Preclinical PET Imaging of Tumor Cell Death following Therapy Using Gallium-68-Labeled C2Am.

There is an unmet clinical need for imaging agents capable of detecting early evidence of tumor cell death, since the timing, extent, and distribution of cell death in tumors following treatment can give an indication of treatment outcome. We describe here 68Ga-labeled C2Am, which is a phosphatidylserine-binding protein, for imaging tumor cell death in vivo using positron emission tomography (PET). A one-pot synthesis of 68Ga-C2Am (20 min, 25 °C, >95% radiochemical purity) has been developed, using a NODAGA-maleimide chelator. The binding of 68Ga-C2Am to apoptotic and necrotic tumor cells was assessed in vitro using human breast and colorectal cancer cell lines, and in vivo, using dynamic PET measurements in mice implanted subcutaneously with the colorectal tumor cells and treated with a TRAIL-R2 agonist. 68Ga-C2Am showed predominantly renal clearance and low retention in the liver, spleen, small intestine, and bone and generated a tumor-to-muscle (T/m) ratio of 2.3 ± 0.4, at 2 h post probe administration and at 24 h following treatment. 68Ga-C2Am has the potential to be used in the clinic as a PET tracer for assessing early treatment response in tumors.

In Vivo Cell Tracking Using PET: Opportunities and Challenges for Clinical Translation in Oncology.

Cell therapy is a rapidly evolving field involving a wide spectrum of therapeutic cells for personalised medicine in cancer. In vivo imaging and tracking of cells can provide useful information for improving the accuracy, efficacy, and safety of cell therapies. This review focuses on radiopharmaceuticals for the non-invasive detection and tracking of therapeutic cells using positron emission tomography (PET). A range of approaches for imaging therapeutic cells is discussed: Direct ex vivo labelling of cells, in vivo indirect labelling of cells by utilising gene reporters, and detection of specific antigens expressed on the target cells using antibody-based radiopharmaceuticals (immuno-PET). This review examines the evaluation of PET imaging methods for therapeutic cell tracking in preclinical cancer models, their role in the translation into patients, first-in-human studies, as well as the translational challenges involved and how they can be overcome.

3D printing 18F radioactive phantoms for PET imaging.

PURPOSE: Phantoms are routinely used in molecular imaging to assess scanner performance. However, traditional phantoms with fillable shapes do not replicate human anatomy. 3D-printed phantoms have overcome this by creating phantoms which replicate human anatomy which can be filled with radioactive material. The problem with these is that small objects suffer to a greater extent than larger objects from the effects of inactive walls, and therefore, phantoms without these are desirable. The purpose of this study was to explore the feasibility of creating resin-based 3D-printed phantoms using 18F. METHODS: Radioactive resin was created using an emulsion of printer resin and 18F-FDG. A series of test objects were printed including twenty identical cylinders, ten spheres with increasing diameters (2 to 20 mm), and a double helix. Radioactive concentration uniformity, printing accuracy and the amount of leaching were assessed. RESULTS: Creating radioactive resin was simple and effective. The radioactive concentration was uniform among identical objects; the CoV of the signal was 0.7% using a gamma counter. The printed cylinders and spheres were found to be within 4% of the model dimensions. A double helix was successfully printed as a test for the printer and appeared as expected on the PET scanner. The amount of radioactivity leached into the water was measurable (0.72%) but not visible above background on the imaging. CONCLUSIONS: Creating an 18F radioactive resin emulsion is a simple and effective way to create accurate and complex phantoms without inactive walls. This technique could be used to print clinically realistic phantoms. However, they are single use and cannot be made hollow without an exit hole. Also, there is a small amount of leaching of the radioactivity to take into consideration.

The role of [68 Ga]Ga-DOTATATE PET/CT in wild-type KIT/PDGFRA gastrointestinal stromal tumours (GIST).

BACKGROUND: [68 Ga]Ga-DOTATATE PET/CT is now recognised as the most sensitive functional imaging modality for the diagnosis of well-differentiated neuroendocrine tumours (NET) and can inform treatment with peptide receptor radionuclide therapy with [177Lu]Lu-DOTATATE. However, somatostatin receptor (SSTR) expression is not unique to NET, and therefore, [68 Ga]Ga-DOTATATE PET/CT may have oncological application in other tumours. Molecular profiling of gastrointestinal stromal tumours that lack activating somatic mutations in KIT or PDGFRA or so-called 'wild-type' GIST (wtGIST) has demonstrated that wtGIST and NET have overlapping molecular features and has encouraged exploration of shared therapeutic targets, due to a lack of effective therapies currently available for metastatic wtGIST. AIMS: To investigate (i) the diagnostic role of [68 Ga]Ga-DOTATATE PET/CT; and, (ii) to investigate the potential of this imaging modality to guide treatment with [177Lu]Lu-DOTATATE in patients with wtGIST. METHODS: [68 Ga]Ga-DOTATATE PET/CT was performed on 11 patients with confirmed or metastatic wtGIST and one patient with a history of wtGIST and a mediastinal mass suspicious for metastatic wtGIST, who was subsequently diagnosed with a metachronous mediastinal paraganglioma. Tumour expression of somatostatin receptor subtype 2 (SSTR2) using immunohistochemistry was performed on 54 tumour samples including samples from 8/12 (66.6%) patients who took part in the imaging study and 46 tumour samples from individuals not included in the imaging study. RESULTS: [68 Ga]Ga-DOTATATE PET/CT imaging was negative, demonstrating that liver metastases had lower uptake than background liver for nine cases (9/12 cases, 75%) and heterogeneous uptake of somatostatin tracer was noted for two cases (16.6%) of wtGIST. However, [68 Ga]Ga-DOTATATE PET/CT demonstrated intense tracer uptake in a synchronous paraganglioma in one case and a metachronous paraganglioma in another case with wtGIST. CONCLUSIONS: Our data suggest that SSTR2 is not a diagnostic or therapeutic target in wtGIST. [68 Ga]Ga-DOTATATE PET/CT may have specific diagnostic utility in differentiating wtGIST from other primary tumours such as paraganglioma in patients with sporadic and hereditary forms of wtGIST.

Preclinical evaluation of [18F]cabozantinib as a PET imaging agent in a prostate cancer mouse model.

INTRODUCTION: Cabozantinib is a tyrosine kinase inhibitor (TKI) approved for the treatment of medullary thyroid cancer, renal cell carcinoma and hepatocellular carcinoma, and is currently in clinical trials for the treatment of prostate cancer and others. It exerts its therapeutic effect mainly through inhibition of the tyrosine kinases MET (hepatocyte growth factor receptor) and VEGFR2 (vascular endothelial growth factor receptor 2), in addition to several other kinases involved in cancer. PET imaging with TKIs such as [18F]cabozantinib could potentially aid in cancer diagnosis and guide treatment. This study aims to evaluate the utility of [18F]cabozantinib as a PET imaging probe in PC3 tumor xenografted mice. METHODS: [18F]cabozantinib was evaluated in non-tumor and tumor bearing (PC3 xenografted) male mice by ex vivo biodistribution studies and in vivo µPET imaging. Pretreatment studies were performed in the tumor bearing mice with the MET inhibitor PF04217903. Mouse plasma was analyzed with HPLC to quantify radiometabolites. To further evaluate the binding specificity of [18F]cabozantinib, in vitro autoradiography studies on heart and PC3 tumor sections were performed in the presence of authentic cabozantinib or specific MET and VEGFR2 inhibitors. RESULTS: Tissue distribution studies in non-tumor bearing mice revealed slow blood clearance, absence of brain uptake and a high myocardial uptake. In the tumor bearing mice, tumor uptake was low (0.58 ± 0.20% ID/g at 30 min post tracer injection), which was confirmed by µPET imaging. No differences in tissue distribution and kinetics were observed in both biodistributions and µPET studies after pretreatment with the MET inhibitor PF04217903. At 30 min post tracer injection, 60 ± 3% of the recovered radioactivity in plasma in non-tumor bearing mice was present as intact tracer. [18F]cabozantinib binding in vitro to heart and tumor tissues was partly blocked in the presence of selective MET and VEGFR2 inhibitors (up to 40% block). The fraction of non-specific binding was relatively high for both tissues (66% for heart and 39% for tumor). CONCLUSION: [18F]cabozantinib exhibits non-favorable properties as a PET imaging probe, demonstrated by slow excretion kinetics along with low tumor uptake and high non-specific binding in tumor and heart tissue. The results reflect cabozantinibs multi-kinase activity, making PET imaging of tumor specific kinase expression with [18F]cabozantinib challenging.

The emerging role of cell surface receptor and protein binding radiopharmaceuticals in cancer diagnostics and therapy.

Targeting specific cell membrane markers for both diagnostic imaging and radionuclide therapy is a rapidly evolving field in cancer research. Some of these applications have now found a role in routine clinical practice and have been shown to have a significant impact on patient management. Several molecular targets are being investigated in ongoing clinical trials and show promise for future implementation. Advancements in molecular biology have facilitated the identification of new cancer-specific targets for radiopharmaceutical development.

