Lgr5+CD44+EpCAM+ Strictly Defines Cancer Stem Cells in Human Colorectal Cancer

Abstract

Background/Aims: Although EpCAM+CD44+ cells exhibit more stem-like properties than did EpCAM-CD44- cells, the specificity of EpCAM combined with CD44 in defining CSCs needs further improvement. Lgr5 is used as a biomarker to isolate cancer stem cells (CSCs) in colorectal cancer. However, it remains unclear whether Lgr5, along with EpCAM and CD44, can further identify and define CSCs in colorectal cancer. Methods: Lgr5+CD44+EpCAM+, Lgr5+CD44+EpCAM-, Lgr5+CD44-EpCAM+, Lgr5-CD44+EpCAM+, and Lgr5-CD44-EpCAM-cells were separately isolated using fluorescence-activated cell sorting (FACS). Colony formation, self-renewal, differentiation, and tumorigenic properties of these cells were investigated through in vitro experiments and in vivo tumor xenograft models. The expression of stemness genes and CSC- and epithelial-mesenchymal transition (EMT)-related genes, such as KLF4, Oct4, Sox2, Nanog, CD133, CD44, CD166, ALDH1, Lgr5, E-cadherin, ZO-1, Vimentin, Snail, Slug, and Twist, was examined using real-time PCR. Results: Lgr5-positive subpopulations exhibited higher capacities for colony formation, self-renewal, differentiation, and tumorigenicity as well as higher expression of stemness genes and mesenchymal genes and lower expression of epithelial genes than did Lgr5-negative subpopulations. Conclusion: Our data revealed that tumorigenic cells were highly restricted to Lgr5-positive subpopulations. Most importantly, Lgr5+CD44+EpCAM+ cells exhibited more pronounced CSC-like traits than did any other subpopulation, indicating that Lgr5 combined with CD44 and EpCAM can further improve the stem-like traits of CSCs in colorectal cancer.

Krüppel‐like factor 4 regulates stemness and mesenchymal properties of colorectal cancer stem cells through the TGF‐β1/Smad/snail pathway

“…2,3 Therefore, CSCs have recently gained increasing attention in the field of cancer research. 17 Other studies demonstrated an association of the CSC phenotype with mesenchymal F I G U R E 5 Effect of SB525334 on KLF4 overexpression-induced stemness and tumorigenesis of Lgr5 + CD44 + EpCAM + cells. [32][33][34] Human colorectal CSCs have been well recognized by their specific markers, such as CD133, CD44, CD24, EpCAM, ALDH and Lgr5 together with the CSC malignant profiles, including spheroid and colony formation.…”

“…Human Lgr5 + CD44 + EpCAM + cells were sorted and cultured according to our previous study 17 Lentivirus carrying KLF4 cDNA or KLF4 shRNA was purchased from Shanghai GeneChem Co., Ltd. The stably transfected cells were generated according to previous studies 14,18 and then treated with 10 μg/mL puromycin for 48 hours.…”

“…17 To delineate the Lgr5 + CD44 + EpCAM + cells in CRC, we measured the percentage of Lgr5 + CD44 + EpCAM + cells in various human CRC cell lines and tissue samples using flow cytometry (Table S3). Moreover, Lgr5 combined with CD44 and EpCAM might assist make strides the stem-like characteristics of colorectal CSCs.…”

Colon cancer therapy by focusing on colon cancer stem cells and their tumor microenvironment

“…year (Ricci-Vitiani et al, 2007). CD44 as a receptor of hyaluronic acid in extracellular matrix (ECM) (Leng et al, 2018), ATP-binding cassette transporter as a drug efflux pump, which promotes tumor cell resistance to chemotherapies (Guo et al, 2018), CD166 as another adhesion molecule to ECM (Shafaei, Sharbatdaran, Kamrani, & Khafri, 2013) are some other robust surface markers of CCSCs (Table 1). Some of these cell surface markers not only are applied in the identification and isolation but also are used in targeted therapy such as in the case of…”

“…Ricci‐Vitiani et al () showed that subcutaneous injection of CD133− colon cancer cells in immunodeficient mice did not have tumor formation ability, whereas CD133+ cells not only reproduced the original tumor but also they could grow as undifferentiated tumorspheres in serum‐free medium for more than 1 year (Ricci‐Vitiani et al, ). CD44 as a receptor of hyaluronic acid in extracellular matrix (ECM) (Leng et al, ), ATP‐binding cassette transporter as a drug efflux pump, which promotes tumor cell resistance to chemotherapies (Guo et al, ), CD166 as another adhesion molecule to ECM (Shafaei, Sharbatdaran, Kamrani, & Khafri, ) are some other robust surface markers of CCSCs (Table ). Some of these cell surface markers not only are applied in the identification and isolation but also are used in targeted therapy such as in the case of LGR5 directed antibody‐drug conjugates.…”