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Single-Cell Sequencing Reveals CD133+CD44- – Originating Evolution and Stemness-Variants in AHNAK2, PLIN4, HLA-B, ALK, CCDC92 and ALMS1 of Human Colorectal Cancer Stem Cells

43 Pages Posted: 18 Jan 2019 Sneak Peek Status: Review Complete

See all articles by Xiaoyan Zhang

Xiaoyan Zhang

Hangzhou Cancer Hospital - Hangzhou Cancer Institute

Ling Yang

Hangzhou Cancer Hospital - Hangzhou Cancer Institute

Wanjun Lei

Beijing Novogene Bioinformatics Technology Co., Ltd - Novogene Bioinformatics Institute

Qiang Hou

Hangzhou Cancer Hospital - Hangzhou Cancer Institute

Ming Huang

Hangzhou Cancer Hospital - Hangzhou Cancer Institute

Hongfang Zhang

Hangzhou Cancer Hospital - Hangzhou Cancer Institute

Ruifei Xie

Hangzhou Cancer Hospital - Information Center

Rongjing Zhou

Hangzhou Cancer Hospital - Department of Pathology

Hongjin Wu

Hangzhou Cancer Hospital - Hangzhou Cancer Institute

Xiaoli Zheng

Hangzhou Cancer Hospital - Hangzhou Cancer Institute

Ying Li

Hangzhou Cancer Hospital - Hangzhou Cancer Institute

Jing Yue

Hangzhou Cancer Hospital - Hangzhou Cancer Institute

Zhenzhen Jiang

Hangzhou Cancer Hospital - Hangzhou Cancer Institute

Shixiu Wu

Hangzhou Cancer Hospital - Hangzhou Cancer Institute

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Abstract

Tumor heterogeneity and internal clonal evolution of human colorectal cancer stem cells (CRCSCs) within cancerous tissues often represents an aggressive feature of cancer progression. For a high-resolution examination of CRCSCs, we performed single cell whole-exome sequencing (scWES) of sorted cells, including three groups of CRCSCs: CD133+CD44+, CD133-CD44+, or CD133+CD44- cells, colorectal cancer cells (CRCCs) lacking these stem-cell markers (EpCAM+), plus one group of normal control cells. We examined and validated the scWES data for stem-specifically somatic alterations, clonal evolution, etc. Clonal evolution analysis showed that all CRCSCs were located at the main clone, and that the CD133+CD44- CRCSCs, which harbored the lowest number of mutations, were the likely origin of colorectal cancer (CRC) before evolving into other groups of CRCSCs/CRCCs. These results were validated by cell-lines culture experiments. Functional analyses validated that stem cell-specific mutated genes, such as GOLGA8J, ARHGAP39, LURAP1L, CDH1, were positively associated with stemness and knocking down each of them could increase the proportion of CD133+ cells. To validating mutations, especially stem cell-related genes, we performed targeted exome sequencing using a panel of all mutated genes to analyze the distribution of them in 96 samples (20 patients) those were also sorted into the same three groups of CRCSCs and CRCCs. We found two mutational sites of AHNAK2 gene (Chr14_105410531 and Chr14_105410533) were stem-cell specific in both scWES data and target sequencing data. PLIN4, HLA-B, ALK, CCDC92 and ALMS1 genes were specifically mutated in stem cells in both data. Another 33 genes tended to mutate in stem cells from all data. In all stem cell-related genes, AHNAK2, PTPRF and NIPPA5 which subcellularly located at plasma membrane had totally eleven predicted neoantigens. These neoantigens might be useful for some CRC patients in immunotherapy. All integrative analyses above suggested new markers for CRCSCs and showed the important roles of CRCSCs in tumorigenesis and progression of CRCs.

Suggested Citation

Zhang, Xiaoyan and Yang, Ling and Lei, Wanjun and Hou, Qiang and Huang, Ming and Zhang, Hongfang and Xie, Ruifei and Zhou, Rongjing and Wu, Hongjin and Zheng, Xiaoli and Li, Ying and Yue, Jing and Jiang, Zhenzhen and Wu, Shixiu, Single-Cell Sequencing Reveals CD133+CD44- – Originating Evolution and Stemness-Variants in AHNAK2, PLIN4, HLA-B, ALK, CCDC92 and ALMS1 of Human Colorectal Cancer Stem Cells (2018). Available at SSRN: https://ssrn.com/abstract=3188496 or http://dx.doi.org/10.2139/ssrn.3188496
This is a paper under consideration at Cell Press and has not been peer-reviewed.

Xiaoyan Zhang (Contact Author)

Hangzhou Cancer Hospital - Hangzhou Cancer Institute

Hangzhou, 310002
China

Ling Yang

Hangzhou Cancer Hospital - Hangzhou Cancer Institute

Hangzhou, 310002
China

Wanjun Lei

Beijing Novogene Bioinformatics Technology Co., Ltd - Novogene Bioinformatics Institute

Beijing, 100083
China

Qiang Hou

Hangzhou Cancer Hospital - Hangzhou Cancer Institute

Hangzhou, 310002
China

Ming Huang

Hangzhou Cancer Hospital - Hangzhou Cancer Institute

Hangzhou, 310002
China

Hongfang Zhang

Hangzhou Cancer Hospital - Hangzhou Cancer Institute

Hangzhou, 310002
China

Ruifei Xie

Hangzhou Cancer Hospital - Information Center

Hangzhou, 310002
China

Rongjing Zhou

Hangzhou Cancer Hospital - Department of Pathology

Hangzhou, 310002
China

Hongjin Wu

Hangzhou Cancer Hospital - Hangzhou Cancer Institute

Hangzhou, 310002
China

Xiaoli Zheng

Hangzhou Cancer Hospital - Hangzhou Cancer Institute

Hangzhou, 310002
China

Ying Li

Hangzhou Cancer Hospital - Hangzhou Cancer Institute

Hangzhou, 310002
China

Jing Yue

Hangzhou Cancer Hospital - Hangzhou Cancer Institute

Hangzhou, 310002
China

Zhenzhen Jiang

Hangzhou Cancer Hospital - Hangzhou Cancer Institute

Hangzhou, 310002
China

Shixiu Wu

Hangzhou Cancer Hospital - Hangzhou Cancer Institute ( email )

Hangzhou, 310002
China

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