Abstract
1046
Objectives: The small peptide with an amino-acid sequence CTPSPFSHC (TCP-1) was identified from orthotopic colorectal cancer (CRC) in mice by phage library selection. Our previous studies have shown that fluorescent and radiolabeled TCP-1 peptides can specifically recognize human CRC xenografts. It is currently unclear whether the TCP-1 probe can target other gastrointestinal malignancies. The objective of this study was to comparatively evaluate the properties of 99mTc-labeled TCP-1 (99mTc-TCP-1) and Cyanine 7 (Cy7)-labeled TCP-1 (Cy7-TCP-1) as molecular probes to detect colorectal and pancreatic cancer by in vitro cell culture studies and in vivo imaging studies in mouse models with xenografted tumors.
Methods: TCP-1 was radiolabeled with 99mTc using 6-hydrazinopyridine-3-carboxylic acid (HYNIC) as a bifunctional chelator and tricine as co-ligand. Labeling took place at 80°C for 30 min followed by HPLC purification to obtain greater than 97% radiochemical purity of 99mTc-TCP-1. Cy7-labeled TCP-1 was prepared by mixing Cy7-N-hydroxysuccinimide (NHS) ester with TCP-1 at a molar ratio of 1:1 in anhydrous N,N-dimethylformamide (DMF). The reaction mixture was purified by semi-preparative HPLC using a C18 column to provide pure Cy7-TCP-1. Cell studies with Cy7-TCP-1 were performed by seeding human Mia PaCa-2 pancreatic cancer cells and HCT116 CRC cells (106 cells/well) on glass cover slips in a 4-well plate and incubating in cell culture media. To verify subcellular localization of Cy7-TCP-1, the cancer cell lines were co-incubated with Cy5.5-labeled hyaluronic acid (HA) at 10kDa molecular weight (Cy5.5-HA) and with Cy7-TCP-1. We have previously demonstrated that Cy5.5-HA can be internalized and distributed in the cytoplasm of Mia PaCa-2 cells. After incubation with the fluorescent agents for 1 h, cells were washed with PBS to remove unbound TCP-1 peptide and HA agent. Cells were fixed and stained with 4',6-diamidino-2-phenylindole (DAPI) to allow fluorescence imaging of nuclei. The Cy7-TCP-1 probe was investigated by fluorescence microscopy. 99mTc-TCP-1 in vivo imaging studies were performed in five SCID mice with xenografted human Mia PaCa-2 cancer and another five mice with HCT116 CRC xenografts. Two hours after radiotracer intravenous injection, 99mTc-TCP-1 images were acquired using an intensified quantum imaging detector [iQID], which is a portable planar imager that offers high spatial resolution (~200 µm) and high sensitivity to low-energy photons. The mice were euthanized for biodistribution measurements and postmortem analyses.
Results: Fluorescence microscopy of Cy7-TCP-1 showed that the TCP-1 peptide was accumulated by Mia PaCa-2 cells and HCT116 cells. Co-registered Cy7-TCP-1, Cy5.5-HA, and DAPI images revealed that the subcellular distribution of TPC-1 fluorescent-probe was associated with the nucleus. In subcutaneous Mia PaCa-2 xenografts, 99mTc-TCP-1 showed detectable accumulation in the pancreatic cancer. However, the radioactive uptake level in the Mia PaCa-2 cancer was lower than that in the HCT116 CRC. Postmortem biodistribution analyses revealed that the tumor radioactivity was 0.16±0.04 %ID/g in the Mia PaCa-2 pancreatic cancer and 1.01±0.15 %ID/g (n=5) in the HCT116 CRC (n=5) (P< 0.01).
Conclusions: Cy7-TCP-1 and 99mTc-TCP-1 showed positive uptake in the Mia PaCa-2 human CRC and pancreatic cancer cells. TCP-1 appears to localize in the cell nucleus. Since the uptake of 99mTc-TCP-1 was more prominent in the CRC model, further studies of the specificity of TCP-1 in pancreatic cancer are warranted.