Suppression Of VEGF Induced Angiogenesis And Tumor Growth By Recombinant T4 Phages Displaying VEGFR2

BackgroundThe occurrence and development of tumor is the result of synergistic action of various carcinogenic factors.Tumor growth,invasion and metastasis are dependent on angiogenesis.The process of tumor angiogenesis is controlled by a variety of pro-and anti-angiogenic factors.VEGF is the most important pro-angiogenic factor in tumor angiogenesis.VEGF mediates its biological function by binding to VEGF receptor(VEGFR)on the membrane of vascular endothelial cells,VEGFR-2 is the main functional receptor of VEGF.VEGFR2 contains multiple tyrosine phosphorylation sites in its intracellular domain which help regulate VEGF-mediated survival,proliferation and migration of vascular endothelial cells.VEGF/VEGFR2 signal transduction pathway has been shown to play a critical role in tumor angiogenesis.Therefore,blocking VEGF/VEGFR2 signaling transduction pathway can inhibit tumor pathogenesis.T4 phage surface display technology is a protein expression system that utilizes two non-essential T4 coat proteins [small outer capsid(SOC)and highly antigenic outer capsid(HOC)] to display foreign protein on the phage surface.Since T4 phage contains a linear double-stranded DNA genome which is larger,compared to the M13,? and T7 phages,it can display foreign proteins with larger molecular weights.Foreign proteins can be displayed on the surface of T4 phage by fusion to SOC or HOC while maintaining a relatively independent structure and the original biological activity.Currently,T4 phage display technology is mainly used for vaccine designing,peptide library construction and protein-protein interaction research.T4 phage display has been used in developing vaccines against human immunodeficiency virus(HIV),foot and mouth disease virus(FMDV)and swine fever virus(CSFV).In the present study,the extracellular domain of VEGFR2 was displayed on the surface of T4 phage to construct T4-VEGFR2 recombinant phages and investigated their anti-angiogenic activity.The role of T4-VEGFR2 recombinant phage in inhibiting tumor growth and metastasis was studied by establishing Lewis lung carcinoma(LLC)and colon carcinoma(CC)murine models.ObjectiveThe extracellular domain of VEGFR2 was displayed on the surface of T4 phage to construct T4-VEGFR2 recombinant phages,and in vitro and in vivo experiments were performed to investigate their anti-angiogenic and anti-tumor potential,which provided scientific basis for further clinical trials.Methods1.The extracellular region nucleic acid encoding sequence of human VEGFR2(h VEGFR2)and mouse VEGFR2(m VEGFR2)was synthesized.The m VEGFR2 and h VEGFR2 gene fragments with different length domains were amplified by PCR and then ligated to the pJKS plasmid to construct pJKS-VEGFR2 recombinant plasmid.2.The pJKS-VEGFR2 recombinant plasmid were transformed into E.coli BL21 chemically competent cells.Then,E.coli BL21 containing the recombinant plasmid were inoculated into T4-e-phage with a multiplicity of infection of 0.5,and the recombinant T4-VEGFR2 phage were constructed by homologous recombination of T4 phage and pJKS-VEGFR2 plasmid.3.The candidate T4-VEGFR2 recombinant phages from single plaques were propagated using the double-layer agar method.The T4-VEGFR2 recombinant phage were screened by plaque PCR and flow cytometry.4.ELISA-Titer and phage-ELISA assays were used to detect the binding ability of T4-VEGFR2 recombinant phages to VEGF.The wild type T4 phage(T4-W)was used as control in subsequent experiments.5.An MTT assay and a trans-well assay were used to investigate the inhibitory effect of T4-VEGFR2 recombinant phages on VEGF-induced proliferation and migration of vascular endothelial cells.To explore the function of the T4-VEGFR2 phages in inhibiting VEGF-induced phosphorylation of VEGFR2 and its downstream signaling in vascular endothelial cells,a phospho-specific flow cytometric analysis was performed.6.To investigate the antitumor function of the T4-VEGFR2 phages in vivo,a Lewis lung cancer(LLC)and a colon cancer(CC)subcutaneous xenograft mouse models were established andrandomly divided into T4-VEGFR2 phage-treated group,T4-W phage-treated group and PBS-treated group.To investigate the anti-angiogenic potential of the T4-VEGFR2 phages in vivo,tumor samples were collected from the LLC models of three groups,and IHC staining of microvessels was performed using anti-CD31 antibody to determine the MVD of the tumor tissues.Results1.The T4-VEGFR2 recombinant phages were successfully constructed.After being identified by plaque PCR and flow cytometry,VEGFR2 gene fragment was successfully inserted into the T4 phage genome,and VEGFR2 protein was successfully expressed and displayed on the surface of T4 bacteriophage.2.ELISA-titer and phage-ELISA assays confirmed that T4-VEGFR2 recombinant phage can specifically bind to VEGF,and T4-VEGFR2-1-7 phage exhibited the strongest binding ability to VEGF.3.VEGF can promote the migration of vascular endothelial cells and the T4-VEGFR2 recombinant phages can inhibit this pro-migratory effect.4.The T4-VEGFR2 recombinant phages can inhibit VEGF-induced phosphorylation of VEGFR2 and its downstream signaling.5.The T4-VEGFR2 recombinant phages can inhibit tumor growth and prolong the survival of tumor-bearing mice.6.The T4-VEGFR2 recombinant phages can reduce the microvessel density in mouse transplanted tumors.ConclusionsThe T4-VEGFR2 recombinant phages could inhibit VEGF-mediated tumor angiogenesis and inhibit tumor progression,providing a new method for anti-angiogenesis therapy.