The Functional Identification Of Short Peptide Binding Specifically To Osteosarcoma-associated Vascular Endothelial Cells

BackgroundOsteosarcoma is a malignant bone tumor originated from mesenchymal tissue, mainly in the children and adolescents. At present, High dose chemotherapy is the main treatment for osteosarcoma, which is effective but non-selective. Chemotherapy is also of serious toxic effect for the normal tissue. Therefore, to enhance the targeting effect of the chemotherapy for osteosarcoma has become an urgent problem. Tumor angiogenesis is an important aspect of tumor development, because tumor needs the new vascular system to provide adequate nutrition to support its expansion. The researchers have been paying more and more attention on some specific molecules on the tumor endothelium, which are expected to play as the new drug targets. In order to get the above-mentioned specific binding ligands, an effective screening tool is needed. Phage display technology is one of the most effective one.Phage-display technology is an effective molecular biologic tool that has been developed and widely used since the 1990s. It has been used to create a directly physical linkage between a vast library of random peptide sequences to the DNA encoding each sequence, allowing rapid identification of peptide ligands for a variety of target molecules (antibodies, enzymes, cell-surface receptors, etc.) by an in vitro selection process, which is called panning.The in vivo phage display technique combining the phage display technology with animal models, is a more effective way to looking for the organ-specific binding peptides. This method can help us to find the target and confirm the domain in the natural environment-tissues or organs, by using the different character of antigen of the phage. In 2007, our team inoculated murine osteosarcoma cells UMR-106 in nude mice, and had established the tumor-bearing animal models successfully. We have screened the endothelial cell of tumor vasculatures of nude mouse for four rounds by in vivo phage display technique without knowing the target. According to the result of in vivo experiment and immunohistochemical staining, we could get conclusion that the TKPDKGY phage can effectively target to the endothelial cell of tumor vasculture of nude mice. The TKPDKGY peptide may became the targeting peptide of vascular targeting therapy, which has laid a good foundation for the following study.In order to provide a new method and clue for the therapy and mechanism of osteosarcoma, we will identify the binding specification of the short peptide (TKPDKGY) to osteosarcoma-associated vascular endothelial cells.ObjectiveTo identify the binding specification of the short peptide (TKPDKGY) to osteosarcoma-associated vascular endothelial cells cultured in vitro and to vascular endothelial of osteosarcoma model in nude mice in vivo.Method1. Primary culture and identification of AECs and OVAECsThe digestion-adherent method is used in Primary culture of AECs and OVAECs. After serial subcultivation, AECs and OVAECs are identified by immunohistochemical method with anti-â…§factor antibody.2. The in-vitro functional identification of short peptide binding specificallyThe peptide (TKPDKGY) was synthesized with the N-teminal marked with fluorescein isothiocyanate (FITC). Fluorescence microscopy and flow cytometry were used to detect the in-vitro binding activity.3. The in-vivo functional identification of short peptide binding specificallyWhole-body optical imaging system for Small Animals and Confocal Microscope were used to detect the in-vivo binding activity.Result1. Primary culture and identification of AECs and OVAECsOAVECs and AECs were derived from osteosarcoma by primary culture and characterization was confirmed based on the immunocytochemical technology.2. The in-vitro functional identification of short peptide binding specificallyFluorescein-conjugated TKPDKGY peptide selectively bound to OAVECs, whereas only negligible binding to AECs and UMR-106 were observed by Fluorescence microscopy. Flow cytometry also showed that the fluorescence positive cell rate was 89.54% in the OAVECs co-incubated with TKPDKGY-FITC, and were 0.27% and 0.22% in the UMR-106 and AECs incubated with TKPDKGY-FITC respectively.3. The in-vivo functional identification of short peptide binding specificallyWhole-body optical imaging system showed that tumor was hypofluorescence enrichment in injection 10 min, fluorescence was increased in 30 min gradually, the strongest fluorescence appeared in 1 hr and fluorescence was subsidised 4hrs later, no residual fluorescence in 24hrs. Confocal microscope also showed that the short peptide homed to vascular endothelium in osteosarcoma but was not detectable in heart, brain, lung and liver.ConclusionThe TKPDKGY peptide has specifically binding activity to vascular endothelium in osteosarcoma. It may be useful as a targeting moiety for selective delivery of therapeutics and as a diagnostic probe for the detection of osteosarcoma. Taken together, we draw the following conclusions:1. We had successfully established the tumor-bearing animal models of osteosarcoma;2. The method established in the present study is simple and easy to handle, and can obtain a certain number of high purity AECs and OAVECs;3. The specifically binding activity to vascular endothelium in osteosarcoma was examined with fluorescent tracer technique and flow cytometer method. The research confirmed that the vascular endothelial cell in osteosarcoma is the target of TKPDKGY.4. Whole-body optical imaging system and laser scanning confocal microscope is used to evaluate the binding specificity of TKPDKGY. In the study, the biological activity of short peptide was stable in vivo. TKPDKGY shows a good specifically binding activity to OAVECs.