Profiles of Cancer Stem Cell Subpopulations in Cholangiocarcinomas
2015; Elsevier BV; Volume: 185; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2015.02.010
ISSN1525-2191
AutoresVincenzo Cardinale, Anastasia Renzi, Guido Carpino, A. Torrice, Maria Consiglia Bragazzi, Felice Giuliante, Agostino Maria De Rose, A. Fraveto, Paolo Onori, Chiara Napoletano, Antonio Franchitto, Alfredo Cantàfora, Gian Luca Grazi, Nicola Caporaso, Giuseppe D’Argenio, Gianfranco Alpini, Lola M. Reid, Eugenio Gaudio, Domenico Alvaro,
Tópico(s)Cancer Diagnosis and Treatment
ResumoCholangiocarcinomas (CCAs) comprise a mucin-secreting form, intrahepatic or perihilar, and a mixed form located peripherally. We characterized cancer stem cells (CSCs) in CCA subtypes and evaluated their cancerogenic potential. CSC markers were investigated in 25 human CCAs in primary cultures and established cell lines. Tumorigenic potential was evaluated in vitro or in xenografted mice after s.c. or intrahepatic injection in normal and cirrhotic (carbon tetrachloride-induced) mice. CSCs comprised more than 30% of the tumor mass. Although the CSC profile was similar between mucin-intrahepatic and mucin-perihilar subtypes, CD13+ CSCs characterized mixed-intrahepatic, whereas LGR5+ characterized mucin-CCA subtypes. Many neoplastic cells expressed epithelial-mesenchymal transition markers and coexpressed mesenchymal and epithelial markers. In primary cultures, epithelial-mesenchymal transition markers, mesenchymal markers (vimentin, CD90), and CD13 largely predominated over epithelial markers (CD133, EpCAM, and LGR5). In vitro, CSCs expressing epithelial markers formed a higher number of spheroids than CD13+ or CD90+ CSCs. In s.c. tumor xenografts, tumors dominated by stromal markers were formed primarily by CD90+ and CD13+ cells. By contrast, in intrahepatic xenografts in cirrhotic livers, tumors were dominated by epithelial traits reproducing the original human CCAs. In conclusion, CSCs were rich in human CCAs, implicating CCAs as stem cell–based diseases. CSC subpopulations generate different types of cancers depending on the microenvironment. Remarkably, CSCs reproduce the original human CCAs when injected into cirrhotic livers. Cholangiocarcinomas (CCAs) comprise a mucin-secreting form, intrahepatic or perihilar, and a mixed form located peripherally. We characterized cancer stem cells (CSCs) in CCA subtypes and evaluated their cancerogenic potential. CSC markers were investigated in 25 human CCAs in primary cultures and established cell lines. Tumorigenic potential was evaluated in vitro or in xenografted mice after s.c. or intrahepatic injection in normal and cirrhotic (carbon tetrachloride-induced) mice. CSCs comprised more than 30% of the tumor mass. Although the CSC profile was similar between mucin-intrahepatic and mucin-perihilar subtypes, CD13+ CSCs characterized mixed-intrahepatic, whereas LGR5+ characterized mucin-CCA subtypes. Many neoplastic cells expressed epithelial-mesenchymal transition markers and coexpressed mesenchymal and epithelial markers. In primary cultures, epithelial-mesenchymal transition markers, mesenchymal markers (vimentin, CD90), and CD13 largely predominated over epithelial markers (CD133, EpCAM, and LGR5). In vitro, CSCs expressing epithelial markers formed a higher number of spheroids than CD13+ or CD90+ CSCs. In s.c. tumor xenografts, tumors dominated by stromal markers were formed primarily by CD90+ and CD13+ cells. By contrast, in intrahepatic xenografts in cirrhotic livers, tumors were dominated by epithelial traits reproducing the original human CCAs. In conclusion, CSCs were rich in human CCAs, implicating CCAs as stem cell–based diseases. CSC subpopulations generate different types of cancers depending on the microenvironment. Remarkably, CSCs reproduce the original human CCAs when injected into cirrhotic livers. Cholangiocarcinoma (CCA) is the second most common primary hepatic malignancy and arises from the neoplastic transformation of cells in the cholangiocytic lineage.1Rizvi S. Gores G.J. Pathogenesis, diagnosis, and management of cholangiocarcinoma.Gastroenterology. 