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

Abstract

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 siteinhibited FVII (FVIIai) labeled with 64 Cu for PET imaging of TF expression. Methods: FVIIai was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCNBn-NOTA) and labeled with 64 Cu ( 64 Cu-NOTA-FVIIai). Longitudinal in vivo PET imaging was performed at 1, 4, 15, and 36 h after injection of 64 Cu-NOTA-FVIIai in mice with pancreatic adenocarcinomas (BxPC-3). The specificity of TF imaging with 64 Cu-NOTAFVIIai 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 64 Cu-NOTA-FVIIai and PET/MRI. In vivo imaging data were supported by ex vivo biodistribution, flow cytometry, and immunohistochemistry. Results: Longitudinal PET imaging with 64 Cu-NOTA-FVIIai 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-tonormal-tissue contrast was observed over the imaging time course. Competition with unlabeled FVIIai 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 64 Cu-NOTA-FVIIai was colocalized with viable tumor tissue. Conclusion: 64 Cu-NOTA-FVIIai 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. Ti ssue factor (TF) is a 47-kDa glycosylated transmembrane protein consisting of a large extracellular domain and a short cytoplasmic domain. TF acts as the initiator of the extrinsic coagulation cascade. The zymogen factor VII (FVII) is the natural ligand to TF and gets activated to FVIIa on binding to TF. The TF-FVIIa complex further activates factor IX and factor X, eventually leading to fibrin deposition, platelet aggregation, and formation of a thrombus (1).In addition to its role in the coagulation cascade, TF is associated with cancer (1-3). TF expression has been found in all solid tumors, and its expression has been linked to mutation of the K-ras oncogene, loss...

Crosstalk between TF/FVIIa and EGFR signaling in colorectal cancer cells

“…Therefore, understanding the crosstalk between EGFR pathway and TF/FVIIa pathway will help design better cancer therapy strategies targeting TF. [18][19][20][21][22] In addition, KRAS mutation results in constitutive active Ras independent of the activation of EGFR, causing the failure of anti-EGFR therapy. 23,24 In this study we found that the activation of EGFR pathway in LoVo cells with mutant KRAS could activate TF/FVIIa signaling, on the one hand, the activation of TF/FVIIa signaling could upregulate the expression of EGFR ligands to activate EGFR pathway, thus forming a positive feedback loop to promote malignant phenotypes of cancer cells.…”

Molecular imaging using an anti-human tissue factor monoclonal antibody in an orthotopic glioma xenograft model

“…Previous studies have demonstrated successful visualization of TF expressed in pancreatic or breast cancer models by PET imaging with a full-length anti-human TF antibody 48 , its antigen-binding fragment (Fab) 49 , a bispecific heterodimer composed of anti-human TF and anti-human/murine CD105 Fab fragments 50 , 51 and an active site-inhibited factor VIIa 52 , 53 , and proposed that PET imaging of TF can be used for assessing disease stage and progression in patients with malignancy and used as a companion diagnostics for TF-targeted therapies. In the present study, we succeeded in imaging TF expression in tumours of the orthotopic glioma model mice using our original mAb.…”

Overview and Future Perspectives on Tumor-Targeted Positron Emission Tomography and Fluorescence Imaging of Pancreatic Cancer in the Era of Neoadjuvant Therapy

“…Low/moderate expression is seen in pancreatitis, whereas normal pancreatic parenchyma does only minimally express TF [ 127 ]. Further evaluation with the available TF-targeted small molecule inhibitors is warranted to address the potential for early detection and monitoring of PDAC [ 130 , 131 , 132 ].…”

“…Nielsen et al constructed and evaluated a TF targeted antibody PET-tracer ( 64 Cu-NOTA-FVIIai) in a subcutaneous PDAC mouse model. PET/MRI imaging resulted in adequate visualization of PDAC with a tumor-to-muscle ratio (T/M ratio) >30, intermediate/high tracer uptake in the liver was observed [ 131 ].…”

“… Delayed imaging of 64Cu-NOTA-FVIIai improved the tumor–to–background ratio, and subcutaneous tumors were clearly visible 15 h after injection. [ 131 ] After 15 h, 36 h Tumor-to-muscle ratio After 15 h: 20 Tumor-to-pancreas ratio After 36 h: 36 After 15 h: 10 After 36 h: 13 Maximum tumor uptake/biodistribution in % (SD) of injected dosage/g BW PANC-1 (low TF+): 2.2 (±0.1) AsPC-1 (intermed. TF+): 4.1 (±0.1) BxPC-3 (high TF+): 7.5 (±0.5) TF/ Endoglin (CD105) 64 Cu-NOTA-ALT-836/TRC105 Dual- targeted antibody fragment PET/CT In vitro/In vivo Preclinical probe construction and target validation in mouse model Subcutaneous PDAC mouse model (BXPC-3, TF/CD105+/+) After 30 h Tumor uptake/biodistribution in % of injected dosage/g BW (SD) Subcutaneous: PDAC: 17.1 (±4.9) Pancreas: <1 Liver: 8.5 Proof-of-concept, high target binding affinity for dual-TF+/Endoglin+ PDAC, allowing tumor visualization with targeted PET/CT-imaging.…”