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• W2063660107 abstract "Cancer stem-like cell subpopulations, referred to as “side-population” (SP) cells, have been identified in several tumors based on their ability to efflux the fluorescent dye Hoechst 33342. Although SP cells have been identified in the normal human endometrium and endometrial cancer, little is known about their characteristics. In this study, we isolated and characterized the SP cells in human endometrial cancer cells and in rat endometrial cells expressing oncogenic human K-Ras protein. These SP cells showed i) reduction in the expression levels of differentiation markers; ii) long-term proliferative capacity of the cell cultures; iii) self-renewal capacity in vitro; iv) enhancement of migration, lamellipodia, and, uropodia formation; and v) enhanced tumorigenicity. In nude mice, SP cells formed large, invasive tumors, which were composed of both tumor cells and stromal-like cells with enriched extracellular matrix. The expression levels of vimentin, α-smooth muscle actin, and collagen III were enhanced in SP tumors compared with the levels in non-SP tumors. In addition, analysis of microdissected samples and fluorescence in situ hybridization of Hec1-SP-tumors showed that the stromal-like cells with enriched extracellular matrix contained human DNA, confirming that the stromal-like cells were derived from the inoculated cells. Moreober, in a Matrigel assay, SP cells differentiated into α-smooth muscle actin-expressing cells. These findings demonstrate that SP cells have cancer stem-like cell features, including the potential to differentiate into the mesenchymal cell lineage. Cancer stem-like cell subpopulations, referred to as “side-population” (SP) cells, have been identified in several tumors based on their ability to efflux the fluorescent dye Hoechst 33342. Although SP cells have been identified in the normal human endometrium and endometrial cancer, little is known about their characteristics. In this study, we isolated and characterized the SP cells in human endometrial cancer cells and in rat endometrial cells expressing oncogenic human K-Ras protein. These SP cells showed i) reduction in the expression levels of differentiation markers; ii) long-term proliferative capacity of the cell cultures; iii) self-renewal capacity in vitro; iv) enhancement of migration, lamellipodia, and, uropodia formation; and v) enhanced tumorigenicity. In nude mice, SP cells formed large, invasive tumors, which were composed of both tumor cells and stromal-like cells with enriched extracellular matrix. The expression levels of vimentin, α-smooth muscle actin, and collagen III were enhanced in SP tumors compared with the levels in non-SP tumors. In addition, analysis of microdissected samples and fluorescence in situ hybridization of Hec1-SP-tumors showed that the stromal-like cells with enriched extracellular matrix contained human DNA, confirming that the stromal-like cells were derived from the inoculated cells. Moreober, in a Matrigel assay, SP cells differentiated into α-smooth muscle actin-expressing cells. These findings demonstrate that SP cells have cancer stem-like cell features, including the potential to differentiate into the mesenchymal cell lineage. Recently, adult stem cells have been identified in several mature tissues, such as the adult intestine,1Bjerknes M Cheng H Clonal analysis of mouse intestinal epithelial progenitors.Gastroenterology. 1999; 116: 7-14Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar skin,2Alonso L Fuchs E Stem cells of the skin epithelium.Proc Natl Acad Sci USA. 2003; 100: 11830-11835Crossref PubMed Scopus (374) Google Scholar muscle,3Jankowski RJ Deasy BM Huard J Muscle-derived stem cells.Gene Ther. 2002; 9: 642-647Crossref PubMed Scopus (273) Google Scholar blood,4Spangrude GJ Smith L Uchida N Ikuta K Heimfeld S Friedman J Weissman IL Mouse hematopoietic stem cells.Blood. 