2013; 145: 1215-1229Abstract Full Text Full Text PDF PubMed Scopus (858) Google Scholar CCA is associated with a very bad prognosis with virtually no response to current chemotherapeutics or radiation therapies.1Rizvi S. Gores G.J. Pathogenesis, diagnosis, and management of cholangiocarcinoma.Gastroenterology. 2013; 145: 1215-1229Abstract Full Text Full Text PDF PubMed Scopus (858) Google Scholar CCA is classified as intrahepatic (IHCCA), perihilar (pCCA), or distal, characterized by significant differences in terms of epidemiology, pathobiology, and molecular biology.1Rizvi S. Gores G.J. Pathogenesis, diagnosis, and management of cholangiocarcinoma.Gastroenterology. 2013; 145: 1215-1229Abstract Full Text Full Text PDF PubMed Scopus (858) Google Scholar Recent studies reveal that IHCCA comprises two different forms: mucin-IHCCA constituted by pure mucin-secreting cells and displaying similarities with pCCA, and mixed-IHCCA comprising areas of hepatocytic differentiation and neoplastic ductular reaction.2Komuta M. Govaere O. Vandecaveye V. Akiba J. Van Steenbergen W. Verslype C. Laleman W. Pirenne J. Aerts R. Yano H. Nevens F. Topal B. Roskams T. Histological diversity in cholangiocellular carcinoma reflects the different cholangiocyte phenotypes.Hepatology. 2012; 55: 1876-1888Crossref PubMed Scopus (233) Google Scholar The cancer stem cell (CSC) hypothesis has been validated recently by the identification of a subpopulation of self-renewing stem cells that give rise to maturational lineages with a hierarchical organization and are able to divide symmetrically and asymmetrically to generate the tumor mass.3Yamashita T. Wang X.W. Cancer stem cells in the development of liver cancer.J Clin Invest. 2013; 123: 1911-1918Crossref PubMed Scopus (399) Google Scholar, 4Magee J.A. Piskounova E. Morrison S.J. Cancer stem cells: impact, heterogeneity, and uncertainty.Cancer Cell. 2012; 21: 283-296Abstract Full Text Full Text PDF PubMed Scopus (892) Google Scholar CSCs, also referred to as tumor-initiating cells or tumor-propagating cells, are tumorigenic, metastatic, resistant to chemoradio therapies, and responsible for tumor recurrence.3Yamashita T. Wang X.W. Cancer stem cells in the development of liver cancer.J Clin Invest. 2013; 123: 1911-1918Crossref PubMed Scopus (399) Google Scholar, 4Magee J.A. Piskounova E. Morrison S.J. Cancer stem cells: impact, heterogeneity, and uncertainty.Cancer Cell. 2012; 21: 283-296Abstract Full Text Full Text PDF PubMed Scopus (892) Google Scholar For all these reasons, CSCs represent a primary therapeutic target.3Yamashita T. Wang X.W. Cancer stem cells in the development of liver cancer.J Clin Invest. 2013; 123: 1911-1918Crossref PubMed Scopus (399) Google Scholar, 4Magee J.A. Piskounova E. Morrison S.J. Cancer stem cells: impact, heterogeneity, and uncertainty.Cancer Cell. 2012; 21: 283-296Abstract Full Text Full Text PDF PubMed Scopus (892) Google Scholar Recently, several CSC markers have been reported in human CCA, including CD133,5Leelawat K. Thongtawee T. Narong S. Subwongcharoen S. Treepongkaruna S.A. Strong expression of CD133 is associated with increased cholangiocarcinoma progression.World J Gastroenterol. 