Development of Immortalized Human Tumor Endothelial Cells from Renal Cancer
Abstract
:1. Introduction
2. Results
2.1. Establishment of Immortalized ECs
2.2. Extended Life Span of ECs by Ectopic SV40 Large T Antigen and hTERT Expression
2.3. Maintenance of Endothelial Characteristics in Immortalized ECs
2.4. Maintenance of TEC-Specific Characteristics in Immortalized ECs
3. Discussion
4. Materials and Methods
4.1. Human Tissue Samples
4.2. Isolation of hTECs and hNECs and Cell Culture
4.3. Plasmids and Transfection
4.4. Isolation of RNA, Reverse Transcription-PCR (RT-PCR), and Quantitative PCR
4.5. Determination of Population Doubling (PD) Time
4.6. Senescence-Associated β-Galactosidase (SA-β-Gal)
4.7. Tube Formation Assay
4.8. Soft Agar Colony Formation Assay
4.9. Karyotype Analysis
4.10. Cell Survival Assay
4.11. Wound Healing Assay
4.12. Statistics
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
ALDH | aldehyde dehydrogenase |
ATCC | American Type Culture Collection |
BGN | bigycan |
EC | endothelial cell |
FBS | fetal bovine serum |
GAPDH | glyceraldehyde 3-phosphate dehydrogenase |
h-imTEC | immortalized human tumor endothelial cell |
h-imNEC | immortalized human normal endothelial cell |
HMVEC | human microvascular endothelial cell |
hNEC | human normal endothelial cell |
hTEC | human tumor endothelial cell |
hTERT | human telomerase reverse transcriptase |
HUVEC | human umbilical vein endothelial cell |
imHMVEC | immortalized HMVEC |
LOX | lysyl oxidase |
MDR1 | multidrug resistance 1 |
MTS | 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt |
NEC | normal endothelial cell |
PD | population doubling |
RCC | renal cell carcinoma |
RT-PCR | reverse transcription-PCR |
SA-β-Gal | Senescence-associated β-galactosidase |
SV40 | simian virus 40 |
TEC | tumor endothelial cell |
References
- Jayson, G.C.; Kerbel, R.; Ellis, L.M.; Harris, A.L. Antiangiogenic therapy in oncology: Current status and future directions. Lancet 2016, 388, 518–529. [Google Scholar] [CrossRef]
- Chen, H.X.; Cleck, J.N. Adverse effects of anticancer agents that target the VEGF pathway. Nat. Rev. Clin. Oncol. 2009, 6, 465–477. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Dudley, A.C. Tumor endothelial cells. Cold. Spring Harb. Perspect. Med. 2012, 2, a006536. [Google Scholar] [CrossRef]
- St Croix, B.; Rago, C.; Velculescu, V.; Traverso, G.; Romans, K.E.; Montgomery, E.; Lal, A.; Riggins, G.J.; Lengauer, C.; Vogelstein, B.; et al. Genes expressed in human tumor endothelium. Science 2000, 289, 1197–1202. [Google Scholar] [CrossRef] [PubMed]
- Bussolati, B.; Deambrosis, I.; Russo, S.; Deregibus, M.C.; Camussi, G. Altered angiogenesis and survival in human tumor-derived endothelial cells. FASEB J. 2003, 17, 1159–1161. [Google Scholar] [CrossRef]
- Hida, K.; Hida, Y.; Amin, D.N.; Flint, A.F.; Panigrahy, D.; Morton, C.C.; Klagsbrun, M. Tumor-associated endothelial cells with cytogenetic abnormalities. Cancer Res. 2004, 64, 8249–8255. [Google Scholar] [CrossRef]
- Akino, T.; Hida, K.; Hida, Y.; Tsuchiya, K.; Freedman, D.; Muraki, C.; Ohga, N.; Matsuda, K.; Akiyama, K.; Harabayashi, T.; et al. Cytogenetic abnormalities of tumor-associated endothelial cells in human malignant tumors. Am. J. Pathol. 2009, 175, 2657–2667. [Google Scholar] [CrossRef]