1991; 78: 1395-1402PubMed Google Scholar and the nervous system5Morrison SJ White PM Zock C Anderson DJ Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells.Cell. 1999; 96: 737-749Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar, 6Uchida N Buck DW He D Reitsma MJ Masek M Phan TV Tsukamoto AS Gage FH Weissman IL Direct isolation of human central nervous system stem cells.Proc Natl Acad Sci USA. 2000; 97: 14720-14725Crossref PubMed Scopus (1500) Google Scholar, 7Rietze RL Valcanis H Brooker GF Thomas T Voss AK Bartlett PF Purification of a pluripotent neural stem cell from the adult mouse brain.Nature. 2001; 412: 736-739Crossref PubMed Scopus (574) Google Scholar A stem cell is an undifferentiated cell that is defined by its ability to both self-renew and to produce mature progeny cells.8Reya T Morrison SJ Clarke MF Weissman IL Stem cells, cancer, and cancer stem cells.Nature. 2001; 414: 105-111Crossref PubMed Scopus (7349) Google Scholar Stem cells are classified based on their developmental potential as totipotent, pluripotent, oligopotent, and unipotent. Adult somatic stem cells were originally thought to be tissue specific and only able to give rise to progeny cells corresponding to their tissue of origin. Recent studies, however, have shown that adult mammalian stem cells are able to differentiate across tissue lineage boundaries,9Herzog EL Chai L Krause DS Plasticity of marrow-derived stem cells.Blood. 2003; 102: 3483-3493Crossref PubMed Scopus (661) Google Scholar, 10Goodell MA Stem-cell “plasticity”: befuddled by the muddle.Curr Opin Hematol. 2003; 10: 208-213Crossref PubMed Scopus (91) Google Scholar although this “plasticity” of adult somatic stem cells remains controversial. Stem cell subpopulations (“side-population” (SP) cells) have been identified in many mammals, including humans, based on the ability of these cells to efflux the fluorescent dye Hoechst 33342.11Goodell MA Brose K Paradis G Conner AS Mulligan RC Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.J Exp Med. 1996; 183: 1797-1806Crossref PubMed Scopus (2407) Google Scholar Recent evidence suggests that the SP phenotype is associated with a high expression level of the ATP-binding cassette transporter protein ABCG2/Bcrp1.12Zhou S Schuetz JD Bunting KD Colapietro AM Sampath H Sorrentino BP The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype.Nat Med. 2001; 7: 1028-1034Crossref PubMed Scopus (1918) Google Scholar Most recently, established malignant cell lines, which have been maintained for many years in culture, have also been shown to contain SP cells as a minor subpopulation.13Kondo T Setoguchi T Taga T Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line.Proc Natl Acad Sci USA. 2004; 101: 781-786Crossref PubMed Scopus (913) Google Scholar The human endometrium is a highly dynamic tissue undergoing cycles of growth, differentiation, shedding, and regeneration throughout the reproductive life of women. Endometrial adult stem/progenitor cells are likely responsible for endometrial regeneration.14Gargett CE Review article: stem cells in human reproduction.Reprod Sci. 2007; 14: 405-424Crossref PubMed Scopus (30) Google Scholar Rare populations of human endometrial epithelial and stromal colony-forming cells15Chan RW Schwab KE Gargett CE Clonogenicity of human endometrial epithelial and stromal cells.Biol Reprod. 2004; 70: 1738-1750Crossref PubMed Scopus (439) Google Scholar and SP cells16Kato K Yoshimoto M Kato K Adachi S Yamayoshi A Arima T Asanoma K Kyo S Nakahata T Wake N Characterization of side-population cells in human normal endometrium.Hum Reprod. 2007; 22: 1214-1223Crossref PubMed Scopus (176) Google Scholar, 17Tsuji S Yoshimoto M Takahashi K Noda Y Nakahata T Heike T Side population cells contribute to the genesis of human endometrium.Fertil Steril. 2008; 90: 1528-1537Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar have been identified. Although coexpression of CD146 and PDGFRβ isolates a population of mesenchymal stem like cells from human endometrium,18Schwab KE Gargett CE Co-expression of two perivascular cell markers isolates mesenchymal stem-like cells from human endometrium.Hum Reprod. 2007; 22: 2903-2911Crossref PubMed Scopus (380) Google Scholar specific stem cell markers of endometrium remain unclear. Recently, Gotte et al19Gotte M Wolf M Staebler A Buchweitz O Kelsch R Schuring AN Kiesel L Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.J Pathol. 