2011; 17: 1192-1198Crossref PubMed Scopus (28) Google Scholar epithelial cell adhesion molecule (EpCAM),6Sulpice L. Rayar M. Turlin B. Boucher E. Bellaud P. Desille M. Meunier B. Clément B. Boudjema K. Coulouarn C. Epithelial cell adhesion molecule is a prognosis marker for intrahepatic cholangiocarcinoma.J Surg Res. 2014; 192: 117-123Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar CD44,7Gu M.J. Jang B.I. Clinicopathologic significance of Sox2, CD44 and CD44v6 expression in intrahepatic cholangiocarcinoma.Pathol Oncol Res. 2014; 20: 655-660Crossref PubMed Scopus (26) Google Scholar CD13,8Haraguchi N. Ishii H. Mimori K. Tanaka F. Ohkuma M. Kim H.M. kita H. Takiuchi D. Hatano H. Nagano H. Barnard G.F. Doki Y. Mori M. CD13 is a therapeutic target in human liver cancer stem cells.J Clin Invest. 2010; 120: 3326-3339Crossref PubMed Scopus (492) Google Scholar and CD90.9Sukowati C.H. Anfuso B. Torre G. Francalanci P. Crocè L.S. Tiribelli C. The expression of CD90/Thy-1 in hepatocellular carcinoma: an in vivo and in vitro study.PLoS One. 2013; 8: e76830Crossref PubMed Scopus (67) Google Scholar In addition, most cells in human CCAs have been demonstrated to coexpress cytokeratin (K)19 and albumin, a feature characterizing hepatobiliary stem/progenitor cells.10Thanan R. Pairojkul C. Pinlaor S. Khuntikeo N. Wongkham C. Sripa B. Ma N. Vaeteewoottacharn K. Furukawa A. Kobayashi H. Hiraku Y. Oikawa S. Kawanishi S. Yongvanit P. Murata M. Inflammation-related DNA damage and expression of CD133 and Oct3/4 in cholangiocarcinoma patients with poor prognosis.Free Radic Biol Med. 2013; 65: 1464-1472Crossref PubMed Scopus (47) Google Scholar Recent reports support further investigations on the role of CSCs in CCA. Unfortunately, very little information exists with respect to CSCs in CCA and its subtypes. Our aim was to analyze CSCs in different human CCA subtypes, primary cultures obtained from human CCA, and established CCA cell lines. Reagents were purchased from Sigma Chemical (St. Louis, MO) unless otherwise indicated. Multiple established human cell lines of IH- and pCCA were used: CCLP-1 and HUCCT-1, derived from IHCCA; HuH-28, derived from IHCCA; TFK-1, derived from pCCA; and Mz-ChA1, derived from a gallbladder cancer. CCLP-1 and HUCCT-1 cell lines were obtained from Dr. Anthony J. Demetris (University of Pittsburgh, Pittsburgh, PA). HuH-28 cells were acquired from the Cancer Cell Repository (Tohoku University, Sendai, Japan). TFK-1 cells were kindly provided by Dr. Yoshiyuki Ueno (the Cancer Cell Repository, Tohoku University, Sendai, Japan). Mz-ChA1 cells were a gift from Dr. J. Gregory Fitz (University of Texas Southwestern, Dallas, TX). HuH-28 cells and Mz-ChA-1 cells were maintained in CMRL 1066 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin-glutamine (Gibco/BRL; Life Technologies, Italia srl., Milan, Italy). TFK-1 cells and HUCCT-1 cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin-glutamine (Gibco/BRL; Life Technologies). CCLPI cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin-glutamine (Gibco/BRL; Life Technologies). All human cell lines were maintained at 37°C in a humidified atmosphere of 5% CO2 in air. Samples of CCA and peritumoral noncancerous liver were obtained from patients submitted to surgical resection, specifically: 17 patients (eight women, aged 50 to 83 years and nine men, aged 57 to 75 years) with IHCCA presenting as a single mass lesion within the liver; eight patients affected by pCCA (Klatskin tumor), (five men, three women, aged 66 to 83 years). Distinctions between IHCCA or pCCA were based on clinical records including surgery reports.1Rizvi S. Gores G.J. Pathogenesis, diagnosis, and management of cholangiocarcinoma.Gastroenterology. 2013; 145: 1215-1229Abstract Full Text Full Text PDF PubMed Scopus (858) Google Scholar Specimens were fixed in 10% buffered formalin for 2 to 4 hours, embedded in low-temperature-fusion paraffin (55°C to 57°C), and 3- to 4-μm sections were stained with H&E, periodic acid-Schiff (PAS) staining system, Sirius Red/Fast Green, according to standard protocols. For immunohistochemistry (IHC), endogenous peroxidase activity was blocked by 30 minutes incubation in methanolic hydrogen peroxide (2.5%). Antigens were retrieved by applying Proteinase K (code S3020; Dako, Glostrup, Denmark) for 10 minutes at room temperature in a protocol indicated by the vendor. Sections were then incubated overnight at 4°C with primary antibodies (Table 1). Samples were rinsed twice with phosphate-buffered saline (PBS) for 5 minutes, incubated for 20 minutes at room temperature with secondary biotinylated antibody (LSAB+ System-HRP, code K0690; Dako), and then incubated with streptavidin–horseradish peroxidase (LSAB+ System-HRP, code K0690; Dako). Diaminobenzidine (Dako) was used as substrate, and sections were counterstained with hematoxylin. For all immunoreactions, negative controls (the primary antibody was replaced with preimmune serum) were also included. Table 1 shows the details of antibodies used in the study.Table 1List of Antibodies Used for IHC and IFNameHost/isotypeSourceCatalog#DilutionCD326/EpCAMMouse IgG1Santa Cruz Biotechnology (Dallas, TX)sc-597821:50K7 (cytokeratin 7)Mouse IgG1DakoM70181:100K19 (cytokeratin 19)Mouse IgG1Abcam (Cambridge, UK)ab870141:50K19 (cytokeratin 19)Mouse IgG1DakoM08881:100HepPar-1Mouse IgG1DakoM71581:50CD133/Prominin 1Rabbit IgGAbnova (Taipei, Taiwan)PAB126631:100CD133/PROM1Mouse IgG1OriGene (Unimed Scientifica, Rome, Italy)TA3099431:50CD90/Thy1Rabbit IgGAbcamab925741:100CD13Mouse IgG1Novacastra Reagents (Leica Biosystems, Buffalo Grove, IL)NCL-CD13-3041:100LGR5Goat IgGSanta Cruz BiotechnologySC-685801:50DesminMouse IgG1DakoM07601:100VimentinMouse IgG1Santa Cruz Biotechnologysc-323221:100NestinMouse IgG1Santa Cruz Biotechnologysc-239271:100α-SMAMouse IgG1DakoM08511:50S100A4Rabbit IgGDakoA51141:100SNAILRabbit IgGSanta Cruz Biotechnologysc-281991:50TWISTRabbit IgGSanta Cruz Biotechnologysc-153931:50LGR5Rabbit IgGOriGeneTA3013231:50NCAM-PEMouse IgG1BD Pharmingen (Milan, Italy)555.5161:50E-cadherinMouse IgG1Santa Cruz Biotechnologysc-217911:50P-cadherinRabbit IgGSanta Cruz Biotechnologysc-78931:50GFAPMouse IgG1DakoM07611:50CD163Mouse IgG1OriGeneTA5063821:50CD31Mouse IgG1DakoM08231:50CD13Mouse IgG1AbcamAb74171:100SDF1Rabbit IgGSanta Cruz Biotechnologysc-288761:50FAPMouse IgG1Santa Cruz Biotechnologysc-653981:50PeriostinGoat IgGSanta Cruz Biotechnologysc-494801:50PeriostinRabbit IgGSanta Cruz Biotechnologysc-672331:50CD90-FITCHumanMiltenyi Biotec (Cologne, Germany)130-095-4031:10CD326 (EPCAM)-FITCHumanMiltenyi Biotec130-080-3011:10Goat anti-rabbit FITCIgGAbcamab-67171:400Goat anti-rabbit TRITCIgGAbcamab-67181:400Goat anti-mouse FITCIgGAbcamab-67851:400Goat anti-mouse TRITCIgGAbcamab-67861:400 Open table in a new tab Sections were examined in a coded fashion by Leica Microsystems DM 4500 B Light and Fluorescence Microscopy (Leica Microsystems, Weltzlar, Germany) equipped with a Jenoptik ProgRes C10 Plus Videocam (Jenoptik, Jena, Germany). Light microscopy and observations were processed with an Image Analysis System (Delta Sistemi, Roma, Italy) and were independently performed by two researchers in a blinded fashion (A.R. and G.C.). For IHC staining, the number of positive cells was counted in a random, blinded fashion in six nonoverlapping fields (magnification ×20) for each slide and semiquantitatively scored as previously (0, <1%; 1, 1% to 5%; 2, 6% to 30%; 3, 31% to 50%; and 4 = >50%).11Carpino G. Cardinale V. Onori P. Franchitto A. Berloco P.B. Rossi M. Wang Y. Semeraro R. Anceschi M. Brunelli R. Alvaro D. Reid L.M. Gaudio E. Biliary tree stem/progenitor cells in glands of extrahepatic and intrahepatic bile ducts: an anatomical in situ study yielding evidence of maturational lineages.J Anat. 2012; 220: 186-199Crossref PubMed Scopus (168) Google Scholar Necrosis and stromal areas were evaluated, respectively, in hematoxylin and eosin and Sirius Red stains. Necrotic areas were measured by an Image Analysis System (Delta Sistemi), and the total area occupied by necrosis was calculated and expressed as the percentage with respect to the total area.11Carpino G. Cardinale V. Onori P. Franchitto A. Berloco P.B. Rossi M. Wang Y. Semeraro R. Anceschi M. Brunelli R. Alvaro D. Reid L.M. Gaudio E. Biliary tree stem/progenitor cells in glands of extrahepatic and intrahepatic bile ducts: an anatomical in situ study yielding evidence of maturational lineages.J Anat. 2012; 220: 186-199Crossref PubMed Scopus (168) Google Scholar Sirius Red–stained slides were scanned by a digital scanner (Aperio Scanscope CS System; Aperio Technologies, Oxford, UK) processed by ImageScope. An image analysis algorithm was used to quantify the proportion of picrosirius-stained area. The algorithm was applied to the entire sections of at least three different slides from each specimen. The extent of stromal area was expressed as the proportion (%) of picrosirius-stained area with respect to the total area. CCA samples were subjected to mechanical and enzymatic dissociation. In detail, tumor specimens were transferred into fresh, sterile Dulbeccos’ PBS in a 9-cm Petri dish and rinsed. The tissue was transferred to a second dish and minced in a small volume of DMEM high glucose/DMEM:F12 mixture (1:1, growth medium, Gibco/BRL; Life Technologies) supplemented with 1.8 × 10−4 mol/L adenine, 5 μg/mL insulin, 5 μg/mL transferrin, 2 × 10−9 mol/L triiodothyronine, 1.7 × 10−6 mol/L hydrocortisone, 1.0 × 10−6 mg/L human epidermal growth factor, 5.5 × 10−6 mg/L epinephrine (Sigma-Aldrich, Milan, Italy), 10% fetal bovine serum (Gibco/BRL; Life Technologies), 100 U/mL penicillin, and 100 μg/mL streptomycin. The tissue was minced with surgical scalpels into fragments of approximately 1 mm.3Yamashita T. Wang X.W. Cancer stem cells in the development of liver cancer.J Clin Invest. 2013; 123: 1911-1918Crossref PubMed Scopus (399) Google Scholar After cutting, the tissue was washed with Dulbeccos’ PBS by bench centrifugation (300 × g for 3 minutes) and resuspended. After washing, fragments were transferred into digestion solution (growth medium with 1 mg/mL collagenase type IV, 0.1 mg/mL hyaluronidase, and 0.1 mg/mL DNase), and incubated for 12 to 16 hours at 37°C in a humidified atmosphere of 5% CO2 in air. The effective disaggregation was checked by gently moving the dish; single cells and small clusters were dispersed by gentle pipetting. The cell suspension was filtered with a 100-μm cell strainer placed on a 50-mL tube. The cell strainer was washed with 5 mL of growth medium. The cell suspension was then filtered with a 70-μm cell strainer placed on a 50-mL tube. The cell strainer was washed with 5 mL of growth medium. The cell suspension was then centrifuged at 300 × g for 10 minutes, and the supernatant was discarded. To remove erythrocytes or dead cells, sterile water for injection was used. The cells were resuspended in growth medium and placed into 6-well dish at 37°C in a humidified atmosphere of 5% CO2 in air. When the cells in the dish were expanded, they were transferred into a fresh growth medium supplemented with 10% fetal bovine serum (Gibco/BRL; Life Technologies), 100 U/mL penicillin, and 100 μg/mL streptomycin. For IHC and immunofluorescence (IF), the medium of the CCA cell cultures was removed, and cells were fixed in 4% paraformaldehyde solution for 10 minutes at room temperature. Cells were rinsed twice with PBS buffer for 2 minutes, blocked, and then incubated 1 hour with the primary antibody at room temperature. After rinsing twice with PBS for 2 minutes, cells were incubated for 40 minutes at room temperature with secondary biotinylated antibody (Vector Laboratories, Milan, Italy), rinsed twice with PBS, and then incubated with Vectastain ABC reagent (Vector Laboratories) for 20 minutes. Diaminobenzidine (DAB substrate kit; Vector Laboratories) was used as substrate, and sections were counterstained with hematoxylin. Slides were examined by DM 2000 Light and/or Fluorescence Microscopy (Leica Microsystems) equipped with a DFC450 C Videocam (Leica Microsystems). Table 1 shows the details of antibodies used in the study. Human CCA cell cultures were extracted for total RNA by using TRI Reagent (Sigma-Aldrich, St. Louis, MO) and 1-bromo-3-chloropropane in substitution of chloroform, according to the procedure of Chomczynski and Sacchi.12Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63169) Google Scholar The isolated RNA was dissolved in 55 μL of RNase-free water. RNA quality and quantity was controlled by the Experion Automated Electrophoresis System equipped with the RNA StSens Analysis Chip (Bio-Rad Laboratories, Hercules, CA). The reverse transcription primed by the random hexamer (Invitrogen s.r.l., S. Giuliano Milanese, Italy) was conducted in a 20-μL volume with an amount of 2.5 μg of total RNA and the M-MLV reverse transcriptase (Invitrogen s.r.l.) according to the manufacturer’s directions. Gene expression was determined by real-time PCR with a MX3000P instrument (Agilent, La Jolla, CA) using the averaged cycle threshold (CT) automatically computed by the built-in software from three replicas of each sample. PCR amplifications were conducted into a volume of 25 μL, with 1.0 μL of cDNA template, 12.5 μL of 2× SYBR Green Brilliant QPCR Master Mix (Stratagene, La Jolla, CA), 3 pmoles each of upstream and downstream primer for the gene analyzed, and 0.3 μL of diluted reference dye (ROX at a final concentration 30 nmol/L). All real-time PCR amplifications were conducted with the cycling program: 10 minutes at 95°C followed by 40 cycles (30 seconds at 95°C, 30 seconds at 58°C, 30 seconds at 72°C). The fluorescence detection was performed during the extension step of each cycle. The following genes of interest were measured: CD13 (ANPEP), CD44, CD90 (THY1), CD133 (PROM1), VIM, NANOG, OCT4 (POU5F1), SOX2, SALL4, NIS (SLC5A5, sodium/iodide symporter), E-cadherin (CDH1), P-cadherin (CDH3), EPCAM, and LGR5. All expression levels were normalized to the expression of GAPDH housekeeping gene. Table 2 shows the details of primers used in the study (ProbeFinder software version 2.50; Roche, Mannheim, Germany).13Turner R.A. Mendel G. Wauthier E. Barbier C. Reid L.M. Hyaluronan-supplemented buffers preserve adhesion mechanisms facilitating cryopreservation of human hepatic stem/progenitor cells.Cell Transplant. 2012; 21: 2257-2266Crossref PubMed Scopus (21) Google Scholar, 14Walker F. Zhang H.H. Odorizzi A. Burgess A.W. LGR5 is a negative regulator of tumourigenicity, antagonizes Wnt signalling and regulates cell adhesion in colorectal cancer cell lines.PLoS One. 2011; 6: e22733Crossref PubMed Scopus (114) Google Scholar, 15Oikawa T. Kamiya A. Zeniya M. Chikada H. Hyuck A.D. Yamazaki Y. Wauthier E. Tajiri H. Miller L.D. Wang X.W. Reid L.M. Nakauchi H. Sal-like protein 4 (SALL4), a stem cell biomarker in liver cancers.Hepatology. 2013; 57: 1469-1483Crossref PubMed Scopus (161) Google Scholar, 16Li H. Richard K. McKinnon B. Mortimer R.H. Effect of iodide on human choriogonadotropin, sodium-iodide symporter expression, and iodide uptake in BeWo choriocarcinoma cells.J Clin Endocrinol Metab. 