Quantitative PET Imaging of Tissue Factor Expression Using ¹⁸F-Labeled Active Site–Inhibited Factor VII

Abstract

Tissue factor (TF) is upregulated in many solid tumors, and its expression is linked to tumor angiogenesis, invasion, metastasis, and prognosis. A noninvasive assessment of tumor TF expression status is therefore of obvious clinical relevance. Factor VII is the natural ligand to TF. Here we report the development of a new PET tracer for specific imaging of TF using an 18 F-labeled derivative of factor VII. Methods: Active site-inhibited factor VIIa (FVIIai) was obtained by inactivation with phenylalanine-phenylalanine-arginine-chloromethyl ketone. FVIIai was radiolabeled with N-succinimidyl 4-18 F-fluorobenzoate and purified. The corresponding product, 18 F-FVIIai, was injected into nude mice with subcutaneous human pancreatic xenograft tumors (BxPC-3) and investigated using small-animal PET/CT imaging 1, 2, and 4 h after injection. Ex vivo biodistribution was performed after the last imaging session, and tumor tissue was preserved for molecular analysis. A blocking experiment was performed in a second set of mice. The expression pattern of TF in the tumors was visualized by immunohistochemistry and the amount of TF in tumor homogenates was measured by enzyme-linked immunosorbent assay and correlated with the uptake of 18 F-FVIIai in the tumors measured in vivo by PET imaging. Results: The PET images showed high uptake of 18 F-FVIIai in the tumor regions, with a mean uptake of 2.5 ± 0.3 percentage injected dose per gram (%ID/g) (mean ± SEM) 4 h after injection of 7.3-9.3 MBq of 18 F-FVIIai and with an average maximum uptake in the tumors of 7.1 ± 0.7 %ID/g at 4 h. In comparison, the muscle uptake was 0.2 ± 0.01 %ID/g at 4 h. At 4 h, the tumors had the highest uptake of any organ. Blocking with FVIIai significantly reduced the uptake of 18 F-FVIIai from 2.9 ± 0.1 to 1.4 ± 0.1 %ID/g (P , 0.001). The uptake of 18 F-FVIIai measured in vivo by PET imaging correlated (r 5 0.72, P , 0.02) with TF protein level measured ex vivo. Conclusion: 18 F-FVIIai is a promising PET tracer for specific and noninvasive imaging of tumor TF expression. The tracer merits further development and clinical translation, with potential to become a companion diagnostics for emerging TF-targeted therapies.

PET Imaging of Tissue Factor in Pancreatic Cancer Using 64Cu-Labeled Active Site–Inhibited Factor VII

“…We have previously demonstrated proof of concept for PET imaging of TF expression with 18 F-labeled FVIIai (19). The results showed improved image contrast at the late time points, with the highest tumor uptake and image contrast obtained 4 h after injection of 18 F-FVIIai.…”

“…Cells tested negative for Mycoplasma and a panel of murine pathogens. Cell surface expression of TF was measured by flow cytometry as previously described (19).…”

“…Staining was performed as previously described (19); in brief: formalin-fixed paraffin-embedded tumors were sectioned at 4 mm, deparaffinized, rehydrated, and microwaved in citrate buffer, pH 6, for epitope retrieval. Sections were blocked with 2% bovine serum albumin in phosphate-buffered saline, and primary antibody against human tissue factor CD142 (4508, American Diagnostica Inc.) was added at 1:800 dilution for 1 h at room temperature.…”

“…Here, we report the development and preclinical evaluation of a 64 Cu-labeled PET tracer for noninvasive imaging of tumor TF status. Active site-inhibited FVII (FVIIai), with a molecular weight of 50 kDa, which binds to TF with an affinity of approximately 1 nM (19,20), was conjugated with the 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) chelator. In vivo PET imaging using 64 Cu-NOTA-FVIIai was performed in a panel of pancreatic adenocarcinoma xenograft mouse models established subcutaneously and orthotopically to demonstrate the ability of noninvasive imaging of tumor TF status.…”

Coagulation Factor and Theranostics: A New Paradigm in Molecular Imaging

“…Int he current issue of The Journal of Nuclear Medicine, Nielsen et al (1) illustrate the potentials and significant challenges of targeting cancer cells' surface receptors for imaging and therapy.…”