P75 Neurotrophin Receptor Expression Is a Characteristic of the Mitotically Quiescent Cancer Stem Cell Population Present in Esophageal Squamous Cell Carcinoma
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INTERNATIONAL JOURNAL OF ONCOLOGY 48: 1943-1954, 2016 p75 neurotrophin receptor expression is a characteristic of the mitotically quiescent cancer stem cell population present in esophageal squamous cell carcinoma TETSUJI YAMAGUCHI1, TOMOYUKI OKUMURA1, Katsuhisa HIRANO1, TORU Watanabe1, Takuya Nagata1, Yutaka SHIMADA2 and KAZUHIRO TSUKADA1 1Department of Surgery and Science, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama City, Toyama 930-0194; 2Department of Nanobio Drug Discovery, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan Received December 8, 2015; Accepted January 11, 2016 DOI: 10.3892/ijo.2016.3432 Abstract. Mitotically quiescent cancer stem cells (CSC) are identified and isolated based on their p75NTR expression, hypothesized to exhibit a more aggressive phenotype involving providing researchers with a novel diagnostic and therapeutic greater therapeutic resistance and metastasis. The aim of our target. study was to develop a method for identifying quiescent CSC in esophageal squamous cell carcinoma (ESCC) based on Introduction their expression of the p75 neurotrophin receptor (p75NTR) and other proposed CSC markers, such as CD44 and CD90. Recent studies have demonstrated that some cancer stem Double immunostaining of surgical ESCC specimens cells (CSC) remain in a mitotically quiescent state, which revealed that the mean Ki-67-labeling index of the p75NTR- enhances their chemoresistance and results in a more inva- positive cells was significantly lower than that of the sive and aggressive phenotype (1,2). The p75 neurotrophin p75NTR-negative cells. Real-time PCR analysis of sorted receptor (p75NTR) is expressed in the quiescent basal layer of fractions of ESCC cell lines (KYSE cells) revealed that stem the esophageal epithelium (3,4), and in esophageal squamous cell-related genes (Nanog, p63 and Bmi-1) and epithelial- cell carcinoma (ESCC). It is also expressed in populations of mesenchymal transition (EMT)-related genes (N-cadherin cells that exhibit enhanced colony-forming abilities and xeno- and fibronectin) were expressed at significantly higher levels graft tumorigenicity (5,6). CD44 (7) and CD90 (8) have also in the p75NTR-positive fractions than in the CD44-positive been reported to be markers of populations of esophageal CSC or CD90-positive fractions. In addition, the p75NTR-positive that display enhanced xenograft tumorigenicity and/or meta- fractions exhibited significantly higher colony formation static potential. However, none of these studies investigated in vitro, significantly enhanced tumor formation in mice, the mitotic status of the examined CSC populations. The aim and significantly greater chemoresistance against cisplatin of this study was to develop a method for identifying mitoti- (CDDP) than the CD44-positive or CD90-positive fractions. cally quiescent CSC in ESCC based on their p75NTR, CD44 Furthermore, in both the cultured cells and those from and CD90 expression patterns. the mouse xenograft tumors, the p75NTR-positive/CD44- negative and p75NTR-positive/CD90-negative KYSE Materials and methods cell fractions contained significantly higher proportions of mitotically quiescent cells. These results suggest that Tissue microarray. A total of 56 tumor specimens from the mitotically quiescent CSC population in ESCC can be ESCC patients who had undergone surgery at our hospital from 1990 to 2008 were analyzed using a tissue microarray, as described previously (9). All of the patients underwent R0 resections, and none of them died in hospital. The median follow-up time was 29 months. The subjects included 50 male Correspondence to: Dr Tomoyuki Okumura, Department of Surgery and 6 female patients, and their mean age was 62.8 years. and Science, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama City, The patients' TNM stages (ver. 6) were as follows: stage I, Toyama 930-0194, Japan 6 patients; stage IIA, 15 patients; stage IIB, 5 patients; E-mail: [email protected] stage III, 24 patients; and stage IV, 6 patients. All of the M1 cases involved only distant lymph node metastases, which Key words: esophageal cancer, cancer stem cell, p75NTR, CD271, were surgically removed. Thirty-six patients underwent CD44, CD90 postoperative cisplatin-based chemotherapy. The institu- tional review board at the University of Toyama approved this study (#20-57). 