Experimental Study Of Human Colorectal Cancer Peptide Probe

1. BackgroundColorectal cancer is one of the most common malignant neoplasms in the digestive system. With the development of economy, people’s living standard, life-style and diet structure are changing. In recent years, the incidence and mortality of colorectal cancer is growing at a fast rate in China.At present, imaging examinations still play important roles in the early diagnosis of colorectal cancer, including computed tomography, fibrocolonoscopy, ultrasonography, CT-guided needle biopsy and sigmoidoscopy. However, limited sensitivity may occasionally lead to misdiagnosis. Cancer survival tends to be poorer in developing countries, most likely because of a combination of a late stage at diagnosis and limited access to timely and standard treatment. Thus, it is necessary to develop some more sensitive and safe methods for the early diagnosis of colorectal cancer.Advances have been made in the development of novel endoscopic instruments that are sensitive to fluorescence. Colon is a hollow organ so that it is convenient to be visualized directly by colonoscopy and performed targeted chemotherapy. Targeting imaging requires the detection of molecular targets. However, such targets are too small to be visualized directly by fluorescence colonoscopy, thus probes that are fluorescence labeled are needed to be developed.Currently, some targeting peptides selected from phage display have aroused wide concern due to their better properties, including better targeting, low immunogenicity and toxicity.2. PurposeTo select a novel peptide for the development of sensitive peptide probe, we used an improved subtractive phage display technology to identify positive phage clones that can bind specifically to Caco-2cells and colorectal cancer tissues. The synthetic peptide, when conjugated to fluorescein, has great potential to serve as a novel probe for the detection of CRC.3. MethodsA12-mer phage display library was used to select peptides that bind specifically to the human colorectal cancer cell line Caco-2. The human embryonic kidney cell line HEK293was used as negative cells. After four rounds of panning,30positive phage clones were selected and sequenced. Moreover, we performed various systematic approaches for the selection, characterization and validation of an affinity peptide that target Caco-2cells, including ELISA, immunofluorescent staining, competitive inhibition assay and fluorescence imaging. Positive phage clone SP-2was identified and its displayed peptide was deduced. A novel peptide probe named Caco probe was produced when combined with FITC. The specificity of peptide probe to Caco-2cells and colorectal cancer tissues was further studied by using fluorescence imaging.4. Results1. The novel phage clone SP-2that is sensitive and specific to Caco-2cells and colorectal cancer tissues has been selected via a modified assay.2. This peptide probe exhibited a high specificity to Caco-2cells and colorectal cancer tissues via multi-level and multi-type examinations.3. The peptide probe can specifically bind to the membrane of Caco-2cells.4. A tissue chip containing24cases tissue samples were performed to further evaluate its binding specificity. The results showed that the binding rate was approx.70%, which demonstrated this peptide probe had higher specificity to colorectal cancer tissues.5. Conclusions1. The phage clone SP-2was identified as the best positive candidate that specifically bind to Caco-2cells and colorectal cancer tissues.2. The FITC-labeled peptide probe can specifically bind to Caco-2cells and colorectal cancer tissues.3. These studies suggest that this peptide may be a promising lead candidate in the development of a useful colorectal cancer diagnostic and targeted drug delivery agent.