Rational Identification of a Colorectal Cancer Targeting Peptide through Phage Display

Abstract

Colorectal cancer is frequently diagnosed at an advanced stage due to the absence of early clinical indicators. Hence, the identification of new targeting molecules is crucial for an early detection and development of targeted therapies. This study aimed to identify and characterize novel peptides specific for the colorectal cancer cell line RKO using a phage-displayed peptide library. After four rounds of selection plus a negative step with normal colorectal cells, CCD-841-CoN, there was an obvious phage enrichment that specifically bound to RKO cells. Cell-based enzyme-linked immunosorbent assay (ELISA) was performed to assess the most specific peptides leading to the selection of the peptide sequence CPKSNNGVC. Through fluorescence microscopy and cytometry, the synthetic peptide RKOpep was shown to specifically bind to RKO cells, as well as to other human colorectal cancer cells including Caco-2, HCT 116 and HCT-15, but not to the normal non-cancer cells. Moreover, it was shown that RKOpep specifically targeted human colorectal cancer cell tissues. A bioinformatics analysis suggested that the RKOpep targets the monocarboxylate transporter 1, which has been implicated in colorectal cancer progression and prognosis, proven through gene knockdown approaches and shown by immunocytochemistry co-localization studies. The peptide herein identified can be a potential candidate for targeted therapies for colorectal cancer. Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide and the second leading cause of cancer-related deaths 1. The initiation and progression of benign adenoma to malignant adenocarcinoma may be driven by the accumulation of several gene mutations and epigenetic modifications 2. Early stage screening of CRC can potentially reduce both the incidence and mortality from this type of cancer. However, due to limitations of the current screening modalities in CRC (colonoscopy, biopsy and blood tests), several efforts are being conducted to discover new biomarkers that could be used as alternative screening tools for early diagnosis. Amongst these, peptide ligands that specifically recognize cell surface receptors are particularly promising and are being extensively used in cancer research. Peptides have become an attractive alternative, as they are easy to synthesize in large amounts and their smal size improves tissue penetration, with less nonspecific uptake by the reticuloendothelial system 3. Moreover, they can be chemically modified to alter affinity, charge, hydrophobicity, stability, and solubility and have been used to functionalize different nanosystems for improved and targeted therapy 4. Peptides can be selected in a relatively cost-effective manner using phage display 5-9. This powerful technology was first introduced in 1985 10 and has been modified to a rapid high-throughput one step method-Biopanning and Rapid Analysis of Selective Interactive Ligands (BRASIL) 11 , which has enabled the construction of a large number of phage peptide libraries, with a wide range of ap...

Ligand-Targeted Delivery of Photosensitizers for Cancer Treatment

“…Recently, in vitro phage display using gastric cancer cells allowed the identification of DE532 (VETSQYFRGTLS) and GP-5 (IHKDKNAPSLVP) peptides, and specific antibodies to target gastric cancer [ 125 ]. The RKOpep (CPKSNNGVC) peptide was selected for colorectal cancer targeting [ 126 ]. In vivo phage display was first described by Ruoslahti and co-workers in 1996 principally to identify new peptides to target brain blood vessels [ 127 ].…”

Selection of a new peptide homing SK‐BR‐3 breast cancer cells

“…Phage display has been extensively used in the cancer research field (Aghebati-Maleki et al, 2016;Bakhshinejad et al, 2015;Bazan et al, 2012;Ferreira et al, 2019;Nobrega et al, 2016;Shukla & Krag, 2005;Sørensen et al, 2017). It allows, among others, the use of whole cells as targets against a wide range of peptides, without the need of previous knowledge of the cells' membrane biochemical composition.…”

Application of Phage-Displayed Peptides in Tumor Imaging Diagnosis and Targeting Therapy

“…Here, the genotype and phenotype of exogenous peptides are linked by fusing the corresponding gene fragments to the coat proteins without compromising phage infectivity or propagation ability. The technology provides rapid identification of peptide ligands for various target molecules or structures, such as antigens (Lim et al 2019 ), antibodies (Hintz et al 2019 ), cell surface receptors (Ferreira et al 2019 ; Li et al 2017 ), biological, inorganic and organic materials (Martins et al 2016 ) and even tissues in vivo (Wada et al 2019 ). The biopanning procedure is more likely to keep native structure and functional conformation of proteins than purified protein, and requires no previous knowledge of molecular composition at the site of interest.…”