Targeting Inhibition Of The Epidermal Growth Factor Receptor(egfr) Reverses The Malignant Behavior Of Osteosarcoma

Objective: Phenotype analysis of the epidermal growth factor receptor ( EGFR)family, EGFR, HER2/ErbB-2, HER3/ErbB-3, HER4/ErbB-4 and its related variants expression in osteosarcoma.Methods: Phenotype analysis of the epidermal growth factor receptor ( EGFR)family, EGFR, HER2/ErbB-2, HER3/ErbB-3, HER4/ErbB-4 and its related variants expression in classic osteosarcoma cell lines 143B, MG63, TE85, and primary osteosarcoma UCHOS4, 7, 11, 14, 15 using Flow cytometry, reverse transcriptase PCR methods.Results: (1) EGFR over-expression of different levels were found in classic osteosarcoma cell lines 143B, MG63, TE85. (2) the expression of EGFR variant III were found in osteosarcoma cell lines 143B, MG63, TE85, and primary osteosarcoma UCHOS4, 7,14 .Conclusion: The epidermal growth factor receptor (EGFR) over-expression of different levels were confirmed in osteosarcoma ; the expression of EGFR variant III were confirmed in part of osteosarcomas . Objective: Construction of recombinant adenovirus vector Ad5-scFv-EGFRvIII expressing anti-epidermal growth factor receptor variant III (EGFRvIII) single-chain variable fragment (scFv) antibody, harvested the high-titer adenovirus after transfected Ad5-scFv-EGFRvIII in HEK293 packaging cells .Methods: At first the recombinant human anti-EGFRvIII scFv antibody gene was cloned into the pDisplay vector which targets cell membrane localization of eukaryotic expression by DNA recombinant technology. Then constuct the recombinat adenovirus expressing anti- EGFRvIII scFv antibody using adenovirus shuttle vector pAdTrace-TOX by homologous recombination method, and further the construction were transfected into BJ5183 bacteria with pAdeasy, the adenovirus type 5 backbone plasmid .At last ,the recombinant adenovirus construction carrying anti EGFRvIII-ScFv was transfected into 293 cells, ping-pong cross-infection harvest high titer virus. Detect the expression and function of the adenovirus in infection model of osteosarcoma cells by RT-PCR, Western-blot method .Results: (1) successfully constructed recombinant human-derived anti-epidermal growth factor receptor variant III (EGFRvIII) single-chain variable fragment (scFv) antibodies targeting the membrane orientation of eukaryotic expression vector. (2) successfully constructed and carrying anti EGFRvIII-ScFv recombinant adenovirus shuttle plasmid Ad5-scFv-EGFRvIII, homologous recombination and harvest high titer adenovirus. (3) RT-PCR and Western-blot confirmed the harvested adenovirus can inhibit the expression of EGFR.Conclusions: The constructed recombinant expression of anti-epidermal growth factor receptor variant III (EGFRvIII) single-chain variable fragment (scFv) antibody adenovirus vector Ad5-scFv-EGFRvIII, transfected HEK293 packaging cells and then harvested the high titer adenovirus AdR-scFv-EGFRvIII, and further RT-PCR and Western-blot confirmed the harvested adenovirus can inhibit the expression of EGFR . Objective: To evaluate the malignant behavior changes of osteosarcoma in vitro and vivo after the human osteosarcoma cell models were treated by the targeting inhibition of epidermal growth factor receptor EGFR adenovirus, expressing recombinant anti-epidermal growth factor receptor variant III (EGFRvIII) single-chain variable fragment (scFv) antibody.Methods : Harvesting the recombinant adenovirus expressing recombinant anti-epidermal growth factor receptor variant III (EGFRvIII) single-chain variable fragment (scFv) antibody. To explore the malignant behavior changes of osteosarcoma which include cell proliferation, adhesion, migration and invasion by MTT, crystal violet staining, cell scratch assay, cell assay, cell migration, Matrigel experiment methods after targeting inhibition of epidermal growth factor receptor EGFR in osteosarcoma cell model 143B, MG63, TE85 in vitro. Then detect the tumor growth using Xenogen imaging analysis after animal model of osteosarcoma targeted inhibition of EGFR in vivo.Results:(1) The malignant behaviors which include cell proliferation, adhesion, migration and invasion of osteosarcoma 143B, MG63, TE85 were significantly inhibited after targeting inhibition of EGFR. (2) Xenogen Imaging results indicate that targeting inhibition of EGFR expression in osteosarcoma, tumor growth was inhibited, growth slowed down, and the growth of tumor cell proliferation ability could be inhibited.Conclusions: The malignant behaviors of osteosarcoma which include cell proliferation, adhesion, migration and invasion could be significantly decreased and reversed after targeting inhibition of EGFR in osteosarcoma in vitro.And the growth ability of tumor cell proliferation may significantly decreased after targeting inhibition of EGFR in vivo also. EGFR may be the very promising new treatment target of osteosarcoma in the future.