An Immunofluorescence-Assisted Microfluidic Single Cell Quantitative Reverse Transcription Polymerase Chain Reaction Analysis of Tumour Cells Separated from Blood

Kazunori Hoshino; HaeWon Chung; Chun-Hsien Wu; Kaarthik Rajendran; Yu-Yen Huang; Peng Chen; Konstantin V. Sokolov; Jonghwan Kim; John X.J. Zhang

doi:10.33393/jcb.2015.2061

Authors

Kazunori Hoshino Department of Biomedical Engineering, University of Connecticut, Storrs, CT, USA
HaeWon Chung Department of Molecular Biosciences, Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX, USA
Chun-Hsien Wu Department of Biomedical Engineering, University of Texas at Austin, Austin, TX, USA
Kaarthik Rajendran Department of Biomedical Engineering, University of Texas at Austin, Austin, TX, USA
Yu-Yen Huang Thayer School of Engineering, Dartmouth College, Hanover, NH, USA
Peng Chen Department of Biomedical Engineering, University of Texas at Austin, Austin, TX, USA
Konstantin V. Sokolov Department of Imaging Physics, University of Texas MD Anderson Cancer Center, Houston, TX, USA
Jonghwan Kim Department of Molecular Biosciences, Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX, USA
John X.J. Zhang Thayer School of Engineering, Dartmouth College, Hanover, NH, USA

DOI:

https://doi.org/10.33393/jcb.2015.2061

Keywords:

CTC, single cell PCR, breast cancer, immunomagnetic assay, lab on a chip, laser microdissection

Abstract

Circulating tumour cells (CTCs) are important indicators of metastatic cancer and may provide critical information for individualized treatment. As CTCs are usually very rare, the techniques to obtain information from very small numbers of cells are crucial. Here, we propose a method to perform a single cell quantitative reverse transcription polymerase chain reaction (qPCR) analysis of rare tumour cells. We utilized a microfluidic immunomagnetic assay to separate cancer cells from blood. A combination of detailed immunofluorescence and laser microdissection enabled the precise selection of individual cells. Cancer cells that were spiked into blood were successfully separated and picked up for a single cell PCR analysis. The breast cancer cell lines MCF7, SKBR3 and MDAMB231 were tested with 10 different genes. The result of the single cell analysis matched the results from a few thousand cells. Some markers (e.g., ER, HER2) that are commonly used for cancer identification showed relatively large deviations in expression levels. However, others (e.g., GRB7) showed deviations that are small enough to supplement single cell disease profiling.