1944 YAMAGUCHI et al: Esophageal cancer stem cells Immunohistochemistry. The antibodies used for the immuno- Table I. Polymerase chain reaction primer sequences. histochemical staining were as follows: anti-Ki-67 antibody (dilution 1:100), anti-cytokeratin Oscar in vitro diagnostic anti- Gene Size body (dilution 1:200; Abcam Ltd., Cambridge, UK), anti-human product Forward and reverse primers (5'-3') (bp) CD44 monoclonal antibody (156-3C11; dilution 1:400; Cell Signaling Technology, Beverly, MA, USA), anti-human CD90 Nanog F: ATGCCTCACACGGAGACTGT 83 monoclonal antibody (EPR3132; dilution 1:100), and human R: AAGTGGGTTGTTTGCCTTTG p75NTR monoclonal antibody against p75NGER (NGER5; p63 F: CAGACTTGCCAGATCATCC 220 dilution 1:100) (both from Abcam). The immunostaining was R: CAGCATTGTCAGTTTCTTAGC performed using Envision Plus kits, horseradish peroxidase, Bmi-1 F: CCACCTGATGTGTGTGCTTTG 162 or 3,3'-diaminobenzidine (DAB; Dako Cytomation, Kyoto, Japan) as recommended by the supplier. Counterstaining was R: TTCAGTAGTGGTCTGGTCTTGT performed with Mayer's hematoxylin. Involucrin F: TGTTCCTCCTCCAGTCAATACCC 227 The numbers of p75NTR-, CD44- or CD90-positive cells R: ATTCCTCATGCTGTTCCCAGTGC and all tumor cells were counted in three random fields of each E-cadherin F: GTCTGTCATGGAAGGTGCT 370 section. Then, the proportions of each cell type in each tumor R: TACGACGTTAGCCTCGTTC were calculated. We classified tumors as positive when >5% of the tumor cells were stained. N-cadherin F: AGCCAACCTTAACTGAGGAGT 136 Double staining of Ki-67 (brown)/p75NTR (red) or Ki-67 R: GGCAAGTTGATTGGAGGGATG (brown)/CD44 (red) was performed using Bond III automated Fibronectin F: AGGAAGCCGAGGTTTTAACTG 106 immunostainers (Leica Biosystems). The Ki-67 labeling index R: AGGACGCTCATAAGTGTCACC was defined as the number of tumor cells that exhibited posi- tive nuclear immunostaining divided by the total number of DPD F: TCAAGCACACGACTCTTGGTG 205 tumor cells. R: CATACCATTCCACAAGTCAGACC ERCC-1 F: GCCTCCGCTACCACAACCT 313 Human esophageal cancer cell lines and culture conditions. R: TCTTCTCTTGATGCGGCGA Human ESCC cell lines (KYSE-30, KYSE-140, KYSE-150, GAPDH F: ACCACAGTCCATGCCATCAC 452 KYSE-220, KYSE-510, KYSE-520 and KYSE-790) were estab- R: TCCACCACCCTGTTGCTGTA lished by Shimada et al and cultured in Ham's F12/Roswell Park Memorial Institute (RPMI)-1640 medium (Wako, Osaka, F, forward; R, reverse. Japan) supplemented with 2% fetal calf serum (FCS) (Gibco, Grad Island, NY, USA), according to a previously reported method (10). classified cell lines as positive when >1% of their cells were Analysis of cancer stem cell surface antigens and cell sorting. stained. The surface antigen markers of the samples were analyzed The KYSE-30 cells were sorted into p75NTR-positive/ using a FACSCant II flow cytometer and were sorted using CD44-negative, p75NTR-negative/CD44-positive, and p75NTR- a FACSAria II cell sorter and BD FACSDiva software (both negative/CD44-negative fractions. The KYSE-140 cells were from BD Biosciences, San Jose, CA, USA). Fluorescein sorted into p75NTR-positive/CD90-positive, p75NTR-positive/ isothiocyanate (FITC) or allophycocyanin (APC)-conjugated CD90-negative, p75NTR-negative/CD90-positive and p75NTR- monoclonal mouse anti-human CD44 (clone BD105) and negative/CD90-negative fractions. p75NTR (clone ME20.4-1.H4) antibodies were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). RNA extraction, cDNA synthesis and real-time PCR. Total RNA FITC-conjugated monoclonal mouse anti-human CD90 was extracted using the TRIzol reagent (Invitrogen, Carlsbad, (clone 5E-10) antibodies were obtained from BD Biosciences. CA, USA). cDNA was synthesized using the PrimeScript First FITC-conjugated antibodies were used to detect CSC surface Strand cDNA synthesis kit (Takara Inc., Kyoto, Japan) and markers. Isotype-matched APC- or FITC-conjugated anti- used for the quantitative polymerase chain reaction (qPCR) bodies (Miltenyi Biotec) were used as controls. Cultured cells analysis. The cDNA samples were amplified in an Mx3000P were washed once with phosphate-buffered saline (PBS) (-), real-time qPCR system (Agilent Technologies, Santa Clara, before being dissociated from the culture plates using 0.25% CA, USA) using SYBR Premix Ex Taq II (Takara Inc.), as trypsin-ethylenediaminetetraacetic acid (Invitrogen, Carlsbad, described previously (11). Each mRNA expression level was CA, USA) and centrifuged. Single cells were resuspended in normalized to that of the reference gene GAPDH. The primer PBS (-) containing 2% FBS, and then the FITC-conjugated sequences are shown in Table I. The expression level of each antibody or the isotype control antibody was added, before mRNA molecule was evaluated using the ΔΔCt method. the cells were incubated at 4˚C for 30 min. After being washed twice with PBS (-) containing 2% FBS, the cells were Cell cycle analysis. The cell cycle analysis was performed using resuspended in PBS (-) containing 2% FBS. The cells were the CycleTest Plus DNA reagent kit (Becton-Dickinson Inc., also stained with 7-aminoactinomycin D (7-AAD; Bio-Rad