Screening Of Heptapeptides Specifically Binding To Hepatoma Microspheres

Objective: To screen cyclic heptapepetides which binding specifically to microsphere of HCCLM3,a high metastasis hepatoma cell line,for it’s future development in biomedicine applying as a target molecular to metastatic hepatoma..Methods: Using ultra-low attachment flask and serum-free medium system,HCCLM3 was transformed into hepatoma microspheres,which were used as target cells to perform whole-cell peptide screening by phage-display library,with HCCLM3 and LO2 as adsorbing cells.After four-round subtractive screening,positive phage clones were selected out randomly to be sequenced.According to the sequencing results,the phages were detected by cell-ELISA to determine the affinity and specificity of the candidate peptide binding to hepatoma microspheres.The candidate cyclic heptapeptide was synthesized according to the fusion protein motif of the most specific phage clone.The binding ablity of synthetic peptides to target cells compared with correlated phages was examined using competitive inhibition assays.The binding specificity of synthetic peptides to hepatoma microspheres was further verified by laser confocal microscopy detection.The cytotoxicity assay was used to determine the biological activity of the peptide.Results: HCCLM3 microspheres were successfully formed by using suspension cultural system.The enrichment rate of phage clones in hepatoma microspheres increased from(8.50±1.70)×10-5to(1.0±4.08)×10-4(P<0.001)after four-round screening.The results of cell-ELISA testing binding specificity showes that P26 phage clone was the most powerful one whichbinds to hepatoma microspheres.The amino acid sequence of the displayed polypeptide is NTGSPYE(named NTG peptide).NTG peptide can inhibit the binding of P26 phage to hepatoma microspheres(P<0.01 or P<0.001).Immunocytochemistry assay by laser scanning confocal microscopy show that NTG peptides labeled with FITC can bind specifically to hepatoma microspheres,but not to unrelated cells.The cytotoxicity test shows that the NTG peptide has no activity to promote proliferation of LM3 liver cancer cell.Conclusions: Hepatoma microspheres can be stably obtained by suspension culturing with stem cell culture system.P26,a candidate phage,has the highest specificity on binding to hepatoma microspheres.The displayed cyclic heptapeptide sequence is N-T-G-S-P-Y-E.The synthesized NTG peptide has the ability of targeting to hepatoma microspheres and had no effect on cell growth.Therefore,NTG peptide is worth developing to be a vector in targeting hepatocellular carcinoma stem cell,which can be used in clinic diagnosis and therapy.