2007; 92: 4046-4051Crossref PubMed Scopus (22) Google ScholarTable 2Sequences of Primer Pairs (Sense and Antisense, Respectively) Used for Amplifying the Genes of Interest and the Internal Reference Gene (GAPDH) Used for Their NormalizationGene MessengerAccession No.Sequences of primer pairsLength (nt)Amplicon (bp)SourceGAPDHNM_0020465′-AGCCACATCGCTCAGACAC-3′5′-GCCCAATACGACCAAATCC-3′191966∗ProbeFinder software, Roche, Mannheim, Germany.CD13NM_0011505′-CAGTGACACGACGATTCTCC-3′5′-CCTGTTTCCTCGTTGTCCTT-3′202076∗ProbeFinder software, Roche, Mannheim, Germany.CD44NM_0006105′-TGCCGCTTTGCAGGTGTAT-3′5′-GGCCTCCGTCCGAGAGA-3′19176513Turner R.A. Mendel G. Wauthier E. Barbier C. Reid L.M. Hyaluronan-supplemented buffers preserve adhesion mechanisms facilitating cryopreservation of human hepatic stem/progenitor cells.Cell Transplant. 2012; 21: 2257-2266Crossref PubMed Scopus (21) Google ScholarCD90NM_0062885′-AGGACGAGGGCACCTACAC-3′5′-GCCCTCACACTTGACCAGTT-3′1920107∗ProbeFinder software, Roche, Mannheim, Germany.CD133NM_0023545′-CCTGGGGCTGCTGTTTATTA-3′5′-ATCACCAACAGGGAGATTGC-3′2020161∗ProbeFinder software, Roche, Mannheim, Germany.EpCamNM_0023545′-ATAACCTGCTCTGAGCGAGTG-3′5′-TGAAGTGCAGTCCGCAAACT-3′212010414Walker F. Zhang H.H. Odorizzi A. Burgess A.W. LGR5 is a negative regulator of tumourigenicity, antagonizes Wnt signalling and regulates cell adhesion in colorectal cancer cell lines.PLoS One. 2011; 6: e22733Crossref PubMed Scopus (114) Google ScholarVIMENTINNM_0033805′-CTGCCAACCGGAACAATGA-3′5′-GTACTCAGTGGACTCCTGCTTT-3′19225614Walker F. Zhang H.H. Odorizzi A. Burgess A.W. LGR5 is a negative regulator of tumourigenicity, antagonizes Wnt signalling and regulates cell adhesion in colorectal cancer cell lines.PLoS One. 2011; 6: e22733Crossref PubMed Scopus (114) Google ScholarLGR5NM_0036675′-CTTCCAACCTCAGCGTCTTC-3′5′-TTTCCCGCAAGACGTAACTC-3′202011814Walker F. Zhang H.H. Odorizzi A. Burgess A.W. LGR5 is a negative regulator of tumourigenicity, antagonizes Wnt signalling and regulates cell adhesion in colorectal cancer cell lines.PLoS One. 2011; 6: e22733Crossref PubMed Scopus (114) Google ScholarNANOGNM_0248655′-AGATGCCTCACACGGAGACT-3′5′-TTTGCGACACTCTTCTCTGC-3′2020127∗ProbeFinder software, Roche, Mannheim, Germany.OCT4NM_0027015′-TCGAGAACCGAGTGAGAGG-3′5′-GAACCACACTCGGACCACA-3′1919125∗ProbeFinder software, Roche, Mannheim, Germany.SOX2NM_0031065′-TCGAGAACCGAGTGAGAGG-3′5′-GCAAAGCTCCTACCGTACCA-3′192085∗ProbeFinder software, Roche, Mannheim, Germany.SALL4NM_02,043.35′-CGCCCGTGTGTCATGTAGTGAAC-3′5′-TCCGAGAACAGCCGCACTGAGATGGAAG-3′242812315Oikawa T. Kamiya A. Zeniya M. Chikada H. Hyuck A.D. Yamazaki Y. Wauthier E. Tajiri H. Miller L.D. Wang X.W. Reid L.M. Nakauchi H. Sal-like protein 4 (SALL4), a stem cell biomarker in liver cancers.Hepatology. 2013; 57: 1469-1483Crossref PubMed Scopus (161) Google ScholarNISNM_000453.25′-TGCGGGACTTTGCAGTACATT-3′5′-TGCAGATAATTCCGGTGGACA-3′212113316Li H. Richard K. McKinnon B. Mortimer R.H. Effect of iodide on human choriogonadotropin, sodium-iodide symporter expression, and iodide uptake in BeWo choriocarcinoma cells.J Clin Endocrinol Metab. 2007; 92: 4046-4051Crossref PubMed Scopus (22) Google ScholarCDH1NM_0043605′-TCACAGTCACTGACACCAACGA-3′5′-GGCACCTGACCCTTGTAGTACGT-3′222067∗ProbeFinder software, Roche, Mannheim, Germany.CDH3NM_001793.45′-GGAGGTTTCATCCTCCCTGT-3′5′-TCCGCTTCTTTCTCACCAAC-3′202088∗ProbeFinder software, Roche, Mannheim, Germany.