Issue 12, 2018

Isolation and genome sequencing of individual circulating tumor cells using hydrogel encapsulation and laser capture microdissection

Abstract

Circulating tumor cells (CTCs) are malignant cells released into the bloodstream with the potential to form metastases in secondary sites. These cells, acquired non-invasively, represent a sample of highly relevant tumor tissue that is an alternative to difficult and low-yield tumor biopsies. In recent years, there has been growing interest in genomic profiling of CTCs to enable longitudinal monitoring of the tumor's adaptive response to therapy. However, due to their extreme rarity, genotyping CTCs has proved challenging. Relevant mutations can be masked by leukocyte contamination in isolates. Heterogeneity between subpopulations of tumor cells poses an additional obstacle. Recent advances in single-cell sequencing can overcome these limitations but isolation of single CTCs is prone to cell loss and is prohibitively difficult and time consuming. To address these limitations, we developed a single cell sample preparation and genome sequencing pipeline that combines biophysical enrichment and single cell isolation using laser capture microdissection (LCM). A key component of this process is the encapsulation of enriched CTC sample in a hydrogel matrix, which enhances the efficiency of single-cell isolation by LCM, and is compatible with downstream sequencing. We validated this process by sequencing of single CTCs and cell free DNA (cfDNA) from a single patient with castration resistant prostate cancer. Identical mutations were observed in prostate cancer driver genes (TP53, PTEN, FOXA1) in both single CTCs and cfDNA. However, two independently isolated CTCs also had identical missense mutations in the genes for ATR serine/threonine kinase, KMT2C histone methyltransferase, and FANCC DNA damage repair gene. These mutations may be missed by bulk sequencing libraries, whereas single cell sequencing could potentially enable the characterization of key CTC subpopulations that arise during metastasis.

Graphical abstract: Isolation and genome sequencing of individual circulating tumor cells using hydrogel encapsulation and laser capture microdissection

Supplementary files

Article information

Article type
Paper
Submitted
15 2 2018
Accepted
07 5 2018
First published
15 5 2018

Lab Chip, 2018,18, 1736-1749

Isolation and genome sequencing of individual circulating tumor cells using hydrogel encapsulation and laser capture microdissection

E. S. Park, J. P. Yan, R. A. Ang, J. H. Lee, X. Deng, S. P. Duffy, K. Beja, M. Annala, P. C. Black, K. N. Chi, A. W. Wyatt and H. Ma, Lab Chip, 2018, 18, 1736 DOI: 10.1039/C8LC00184G

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements