Positron emission tomography (PET) constitutes a functional imaging technique that is harnessed to probe biological processes in vivo. PET imaging has been used to diagnose and monitor the progression of diseases, as well as to facilitate drug development efforts at both preclinical and clinical stages. The wide applications and rapid development of PET have ultimately led to an increasing demand for new methods in radiochemistry, with the aim to expand the scope of synthons amenable for radiolabeling. In this work, we provide an overview of commonly used chemical transformations for the syntheses of PET tracers in all aspects of radiochemistry, thereby highlighting recent breakthrough discoveries and contemporary challenges in the field. We discuss the use of biologicals for PET imaging and highlight general examples of successful probe discoveries for molecular imaging with PET – with a particular focus on translational and scalable radiochemistry concepts that have been entered to clinical use.

Pretargeted nuclear imaging for the diagnosis of various cancers is an emerging and fast developing field. The tetrazine ligation is currently considered the most promising reaction in this respect. Monoclonal antibodies are often the preferred choice as pretargeting vector due to their outstanding targeting properties. In this work, we evaluated the performance of [⁶⁴Cu]Cu-NOTA-PEG₇-H-Tz using a setup we previously used for [¹¹¹In]In-DOTA-PEG₁₁-BisPy-Tz, thereby allowing for comparison of the performance of these two promising pretargeting imaging agents. The evaluation included a comparison of the physicochemical properties of the compounds and their performance in an ex vivo blocking assay. Finally, [⁶⁴Cu]Cu-NOTA-PEG₇-H-Tz was evaluated in a pretargeted imaging study and compared to [¹¹¹In]In-DOTA-PEG₁₁-BisPy-Tz. Despite minor differences, this study indicated that both evaluated tetrazines are equally suited for pretargeted imaging.

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 micro vessel density, metastasis and poor overall survival. Imaging of TF expression is of clinical relevance as a prognostic biomarker, and as a companion diagnostics 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 (FVIIai) labeled with ⁶⁴Cu 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-SCN-Bn-NOTA) and labeled with ⁶⁴Cu (⁶⁴Cu-NOTA-FVIIai). Longitudinal in vivo PET imaging was performed at 1, 4, 15 and 36 hours after injection of ⁶⁴Cu-NOTA-FVIIai in mice with pancreatic adenocarcinomas (BxPC-3). The specificity of TF imaging with ⁶⁴Cu-NOTA-FVIIai was investigated in subcutaneous pancreatic tumor models with different levels of TF expression and in a competition experiment. In addition, imaging of orthotopic pancreas tumors was performed using ⁶⁴Cu-NOTA-FVIIai and PET/MR imaging. In vivo imaging data were supported by ex vivo biodistribution, flow cytometry and immunohistochemistry. Results: Longitudinal PET imaging with ⁶⁴Cu-NOTA-FVIIai showed a tumor uptake of 2.3 ± 0.2, 3.7 ± 0.3, 3.4 ± 0.3 and 2.4 ± 0.3 % injected dose per gram at 1, 4, 15 and 36 hours after injection, respectively. An increase in tumor to normal 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 ⁶⁴Cu-NOTA-FVIIai was co-localized with viable tumor tissue. Conclusion: ⁶⁴Cu-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.

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 ¹⁸F-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 4-¹⁸F-fluorobenzoate and purified. The corresponding product, ¹⁸F-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 ¹⁸F-FVIIai in the tumors measured in vivo by PET imaging. Results: The PET images showed high uptake of ¹⁸F-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 ¹⁸F-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 ¹⁸F-FVIIai from 2.9 ± 0.1 to 1.4 ± 0.1 %ID/g (P < 0.001). The uptake of ¹⁸F-FVIIai measured in vivo by PET imaging correlated (r = 0.72, P < 0.02) with TF protein level measured ex vivo. Conclusion: ¹⁸F-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.