∗ ProbeFinder software, Roche, Mannheim, Germany. Open table in a new tab Primary CCA cell cultures were characterized at cell culture passages 2 to 3 and 20 to 30 by using the following antibodies: PE-mouse anti-human CD13 (BD Pharmingen, Milan, Italy), CD90-FITC human, CD133-APC human, CD44-APC human, CD45-PE human, EpCAM-FITC human (Miltenyi Biotec, Milan, Italy), anti-LGR5 mouse monoclonal antibody PE conjugate (OriGene, Unimed Scientifica, Rome, Italy). The fluorescence threshold between negative and positive cells was set on the basis of the reactivity of appropriate nonspecific fluorochrome-conjugated isotypical controls. At least 5 × 105 cells were analyzed using a FACSDiva software version (BD). For magnetic cell sorting, cells were labeled with CD133 (AC133), CD326 (EpCAM), and CD90 MicroBeads, and sorted using the Miltenyi Biotec Cell Isolation Kit, according to the manufacturer’s instruction. For magnetic cell sorting of CD13+ and leucine-rich repeat-containing G-protein coupled receptor 5-positive (LGR5+) cells, primary CCA cells were labeled with PE-mouse anti-human CD13 (BD Pharmingen), and anti-Lgr5 mouse monoclonal antibody PE conjugate (OriGene, Unimed Scientifica). They were then labeled with anti-PE MicroBeads and sorted using the Miltenyi Biotec Cell Isolation Kit, according to the manufacturer’s instruction. Isotype-matched mouse immunoglobulins served as controls. For spheroid formation, primary CCA cell cultures at passages >20 were labeled with CD133 (AC133), CD326 (EpCAM), and CD90 MicroBeads, and sorted using the Miltenyi Biotec Cell Isolation Kit, according to the manufacturer’s instruction. For magnetic cell sorting of CD13+ and LGR5+ cells, primary CCA cells were labeled with PE-mouse anti-human CD13 (BD Pharmingen) and anti-Lgr5 mouse monoclonal antibody PE conjugate (OriGene, Unimed Scientifica). They were then labeled with anti-PE MicroBeads and sorted using the Miltenyi Biotec Cell Isolation Kit, according to the manufacturer’s instruction. Two thousand sorted cells (CD90+/CD90−, CD13+/CD13−, CD133+/CD133−, LGR5+/LGR5−; EPCAM+/EPCAM−) were cultured in a serum-free medium of DMEM with high glucose/DMEM:F12 mixture (1:1) (Gibco/BRL; Life Technologies) supplemented with 20 ng/mL EGF, 10 ng/mL FGF-2, and 1× B27 (Gibco/BRL; Life Technologies) into each well of 6-well Ultra-Low Attachment plates (Corning, Lowell, MA). After 7 days, spheres were visualized, counted, and sized using light microscopy. For IF experiments, spheres were incubated with human monoclonal CD326 (EpCAM)–FITC conjugated (1:10; Miltenyi Biotec), human monoclonal CD90-FITC conjugated (1:10; Miltenyi Biotec), human monoclonal CD133/2 (293C3)–PE (1:10; Miltenyi Biotec), Anti-LGR5 mouse monoclonal antibody, clone 2A2, PE conjugated (1:50; OriGene), anti-human CD13 mouse PE conjugated (1:10; BD Pharmingen), or vimentin-FITC conjugated antibodies (1:50; Santa Cruz Biotechnology, Dallas, TX) for 15 minutes at 4°C, rinsed with PBS, and then fixed in 4% paraformaldehyde. Anti-SNAIL rabbit polyclonal (1:50; Santa Cruz Biotechnology), anti-TWIST rabbit polyclonal (1:50; Santa Cruz Biotechnology), anti-P-cadherin (1:50; Santa Cruz Biotechnology) antibodies were incubated for 60 minutes at 4°C, then the cells were rinsed with PBS and incubated for 40 minutes at room temperature with goat anti-rabbit TRITC (1:400; Abcam, Cambridge, UK) secondary antibody, rinsed with PBS, and fixed in 4% paraformaldehyde. For all immunoreactions, the cells were counterstained with Vectashield Mounting Medium with DAPI (Vector Laboratories), observed and photographed in a Leica TCS SP2 confocal microscope. Spheroids prepared from CSCs immunosorted from human CCA primary cultures were injected (approximately 10,000 cells) s.c. or in the liver of normal or cirrhotic (carbon tetrachloride-induced) mice. Male NOD/SCID mice, 4 to 6 wk old, purchased from Charles River Laboratories (Wilmington, MA) were maintained under standard conditions and cared according to the institutional guidelines for animal care. Spheroids prepared from CSCs immunosorted from human CCA primary cultures were injected (approximately 10,000 cells) s.c. into the right and left mid-abdominal areas. Tumor x
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