Helge Thisgaard

The use of radiocobalt as a label improves imaging of EGFR using DOTA-conjugated Affibody molecule

Several anti-cancer therapies target the epidermal growth factor receptor (EGFR). Radionuclide imaging of EGFR expression in tumours may aid in selection of optimal cancer therapy. The 111In-labelled DOTA-conjugated ZEGFR:2377 Affibody molecule was successfully used for imaging of EGFR-expressing xenografts in mice. An optimal combination of radionuclide, chelator and targeting protein may further improve the contrast of radionuclide imaging. The aim of this study was to evaluate the targeting properties of radiocobalt-labelled DOTA-ZEGFR:2377. DOTA-ZEGFR:2377 was labelled with 57Co (T1/2 = 271.8 d), 55Co (T1/2 = 17.5 h), and, for comparison, with the positron-emitting radionuclide 68Ga (T1/2 = 67.6 min) with preserved specificity of binding to EGFR-expressing A431 cells. The long-lived cobalt radioisotope 57Co was used in animal studies. Both 57Co-DOTA-ZEGFR:2377 and 68Ga-DOTA-ZEGFR:2377 demonstrated EGFR-specific accumulation in A431 xenografts and EGFR-expressing tissues in mice. Tumour-to-organ ratios for the radiocobalt-labelled DOTA-ZEGFR:2377 were significantly higher than for the gallium-labelled counterpart already at 3 h after injection. Importantly, 57Co-DOTA-ZEGFR:2377 demonstrated a tumour-to-liver ratio of 3, which is 7-fold higher than the tumour-to-liver ratio for 68Ga-DOTA-ZEGFR:2377. The results of this study suggest that the positron-emitting cobalt isotope 55Co would be an optimal label for DOTA-ZEGFR:2377 and further development should concentrate on this radionuclide as a label.

Evaluation of Cobalt-Labeled Octreotide Analogs for Molecular Imaging and Auger Electron–Based Radionuclide Therapy

The somatostatin receptor, which is overexpressed by many neuroendocrine tumors, is a well-known target for molecular imaging and peptide receptor radionuclide therapy. Recently, 57Co-labeled DOTATOC, an octreotide analog, was shown to have the highest affinity yet found for somatostatin receptor subtype 2. The aim of this study was to evaluate the biologic effects of novel cobalt-labeled octreotide analogs targeting the somatostatin receptor to identify promising candidates for molecular imaging and Auger electron–based radionuclide therapy. Methods: Cobalt-labeled DOTATATE, DOTATOC, and DOTANOC were prepared with 57Co or 58mCo for SPECT or Auger electron–based therapy, respectively. The cellular uptake and intracellular distribution of the radioligands were characterized with the pancreatic tumor cell line AR42J in vitro, including assessment of the therapeutic effects of 58mCo-DOTATATE via DNA double-strand break and proliferation assays. Comparisons with the therapeutic effects of 111In- and 177Lu-DOTATATE were also performed. Tumor uptake and normal tissue uptake were characterized in a subcutaneous pancreatic tumor mouse model. Results: All 3 cobalt-conjugated peptides resulted in time-dependent and receptor-specific uptake, with a high level (≥88%) of cellular internalization in vitro of the total cell-associated radioactivity. The DNA double-strand break yield showed a dose-dependent increase with activity, whereas cell survival showed a dose-dependent decrease. 58mCo-DOTATATE was significantly more efficient in cell killing per cumulated decay than 111In- and 177Lu-DOTATATE. The in vivo pharmacokinetic studies showed a high level of receptor-specific tumor uptake. Conclusion: All cobalt-labeled radioligands showed a high level of receptor-specific uptake both in vitro and in vivo in tumor-bearing mice. Furthermore, 58mCo-DOTATATE showed considerable therapeutic effects in vitro and, thus, could be an effective agent for eradicating disseminated tumor cells and micrometastases.

