Basic Study
Copyright ©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Apr 21, 2021; 27(15): 1578-1594
Published online Apr 21, 2021. doi: 10.3748/wjg.v27.i15.1578
Leucine-rich repeat-containing G protein-coupled receptor 5 marks different cancer stem cell compartments in human Caco-2 and LoVo colon cancer lines
Samah Abdulaali Alharbi, Dmitry A Ovchinnikov, Ernst Wolvetang
Samah Abdulaali Alharbi, Physiology Department, College of Medicine, Umm Al-Qura University, Makkah 24231, Saudi Arabia
Samah Abdulaali Alharbi, Ernst Wolvetang, Department of Stem Cell Engineering Group, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane 4072, QLD, Australia
Dmitry A Ovchinnikov, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane 4072, QLD, Australia
Author contributions: Alharbi SA contributed investigation, methodology, formal analysis, visualization, preparation and original draft preparation; Ovchinnikov DA contributed conceptualization, methodology and supervision; Wolvetang E contributed conceptualization, supervision, funding, acquisition, project administration and resources and reviewed and edited the manuscript.
Institutional review board statement: The study was reviewed and approved by the Institutional Review Board at The University of Queensland (approval No. 2019000159).
Institutional animal care and use committee statement: All animal experiments conformed to the internationally accepted principles for the care and use of laboratory animals, The Australian Institute of Bioengineering and Nanotechnology, Brisbane; Australia (approval No. AIBN/065/12/SCA/LEJEUNE/KACST).
Conflict-of-interest statement: All authors have nothing to disclose.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE Guidelines, and the manuscript was prepared and revised according to the ARRIVE Guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Samah Abdulaali Alharbi, PhD, Assistant Professor, Senior Scientist, Physiology Department, College of Medicine, Umm Al-Qura University, Prince Sultan bin AbdulAziz road, Makkah 24231, Saudi Arabia. saaharbi@uqu.edu.sa
Received: December 1, 2020
Peer-review started: December 1, 2020
First decision: December 21, 2020
Revised: January 22, 2021
Accepted: February 24, 2021
Article in press: February 24, 2021
Published online: April 21, 2021
Abstract
BACKGROUND

Colon cancer cell lines are widely used for research and for the screening of drugs that specifically target the stem cell compartment of colon cancers. It was reported that colon cancer carcinoma specimens contain a subset of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)-expressing stem cells, these so-called “tumour-initiating” cells, reminiscent in their properties of the normal intestinal stem cells (ISCs), may explain the apparent heterogeneity of colon cancer cell lines. Also, colon cancer is initiated by aberrant Wnt signaling in ISCs known to express high levels of LGR5. Furthermore, in vivo reports demonstrate the clonal expansion of intestinal adenomas from a single LGR5-expressing cell.

AIM

To investigate whether colon cancer cell lines contain cancer stem cells and to characterize these putative cancer stem cells.

METHODS

A portable fluorescent reporter construct based on a conserved fragment of the LGR5 promoter was used to isolate the cell compartments expressing different levels of LGR5 in two widely used colon cancer cell lines (Caco-2 and LoVo). These cells were then characterized according to their proliferation capacity, gene expression signatures of ISC markers, and their tumorigenic properties in vivo and in vitro.

RESULTS

The data revealed that the LGR5 reporter can be used to identify and isolate a classical intestinal crypt stem cell-like population from the Caco-2, but not from the LoVo, cell lines, in which the cancer stem cell population is more akin to B lymphoma Moloney murine leukemia virus insertion region 1 homolog (+4 crypt) stem cells. This sub-population within Caco-2 cells exhibits an intestinal cancer stem cell gene expression signature and can both self-renew and generate differentiated LGR5 negative progeny. Our data also show that cells expressing high levels of LGR5/enhanced yellow fluorescent protein (EYFP) from this cell line exhibit tumorigenic-like properties in vivo and in vitro. In contrast, cell compartments of LoVo that are expressing high levels of LGR5/EYFP did not show these stem cell-like properties. Thus, cells that exhibit high levels of LGR5/EYFP expression represent the cancer stem cell compartment of Caco-2 colon cancer cells, but not LoVo cells.

CONCLUSION

Our findings highlight the presence of a spectrum of different ISC-like compartments in different colon cancer cell lines. Their existence is an important consideration for their screening applications and should be taken into account when interpreting drug screening data. We have generated a portable LGR5-reporter that serves as a valuable tool for the identification and isolation of different colon cancer stem cell populations in colon cancer lines.

Keywords: Colorectal cancer, Colon cancer cell lines, Intestinal stem cell, Cancer stem cell, Leucine-rich repeat-containing G protein-coupled receptor 5, Heterogenicity

Core Tip: The intestinal epithelium harbors two distinct pools of putative stem cells, the leucine-rich repeat-containing G protein-coupled receptor 5+ (LGR5) stem cell population and the B lymphoma Moloney murine leukemia virus insertion region 1 homolog+ stem cell population. Colon cancer cell lines such as Caco-2 and LoVo are extensively used in colon cancer research, and express high levels of LGR5. Here, we aimed to investigate whether colon cancer cell lines contain cancer stem cells and characterized these cells. Using an LGR5 reporter, we characterized LGR5+ cells and revealed that Caco-2 cell line contains a classical intestinal stem cell-like population (LGR5+). However, in LoVo cell lines, stem cell-like population is more akin to the B lymphoma Moloney murine leukemia virus insertion region 1 homolog+ stem cells.