title: A novel method to generate single-cell-derived cancer-associated fibroblast clones.
abstract: BACKGROUND
  Cancer-associated fibroblasts (CAFs) communicate with cancer cells to play important roles in tumor progression. However, CAFs have heterogeneous phenotypes and functions. To understand how much of this heterogeneity relates to different biological responses, a more efficient method of generating single-cell-derived CAF clones is required.
  
  
  METHODS
  We transduced two primary CAF cultures (CAFs-608 and CAFs-621) from lung adenocarcinoma with human telomerase reverse transcriptase (hTERT), mutant forms of cyclin dependent kinase 4 (CDK4R24C) independently and in combination (hTERT/CDK4R24C). After live imaging of each sorted-single cell, we evaluated the numbers of successfully established clones from CAFs-hTERT, CAFs-CDK4R24C, and CAFs-hTERT/CDK4R24C. Furthermore, we examined the expression levels of genes associated with tumor promoting pathways in established clones by qRT-PCR.
  
  
  RESULTS
  Overexpression of hTERT and CDK4R24C efficiently extended the lifespan of both CAFs-608 and CAFs-621. The number of established CAF clones was highest for CAFs-hTERT/CDK4R24C, with 57 and 62 clones established from CAFs-608 and CAFs-621, respectively. Conversely, 16 and 11 CAFs-hTERT clones were derived from CAFs-608 and CAFs-621, respectively and 10 and 8 CAFs-CDK4R24C clones were from CAFs-608 and CAFs-621, respectively. TGF-b, ATCA2, and HSF1 mRNA levels differed in individual clones established from CAFs-hTERT/CDK4R24C. The expression levels of ATCA2 and HSF1 were much higher in one clone than in the other established clones and the parental CAFs.
  
  
  CONCLUSIONS
  Our results show that combined exogenous expression of hTERT and mutant CDK4 is an effective method to generate single-cell-derived CAF clones. This provides an innovative and suitable approach to investigate the heterogeneous function and phenotype of CAFs.
authors.affiliation: Division of Pathology, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Kashiwa, 277-8577, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Hashimoto
authors.affiliation: Division of Pathology, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Kashiwa, 277-8577, Japan.|Laboratory of Cancer Biology, Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan.
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Suda
authors.affiliation: Division of Pathology, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Kashiwa, 277-8577, Japan.|Laboratory of Cancer Biology, Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Miyashita
authors.affiliation: Division of Pathology, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Kashiwa, 277-8577, Japan.|Laboratory of Cancer Biology, Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Ochiai
authors.affiliation: Division of Thoracic Surgery, National Cancer Center Hospital East, Kashiwa, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Tsuboi
authors.affiliation: Division of Cancer Stem Cell, National Cancer Center Research Institute, Tsukiji, Japan.
authors.forename: Kenkichi
authors.initials: K
authors.lastname: Masutomi
authors.affiliation: Division of Carcinogenesis and Cancer Prevention, National Cancer Center Research Institute, Tsukiji, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Kiyono
authors.affiliation: Division of Pathology, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Kashiwa, 277-8577, Japan. gishii@east.ncc.go.jp.|Laboratory of Cancer Biology, Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan. gishii@east.ncc.go.jp.
authors.forename: Genichiro
authors.initials: G
authors.lastname: Ishii
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology; D000077192:Adenocarcinoma of Lung; D000072645:Cancer-Associated Fibroblasts / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation / Q000235:genetics; D002999:Clone Cells / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4 / Q000235:genetics*; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic; D018740:Genetic Heterogeneity; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D009154:Mutation; D019098:Telomerase / Q000235:genetics*
chemical_list: C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; C509186:TERT protein, human; D019098:Telomerase
keywords: CDK4R24C; Cancer associated fibroblast; Live imaging; Single-cell-derived clones; hTERT