Journal of Cancer Research and Clinical Oncology

, Volume 143, Issue 8, pp 1409–1419 | Cite as

A novel method to generate single-cell-derived cancer-associated fibroblast clones

  • Hiroko Hashimoto
  • Yoshitaka Suda
  • Tomoyuki Miyashita
  • Atsushi Ochiai
  • Masahiro Tsuboi
  • Kenkichi Masutomi
  • Tohru Kiyono
  • Genichiro IshiiEmail author
Original Article – Cancer Research



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.


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.


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.


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.


Cancer associated fibroblast hTERT CDK4R24C Single-cell-derived clones Live imaging 



This work was supported by the National Cancer Center Research and Development Fund (23-A-12 and 23-K-18), the Princess (Takamatsunomiya Cancer Research Fund), and JSPS KAKENHI (24659185).

Compliance with ethical standards

Conflict of interest

The authors have no conflicts of interest to disclose.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Comprehensive informed consent was obtained in the study.

Supplementary material

432_2017_2409_MOESM1_ESM.tif (15.4 mb)
Supplementary Fig. 1 mRNA expression in CAFs, CAFs-hTERT, CAFs-CDK4R24C and CAFs-hTERT/CDK4R24C measured by qRT-PCR. Vertical axis shows target gene/GAPDH*100. (TIF 15805 KB)
432_2017_2409_MOESM2_ESM.tif (6.3 mb)
Supplementary Fig. 2 Morphological appearance of established clones from CAFs-hTERT and CAFs-CDK4R24C. Immunofluorescence microscopic images of established clones from CAFs-608-hTERT (upper) and CAFs-608-CDK4R24C (lower). (TIF 6482 KB)
432_2017_2409_MOESM3_ESM.tif (30.6 mb)
Supplementary Fig. 3 Morphological appearance of established clones from CAFs-hTERT/CDK4R24C. Immunofluorescence microscopic images of established clones from CAFs-608- hTERT/CDK4R24C. The morphology of each clone is divided into 4 types; 1) cells with a typical fibroblast morphology (clone 10, 18, 32, 39, 46, et al.), 2) cells with a more short spindle shape (clone 3, 19, 29, et al.), 3) cells with a more stellate shape (clone 1, 16, 42, 57, et al.), and 4) cell with a more elongated shape (clone 50, 56, et al.). (TIF 31304 KB)
432_2017_2409_MOESM4_ESM.tif (5.6 mb)
Supplementary Fig. 4 Scatter plot of hTERT/CDK4R24C mRNA levels vs TGFB/ATCA2/HSF1mRNA levels. Spearman’s rank correlation scatter plot of hTERT/CDK4R24C mRNA levels (Y axis) and TGFB/ATCA2/ levels (X axis) in established 14 clones. (TIF 5721 KB)
432_2017_2409_MOESM5_ESM.pdf (92 kb)
Supplementary material 5 (PDF 92 KB)
432_2017_2409_MOESM6_ESM.pdf (21 kb)
Supplementary material 6 (PDF 21 KB)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Hiroko Hashimoto
    • 1
  • Yoshitaka Suda
    • 1
    • 2
  • Tomoyuki Miyashita
    • 1
    • 2
  • Atsushi Ochiai
    • 1
    • 2
  • Masahiro Tsuboi
    • 3
  • Kenkichi Masutomi
    • 4
  • Tohru Kiyono
    • 5
  • Genichiro Ishii
    • 1
    • 2
    Email author
  1. 1.Division of Pathology, Exploratory Oncology Research and Clinical Trial CenterNational Cancer CenterKashiwaJapan
  2. 2.Laboratory of Cancer Biology, Department of Integrated Biosciences, Graduate School of Frontier SciencesThe University of TokyoKashiwaJapan
  3. 3.Division of Thoracic SurgeryNational Cancer Center Hospital EastKashiwaJapan
  4. 4.Division of Cancer Stem CellNational Cancer Center Research InstituteTsukijiJapan
  5. 5.Division of Carcinogenesis and Cancer PreventionNational Cancer Center Research InstituteTsukijiJapan

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