2008; 215: 317-329Crossref PubMed Scopus (149) Google Scholar demonstrated that the adult stem cell marker Musashi-1 was coexpressed with Notch-1 in a subpopulation of endometrial cells. Furthermore, they showed that telomerase and Musashi-1-expressing cells were significantly increased in proliferative endometrium, endometriosis, and endometrial carcinoma tissue, compared with secretary endometrium, suggesting the concept of a stem cell origin of endometriosis and endometrial carcinoma. Recent evidence suggests that cancer stem-like cells exist in several malignant tumors, such as leukemia20Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.Nat Med. 1997; 3: 730-737Crossref PubMed Scopus (5089) Google Scholar, 21Lapidot T Sirard C Vormoor J Murdoch B Hoang T Caceres-Cortes J Minden M Paterson B Caligiuri MA Dick JE A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.Nature. 1994; 367: 645-648Crossref PubMed Scopus (3400) Google Scholar breast cancer,22AI-Hajj M Wicha MS Benito-Hernandez A Morrison SJ Clarke MF Prospective identification of tumorigenic breast cancer cells.Proc Natl Acad Sci USA. 2003; 100: 3983-3988Crossref PubMed Scopus (7851) Google Scholar and brain tumors,23Singh SK Clarke ID Terasaki M Bonn VE Hawkins C Squire J Dirks PB Identification of a cancer stem cell in human brain tumors.Cancer Res. 2003; 63: 5821-5828PubMed Google Scholar and that these stem cells express surface markers similar to those expressed by normal stem cells in each tissue.20Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.Nat Med. 1997; 3: 730-737Crossref PubMed Scopus (5089) Google Scholar, 24Bhatia M Wang JC Kapp U Bonnet D Dick JE Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice.Proc Natl Acad Sci USA. 1997; 94: 5320-5325Crossref PubMed Scopus (698) Google Scholar Development of endometrial carcinoma is associated with a variety of genetic alterations. For example, increased expression and activity of telomerase25Kyo S Kanaya T Takakura M Tanaka M Inoue M Human telomerase reverse transcriptase as a critical determinant of telomerase activity in normal and malignant endometrial tissues.Int J Cancer. 1999; 80: 60-63Crossref PubMed Google Scholar, 26Lehner R Enomoto T McGregor JA Shroyer AL Haugen BR Pugazhenthi U Shroyer KR Quantitative analysis of telomerase hTERT mRNA and telomerase activity in endometrioid adenocarcinoma and in normal endometrium.Gynecol Oncol. 2002; 84: 120-125Abstract Full Text PDF PubMed Scopus (49) Google Scholar and frequent dysregulation of signaling pathways have been observed in endometrial carcinoma. Some of these pathways are important determinants of stem cell activity (Wnt-β-catenin and PTEN).27Moreno-Bueno G Cubillo E Sarrio D Peinado H Rodriguez-Pinilla SM Villa S Bolos V Jorda M Fabra A Portillo F Palacios J Cano A Genetic profiling of epithelial cells expressing E-cadherin repressors reveals a distinct role for Snail. Slug, and E47 factors in epithelial-mesenchymal transition.Cancer Res. 2006; 66: 9543-9556Crossref PubMed Scopus (249) Google Scholar, 28Zhou C Bae-Jump VL Whang YE Gehrig PA Boggess JF The PTEN tumor suppressor inhibits telomerase activity in endometrial cancer cells by decreasing hTERT mRNA levels.Gynecol Oncol. 2006; 101: 305-310Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 29Rossi DJ Weissman IL Pten, tumorigenesis, and stem cell self-renewal.Cell. 2006; 125: 229-231Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar These suggest a stem cell contribution to endometrial carcinoma development. Recently, we isolated SP cells from the human endometrium. These SP cells showed long-term proliferating capacity in cultures and produced both gland and stromal-like cells. Additionally, they were able to function as progenitor cells.16Kato K Yoshimoto M Kato K Adachi S Yamayoshi A Arima T Asanoma K Kyo S Nakahata T Wake N Characterization of side-population cells in human normal endometrium.Hum Reprod. 