Screening And Identification Of Hepatocellular Carcinoma-targeting Peptides By Phage Display

Objective:Hepatocellular carcinoma (HCC) is one of the most commonly malignancies with a high mortality rate. Currently, there is no effective diagnosis and treatment available for liver cancer. The anticancer drugs currently in use show side effects due to the lack of tumor-targeting property. To improve the delivery efficiency and to decrease the drug-related toxicity in non-cancerous tissues, in this study, we try to use phage display screening and live slice technology to identify novel tumor-specific peptides.Methods:1.1×1011pfu12-mer phage library is incubated with three human HCC liver cells, e.g. SMMC-7721. HepG2. MHCC-LM3for screening HCC-targeting peptides. Thirty-fifty random phage clones are selected from final round of phage recovery and used for sequencing.2. Four different assays are applied for evaluating the viability of liver slices derived from normal C57BL/6mice including morphology, propidium iodide staining. ATP and LDH activity.3.1×1011pfu phage library is incubated with human HCC liver slices for screening tumor-targeting peptides. After four rounds of biopanning,30phage clones are randomly selected from last round for conventional sequencing and the entire recovered phage from second round are used for next-generation sequencing.4. To validate the tissue specificity of candidate phage clones,1×1011pfu candidate and empty phage clones are incubated with human HCC liver slices for2hrs.5. To examine the uptake of candidate peptides in human HCC cells, six candidate peptides are labeled with FAM followed by incubation with human normal liver cell and three human liver cancer cells at the concentration of5μM.6. To evaluate the cell-specificity of candidate peptides, FAM-labeled peptides are incubated with other human cancer cells, e.g. cervical cancer cell line Hela. breast cancer cell line MDA-MB-231, kidney cancer cell line CRL-1932and lung cancer cell line A549at the concentration of5μM.7. To establish the in situ HCC model,1×10/HepG2cell are injected to BabL/c nude mice subcutaneously and transplanted to the upper liver lobe when the ectopic tumor is big enough.8. Top candidate peptides P46and P47are chosen for systemic injection at the dose of25mg/kg in nude mice to assess the tissue-specificity and distribution. Body-wide tissues are harvested30min after injection and examined the fluorescence.Results:1. No conserved peptide motif is found from biopanning in three human HCC cells.2. Four different parameters demonstrate the live slice can survive in12hrs.3. Six top candidate peptides stand out from phage screening in human HCC slices.4. The phage uptake results show that P1, P5and P20bear higher binding affinity to human HCC slice than empty control phages.5. The cellular uptake results with six candidate peptides reveal that higher uptake is observed in human HCC cell MHCC-LM3than in normal liver cells.6. The tumor-specificity results demonstrate candidate peptides P46, P47and P48show higher binding affinity to human liver cancer cells MHCC-LM3than to other cancer cells.7. H&E staining confirm the establishment of in situ HCC animal model.8. The tissue distribution results demonstrate that candidate peptides P46and P47bear higher binding affinity to tumors than to normal liver and other tissues.Conclusion:We have successfully established the live liver slice system and proved that it is a viable in vitro model. Six top candidate peptides have been obtained from phage display screening in human HCC slices. Among them, peptide P46and P47show the highest binding affinity to tumor than to liver and other tissues.