PET imaging with the non-pure positron emitters: 55Co, 86Y and 124I

PET/CT with non-pure positron emitters is a highly valuable tool in immuno-PET and for pretherapeutic dosimetry. However, imaging is complicated by prompt gamma coincidences (PGCs) that add an undesired background activity to the images. Time-of-flight (TOF) reconstruction improves lesion detectability in (18)F-PET and can potentially also improve the signal-to-noise ratio in images acquired with non-pure positron emitters. Using the GE Discovery 690 PET/CT system, we evaluated the image quality with (55)Co, (86)Y and (124)I, and the effect of PGC-correction and TOF-reconstruction on image quality and quantitation in a series of phantom studies. PET image quality and quantitation for all isotopes were significantly affected by PGCs. The effect was most severe with (86)Y, and less, but comparable, with (55)Co and (124)I. PGC-correction improved the image quality and the quantitation accuracy dramatically for all isotopes, especially when the activity was limited to a few hot lesions in a warm background. In imaging situations, where high levels of activity were present in the background, activity concentrations were overestimated. TOF-reconstruction improved image quality in isolated lesions but worsened the accuracy of quantitation and uniformity in homogeneous activity distributions. Better modelling of PGCs in the scatter correction can potentially improve the situation.

Evaluation of somatostatin and nucleolin receptors for therapeutic delivery in non-small cell lung cancer stem cells applying the somatostatin-analog DOTATATE and the nucleolin-targeting aptamer AS1411

Cancer stem cells represent the putative tumor-driving subpopulation thought to account for drug resistance, relapse, and metastatic spread of epithelial and other cancer types. Accordingly, cell surface markers for therapeutic delivery to cancer stem cells are subject of intense research. Somatostatin receptor 2 and nucleolin are known to be overexpressed by various cancer types, which have elicited comprehensive efforts to explore their therapeutic utilization. Here, we evaluated somatostatin receptor 2 targeting and nucleolin targeting for therapeutic delivery to cancer stem cells from lung cancer. Nucleolin is expressed highly but not selectively, while somatostatin receptor 2 is expressed selectively but not highly by cancer cells. The non-small cell lung cancer cell lines A549 and H1299, displayed average levels of both surface molecules as judged based on analysis of a larger cell line panel. H1299 compared to A549 cells showed significantly elevated sphere-forming capacity, indicating higher cancer stem cell content, thus qualifying as suitable test system. Nucleolin-targeting 57Co-DOTA-AS1411 aptamer showed efficient internalization by cancer cells and, remarkably, at even higher efficiency by cancer stem cells. In contrast, somatostatin receptor 2 expression levels were not sufficiently high in H1299 cells to confer efficient uptake by either non-cancer stem cells or cancer stem cells. The data provides indication that the nucleolin-targeting AS1411 aptamer might be used for therapeutic delivery to non-small cell lung cancer stem cells.

Seeing the Unseen—Bioturbation in 4D: Tracing Bioirrigation in Marine Sediment Using Positron Emission Tomography and Computed Tomography

Understanding spatial and temporal patterns of bioirrigation induced by benthic fauna ventilation is critical given its significance on benthic nutrient exchange and biogeochemistry in coastal ecosystems. The quantification of this process challenges marine scientists because faunal activities and behaviors are concealed in an opaque sediment matrix. Here, we use a hybrid medical imaging technique, positron emission tomography and computed tomography (PET/CT) to provide a qualitative visual and fully quantitative description of bioirrigation in 4D (space and time). As a study case, we present images of porewater advection induced by the well-studied lugworm (Arenicola marina). Our results show that PET/CT allows more comprehensive studies on ventilation and bioirrigation than possible using techniques traditionally applied in marine ecology. We provide a dynamic three-dimensional description of bioirrigation by the lugworm at very high temporal and spatial resolution. Results obtained with the PET/CT are in agreement with literature data on lugworm ventilation and bioirrigation. Major advantages of PET/CT over methods commonly used are its non-invasive and non-destructive approach and its capacity to provide information that otherwise would require multiple methods. Furthermore, PET/CT scan is versatile as it can be used for a variety of benthic macrofauna species and sediment types and it provides information on burrow morphology or animal behavior. The lack of accessibility to the expensive equipment is its major drawback which can only be overcome through collaboration among several institutions.