18F-C2Am: a targeted imaging agent for detecting tumor cell death in vivo using positron emission tomography.

INTRODUCTION: Trialing novel cancer therapies in the clinic would benefit from imaging agents that can detect early evidence of treatment response. The timing, extent and distribution of cell death in tumors following treatment can give an indication of outcome. We describe here an 18F-labeled derivative of a phosphatidylserine-binding protein, the C2A domain of Synaptotagmin-I (C2Am), for imaging tumor cell death in vivo using PET. METHODS: A one-pot, two-step automated synthesis of N-(5-[18F]fluoropentyl)maleimide (60 min synthesis time, > 98% radiochemical purity) has been developed, which was used to label the single cysteine residue in C2Am within 30 min at room temperature. Binding of 18F-C2Am to apoptotic and necrotic tumor cells was assessed in vitro, and also in vivo, by dynamic PET and biodistribution measurements in mice bearing human tumor xenografts treated with a TRAILR2 agonist or with conventional chemotherapy. C2Am detection of tumor cell death was validated by correlation of probe binding with histological markers of cell death in tumor sections obtained immediately after imaging. RESULTS: 18F-C2Am showed a favorable biodistribution profile, with predominantly renal clearance and minimal retention in spleen, liver, small intestine, bone and kidney, at 2 h following probe administration. 18F-C2Am generated tumor-to-muscle (T/m) ratios of 6.1 ± 2.1 and 10.7 ± 2.4 within 2 h of probe administration in colorectal and breast tumor models, respectively, following treatment with the TRAILR2 agonist. The levels of cell death (CC3 positivity) following treatment were 12.9-58.8% and 11.3-79.7% in the breast and colorectal xenografts, respectively. Overall, a 20% increase in CC3 positivity generated a one unit increase in the post/pre-treatment tumor contrast. Significant correlations were found between tracer uptake post-treatment, at 2 h post-probe administration, and histological markers of cell death (CC3: Pearson R = 0.733, P = 0.0005; TUNEL: Pearson R = 0.532, P = 0.023). CONCLUSION: The rapid clearance of 18F-C2Am from the blood pool and low kidney retention allowed the spatial distribution of cell death in a tumor to be imaged during the course of therapy, providing a rapid assessment of tumor treatment response. 18F-C2Am has the potential to be used in the clinic to assess early treatment response in tumors.

Detection limit of 89Zr-labeled T cells for cellular tracking: an in vitro imaging approach using clinical PET/CT and PET/MRI.

PURPOSE: Tracking cells in vivo using imaging can provide non-invasive information to understand the pharmacology, efficacy, and safety of novel cell therapies. Zirconium-89 (t1/2 = 78.4 h) has recently been used to synthesize [89Zr]Zr(oxinate)4 for cell tracking using positron emission tomography (PET). This work presents an in vitro approach to estimate the detection limit for in vivo PET imaging of Jurkat T cells directly labeled with [89Zr]Zr(oxinate)4 utilizing clinical PET/CT and PET/MRI. METHODS: Jurkat T cells were labeled with varying concentrations of [89Zr]Zr(oxinate)4 to generate different cell-specific activities (0.43-31.91 kBq/106 cells). Different concentrations of labeled cell suspensions (104, 105, and 106 cells) were seeded on 6-well plates and into a 3 × 3 cubic-well plate with 1 cm3 cubic wells as a gel matrix. Plates were imaged on clinical PET/CT and PET/MRI scanners for 30 min. The total activity in each well was determined by drawing volumes of interest over each well on PET images. The total cell-associated activity was measured using a well counter and correlated with imaging data. Simulations for non-specific signal were performed to model the effect of non-specific radioactivity on detection. RESULTS: Using this in vitro model, the lowest cell number that could be visualized on 6-well plate images was 6.8 × 104, when the specific activity was 27.8 kBq/106 cells. For the 3 × 3 cubic-well, a plate of 3.3 × 104 cells could be detected on images with a specific activity of 15.4 kBq/106 cells. CONCLUSION: The results show the feasibility of detecting [89Zr]Zr(oxinate)4-labeled Jurkat T cells on clinical PET systems. The results provide a best-case scenario, as in vivo detection using PET/CT or PET/MRI will be affected by cell number, specific activity per cell, the density of cells within the target volume, and non-specific signal. This work has important implications for cell labeling studies in patients, particularly when using radiosensitive cells (e.g., T cells), which require detection of low cell numbers while minimizing radiation dose per cell.

