| Hepatocellular carcinoma (HCC), which is the second leading cause of cancer death in China, is unique in its hypervascularity. It has been accepted that the growth and metastasis of HCC depend on angiogenesis, anti-angiogenic therapy inhibited growth and metastasis of HCC. Endothelial cells (ECs) are readily accessible to systemically delivered agents and less prone to developing resistance mechanisms. Therefore, tumor-associated blood vessels and angiogenesis based research have become one of the most promising and actively explored fields for the development of therapeutic agents against HCC and a variety of other tumor types.It has become generally accepted that the vascular endothelium are heterogeneous. Structural and functional differences exist among blood vessels in different tissues and tumor types. Vascular beds in different locations, particularly in tumors, express surface proteins that are specific, can be identified and served as a target. A powerful tool for identifying ligands for molecules of biological interest is offered by random peptide phage display, which can display billions of different peptide sequences fused with coat proteins on the surface of filamentous bacteriophage. Phage panning has been adapted in vivo for selectively recovering enriched phage populations from defined organs. Because ECs are prone to phenotypic changes when they are removed from their microenvironment and cultured in vitro, in vivo phage display is especially appropriate for the identification of ligands for molecules expressed on the luminal surface of different vascular endothelia in their physiological environment. In vivo phage display screening system has allowed the selection of a variety of peptides targeting normal tissues, cancer and other angiogenesis related diseases. However, identification of homing phage to tumor vasculature in HCC has not been reported. The ECs lining the vasculature of liver, which belong to the reticuloendothelial system (RES), have resisted selection of specific phage homing to liver.In this study, we showed that the non-specific phage trapped by liver can be circumvented by a series of strategies such as fl-tet phage pre-saturation, well perfusion and laser capture microdissection (LCM). After six rounds of in vivo phage display, we identified a specific phage displaying a Leu-Pro-Asn-Ile-Ser-Lys-Pro(LPNISKP, named as LCI-X?) peptide, which can home to the vasculature of primary HCC and its metastatic foci in nude mice. In addition, LCI-Xv phage and its cognate peptide can also bind to the vessels in 65% (26/40) of human HCC specimens.Part One Optimizing of in-vivo phage display specific for panning in the liverTo date, the in vivo phage display screening system has allowed the selection of a variety of peptides targeting normal tissues, cancer (even in different tumorigenesis stage) and other angiogenesis-related diseases. But liver (or HCC) has never been reported as the target organ for in vivo phage display screening. For organs like liver, spleen, lung and bone marrow, which belong to the reticuloendothelial system (RES), capture too many phages to be used as target organs for selection. The aim of this part is to optimize in-vivo phage display panning specially for the panning in liver cancer. 1. Study of the distribution of filamentous phage in orthotopic HCC in nude miceTo investigate the distribution of filamentous phage in orthotopic HCC in nude mice, we injected Ml3 insertless phage intravenously, then harvested the liver, tumor and control tissues at 1 min, 4 min, 10 min, 30 min, 1 h, 2 h, 6 h, 12 h, 24 h, 48 h and 72 h after heart perfusion with PBS. Quantitative distribution study of phage in tissues was performed by phage titering and anti-phage immunohistochemistry. It showed that phages captured by the liver were overwhelming compared with the other tissues. Phage titre in the liver was 10-1000 folds over the titres in tumor, lung, kidney or brain within 1 h. Immunohistochemistry study showed that phages were found in the parenchyma of the liver and t...
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