18F-Labeled Single-Stranded DNA Aptamer for PET Imaging of Protein Tyrosine Kinase-7 Expression

Abstract

Protein tyrosine kinase-7 (PTK7), a member of receptor tyrosine kinase superfamily initially identified as colon carcinoma kinase-4, is highly expressed in various human malignancies. Its expression was found to correlate with aggressive biologic behaviors such as increased cell proliferation, invasiveness, and migration. Despite the importance and unmet need of imaging PTK7 in vivo, there is currently no clinically relevant method to visualize tumoral PTK7 expression noninvasively such as PET or SPECT. This study aimed to develop a specific, selective, and high-affinity PET radioligand based on single-stranded DNA aptamer to address this challenge.

Methods: Sgc8, a 41-oligonucleotide that targets to PTK7, was labeled with (18)F using a 2-step radiochemical synthesis, which featured a direct 1-step radiofluorination on the distinctive spirocyclic hypervalent iodine(III) precursor to give (18)F-fluorobenzyl azide followed by copper-mediated click conjugation with Sgc8-alkyne. (18)F-Sgc8 was evaluated in vitro and in vivo in 2 cell lines, HCT116 and U87MG, which express high and low amounts of PTK7, respectively.

Results: Sgc8 was labeled efficiently with (18)F in an isolated radiochemical yield of 62% ± 2%, non-decay-corrected based on (18)F-fluorobenzyl azide. (18)F-Tr-Sgc8 was found to possess high-affinity binding to both cell lines, with binding affinity values of 2.7 ± 0.6 nM for HCT116 and 16.9 ± 2.1 nM for U87MG. In vivo PET imaging clearly visualized PTK7 expression in HCT116 xenografted mice, with tumor uptake of 0.76 ± 0.09 percentage injected dose per gram (%ID/g) at 30 min after injection for the subcutaneous tumor model and greater than 1.5 %ID/g for the liver metastasis model. U87MG xenograft tumors had much lower tracer accumulation (0.13 ± 0.06 %ID/g at 30 min after injection), which was consistent with the lower expression of PTK7 in this tumor model. The labeled aptamer was rapidly cleared from the blood through the kidneys and bladder to give high tumor-to-blood and tumor-to-muscle ratios of 7.29 ± 1.51 and 10.25 ± 2.08, respectively.

Conclusion: The (18)F-radiolabeling methodology shown here is a robust procedure for labeling aptamers and similar chemical moieties and can be applied to many different targets. Quantification of PTK7 using (18)F-Tr-Sgc8 may be suitable for clinical translation and might help in the future to select and monitor appropriate therapies.

Keywords: 18F; PET imaging; PTK7; click chemistry; ssDNA aptamer.

Figure 1

Radiosynthesis of ¹⁸F-Tr-Sgc8.

Figure 2

(A) Flow cytometry of PTK7 expression results for HCT116 and U87MG cell lines. (B) Western blot analysis of PTK7 and β-actin expression by cells (C) Binding affinities of ¹⁸F-Tr-Sgc8 in HCT116 and U87MG cells. (D) Representative Western blot analysis of PTK7 and β-actin expression by liver (1), intestine (2), U87MG tumor (3), HCT116 peritoneal metastasis (4), subcutaneous HCT116 tumor (5), HCT116 kidney metastasis (6), HCT116 liver tumor (7) and HCT116 peritoneal wall metastasis (8) (two upper panels) and quantitative results of PTK7/β-actin ratio from three Western blots (lower panel).

Figure 3

(A) Representative coronal (upper) and transaxial (lower) PET images of mice bearing HCT116 xenografts injected with ¹⁸F-Tr-Sgc8 at 30 min (right panel), 1 h (middle panel) and 1 h co-injection with an excess amount of unlabeled aptamer (left panel). (B) Representative coronal (upper) and transaxial (lower) PET images of mice bearing U87MG tumors at 30 min and 1 h p.i. of ¹⁸F-Tr-Sgc8 (100 μCi) and (C) Biodistribution of ¹⁸F-Tr-Sgc8 at 1 h post-injection without additional of unlabeled aptamer (grey column) and with addition of unlabeled aptamer (white columns).

Figure 4

(A, B) Representative PET and MR images of mice with metastatic HCT116 tumors at 1 h p.i. of ¹⁸F-Tr-Sgc8. (C, D) PET image and necropsy of the indicated tumors. Arrows and numbers indicate the different tumors shown in the PET image and matching MRI / necropsy.

Figure 5

(A) Representative agarose gel electrophoresis of ¹⁸F-Tr-Sgc8 post mouse serum incubation and extraction as described in materials and methods. Lane 1 – ladder; lane 2 – aptamer standard; lane 3 – 30 min incubation; lane 4 – 1 h incubation post NAP5; lane 5 – 1 h incubation. (B) In vivo blood stability analysis of ¹⁸F-Tr-Sgc8 by gamma counter.