSA-EPISPOT, and immunofluorescence of CellCollector in vivo-captured CTCs-using the same blood draw.

Multiplex Gene Expression Profiling of In Vivo Isolated Circulating Tumor Cells in High-Risk Prostate Cancer Patients.

Liquid biopsies can be used in castration-resistant prostate cancer (CRPC) to detect androgen receptor splice variant 7 (AR-V7), a splicing product of the androgen receptor. Patients with AR-V7-positive circulating tumor cells (CTCs) have greater benefit of taxane chemotherapy compared with novel hormonal therapies, indicating a treatment-selection biomarker. Likewise, in those with pancreatic cancer (PaCa),  mutations act as prognostic biomarkers. Thus, there is an urgent need for technology investigating the expression and mutation status of CTCs. Here, we report an approach that adds AR-V7 or KRAS status to CTC enumeration, compatible with multiple CTC-isolation platforms.

Shukun Chen

Institute of Cell Biology,
Histology and Embryology,
Institute of Cell Biology, Histology and Embryology

Medical University of Graz

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Shukun Chen

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute of Cell Biology,Histology and Embryology,Institute of Cell Biology, Histology and Embryology

Medical University of Graz

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Institute of Cell Biology,
Histology and Embryology,
Institute of Cell Biology, Histology and Embryology