Comparison of EpCAM^highCD44⁺ cancer stem cells with EpCAM^highCD44⁻ tumor cells in colon cancer by single-cell sequencing

ABSTRACT

Cancer stem cells (CSCs) are considered to be responsible for tumorigenesis and cancer relapse. EpCAM^highCD44⁺ tumor cells are putative colorectal CSCs that express high levels of stem cell genes, while the EpCAM^highCD44⁻ population mostly contains differentiated tumor cells (DTCs). This study aims to determine whether single CSC (EpCAM^highCD44⁺) and DTC (EpCAM^highCD44⁻) can be distinguished in terms of somatic copy number alterations (SCNAs). We applied fluorescence-activated cell sorting to isolate the CD45⁻EpCAM^highCD44⁺ and CD45⁻EpCAM^highCD44⁻ populations from two primary colon tumors, on which low-coverage single-cell whole-genome sequencing (WGS) was then performed ∼0.1x depth. We compared the SCNAs of the CSCs and DTCs at single-cell resolution. In total, 47 qualified single cells of the two populations underwent WGS. The single-cell SCNA profiles showed that there were obvious SCNAs in both the CSCs and DTCs of each patient, and each patient had a specific copy number alteration pattern. Hierarchical clustering and correlation analysis both showed that the SCNA profiles of CSCs and DTCs from the same patient had similar SCNA pattern, while there were regional differences in the CSCs and DTCs in certain patient. SCNAs of CSCs in the same patient were highly reproducible. Our data suggest that major SCNAs occurred at an early stage and were inherited steadily. The similarity of ubiquitous SCNAs between the CSCs and DTCs might have arisen from lineage differentiation. CSCs from the same patient had reproducible SCNA profiles, indicating that gain or loss in certain chromosome is required for colon cancer development.

KEYWORDS:

• Cancer stem cells
• Colon cancer
• Differentiated tumor cells
• Punctuated evolution
• Single-cell sequencing
• Somatic copy number alterations
• Tumorigenesis

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Mr. Zhonglin Fu and Ms. Xuefang Zhang from the National Center for Protein Sciences Beijing (Peking University) for assistance with FACS; Ms. Yu Hou from BIOPIC in Peking University for academic assistance, and Dr. Ying Hu, Yixue Wang, Lijie Song and Meng Chen from the Department of Biobank, Peking University Cancer Hospital & Institute for collecting specimens.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Additional information

Funding

This study was supported by Peking University (PKU) 985 Special Funding for Collaborative Research with PKU Hospitals (to XS), the National Natural Science Foundation of China (No. 81502137 to JD, No. 81272766, No. 81450028, and No. 81672439 to XS), the Beijing Natural Science Foundation (No. 7162039 to XS), and the Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support (No. XM201309 to XS and No. ZYLX201701).