Screening of specific binding peptide targeting blood vessel of human esophageal cancer in vivo in mice.

Background Cancer of the esophagus and gastroesophageal junction remains a virulent malignancy with poor prognosis. Rapid progresses were made in chemotherapeutic agents and the development of molecular markers allowed better identification of candidates for targeted therapy. This study aimed to identify the candidate peptides used for anti-angiogenic therapy of esophageal cancer by in vivo screening C7C peptide library for peptides binding specifically to blood vessels of human esophageal cancer. Methods The phage displayed C7C peptide library was injected intravenously into mice bearing human esophageal tumor xenografts under renal capsule. After 5 rounds of screening, 13 clones were picked up individually and sequenced. During each round of screening, titers of phage recovery were calculated from tumor xenograft and control tissues. Homing of these 9 peptides to tumor vessel was detected by calculating phage titers in the tumor xenograft and control tissues (lung and spleen) after each phage was injected into mice model, and compared with the distribution of phage M13 and VIII-related antigen in tumor xenograft by immunohistochemical staining. Comparisons among groups of data were made using one-way analysis of variance (ANOVA), followed by the Bonferroni multiple comparisons test. Results The number of phage recovered from tumor tissue of each round increased gradually in tumor group while decreased in control groups (P <0.01 in tumor and spleen, P <0.05 in lung). Immunohistochemical staining showed similar staining pattern with M13 antibody or VIII-related antigen antibody, suggesting that phages displaying the selected peptides could home to blood vessel of human esophageal cancer. According to their DNA, 9 corresponding peptide sequences were deduced. And the homing ability to blood vessel of phages displaying the selected peptides was confirmed by comparing with their recovery in tumor and control tissues. Two motifs, YSXNXW and PXNXXN, were also obtained by analyzing the homology of these peptide sequences. The staining distribution of phage with the sequence of PNPNNST was similar to that of the blood vessel marker factor VIII-related antigen staining. After sequencing, each phage with the selected peptide of PNPNNST with 1.0×10 11 pfu/ml was injected intravenously into mice. The homing ability to tumor vessel of these 9 kinds of peptides in the xenograft was higher than control tissues (lung and spleen). Conclusion Nine peptides obtained from in vivo screening homed to the blood vessel of human esophageal cancer, and the two motifs of YSXNXW and PXNXXN are the possible biochemical recognition units binding to vascular endothelial cells of esophageal cancer. Chin Med J 2011;124(4):581-585 ancer of the esophagus and gastroesophageal junction remains a virulent malignancy with an overall poor prognosis. The overall number of cases each year is steadily increasing in China. In some places, like northern China, it is 10 times more common than in North America. 1-5 Over the last two decades, surgery has become the mainstay of treatment. Most patients present with advanced disease, and curative surgery requires considerable resources in the operating theatre and in critical care. Published data show large variations in practice and outcome from surgical treatment. 6-8 Therefore, there are rapid progresses on new researches about chemotherapeutic agents and the development of molecular markers that allow better identification of candidates. The usage of phage display library brings a gleam of sunlight for finding new exciting agents towards therapy of esophageal cancer. In the present study, phage display C7C library was used for screening in vivo to find peptides which may specific bind to endothelial cells of esophageal cancer. METHODS