Suppression of angiogenesis and tumor growth by recombinant T4 phages displaying extracellular domain of vascular endothelial growth factor receptor 2
Tumor growth, invasion and metastasis are dependent on angiogenesis. The Vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) signaling pathway plays a pivotal role in tumor angiogenesis and therefore represents a reasonable target for anti-angiogenesis/anti-tumor therapy. In the present study, we generated T4 recombinant phages expressing the extracellular domain of VEGFR2 (T4-VEGFR2) and investigated their anti-angiogenic activity. The T4-VEGFR2 phages were able to bind to VEGF specifically and inhibit VEGF-mediated phosphorylation of VEGFR2 and its downstream kinases such as extracellular signal-regulated kinase (ERK) and p38 mitogen activated protein kinase (MAPK). The in vitro experiments showed that the T4-VEGFR2 phages could inhibit VEGF-stimulated cell proliferation and migration of endothelial cells. Finally, administration of T4-VEGFR2 phages was able to suppress tumor growth and decrease microvascular density in murine models of Lewis lung carcinoma and colon carcinoma, and prolong the survival of tumor bearing mice. In conclusion, this study reveals that the recombinant T4-VEGFR2 phages generated using T4-based phage display system can inhibit VEGF-mediated tumor angiogenesis and the T4 phage display technology can therefore be used for the development of novel anti-cancer strategies.
Analysis of variance
American Type Culture Collection
Bovine serum albumin
Swine fever virus
Endothelial cell growth supplement
Enzyme-linked immunosorbent assay
Fetal bovine serum
Fetal liver kinase 1
fms-like tyrosine kinase-1
fms-like tyrosine kinase-4
Foot and mouth disease virus
Human immunodeficiency virus
Highly antigenic outer protein capsid
Human umbilical vein endothelial cell
Kinase insert domain receptor
Lewis lung carcinoma
Mitogen-activated protein kinase
Mean fluorescence intensity
Multiplicity of infection
Polymerase chain reaction
Protein kinase B
Placental growth factor
Phospho-vascular endothelial growth factor receptor 2
Small outer capsid
Statistical Package for Social Sciences
Vascular endothelial growth factor
Vascular endothelial growth factor receptor
We thank Dr. Zhaojun Ren (VersatileBio LLC, MD, USA) for providing the pJKS plasmid and the T4-e-phage. We also thank MogoEdit (http://www.mogoedit.com) for its linguistic assistance during the preparation of this manuscript.
SZ, GD and LW contributed equally to this work. SZ and XR designed the study. SZ analyzed the data, and wrote the manuscript. GD, LW and YW performed the experiments. ZH and WY provided critical review and comments. WM supervised the study.
This work was supported by Science and Technology Development Program of Henan Province (No. 132300410274), National Natural Science Foundation of China (No. 81301963), Natural Science Foundation of Henan Province (No. 162300410040) and Outstanding Youth Science Foundation of Henan University (No. yqpy20140036).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
- 8.Makinen T, Veikkola T, Mustjoki S, Karpanen T, Catimel B, Nice EC, Wise L, Mercer A, Kowalski H, Kerjaschki D, Stacker SA, Achen MG, Alitalo K (2001) Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3. EMBO J 20(17):4762–4773CrossRefPubMedPubMedCentralGoogle Scholar
- 11.Chatterjee S, Heukamp LC, Siobal M, Schottle J, Wieczorek C, Peifer M, Frasca D, Koker M, Konig K, Meder L, Rauh D, Buettner R, Wolf J, Brekken RA, Neumaier B, Christofori G, Thomas RK, Ullrich RT (2013) Tumor VEGF:VEGFR2 autocrine feed-forward loop triggers angiogenesis in lung cancer. J Clin Invest 123(4):1732–1740CrossRefPubMedPubMedCentralGoogle Scholar
- 18.Ren ZJ, Tian CJ, Zhu QS, Zhao MY, Xin AG, Nie WX, Ling SR, Zhu MW, Wu JY, Lan HY, Cao YC, Bi YZ (2008) Orally delivered foot-and-mouth disease virus capsid protomer vaccine displayed on T4 bacteriophage surface: 100% protection from potency challenge in mice. Vaccine 26(11):1471–1481CrossRefPubMedGoogle Scholar
- 26.Holash J, Davis S, Papadopoulos N, Croll SD, Ho L, Russell M, Boland P, Leidich R, Hylton D, Burova E, Ioffe E, Huang T, Radziejewski C, Bailey K, Fandl JP, Daly T, Wiegand SJ, Yancopoulos GD, Rudge JS (2002) VEGF-Trap: a VEGF blocker with potent antitumor effects. Proc Natl Acad Sci USA 99(17):11393–11398CrossRefPubMedGoogle Scholar
- 29.Huang J, Frischer JS, Serur A, Kadenhe A, Yokoi A, McCrudden KW, New T, O’Toole K, Zabski S, Rudge JS, Holash J, Yancopoulos GD, Yamashiro DJ, Kandel JJ (2003) Regression of established tumors and metastases by potent vascular endothelial growth factor blockade. Proc Natl Acad Sci USA 100(13):7785–7790CrossRefPubMedGoogle Scholar
- 31.Shinkai A, Ito M, Anazawa H, Yamaguchi S, Shitara K, Shibuya M (1998) Mapping of the sites involved in ligand association and dissociation at the extracellular domain of the kinase insert domain-containing receptor for vascular endothelial growth factor. J Biol Chem 273(47):31283–31288CrossRefPubMedGoogle Scholar