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

Tissue factor (TF) is the main initiator of the extrinsic coagulation cascade. However, TF also plays an important role in cancer. TF expression has been reported in 53%-89% of all pancreatic adenocarcinomas, and the expression level of TF has in clinical studies correlated with advanced stage, increased microvessel density, metastasis, and poor overall survival. Imaging of TF expression is of clinical relevance as a prognostic biomarker and as a companion diagnostic for TF-directed therapies currently under clinical development. Factor VII (FVII) is the natural ligand to TF. The purpose of this study was to investigate the possibility of using active site inhibited FVII (FVllai) labeled with 64Cu for PET imaging of TF expression. Methods: FVllai was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and labeled with Cu-64 (Cu-64-NOTA-FVIlai). Longitudinal in vivo PET imaging was performed at 1, 4, 15, and 36 h after injection of Cu-64-NOTA-FVIlai in mice with pancreatic adenocarcinomas (BxPC-3). The specificity of TF imaging with Cu-64-NOTA-FVllai was investigated in subcutaneous pancreatic tumor models with different levels of TF expression and in a competition experiment. In addition, imaging of orthotopic pancreatic tumors was performed using Cu-64-NOTA-FVllai and PET/MRI. In vivo imaging data were supported by ex vivo biodistribution, flow cytometry, and immunohistochemistry. Results: Longitudinal PET imaging with Cu-64-NOTA-FVllai showed a tumor uptake of 2.3 +/- 0.2, 3.7 +/- 0.3, 3.4 +/- 0.3, and 2.4 +/- 0.3 percentage injected dose per gram at 1, 4, 15, and 36 h after injection, respectively. An increase in tumor-to normal -tissue contrast was observed over the imaging time course. Competition with unlabeled FVllai significantly (P < 0.001) reduced the tumor uptake. The tumor uptake observed in models with different TF expression levels was significantly different from each other (P < 0.001) and was in agreement with the TF level evaluated by TF immunohistochemistry staining. Orthotopic tumors were clearly visible on the PET/MR images, and the uptake of Cu-64-NOTA-FVllai was colocalized with viable tumor tissue. Conclusion: Cu-64-NOTA-FVllai is well suited for PET imaging of tumor TF expression, and imaging is capable of distinguishing the TF expression level of various pancreatic tumor models.