TSPO Versus P2X7 as a Target for Neuroinflammation: An In Vitro and In Vivo Study.

Neuroinflammation is important in amyotrophic lateral sclerosis (ALS). The P2X7 receptor (P2X7R) is a promising target for neuroinflammation. The objective of this study was to compare 18F-DPA714, a second-generation translocator protein tracer, with 11C-JNJ717, a novel P2X7R tracer, in vitro and in vivo in ALS. Methods: For the in vitro portion of the study, autoradiography with 18F-DPA714 and 11C-JNJ717 was performed on human ALS brain sections in comparison to immunofluorescence with Iba1 and GFAP. For the in vivo portion, 3 male patients with early-stage ALS (59.3 ± 7.2 y old) and 6 healthy volunteers (48.2 ± 16.5 y old, 2 men and 4 women) underwent dynamic PET/MR scanning with 18F-DPA714 and 11C-JNJ717. Volume-of-distribution images were calculated using Logan plots and analyzed on a volume-of-interest basis. Results: Autoradiography showed no difference in 11C-JNJ717 binding but did show increased 18F-DPA714 binding in the motor cortex correlating with Iba1 expression (glial cells). Similar findings were observed in vivo, with a 13% increase in 18F-DPA714 binding in the motor cortex. Conclusion: In symptomatic ALS patients, 18F-DPA714 showed increased signal whereas 11C-JNJ717 was not elevated.

Regulating membrane lipid levels at the synapse by small-molecule inhibitors of monoacylglycerol lipase: new developments in therapeutic and PET imaging applications.

Monoacylglycerol lipase (MAGL) is a major endocannabinoid hydrolyzing enzyme and can be regulated to control endogenous lipid levels in the brain. This review highlights the pharmacological roles and in vivo PET imaging of MAGL in brain.

Evaluation of [11C]NMS-E973 as a PET tracer for in vivo visualisation of HSP90.

Heat shock protein 90 is an ATP-dependent molecular chaperone important for folding, maturation and clearance of aberrantly expressed proteins and is abundantly expressed (1-2% of all proteins) in the cytosol of all normal cells. In some tumour cells, however, strong expression of HSP90 is also observed on the cell membrane and in the extracellular matrix and the affinity of tumoural HSP90 for ATP domain inhibitors was reported to increase over 100-fold compared to that of HSP90 in normal cells. Here, we explore [11C]NMS-E973 as a PET tracer for in vivo visualisation of HSP90 and as a potential tool for in vivo quantification of occupancy of HSP90 inhibitors. Methods: HSP90 expression was biochemically characterized in a panel of established cell lines including the melanoma line B16.F10. B16.F10 melanoma xenograft tumour tissue was compared to non-malignant mouse tissue. NMS-E973 was tested in vitro for HSP90 inhibitory activity in several tumour cell lines. HSP90-specific binding of [11C]NMS-E973 was evaluated in B16.F10 melanoma cells and B16.F10 melanoma, prostate cancer LNCaP and PC3, SKOV-3 xenograft tumour slices and in vivo in a B16.F10 melanoma mouse model. Results: Strong intracellular upregulation and abundant membrane localisation of HSP90 was observed in the different tumour cell lines, in the B16.F10 tumour cell line and in B16.F10 xenograft tumours compared to non-malignant tissue. NMS-E973 showed HSP90-specific inhibition and reduced proliferation of cells. [11C]NMS-E973 showed strong binding to B16.F10 melanoma cells, which was inhibited by 200 µM of PU-H71, a non-structurally related HSP90 inhibitor. HSP90-specific binding was observed by in vitro autoradiography of murine B16.F10 melanoma, LNCaP and PC3 prostate cancer and SKOV-3 ovary carcinoma tissue slices. Further, B16.F10 melanoma-inoculated mice were subjected to a µPET study, where the tracer showed fast and persistent tumour uptake. Pretreatment of B16.F10 melanoma mice with PU-H71 or Ganetespib (50 mg/kg) completely blocked tumour accumulation of [11C]NMS-E973 and confirmed in vivo HSP90 binding specificity. HSP90-specific binding of [11C]NMS-E973 was observed in blood, lungs and spleen of tumour-bearing animals but not in control animals. Conclusion: [11C]NMS-E973 is a PET tracer for in vivo visualisation of tumour HSP90 expression and can potentially be used for quantification of HSP90 occupancy. Further translational evaluation of [11C]NMS-E973 is warranted.

