PEGylated and Non-PEGylated TCP-1 Probes for Imaging of Colorectal Cancer

Purpose: Previous studies indicate that ^99mTc- and fluorescent-labeled c[Cys-Thr-Pro-Ser-Pro-Phe-Ser-His-Cys]OH (TCP-1) peptides were able to detect colorectal cancer (CRC) and tumor-associated vasculature. This study was designed to characterize the targeting properties of PEGylated and non-PEGylated TCP-1 peptides for CRC imaging. Procedures: Cell uptake of cyanine 7 (Cy7)-labeled TCP-1 probes (Cy7-PEG₄-TCP-1 and Cy7-TCP-1) was investigated in three CRC cell lines (human, HCT116 and HT29; mouse, CT26). Xenograft and orthotopic CRC tumor models with HCT116 and CT26 cells were used to characterize biodistribution and CRC tumor-targeting properties of TCP-1 fluorescence and radioligand with and without PEGylation, [^99mTc]Tc-HYNIC-PEG₄-TCP-1 vs. [^99mTc]Tc-HYNIC-TCP-1. Results: Fluorescence images showed that TCP-1 probes were distributed in the cytoplasm and nucleus of CRC cells. When CT26 cells were treated with unlabeled TCP-1 peptide prior to the cell incubation with Cy7-PEG₄-TCP-1, cell fluorescent signals were significantly reduced relative to the cells without blockade. Relative to Cy7-TCP-1, superior brilliance and visibility of fluorescence was observed in the tumor with Cy7-PEG₄-TCP-1 and maintained up to 18 h post-injection. [^99mTc]Tc-HYNIC-PEG₄-TCP-1 images in xenograft and orthotopic CRC models demonstrated that TCP-1 PEGylation preserved tumor-targeting capability of TCP-1, but its distribution (%ID/g) in the liver and intestine was higher than that of [^99mTc]Tc-HYNIC-TCP-1 (1.51 ± 0.29 vs 0.53 ± 0.12, P < 0.01). Better tumor visualization by [^99mTc]Tc-HYNIC-TCP-1 was observed in the orthotopic CRC model due to lower intestinal radioactivity. Conclusions: TCP-1-based probes undergo endocytosis and localize in the cytoplasm and nucleus of human and mouse CRC cells. Tumor detectability of fluorescent TCP-1 peptide with a PEG₄ spacer is promising due to its enhanced tumor binding affinity and rapid clearance kinetics from nontumor tissues. Non-PEGylated [^99mTc]Tc-HYNIC-TCP-1 exhibits lower nonspecific accumulation in the liver and gastrointestinal tract and might have better capability for detecting CRC lesions in clinical sites. TCP-1 may represent an innovative targeting molecule for detecting CRC noninvasively.