- Maishi, N.; Ohga, N.; Hida, Y.; Akiyama, K.; Kitayama, K.; Osawa, T.; Onodera, Y.; Shinohara, N.; Nonomura, K.; Shindoh, M.; et al. CXCR7: A novel tumor endothelial marker in renal cell carcinoma. Pathol. Int. 2012, 62, 309–317. [Google Scholar] [CrossRef]
- Yamada, K.; Maishi, N.; Akiyama, K.; Towfik Alam, M.; Ohga, N.; Kawamoto, T.; Shindoh, M.; Takahashi, N.; Kamiyama, T.; Hida, Y.; et al. CXCL12-CXCR7 axis is important for tumor endothelial cell angiogenic property. Int. J. Cancer 2015, 137, 2825–2836. [Google Scholar] [CrossRef]
- Matsuda, K.; Ohga, N.; Hida, Y.; Muraki, C.; Tsuchiya, K.; Kurosu, T.; Akino, T.; Shih, S.C.; Totsuka, Y.; Klagsbrun, M.; et al. Isolated tumor endothelial cells maintain specific character during long-term culture. Biochem. Biophys. Res. Commun. 2010, 394, 947–954. [Google Scholar] [CrossRef]
- Yamamoto, K.; Ohga, N.; Hida, Y.; Maishi, N.; Kawamoto, T.; Kitayama, K.; Akiyama, K.; Osawa, T.; Kondoh, M.; Matsuda, K.; et al. Biglycan is a specific marker and an autocrine angiogenic factor of tumour endothelial cells. Br. J. Cancer 2012, 106, 1214–1223. [Google Scholar] [CrossRef] [PubMed]
- Osawa, T.; Ohga, N.; Akiyama, K.; Hida, Y.; Kitayama, K.; Kawamoto, T.; Yamamoto, K.; Maishi, N.; Kondoh, M.; Onodera, Y.; et al. Lysyl oxidase secreted by tumour endothelial cells promotes angiogenesis and metastasis. Br. J. Cancer. 2013, 109, 2237–2247. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Otsubo, T.; Hida, Y.; Ohga, N.; Sato, H.; Kai, T.; Matsuki, Y.; Takasu, H.; Akiyama, K.; Maishi, N.; Kawamoto, T.; et al. Identification of novel targets for antiangiogenic therapy by comparing the gene expressions of tumor and normal endothelial cells. Cancer Sci. 2014, 105, 560–567. [Google Scholar] [CrossRef] [PubMed]
- Hida, K.; Maishi, N.; Kawamoto, T.; Akiyama, K.; Ohga, N.; Hida, Y.; Yamada, K.; Hojo, T.; Kikuchi, H.; Sato, M.; et al. Tumor endothelial cells express high pentraxin 3 levels. Pathol. Int. 2016, 66, 687–694. [Google Scholar] [CrossRef] [PubMed]
- Akiyama, K.; Ohga, N.; Hida, Y.; Kawamoto, T.; Sadamoto, Y.; Ishikawa, S.; Maishi, N.; Akino, T.; Kondoh, M.; Matsuda, A.; et al. Tumor endothelial cells acquire drug resistance by MDR1 up-regulation via VEGF signaling in tumor microenvironment. Am. J. Pathol. 2012, 180, 1283–1293. [Google Scholar] [CrossRef] [PubMed]
- Hida, K.; Kikuchi, H.; Maishi, N.; Hida, Y. ATP-binding cassette transporters in tumor endothelial cells and resistance to metronomic chemotherapy. Cancer Lett. 2017, 400, 305–310. [Google Scholar] [CrossRef]
- Hida, K.; Maishi, N.; Akiyama, K.; Ohmura-Kakutani, H.; Torii, C.; Ohga, N.; Osawa, T.; Kikuchi, H.; Morimoto, H.; Morimoto, M.; et al. Tumor endothelial cells with high aldehyde dehydrogenase activity show drug resistance. Cancer Sci. 2017, 108, 2195–2203. [Google Scholar] [CrossRef]
- Yu, S.T.; Yang, Y.B.; Liang, G.P.; Li, C.; Chen, L.; Shi, C.M.; Tang, X.D.; Li, C.Z.; Li, L.; Wang, G.Z.; et al. An optimized telomerase-specific lentivirus for optical imaging of tumors. Cancer Res. 2010, 70, 2585–2594. [Google Scholar] [CrossRef]
- Arbiser, J.L.; Moses, M.A.; Fernandez, C.A.; Ghiso, N.; Cao, Y.; Klauber, N.; Frank, D.; Brownlee, M.; Flynn, E.; Parangi, S.; et al. Oncogenic H-ras stimulates tumor angiogenesis by two distinct pathways. Proc. Natl. Acad. Sci. USA 1997, 94, 861–866. [Google Scholar] [CrossRef] [Green Version]
