Modeling Wnt signaling by CRISPR-Cas9 genome editing recapitulates neoplasia in human Barrett epithelial organoids

Xi Liu; Yulan Cheng; John M Abraham; Zhixiong Wang; Zhe Wang; Xiquan Ke; Rong Yan; Eun Ji Shin; Saowanee Ngamruengphong; Mouen A Khashab; Guanjun Zhang; George McNamara; Andrew J Ewald; DeChen Lin; Zhengwen Liu; Stephen J Meltzer

doi:10.1016/j.canlet.2018.08.017

Modeling Wnt signaling by CRISPR-Cas9 genome editing recapitulates neoplasia in human Barrett epithelial organoids

Cancer Lett. 2018 Nov 1:436:109-118. doi: 10.1016/j.canlet.2018.08.017. Epub 2018 Aug 23.

Authors

Xi Liu¹, Yulan Cheng², John M Abraham², Zhixiong Wang³, Zhe Wang², Xiquan Ke⁴, Rong Yan⁵, Eun Ji Shin², Saowanee Ngamruengphong², Mouen A Khashab², Guanjun Zhang⁶, George McNamara⁷, Andrew J Ewald⁸, DeChen Lin⁹, Zhengwen Liu¹⁰, Stephen J Meltzer¹¹

Affiliations

¹ Department of Pathology, The First Affiliated Hospital of Xi' an Jiaotong University, No. 277 Yanta West Road, Xi' an, 710061, Shaanxi, China; Division of Gastroenterology, Department of Medicine, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, School of Medicine, Baltimore, MD, 21205, USA; Division of Gastroenterology, Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, School of Medicine, Baltimore, MD, 21205, USA.
² Division of Gastroenterology, Department of Medicine, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, School of Medicine, Baltimore, MD, 21205, USA; Division of Gastroenterology, Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, School of Medicine, Baltimore, MD, 21205, USA.
³ Department of Gastrointestinal Surgery, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China.
⁴ Department of Gastroenterology, The First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui, China.
⁵ Department of Surgical Oncology, First Affiliated Hospital of Xi' an Jiaotong University, No. 277 Yanta West Road, Xi' an, 710061, Shaanxi, China.
⁶ Department of Pathology, The First Affiliated Hospital of Xi' an Jiaotong University, No. 277 Yanta West Road, Xi' an, 710061, Shaanxi, China.
⁷ Division of Gastroenterology - Ross Fluorescence Imaging Center, Department of Medicine, Johns Hopkins University, School of Medicine, Baltimore, MD, 21205, USA.
⁸ Department of Cell Biology and Oncology, Center for Cell Dynamics, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
⁹ Division of Hematology and Oncology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA, 90048, USA.
¹⁰ Department of Infectious Diseases, First Affiliated Hospital of Xi' an Jiaotong University, No. 277 Yanta West Road, Xi' an, 710061, Shaanxi, China.
¹¹ Division of Gastroenterology, Department of Medicine, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, School of Medicine, Baltimore, MD, 21205, USA; Division of Gastroenterology, Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, School of Medicine, Baltimore, MD, 21205, USA. Electronic address: smeltzer@jhmi.edu.

Abstract

Primary organoid cultures generated from patient biopsies comprise a novel improved platform for disease modeling, being genetically stable and closely recapitulating in vivo scenarios. Barrett esophagus (BE) is the major risk factor for esophageal adenocarcinoma. There has been a dearth of long-term in vitro expansion models of BE neoplastic transformation. We generated a long-term virus-free organoid expansion model of BE neoplasia from patient biopsies. Both wild-type and paired APC-knockout (APC^KO) BE organoids genome-edited by CRISPR-Cas9 showed characteristic goblet cell differentiation. Autonomous Wnt activation was confirmed in APC^KO organoids by overexpression of Wnt target genes and nuclear-translocated β-catenin expression after withdrawal of Wnt-3A and R-spondin-1. Wnt-activated organoids demonstrated histologic atypia, higher proliferative and replicative activity, reduced apoptosis, and prolonged culturability. Wnt-activated organoids also showed sustained protrusive migration ability accompanied by disrupted basement membrane reorganization and integrity. This CRISPR-Cas9 editing human-derived organoid model recapitulates the critical role of aberrant Wnt/β-catenin signaling activation in BE neoplastic transformation. This system can be used to study other 'driver' pathway alterations in BE-associated neoplasia, avoiding signaling noise present in immortalized or cancer-derived cell lines.

Keywords: Barrett esophagus; CRISPR/Cas9; Neoplastic transformation; Organoids; Wnt signaling.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Adenomatous Polyposis Coli Protein / genetics
Adenomatous Polyposis Coli Protein / metabolism
Apoptosis / genetics
Barrett Esophagus / metabolism*
Barrett Esophagus / pathology
CRISPR-Cas Systems*
Cell Differentiation / genetics
Cell Movement / genetics
Cell Proliferation / genetics
Cell Transformation, Neoplastic / genetics*
Gene Editing / methods*
Gene Knockout Techniques
Humans
Models, Genetic
Organoids / metabolism
Organoids / pathology
Wnt Signaling Pathway / genetics*
beta Catenin / genetics*

Substances

Adenomatous Polyposis Coli Protein
CTNNB1 protein, human
beta Catenin

Abstract

Publication types

MeSH terms

Substances

Grants and funding