Hiroko Hashimoto1, Yoshitaka Suda1,2, Tomoyuki Miyashita1,2, Atsushi Ochiai1,2, Masahiro Tsuboi3, Kenkichi Masutomi4, Tohru Kiyono5, Genichiro Ishii6,7. 1. Division of Pathology, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Kashiwa, 277-8577, Japan. 2. Laboratory of Cancer Biology, Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan. 3. Division of Thoracic Surgery, National Cancer Center Hospital East, Kashiwa, Japan. 4. Division of Cancer Stem Cell, National Cancer Center Research Institute, Tsukiji, Japan. 5. Division of Carcinogenesis and Cancer Prevention, National Cancer Center Research Institute, Tsukiji, Japan. 6. Division of Pathology, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Kashiwa, 277-8577, Japan. gishii@east.ncc.go.jp. 7. Laboratory of Cancer Biology, Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan. gishii@east.ncc.go.jp.
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. METHOD: 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. CONCLUSION: 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.
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. METHOD: 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. CONCLUSION: 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.
Entities:
Keywords:
CDK4R24C; Cancer associated fibroblast; Live imaging; Single-cell-derived clones; hTERT
Authors: Y Bono; S Kyo; M Takakura; Y Maida; Y Mizumoto; M Nakamura; K Nomura; T Kiyono; M Inoue Journal: Br J Cancer Date: 2012-02-21 Impact factor: 7.640