2007; 22: 1214-1223Crossref PubMed Scopus (176) Google Scholar In this study, we isolated and characterized SP cells from human endometrial cancer cells and from rat endometrial cells expressing oncogenic [12Val] human K-Ras protein and demonstrated their cancer stem-like cell phenotypes. pZIP-Neo SV(X)1 containing [12Val] human K-ras 4B cDNA was a gift from Dr. C. Der (University of North Carolina, Chapel Hill, NC).30Casey PJ Solski PA Der CJ Buss JE p21ras is modified by a farnesyl isoprenoid.Proc Natl Acad Sci USA. 1989; 86: 8323-8327Crossref PubMed Scopus (749) Google Scholar, 31Kato K Cox AD Hisaka MM Graham SM Buss JE Der CJ Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity.Proc Natl Acad Sci USA. 1992; 89: 6403-6407Crossref PubMed Scopus (537) Google Scholar The pZeo− vector was purchased from Invitrogen (Carlsbad, CA). We cut the 1.1-kb fragment containing [12Val] human K-ras 4B cDNA from the pZIP-Neo SX (X)1 construct with BamHI and ligated it to the BamHI site of pZeo vector. An endometrial cancer cell line (Hec-1) and a rat endometrial cell line (RENT4) were used in the present study. The Hec-1 cell line was established by Kuramoto et al32Kuramoto H Tamura S Notake Y Establishment of a cell line of human endometrial adenocarcinoma in vitro.Am J Obstet Gynecol. 1972; 114: 1012-1019Abstract Full Text PDF PubMed Scopus (213) Google Scholar from explants of adenocarcinoma of human endometrium. RENT4 cells were established by Wiehle et al33Wiehle RD Helftenbein G Land H Neumann K Beato M Establishment of rat endometrial cell lines by retroviral mediated transfer of immortalizing and transforming oncogenes.Oncogene. 1990; 5: 787-794PubMed Google Scholar and obtained from the European Collection of Cell Cultures. Hec1 cells expressed CD9 and Tie-2 but not CD13 or α-smooth muscle actin (α-SMA). Although Tie-2 is an endothelial cell marker, it has been reported that Tie-2 is expressed in glandular cells of both normal endometrium and endometrial adenocarcinoma.34Saito M Sato Y Watanabe J Kuramoto H Kaba S Fukuda T Angiogenic factors in normal endometrium and endometrial adenocarcinoma.Pathol Int. 2007; 57: 140-147Crossref PubMed Scopus (23) Google Scholar These results demonstrated that Hec-1 cells exhibited the phenotype of endometrial glandular cells and not stromal cells. RENT4 cells expressed Tie2, CD9, and CD13. α-SMA were stained weakly in RENT4 cells (Supplemental Figure S1, see http://ajp.amjpathol.org). RENT4 cells harboring mutant [12Val] versions of K-ras4B were established by transfecting RENT4 cells with pZeo constructs, containing cDNA sequences encoding [12Val] K-Ras using Lipofectoamine (Invitrogen, Tokyo, Japan). Stably transfected cells were selected and isolated in growth medium containing 400 μg/ml Zeocin (Invitrogen) to establish cell lines expressing K-Ras protein, previously described elsewhere.35Ninomiya Y Kato K Takahashi A Ueoka Y Kamikihara T Arima T Matsuda T Kato H Nishida J Wake N K-Ras and H-Ras activation promote distinct consequences on endometrial cell survival.Cancer Res. 2004; 64: 2759-2765Crossref PubMed Scopus (29) Google Scholar Pooled populations were used for the assay. Cells were cultured with Dulbecco's modified Eagle's medium (DMEM) (Nissui, Seika, Japan) supplemented with 20 μg/ml Gly-His-Lys, 2 mmol/L glutamine, 80 IU insulin (Sigma-Aldrich, St. Louis, MO), and 10% fetal calf serum (FCS) (Hyclone, Logan, UT).33Wiehle RD Helftenbein G Land H Neumann K Beato M Establishment of rat endometrial cell lines by retroviral mediated transfer of immortalizing and transforming oncogenes.Oncogene. 1990; 5: 787-794PubMed Google Scholar Endometrial cancer tissues were obtained from uteri after hysterectomy. This study was approved by the ethical committee of Kyushu University, and preoperative informed consent was obtained from each patient. Cell suspensions of endometrial cancer cells were obtained using enzymatic digestion and mechanical means. The endometrial cancer tissues were diced finely and dissociated in HBSS containing HEPES (25 mmol), penicillin (200 U/ml), streptomycin (200 μg/ml), and collagenase (1 mg/ml, 15 U/mg) (Sigma-Aldrich) for 30 minutes at 37°C with agitation. The dispersed endometrial cancer cells were separated by filtration through a wire sieve. To identify and isolate Hec1 and RK12 V SP cells, the cells were dislodged from the culture dishes with trypsin and EDTA, washed, and suspended at a concentration of 106 cells/ml in DMEM containing 2% FCS. The cells were then labeled in the same medium at 37°C for 90 minutes with 2.5 μg/ml Hoechst 33342 dye (Molecular Probes, Eugene, OR), either alone or in combination