Enhanced cancer stem cell properties of a mitotically quiescent subpopulation of p75NTR-positive cells in esophageal squamous cell carcinoma

Kojima,Hirofumi; Okumura,Tomoyuki; Yamaguchi,Tetsuji; Miwa,Takeshi; Shimada,Yutaka; Nagata,Takuya

doi:10.3892/ijo.2017.4001

Enhanced cancer stem cell properties of a mitotically quiescent subpopulation of p75NTR-positive cells in esophageal squamous cell carcinoma

Authors:
- Hirofumi Kojima
- Tomoyuki Okumura
- Tetsuji Yamaguchi
- Takeshi Miwa
- Yutaka Shimada
- Takuya Nagata
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Affiliations: Department of Surgery and Science, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama city, Toyama 930-0194, Japan, Department of Nanobio Drug Discovery, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
Published online on: May 16, 2017 https://doi.org/10.3892/ijo.2017.4001
Pages: 49-62
Copyright: © Kojima et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Mitotically quiescent cancer stem cells (CSCs) possess higher malignant potential than other CSCs, indicating their higher contribution to therapeutic resistance than that of other CSCs. In esophageal squamous cell carcinoma (ESCC), p75 neurotrophin receptor (p75NTR) is expressed in a candidate CSC population showing high tumorigenicity and chemoresistance. In the present study, we isolated and characterized quiescent CSCs population in ESCC based on p75NTR expression and cell cycle status. Expression of p75NTR and Ki-67 in ESCC cell lines (KYSE cells) and surgically resected ESCC specimens was detected by performing immunocytochemical analysis. p75NTR-positive KYSE cells were fractionated into quiescent and proliferating cells by performing flow cytometry with a fluorescent DNA-staining dye to determine their CSC phenotype. Immunocytochemical analysis showed that 21.8 and 36.5% of the p75NTR-positive cells were Ki-67-negative (G0), which accounted for 11.4 and 15.7% of cells in KYSE-30 and KYSE-140 cell lines, respectively. Flow cytometric cell sorting showed that p75NTR-positive cells in the G0-G1 phase (p75NTR-positive/G0-1 cells) but not in the S-G2-M phase (p75NTR-positive/S-G2-M cells) showed strong expression of stem cell-related genes Nanog, BMI-1, and p63; high colony formation ability; high tumorigenicity in a mouse xenograft model; and strong chemoresistance against cisplatin because of the expression of drug resistance genes ABCG2 and ERCC1. Label-retention assay showed that 3.4% p75NTR-positive cells retained fluorescent cell-tracing dye, but p75NTR-negative cells did not. Immunohistochemical analysis of ESCC specimens showed p75NTR expression in 39 of 95 (41.1%) patients, with a median of 13.2% (range, 3.0-80.1%) p75NTR-positive/Ki-67-negative cells, which were found to be associated with poorly differentiated histology. Our results suggest that p75NTR-positive/G0-1 cells represent quiescent CSCs in ESCC and indicate that these cells can be used as targets to investigate molecular processes regulating CSC phenotype and to develop novel therapeutic strategies.

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