Preclinical evaluation of [18 F]MA3: a CB2 receptor agonist radiotracer for PET.

BACKGROUND AND PURPOSE: Non-invasive in vivo imaging of cannabinoid CB2 receptors using PET is pursued to study neuroinflammation. The purpose of this study is to evaluate the in vivo binding specificity of [18 F]MA3, a CB2 receptor agonist, in a rat model with local overexpression of human (h) CB2 receptors. METHODS: [18 F]MA3 was produced with good radiochemical yield and radiochemical purity. The radiotracer was evaluated in rats with local overexpression of hCB2 receptors and in a healthy non-human primate using PET. KEY RESULTS: Ex vivo autoradiography demonstrated CB2 -specific binding of [18 F]MA3 in rat hCB2 receptor vector injected striatum. In a PET study, increased tracer binding in the hCB2 receptor vector-injected striatum compared to the contralateral control vector-injected striatum was observed. Binding in hCB2 receptor vector-injected striatum was blocked with a structurally non-related CB2 receptor inverse agonist, and a displacement study confirmed the reversibility of tracer binding. This study identified the utility of mutated inactive vector model for evaluation of CB2 receptor agonist PET tracers. [18 F]MA3 PET scans in the non-human primate showed good uptake and fast washout from brain, but no CB2 receptor-specific binding was observed. CONCLUSION AND IMPLICATIONS: Evaluation of [18 F]MA3 in a rat model with local overexpression of hCB2 receptors showed CB2 receptor-specific and reversible tracer binding. [18 F]MA3 showed good brain uptake and subsequent washout in a healthy non-human primate, but no specific binding was observed. Further clinical evaluation of [18 F]MA3 in patients with neuroinflammation is warranted. LINKED ARTICLES: This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.

Synthesis and preclinical evaluation of [11C]MA-PB-1 for in vivo imaging of brain monoacylglycerol lipase (MAGL).

MAGL is a potential therapeutic target for oncological and psychiatric diseases. Our objective was to develop a PET tracer for in vivo quantification of MAGL. We report [11C]MA-PB-1 as an irreversible MAGL inhibitor PET tracer. The in vitro inhibitory activity, ex vivo distribution, brain kinetics and specificity of [11C]MA-PB-1 binding were studied. Ex vivo biodistribution and microPET showed good brain uptake which could be blocked by pretreatment with both MA-PB-1 and a structurally non-related MAGL inhibitor MJN110. These initial results suggest that [11C]MA-PB-1 is a suitable tracer for in vivo imaging of MAGL.

[18F]JNJ42259152 binding to phosphodiesterase 10A, a key regulator of medium spiny neuron excitability, is altered in the presence of cyclic AMP.

Phosphodiesterase 10A (PDE10A) is a key regulator of medium spiny neuron excitability. Therefore, it plays an important role in the regulation of motor, reward, and cognitive processes. Despite the interest in PDE10A as a drug and positron emission tomography (PET) imaging target, little is known about the regulation of PDE10A enzymatic activity. This study aimed to further investigate the role of cAMP in the regulation of PDE10A activity and PDE10A PET imaging. Using [18 F]JNJ42259152 as radioligand, we investigated alterations in PDE10A binding secondary to changes in cAMP levels. An in vitro striatum homogenate binding assay was developed to determine KD and Bmax of [18 F]JNJ42259152. Homogenate binding was assessed after addition of increasing concentrations of exogenous cAMP (1, 10, and 100 µM). Rats were treated using JNJ49137530 and rolipram to induce in vivo alterations of cAMP. The effect of the induced cAMP alterations on PDE10A binding was assessed by comparing [18 F]JNJ42259152 microPET studies after treatment to microPET studies acquired at baseline conditions prior to treatment. In vitro binding affinity of [18 F]JNJ42259152 was higher in the presence of cAMP compared to baseline conditions (KD  = 3.17 ± 0.91 nM with 10 µM cAMP vs. KD  = 6.62 ± 0.7 nM at baseline). Inhibition of PDE4 using rolipram significantly increased [18 F]JNJ42259152 binding (BPND  = 2.61 ± 0.50 vs. 1.91 ± 0.36 at baseline). Administration of the PDE2 inhibitor JNJ49137530 significantly increased PDE10A binding potential (BPND  = 2.74 ± 0.22 vs. 2.05 ± 0.16 at baseline). Our data indicate an important role for cAMP in the regulation of PDE10A activity. Additionally, our data show a profound interaction between several PDEs in striatum.