with 50 μmol/L verapamil (Sigma-Aldrich). Finally, the cells were counterstained with 1 μg/ml propidium iodide to label dead cells. The cells were then analyzed in a FACS Vantage fluorescence-activated cell sorter (BD Biosciences, San Jose, CA) or the EPICS ALTRA HyPerSort (Beckman Coulter, Fullerton, CA) using dual wavelength analysis (blue, 424–444 nm; red, 675 nm) after excitation with 350 nm of UV light. Propidium iodide-positive dead cells were excluded from the analysis. The SP cells were separated by FACS from the non-SP (NSP) cells and both fractions were seeded in a mesenchymal stem cell maintenance medium (MF medium) (Toyobo, Osaka, Japan) and 10% FCS on a collagen-coated 24-well plate (2 cm2) (Iwaki, Funabashi, Japan). The cells were cultured for 2 to 4 weeks. The cells were then transferred to collagen-coated plates (60 mm). Cells were plated in a mesenchymal stem cell maintenance medium (MF medium; Toyobo). Cells were passaged during the 2 months of cultivation. Viable cells were counted for 2 months. Cell counts were performed using a hemocytometer. SP cells or NSP cells were plated in 24-well collagen-coated dishes (50 cells/cm2). SP cells, but not NSP cells, formed colonies. Cells from individual colonies of SP cells were reseeded at 300 cells/cm2 in triplicate in 100-mm collagen-coated dishes to generate colonies. Colonies were monitored to ensure they were derived from single cells. The secondary colonies were reseeded in a similar manner to generate tertiary colonies. The cloning plates were stained with the crystal violet solution (Sigma-Aldrich). We inoculated 1 × 104 cells in Matrigel (BD Matrigel Basement Membrane Matrix High Concentration; BD Biosciences, Bedford, MA) into the s.c. connective tissue of 5-week-old nude mice (Balb nu/nu). After 6 weeks (RK12V- SP and NSP cells) or 3 months (Hec1-SP and -NSP cells), mice were sacrificed and the tumors excised. All mouse experiments were approved by the animal ethics committee of Kyushu University. Cells (1 × 105) (Hec1-SP or -NSP cells) were seeded in Matrigel in a Cell Culture Insert (0.4 mm) (BD Falcon, Franklin Lakes, NJ) and incubated with either an MF medium, an ordinary growth medium (DMEM containing 10% FCS), or a smooth muscle cell differentiation medium (BD Biosciences). The smooth muscle cell differentiation medium consisted of MCDB 131 basal medium supplemented with insulin, transferrin, selenous acid, linoleic acid, and bovine serum albumin. After 8 weeks, each Matrigel sample was fixed by treatment with 4% paraformaldehyde and analyzed by immunohistochemistry. Hec1-SP and -NSP cells were incubated with mesenchymal stem cell maintenance medium (MF medium; Toyobo) in a collagen-coated 24-well plate. Cells were observed with a ×10 immersion objective every minute for 48 hours under 5% CO2 at 37°C incubation, using a real-time cultured cell monitoring system (Astec, Fukuoka, Japan). Primary antibodies used in this study were as follows: CD9 polyclonal antibody (H-110), CD13 monoclonal antibody (3D8), vimentin monoclonal antibody (V9) and collagen III polyclonal antibody (H-300), and Tie-2 polyclonal antibody (C-20) (all obtained from Santa Cruz Biotechnology, Santa Cruz, CA). α-SMA monoclonal antibody (1A4) was purchased from MBL (Nagoya, Japan). Formalin-fixed histological tumor sections from nude mice, human endometrial cancer tissues, or cultured cells were used. Cultured cells were incubated on glass chamber slides (Lab-Tek; Nalge Nunc International, Naperville, IL) and fixed by treatment with 10% formalin. Sections or cells were rinsed twice in PBS (pH 7.4) for 5 minutes each. Samples were then incubated with 4% blocking horse serum (Vector Laboratories, Burlingame, CA) for 1 hour at room temperature in a humidified chamber, followed by incubation with the primary antibody (200 μg/ml, 1/100 diluted). We also used nonimmune mouse or rabbit IgG as a control for the primary antibody. Staining with the primary or control antibody was performed overnight at 4°C. Bound antibodies were detected with a biotinylated anti-rabbit IgG secondary antibody (1.5 mg/ml) and an avidin-biotin complex linked to horseradish peroxidase (Vectastain; Vector Laboratories), followed by incubation with diaminobenzidine