title: Identification of a unique hepatocellular carcinoma line, Li-7, with CD13(+) cancer stem cells hierarchy and population change upon its differentiation during culture and effects of sorafenib.
abstract: BACKGROUND
  Cancer stem cell (CSC) research has highlighted the necessity of developing drugs targeting CSCs. We investigated a hepatocellular carcinoma (HCC) cell line that not only has CSC hierarchy but also shows phenotypic changes (population changes) upon differentiation of CSC during culture and can be used for screening drugs targeting CSC.
  
  
  METHODS
  Based on a hypothesis that the CSC proportion should decrease upon its differentiation into progenitors (population change), we tested HCC cell lines (HuH-7, Li-7, PLC/PRF/5, HLF, HLE) before and after 2 months culture for several markers (CD13, EpCAM, CD133, CD44, CD90, CD24, CD166). Tumorigenicity was tested using nude mice. To evaluate the CSC hierarchy, we investigated reconstructivity, proliferation, ALDH activity, spheroid formation, chemosensitivity and microarray analysis of the cell populations sorted by FACS.
  
  
  RESULTS
  Only Li-7 cells showed a population change during culture: the proportion of CD13 positive cells decreased, while that of CD166 positive cells increased. The high tumorigenicity of the Li-7 was lost after the population change. CD13(+)/CD166(-) cells showed slow growth and reconstructed the bulk Li-7 populations composed of CD13(+)/CD166(-), CD13(-)/CD166(-) and CD13(-)/CD166(+) fractions, whereas CD13(-)/CD166(+) cells showed rapid growth but could not reproduce any other population. CD13(+)/CD166(-) cells showed high ALDH activity, spheroid forming ability and resistance to 5-fluorouracil. Microarray analysis demonstrated higher expression of stemness-related genes in CD166(-) than CD166(+) fraction. These results indicated a hierarchy in Li-7 cells, in which CD13(+)/CD166(-) and CD13(-)/CD166(+) cells serve as slow growing CSCs and rapid growing progenitors, respectively. Sorafenib selectively targeted the CD166(-) fraction, including CD13(+) CSCs, which exhibited higher mRNA expression for FGF3 and FGF4, candidate biomarkers for sorafenib. 5-fluorouracil followed by sorafenib inhibited the growth of bulk Li-7 cells more effectively than the reverse sequence or either alone.
  
  
  CONCLUSIONS
  We identified a unique HCC line, Li-7, which not only shows heterogeneity for a CD13(+) CSC hierarchy, but also undergoes a "population change" upon CSC differentiation. Sorafenib targeted the CSC in vitro, supporting the use of this model for screening drugs targeting the CSC. This type of "heterogeneous, unstable" cell line may prove more useful in the CSC era than conventional "homogeneous, stable" cell lines.
authors.affiliation: Division of Gastroenterology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki, 305-8575, Japan. t.yamada718@gmail.com.|Cell Engineering Division, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan. t.yamada718@gmail.com.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yamada
authors.affiliation: Division of Gastroenterology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki, 305-8575, Japan. m-abei@md.tsukuba.ac.jp.
authors.forename: Masato
authors.initials: M
authors.lastname: Abei
authors.affiliation: Functional Evaluation of Genomic Polymorphisms, Tohoku Medical Megabank Organization, Sendai, Japan. inaho.danjoh@megabank.tohoku.ac.jp.
authors.forename: Inaho
authors.initials: I
authors.lastname: Danjoh
authors.affiliation: Cell Engineering Division, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan. ryoko.mail.932102@gmail.com.
authors.forename: Ryoko
authors.initials: R
authors.lastname: Shirota
authors.affiliation: Department of Gastroenterology, Kanazawa University Hospital, 13-1, Takara-machi, Kanazawa, Ishikawa, 920-8641, Japan. taroy@m-kanazawa.jp.
authors.forename: Taro
authors.initials: T
authors.lastname: Yamashita
authors.affiliation: Division of Gastroenterology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki, 305-8575, Japan. ihyodo@md.tsukuba.ac.jp.
authors.forename: Ichinosuke
authors.initials: I
authors.lastname: Hyodo
authors.affiliation: Cell Engineering Division, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan. yukionak@brc.riken.jp.
authors.forename: Yukio
authors.initials: Y
authors.lastname: Nakamura
mesh_terms: D000818:Animals; D018826:CD13 Antigens / Q000096:biosynthesis / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D002454:Cell Differentiation / Q000235:genetics*; D045744:Cell Line, Tumor; D019070:Cell Lineage / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D018740:Genetic Heterogeneity; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D051379:Mice; D014411:Neoplastic Stem Cells; D009536:Niacinamide / Q000008:administration & dosage / Q000031:analogs & derivatives; D010671:Phenylurea Compounds / Q000008:administration & dosage; D000077157:Sorafenib
chemical_list: D010671:Phenylurea Compounds; D009536:Niacinamide; D000077157:Sorafenib; D018826:CD13 Antigens