Abstract _ Full Text (HTML) _ Full Text (PDF) 

Research Article

Cancer Research Frontiers. 2016 May; 2(2): 286-299. doi: 10.17980/2016.286

Comprehensive isolation, identification, and nucleic acid analysis of single breast cancer cells: CTC-isoTECH

Lori M. Millner1,2*, Lindsay N. Strotman1,2, Kevin S. Goudy1,2, Mark W. Linder1,2, Roland Valdes, Jr. 1,2

1University of Louisville, School of Medicine, Department of Pathology and Laboratory Medicine; 511 South Floyd Street, MDR 221, Louisville KY 40202

2PGXL Technologies, 201 E. Floyd St., Suite 306, Louisville KY

*Corresponding author: Lori Millner. Email: lmmill10@louisville.edu

Citation: Lori Millner, et al. Comprehensive isolation, identification, and nucleic acid analysis of single breast cancer cells: CTC-isoTECH. Cancer Research Frontiers. 2016 May; 2(2): 286-299. doi: 10.17980/2016.286

Copyright: @ 2016 Lori Millner, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Competing Interests: The authors declare no competing financial interests.

Received Jan 8, 2016; Revised Mar 18, 2016; Accepted Mar 31, 2016. Published May 27, 2016

 

Abstract

The ability to isolate, phenotypically characterize, and profile the gene signature of single circulating tumor cells (CTCs) will provide deeper insight into cancer metastasis and will lead to improved diagnosis and treatment of cancer patients. Using current methods based on positive selection of epithelial markers, up to 40% of patients with highly aggressive metastatic breast cancer have no CTCs detected. This may be due to CTCs undergoing the process of epithelial to mesenchymal transition (EMT). This process causes epithelial characteristics to be down regulated as more mesenchymal features develop allowing the cells to have higher motility and increased ability to evade immune detection. Methods to enable capture and characterization of heterogeneous CTCs including those with non-epithelial phenotypes are needed. Additionally, having the ability to isolate pure, single CTCs allows for individual cell analysis providing information that is masked by bulk analysis. Here we provide details on an integrated method for isolating and characterizing heterogeneous single breast cancer cells based on a multiantigen, negative depletion strategy followed by sorting with dielectrophoresis (DEPArray). This negative depletion method removes unwanted leukocytes and allows heterogeneous, epithelial and non-epithelial cells to be retained. We were able to visually verify the isolation of single tumor cells with 100% purity. Following sorting, next generation sequencing and real-time PCR of single cells is described. This method allows single, heterogeneous CTCs to be analyzed which will have implications for prognosis and treatment options.

Keywords: Circulating tumor cells (CTCs), breast cancer, negative enrichment, dielectrophoresis, next generation sequencing

 

 

 

 

 

 

 

 

 

Multiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier SchönmannMultiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier Schönmann