tetrahydrochloride as the substrate. In immunofluorescent stain, bound vimentin antibodies were visualized by green fluorescence from secondary antibody staining with anti-mouse IgG conjugated to Alexa 488 (Molecular Probes). Cell nuclei were counterstained with 4′,6-diamidino-2-phenyl-indole. The level of vimentin in a semiquantitative manner was determined by calculating fluorescent intensity per three different positive or negative areas using a BIOREVO BZ-9000 fluorescence microscope (Keyence, Osaka, Japan). Glass slides with an overlay of 4 mm of thin LM Film (PALM Microlaser Technologies, Bernried, Germany) were prepared. Formalin-fixed, paraffin-embedded tumor tissue was cut into 7-μm sections and placed on the slides. The sections were then deparaffinized and stained with H&E. Using a laser microdissection system (Leica Microsystems, Wetzlar, Germany), tumor cells or stromal cells were isolated into the cap of a 0.5-ml microtube. After retrieval of the cells, 50 μl of proteinase K solution (Pico Pure DNA Extraction kit; Arcturus, Mountain View, CA) was added into the cap. The DNA was extracted by overnight incubation at 65°C. The solution was then boiled for 10 minutes to inactivate the proteinase K. To amplify the K-ras gene (human genomic DNA for Hec1-SP tumor cells or cDNA for RK12V-SP tumor), PCR using a T3000 thermal cycler was performed (Biometra, Floral City, FL). The primers used for PCR were as follows: i) 5′-AAAAGGTACTGGTGGAGTATTTGA-3′ (sense), 5′-TTGAAACCCAAGGTACATTTCA-3′ (antisense) for human genomic K-ras DNA; and ii) 5′-GACTGAATATAAACTT-3′ (sense), 5′-CATAATTACACACTTTGTCTT-3′ for human K-ras cDNA. The PCR cycling conditions were as follows: i) preheating for 2 minutes at 94°C, 39 cycles of denaturation for 1 minute at 94°C, annealing for 30 seconds at 59.3°C, and extension for 1 minute at 72°C; ii) preheating for 2 minutes at 94°C, 39 cycles of denaturation for 1 minute at 94°C, annealing for 30 seconds at 59.3°C, and extension for 1 minute at 72°C. After the last cycle, a final extension of 5 minutes at 72°C was added. PCR products were electrophoresed on a 2% agarose gel, and the band corresponding to the desired target was cut from the gel. DNA was extracted using the GFX PCR DNA gel band purification kit (GE Healthcare, Buckingham-Shire, UK). Direct sequencing of the PCR product was then performed using an ABI PRISM Big Dye Termination Ver3.1 Cycle Sequencing Kit according to the manufacturer's instructions and the ABI PRISM 3100 (Applied Biosystems). The primer used for sequencing was as follows: 5′-TTGAAACCCAAGGTACATTTCA-3′ (antisense) for K-ras DNA. Fluorescence in situ hybridization (FISH) studies were performed on paraffin-embedded Hec1-SP tumor tissues. Tissue sections were deparaffinized and rehydrated according to standard protocols. After washing with distilled water, the slides were baked in a high-pressure cooker with distilled water for 3 minutes. After washing with distilled water, the slides were digested in pepsin at 37°C for 30 minutes, washed with distilled water, dehydrated with ethanol, and air-dried for 10 minutes, followed by 50°C oven for 10 minutes. A total of 1.4 μl of the FISH probes (DEP X Spectrum Orange, Vysis, Downers Grove, IL; Mouse Pan-centromeric Chromosome Paint, Cambio, Cambridge, UK) was applied onto the slides, and denaturation was performed at 80°C for 30 minutes. The hybridization was performed at 37°C in a humid chamber for 2 days. The excess of the probes was washed with 0.4× standard saline citrate/0.3% IGEPAL at 72°C for 2 minutes, followed by 2× standard saline citrate/0.1% IGEPAL at room temperature for 1 minute and 2× standard saline citrate for 5 minutes. The slides were mounted with antifade solution with 4′,6-diamidino-2-phenyl-indole and checked under fluorescence microscopy. Data are represented as the means ± SEM and were analyzed with Student's t-test. A value of P < 0.05 was considered statistically significant. We first analyzed primary endometrial cancer cells freshly isolated from endometrial cancer tissues after 7 days of cultivation (n = 7; Table 1) and a human endometrial cancer cell line, Hec1, by FACS. SP cells were present in both cells (0.20 ± 0.09% in primary endometrial cancer