Characterization of TCP-1 probes for molecular imaging of colon cancer

Abstract

Molecular probes capable of detecting colorectal cancer (CRC) are needed for early CRC diagnosis. The objective of this study was to characterize c[CTPSPFSHC]OH (TCP-1), a small peptide derived from phage display selection, for targeting human CRC xenografts using technetium-99m ((99m)Tc)-labeled TCP-1 and fluorescent cyanine-7 (Cy7)-labeled form of the peptide (Cy7-TCP-1). (99m)Tc-TCP-1 was generated by modifying TCP-1 with succinimidyl-6-hydrazino-nicotinamide (S-HYNIC) followed by radiolabeling. In vitro saturation binding experiments were performed for (99m)Tc-TCP-1 in human HCT116 colon cancer cells. SCID mice with human HCT116 cancer xenografts were imaged with (99m)Tc-TCP-1 or control peptide using a small-animal SPECT imager: Group I (n=5) received no blockade; Group II (n=5) received a blocking dose of non-radiolabeled TCP-1. Group III (n=5) were imaged with (99m)Tc-labeled control peptide (inactive peptide). SCID mice with human PC3 prostate cancer xenografts (Group IV, n=5) were also imaged with (99m)Tc-TCP-1. Eight additional SCID mice bearing HCT116 xenografts in dorsal skinfold window chambers (DSWC) were imaged by direct positron imaging of (18)F-fluorodeoxyglucose ((18)F-FDG) and fluorescence microscopy of Cy7-TCP-1. In vitro(99m)Tc-HYNIC-TCP-1 binding assays on HCT 116 cells indicated a mean Kd of 3.04±0.52nM. In cancer xenografts, (99m)Tc-TCP-1 radioactivity (%ID/g) was 1.01±0.15 in the absence of blockade and was reduced to 0.26±0.04 (P<0.01) with blockade. No radioactive uptake was observed in the PC3 tumors with (99m)Tc-TCP-1 or HCT116 tumors with inactive peptide. Cy7-TCP-1 activity localized not only in metabolically active tumors, as defined by (18)F-FDG imaging, but also in peritumoral microvasculature. In conclusion, TCP-1 probes may have a distinct targeting mechanism with high selectivity for CRC and tumor-associated vasculature. Molecular imaging with TCP-1 probes appears promising to detect malignant colorectal lesions.

Keywords: (99m)Tc; Colorectal cancer; Molecular imaging; Mouse xenograft models; Peptide; SPECT.

Fig. 1

Left panel: The lowest-energy conformation of c[CTPSPFSHC] (TCP-1). It shows a β-sheet-like structure. Right panel: Ramachandran plot of TCP-1 demonstrates that the majority of amino acids sit in the red region corresponding to the β-sheet region. His⁸ and Ser⁷ are in the yellow region corresponding to the random coil region, but close to the right handed α-helix. The plot calculation was done with Macro Model program.

Fig. 2

Representative HPLC radiochromatograms of ^99mTc-TCP-1 immediately after purification in saline (a) and after incubation at 37°C in mouse serum for 1 hour (b) and 6 hours (c).

Fig. 3

Saturation curve for binding of ^99mTc-TCP-1 to HCT116 colon cancer cells. The mean Kd was 3.04 ± 0.52 nM.

Fig. 4

Dynamic images (serial transversal slices) of ^99mTc-TCP-1 in a SCID mouse bearing HCT116 colon cancer xenograft. The numbers in the upper left corners represent the minutes after injection. Immediately after radiotracer injection, cardiac blood pool was evident, as outlined in yellow on the 1-minute image, and remained visible up to at least 30 minutes. Tumor implanted on the right shoulder became visible about 10–15 minutes after injection, with increasing ratio of tumor to background activity from 60 to 180 minutes. The tumor is outlined in blue on the 180-minute image.

Fig. 5

Representative volume-rendered data sets in three-dimensional display of SPECT images co-registered with photographs of four mice with cancer xenografts, with each tumor outlined in white. The mouse with HCT116 xenograft (a), which received no blockade, demonstrated higher uptake of ^99mTc-TCP-1 in the tumor compared to the mouse with HCT116 tumor receiving unlabeled TCP-1 blockade (b), xenografted HCT116 tumor imaged by inactive ^99mTc-CVQTAQLLC (c), and the PC3 prostate cancer imaged by ^99mTc-TCP-1 (d). High uptake of radioactivity is also seen in the kidneys as marked by arrows.

Fig. 6

Images of the DSWC model. Top row: (a) White-light photomicrograph of HCT116 cells growing in the window chamber. Underlying blood vessels are clearly visible. The site of implanted tumor cells along with peritumoral microvasculature is defined as the tumor anatomical lesion. (b) ¹⁸F-FDG direct positron image of the window chamber in a showing the area of tumor metabolic activity. ¹⁸F-FDG uptake exhibits annular distribution with smaller size relative to the anatomical lesion observed by microscopy. (a&b) Co-registered image of a and b. (c) Cy7-TCP-1 fluorescence microscopic image corresponding to the window-chamber image in a. (a&c) Co-registered image of a and c. The Cy7 fluorescence signal co-localizes with the anatomical lesion. Cy7-TCP-1 uptake also corresponds to some blood vessels associated with the tumor. Bottom row: (d) White-light photomicrograph of window chamber with implanted HCT116/RFP cells with red fluorescent protein expression. (e) Corresponding red fluorescence image of window chamber in d. (d&e) Co-registered image of d and e. The area of RFP-expressing cells is smaller than the anatomical lesion under white-light illumination. (f) Cy7-TCP-1 fluorescence image in the same window chamber as d. (d&f) Co-registered image of d and f. The area of Cy7 fluorescence corresponds to the anatomical lesion but is larger than the area of RFP expression.

Fig. 7

H&E stained histological sections of mouse skin and tumor tissue within the window chamber corresponding to the two mice in Fig. 6. Top row: DSWC with implanted HCT116 cells (a: 100X; b: 200X). Bottom row: DSWC with HCT116/RFP cells (c: 200X; d: 400X). Cancer cells grew at high density in loose connective tissue. The tumor was surrounded by adipocytes and then a peripheral rim of collagen, stromal cells, and numerous newly formed capillaries.