Abstract
1154
Objectives c[Cys-Thr-Pro-Ser-Pro-Phe-Ser-His-Cys]OH (TCP-1) is a cyclic peptide that was originally identified in orthotopic mouse colorectal cancer (CRC) using phage display selection. Although the receptor for TCP-1 binding has not been identified, our preliminary results in xenografted tumor models showed that TCP-1 labeled with 99mTc was able to specifically target human CRC. This study was designed to characterize the uptake of TCP-1 peptide labeled with near-infrared (NIR) fluorochrome Cy7 (Cy7-TCP-1) in human colon cancer xenografts and tumor-associated vasculature using a mouse dorsal skinfold window chamber (DSWC) model.
Methods Human HCT116 colon cancer xenografts were established inside DSWCs in eight SCID mice. Tumor metabolism in these mice was assessed using 18F-FDG and a direct positron/electron imager. Twenty-four hours later, four of the mice were injected intravenously with 99mTc-HYNIC-3P4-RGD2, a 99mTc-labeled Arg-Gly-Asp (RGD) peptide dimer that targets αvβ3 integrin, to image tumor angiogenesis using the direct positron/electron imager. The other four mice were injected with Cy7-TCP-1. White-light and Cy7-TCP-1 fluorescent images of tissues within the chambers were acquired by a fluorescence microscope at 749 nm excitation (emission 820 nm). To further map the tumor location, size, and vascular network related to Cy7-TCP-1 uptake, Cy7-TCP-1 was injected in four additional mice bearing DSWC HCT116/RFP xenografts expressing red fluorescent protein (RFP). The HCT116/RFP tumors were imaged using the same microscope as described for Cy7 and then imaged at 558 nm excitation (emission 583 nm) for RFP. The animals were sacrificed for histological analysis.
Results Microscopic images showed the tumors as well as underlying blood vessels in the window chamber. The area of active tumor metabolic activity, indicated by 18F-FDG uptake, was smaller than the anatomical lesion size on the microscopic white-light image. 99mTc-HYNIC-3P4-RGD2 predominately localized in peritumoral stromal tissue, particularly on the tumor margin with high microvessel density. Fluorescence microscopy demonstrated high Cy7-TCP-1 uptake in the tumor mass, corresponding to the 18F-FDG positive area, and tumor-associated vasculature in a peripheral connective tissue rim containing numerous newly-formed vessels and capillary-like networks surrounding the tumor mass. The accumulation of Cy7-TCP-1 extended beyond the RFP-positive tumoral lesion and corresponded to the anatomical lesion and tumor-associated vasculature on microscopy. Histological examination showed that cancer cells grew at high density in loose connective tissue inside the window chamber. The tumor was surrounded by adipocytes and then a mixture of collagen, various stromal cells, and abundant newly-formed capillaries.
Conclusions The Cy7-TCP-1 probe is promising for detecting metabolically active CRC lesions and tumor-associated vasculature. The tumor vascular uptake of Cy7-TCP-1 inside the DSWC might represent a specific biomarker expressed on endothelial cell phenotypes promoted by or derived from the colon cancer cells. The mechanism of TCP-1 targeting appears to be different from RGD targeting of integrin αvβ3.