- Maishi, N.; Ohba, Y.; Akiyama, K.; Ohga, N.; Hamada, J.; Nagao-Kitamoto, H.; Alam, M.T.; Yamamoto, K.; Kawamoto, T.; Inoue, N.; et al. Tumour endothelial cells in high metastatic tumours promote metastasis via epigenetic dysregulation of biglycan. Sci. Rep. 2016, 6, 28039. [Google Scholar] [CrossRef]
- Xu, T.; Bianco, P.; Fisher, L.W.; Longenecker, G.; Smith, E.; Goldstein, S.; Bonadio, J.; Boskey, A.; Heegaard, A.M.; Sommer, B.; et al. Targeted disruption of the biglycan gene leads to an osteoporosis-like phenotype in mice. Nat. Genet. 1998, 20, 78–82. [Google Scholar] [CrossRef] [PubMed]
- Kondoh, M.; Ohga, N.; Akiyama, K.; Hida, Y.; Maishi, N.; Towfik, A.M.; Inoue, N.; Shindoh, M.; Hida, K. Hypoxia-induced reactive oxygen species cause chromosomal abnormalities in endothelial cells in the tumor microenvironment. PLoS ONE 2013, 8, e80349. [Google Scholar] [CrossRef]
- Kastan, M.B.; Schlaffer, E.; Russo, J.E.; Colvin, O.M.; Civin, C.I.; Hilton, J. Direct demonstration of elevated aldehyde dehydrogenase in human hematopoietic progenitor cells. Blood 1990, 75, 1947–1950. [Google Scholar] [PubMed]
- Corti, S.; Locatelli, F.; Papadimitriou, D.; Donadoni, C.; Salani, S.; Del Bo, R.; Strazzer, S.; Bresolin, N.; Comi, G.P. Identification of a primitive brain-derived neural stem cell population based on aldehyde dehydrogenase activity. Stem. Cells 2006, 24, 975–985. [Google Scholar] [CrossRef]
- Ohmura-Kakutani, H.; Akiyama, K.; Maishi, N.; Ohga, N.; Hida, Y.; Kawamoto, T.; Iida, J.; Shindoh, M.; Tsuchiya, K.; Shinohara, N.; et al. Identification of tumor endothelial cells with high aldehyde dehydrogenase activity and a highly angiogenic phenotype. PLoS ONE 2014, 9, e113910. [Google Scholar] [CrossRef]
- Zhou, W.; Yang, Y.; Gu, Z.; Wang, H.; Xia, J.; Wu, X.; Zhan, X.; Levasseur, D.; Zhou, Y.; Janz, S.; et al. ALDH1 activity identifies tumor-initiating cells and links to chromosomal instability signatures in multiple myeloma. Leukemia 2014, 28, 1155–1158. [Google Scholar] [CrossRef] [PubMed]
- Akiyama, K.; Maishi, N.; Ohga, N.; Hida, Y.; Ohba, Y.; Alam, M.T.; Kawamoto, T.; Ohmura, H.; Yamada, K.; Torii, C.; et al. Inhibition of multidrug transporter in tumor endothelial cells enhances antiangiogenic effects of low-dose metronomic paclitaxel. Am. J. Pathol. 2015, 185, 572–580. [Google Scholar] [CrossRef]
- Fontijn, R.; Hop, C.; Brinkman, H.J.; Slater, R.; Westerveld, A.; van Mourik, J.A.; Pannekoek, H. Maintenance of vascular endothelial cell-specific properties after immortalization with an amphotrophic replication-deficient retrovirus containing human papilloma virus 16 E6/E7 DNA. Exp. Cell Res. 1995, 216, 199–207. [Google Scholar] [CrossRef]
- Ades, E.W.; Candal, F.J.; Swerlick, R.A.; George, V.G.; Summers, S.; Bosse, D.C.; Lawley, T.J. HMEC-1: Establishment of an immortalized human microvascular endothelial cell line. J. Invest. Dermatol. 1992, 99, 683–690. [Google Scholar] [CrossRef]
- Salmon, P.; Oberholzer, J.; Occhiodoro, T.; Morel, P.; Lou, J.; Trono, D. Reversible immortalization of human primary cells by lentivector-mediated transfer of specific genes. Mol. Ther. 2000, 2, 404–414. [Google Scholar] [CrossRef]