cells from seven cases and 0.63 ± 0.55% in Hec1 cells from 10 independent experiments). Verapamil blocked the dye efflux, increased staining, and made the SP cells undetectable by FACS (Figure 1A). Next, both Hec1-SP cells and -NSP cells were cultured for 2 weeks, stained with Hoechst 33342, and then reanalyzed by FACS. Hec1-SP cell cultures generated both SP and NSP subpopulations. In contrast, NSP cell cultures produced NSP cells but never produced SP cells (Figure 1B).Table 1Patient CharacteristicsCase no.Age (yr)Histology (grade)Stage152Endometrioid (G1)Ia234Endometrioid (G1)Ia356Endometrioid (G1)IIIc440Endometrioid (G1)IIIc572Endometrioid (G2)IV661Endometrioid (G2)Ic777SerousIV Open table in a new tab We previously demonstrated that CD9 is expressed in glandular cells, and CD13 is expressed in both glandular and stromal cells that was stained more intensely in stromal cells than that in glandular cells in the human endometrium.16Kato K Yoshimoto M Kato K Adachi S Yamayoshi A Arima T Asanoma K Kyo S Nakahata T Wake N Characterization of side-population cells in human normal endometrium.Hum Reprod. 2007; 22: 1214-1223Crossref PubMed Scopus (176) Google Scholar Most previously characterized SP cells from human endometrium are negative for both CD9 and CD13.16Kato K Yoshimoto M Kato K Adachi S Yamayoshi A Arima T Asanoma K Kyo S Nakahata T Wake N Characterization of side-population cells in human normal endometrium.Hum Reprod. 2007; 22: 1214-1223Crossref PubMed Scopus (176) Google Scholar We investigated whether this was also the case in the endometrial cancer cell lines by analyzing the expression profiles of CD9 and CD13 in SP and NSP cells isolated from Hec1 cells by immunohistochemistry following 3 days of culture. The expression levels of both CD9 and CD13 were lower in SP cells than in NSP cells (Figure 1C). SP cells and NSP cells derived from Hec1 cells were cultured on collagen-coated plates in mesenchymal stem cell maintenance medium (MF medium). Cell growth rate was analyzed for 2 months. Both cell types grew after 2 weeks of culture. SP cells lost contact inhibition, continued to divide for 2 months, and accumulated in colonies atop the confluent cell layer. In contrast, NSP cells stopped growing following rapid growth for 2 weeks cultures and the total cell number decreased. Finally, the cells became flat and enlarged after 2 months (Figure 2A). These results show that SP cells, but not NSP cells, maintain the capacity for long-term proliferating capacity of the cell culture. When Hec1-SP cells were plated in 24-well collagen-coated dishes (50 cells/cm2), they proliferated and formed colonies (Figure 2Ba). Hec1-NSP cells plated in a similar fashion. The cells were more loosely arranged and did not form well-separated individual colonies (Figure 2Ba). To test the self-renewal capability of SP cells within each colony, we dissociated the primary colonies into single cells and then cultured these cells in 100-mm collagen-coated dishes (300 cells/cm2). A single cell formed a secondary colony (Figure 2B, b and c). The secondary colonies were reseeded in a similar manner to generate tertiary colonies. We showed the stained secondary cloning plates. SP cells produced two types of colonies, large densely packed and small colonies (Figure 2C, upper panel). Large colonies were picked up for the serial cloning. On average, 520 secondary colonies were formed per 2 × 104 seeded single cells (2.6%), and an average of 390 tertiary colonies were formed per 2 × 104 seeded single cells from the secondary colonies (1.9%) (Figure 2C, lower panel). This indicates that the colony-forming cells isolated from existing colonies retain the same colony-forming potential and self-renewal capability of the primary SP cells. NSP cells also produced secondary clones, but there was much overlap. They did not form well-" @default.
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• W2063660107 date "2010-01-01" @default.
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• W2063660107 title "Endometrial Cancer Side-Population Cells Show Prominent Migration and Have a Potential to Differentiate into the Mesenchymal Cell Lineage" @default.
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