Modeling colorectal tumorigenesis using the organoids derived from conditionally immortalized mouse intestinal crypt cells (ciMICs)

Xiaoxing Wu; Zhaoxia Li; Hongyu Zhang; Fang He; Min Qiao; Huaxiu Luo; Jing Zhang; Meng Zhang; Yukun Mao; William Wagstaff; Yongtao Zhang; Changchun Niu; Xia Zhao; Hao Wang; Linjuan Huang; Deyao Shi; Qing Liu; Na Ni; Kai Fu; Rex C Haydon; Russell R Reid; Hue H Luu; Tong-Chuan He; Ziwei Wang; Houjie Liang; Bing-Qiang Zhang; Ning Wang

doi:10.1016/j.gendis.2021.01.004

Modeling colorectal tumorigenesis using the organoids derived from conditionally immortalized mouse intestinal crypt cells (ciMICs)

Genes Dis. 2021 Jan 28;8(6):814-826. doi: 10.1016/j.gendis.2021.01.004. eCollection 2021 Nov.

Authors

Xiaoxing Wu^{1

2}, Zhaoxia Li³, Hongyu Zhang^{1

2}, Fang He^{1

2}, Min Qiao^{1

2}, Huaxiu Luo^{2

4}, Jing Zhang^{1

2}, Meng Zhang^{2

5}, Yukun Mao^{2

6}, William Wagstaff², Yongtao Zhang^{2

7}, Changchun Niu^{2

8}, Xia Zhao^{2

7}, Hao Wang^{2

9}, Linjuan Huang^{1

2}, Deyao Shi^{2

10}, Qing Liu^{2

11}, Na Ni^{2

9}, Kai Fu^{2

6}, Rex C Haydon², Russell R Reid², Hue H Luu², Tong-Chuan He², Ziwei Wang¹, Houjie Liang¹², Bing-Qiang Zhang^{1

2}, Ning Wang^{2

3}

Affiliations

¹ Departments of Gastrointestinal Surgery, Medicine/Gastroenterology, and Obstetrics and Gynecology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, PR China.
² Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 606037, USA.
³ Department of Oncology, The PLA Rocket Force Characteristic Medical Center, Beijing, 100088, PR China.
⁴ Departments of Burn & Plastic Surgery, West China Hospital of Sichuan University, Chengdu, Sichuan Province, 610041, PR China.
⁵ Department of Orthopaedic Surgery, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, Guangdong Province, 510405, PR China.
⁶ Departments of Orthopaedic Surgery, The Affiliated Zhongnan Hospital of Wuhan University, Wuhan, Hubei Province, 430071, PR China.
⁷ Department of Orthopaedic Surgery, The Affiliated Hospital of Qingdao University, Qingdao, Shandong Province, 266000, PR China.
⁸ Department of Clinical Laboratory Medicine, Chongqing General Hospital Affiliated with the University of Chinese Academy of Sciences, Chongqing, 400013, PR China.
⁹ Ministry of Education Key Laboratory of Diagnostic Medicine, and the School of Laboratory Diagnostic Medicine, Chongqing Medical University, Chongqing, 400016, PR China.
¹⁰ Department of Orthopaedics, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430022, PR China.
¹¹ Department of Spine Surgery, Second Xiangya Hospital, Central South University, Changsha, Hunan Province, 410008, PR China.
¹² Department of Oncology and Southwest Cancer Center, Southwest Hospital, Army Medical University, Chongqing, 400038, PR China.

Abstract

Intestinal cancers are developed from intestinal epithelial stem cells (ISCs) in intestinal crypts through a multi-step process involved in genetic mutations of oncogenes and tumor suppressor genes. ISCs play a key role in maintaining the homeostasis of gut epithelium. In 2009, Sato et al established a three-dimensional culture system, which mimicked the niche microenvironment by employing the niche factors, and successfully grew crypt ISCs into organoids or Mini-guts in vitro. Since then, the intestinal organoid technology has been used to delineate cellular signaling in ISC biology. However, the cultured organoids consist of heterogeneous cell populations, and it was technically challenging to introduce genomic changes into three-dimensional organoids. Thus, there was a technical necessity to develop a two-dimensional ISC culture system for effective genomic manipulations. In this study, we established a conditionally immortalized mouse intestinal crypt (ciMIC) cell line by using a piggyBac transposon-based SV40 T antigen expression system. We showed that the ciMICs maintained long-term proliferative activity under two-dimensional niche factor-containing culture condition, retained the biological characteristics of intestinal epithelial stem cells, and could form intestinal organoids in three-dimensional culture. While in vivo cell implantation tests indicated that the ciMICs were non-tumorigenic, the ciMICs overexpressing oncogenic β-catenin and/or KRAS exhibited high proliferative activity and developed intestinal adenoma-like pathological features in vivo. Collectively, these findings strongly suggested that the engineered ciMICs should be used as a valuable tool cell line to dissect the genetic and/or epigenetic underpinnings of intestinal tumorigenesis.

Keywords: Cancer modeling; Conditional immortalization; Mini-gut organoids; Mouse intestinal crypt (MIC) cells; Tumorigenesis.