- Yang, J.; Chang, E.; Cherry, A.M.; Bangs, C.D.; Oei, Y.; Bodnar, A.; Bronstein, A.; Chiu, C.P.; Herron, G.S. Human endothelial cell life extension by telomerase expression. J. Biol. Chem. 1999, 274, 26141–26148. [Google Scholar] [CrossRef] [PubMed]
- Bodnar, A.G.; Ouellette, M.; Frolkis, M.; Holt, S.E.; Chiu, C.P.; Morin, G.B.; Harley, C.B.; Shay, J.W.; Lichtsteiner, S.; Wright, W.E. Extension of life-span by introduction of telomerase into normal human cells. Science 1998, 279, 349–352. [Google Scholar] [CrossRef] [PubMed]
- Wen, V.W.; MacKenzie, K.L. Modeling human endothelial cell transformation in vascular neoplasias. Dis Model. Mech. 2013, 6, 1066–1079. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Wright, W.E.; Piatyszek, M.A.; Rainey, W.E.; Byrd, W.; Shay, J.W. Telomerase activity in human germline and embryonic tissues and cells. Dev. Genet. 1996, 18, 173–179. [Google Scholar] [CrossRef]
- Lee, H.W.; Blasco, M.A.; Gottlieb, G.J.; Horner, J.W., 2nd; Greider, C.W.; DePinho, R.A. Essential role of mouse telomerase in highly proliferative organs. Nature 1998, 392, 569–574. [Google Scholar] [CrossRef] [PubMed]
- Ohga, N.; Ishikawa, S.; Maishi, N.; Akiyama, K.; Hida, Y.; Kawamoto, T.; Sadamoto, Y.; Osawa, T.; Yamamoto, K.; Kondoh, M.; et al. Heterogeneity of tumor endothelial cells: Comparison between tumor endothelial cells isolated from high- and low-metastatic tumors. Am. J. Pathol 2012, 180, 1294–1307. [Google Scholar] [CrossRef] [PubMed]
- Ray, F.A.; Peabody, D.S.; Cooper, J.L.; Cram, L.S.; Kraemer, P.M. SV40 T antigen alone drives karyotype instability that precedes neoplastic transformation of human diploid fibroblasts. J. Cell Biochem. 1990, 42, 13–31. [Google Scholar] [CrossRef] [PubMed]
- Kurosu, T.; Ohga, N.; Hida, Y.; Maishi, N.; Akiyama, K.; Kakuguchi, W.; Kuroshima, T.; Kondo, M.; Akino, T.; Totsuka, Y.; et al. HuR keeps an angiogenic switch on by stabilising mRNA of VEGF and COX-2 in tumour endothelium. Br. J. Cancer 2011, 104, 819–829. [Google Scholar] [CrossRef] [PubMed]
- Hashimoto, H.; Suda, Y.; Miyashita, T.; Ochiai, A.; Tsuboi, M.; Masutomi, K.; Kiyono, T.; Ishii, G. A novel method to generate single-cell-derived cancer-associated fibroblast clones. J. Cancer Res. Clin. Oncol. 2017, 143, 1409–1419. [Google Scholar] [CrossRef]
© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Maishi, N.; Kikuchi, H.; Sato, M.; Nagao-Kitamoto, H.; Annan, D.A.; Baba, S.; Hojo, T.; Yanagiya, M.; Ohba, Y.; Ishii, G.; et al. Development of Immortalized Human Tumor Endothelial Cells from Renal Cancer. Int. J. Mol. Sci. 2019, 20, 4595. https://doi.org/10.3390/ijms20184595
Maishi N, Kikuchi H, Sato M, Nagao-Kitamoto H, Annan DA, Baba S, Hojo T, Yanagiya M, Ohba Y, Ishii G, et al. Development of Immortalized Human Tumor Endothelial Cells from Renal Cancer. International Journal of Molecular Sciences. 2019; 20(18):4595. https://doi.org/10.3390/ijms20184595
Chicago/Turabian StyleMaishi, Nako, Hiroshi Kikuchi, Masumi Sato, Hiroko Nagao-Kitamoto, Dorcas A. Annan, Shogo Baba, Takayuki Hojo, Misa Yanagiya, Yusuke Ohba, Genichiro Ishii, and et al. 2019. "Development of Immortalized Human Tumor Endothelial Cells from Renal Cancer" International Journal of Molecular Sciences 20, no. 18: 4595. https://doi.org/10.3390/ijms20184595