Estimation of Tumor Volumes by 11C-MeAIB and 18F-FDG PET in an Orthotopic Glioblastoma Rat Model

Brain tumor volume assessment is a major challenge. Molecular imaging using PET may be a promising option because it reflects the biologically active cells. We compared the agreement between PET- and histology-derived tumor volumes in an orthotopic glioblastoma rat model with a noninfiltrating (U87MG) and an infiltrating (T87) tumor phenotype using 2 different radiotracers, 2 different image reconstruction algorithms, parametric imaging, and 2 different image segmentation techniques. Methods: Rats with U87MG- and T87-derived glioblastomas were continuously scanned with PET for 1 h starting immediately after the injection of 11C-methylaminoisobutyric acid (11C-MeAIB). One hour later, 18F-FDG was injected, followed by a 3-h dynamic PET scan. Images were reconstructed using 2-dimensional ordered-subsets expectation maximization and 3-dimensional maximum a posteriori probability (MAP3D) algorithms. In addition, a parametric image, encompassing the entire tumor kinetics in a single image, was calculated on the basis of the 11C-MeAIB images. All reconstructed images were segmented by fixed thresholding of maximum voxel intensity (VImax) and mean background intensity. The agreement between PET- and histology-derived tumor volumes and intra- and interobserver agreement of the PET-derived volumes were evaluated using Bland–Altman plots. Results: By PET, the mean U87MG tumor volume was 35.0 mm3 using 18F-FDG and 34.1 mm3 with 11C-MeAIB, compared with 33.7 mm3 by histology. Corresponding T87 tumor volumes were 122.1 mm3 using 18F-FDG, 118.3 mm3 with 11C-MeAIB, and 125.4 mm3 by histology. None of these volumes were significantly different. The best agreement between PET- and histology-derived U87MG tumor volumes was achieved with 11C-MeAIB, MAP3D reconstruction, and fixed thresholding of VImax. The intra- and interobserver agreement was high using this method. For T87 tumors, the best agreement between PET- and histology-derived volumes was obtained using 18F-FDG, MAP3D reconstruction, and fixed thresholding of mean background intensity. The agreement using 11C-MeAIB, parametric imaging, and fixed thresholding of VImax was slightly inferior, but the intra- and interobserver agreement was clearly superior. Conclusion: Estimation of tumor volume by PET of noninfiltrating brain tumors was accurate and reproducible. In contrast, tumor volume estimation by PET of infiltrating brain tumors was difficult and hard to reproduce. On the basis of our results, PET evaluation of highly infiltrating brain tumors should be further developed.

Novel radioisotope-based nanomedical approaches

Abstract Radioisotope therapy of cancer is on the rise applying mainly β-emitting radionuclides. However, due to exposure of healthy tissues, the maximum achievable radiation dose with these is limited. Auger-electron emitters (AEs) represent a promising alternative because of their mode of decay within a short nanometer range. The challenge is that their therapeutic efficacy relies on a close vicinity to DNA. To overcome this and to minimize toxicity, the construction of smart nanomedical devices is required, which ascertain tumor cell targeting with succeeding cellular uptake and nuclear translocation. In this review we describe the potential of AEs with focus on their delivery down to the DNA level and their cellular effects. Reported efforts comprise different tumor-targeting strategies, including the use of antibodies or peptides with nuclear localizing sequences. Recently, attention has shifted to various nanoparticle formats for overcoming delivery problems. To this end, these approaches have mostly been tested in cell lines in vitro applying AEs more suited for imaging than therapy. This defines a demand for nanomedical formulations with documented in vivo activity, using AEs selected for their therapeutic potential to come closer to real clinical settings.

High Contrast PET Imaging of GRPR Expression in Prostate Cancer Using Cobalt-Labeled Bombesin Antagonist RM26

High gastrin releasing peptide receptor (GRPR) expression is associated with numerous cancers including prostate and breast cancer. The aim of the current study was to develop a 55Co-labeled PET agent based on GRPR antagonist RM26 for visualization of GRPR-expressing tumors. Labeling with 57Co and 55Co, stability, binding specificity, and in vitro and in vivo characteristics of 57Co-NOTA-PEG2-RM26 were studied. NOTA-PEG2-RM26 was successfully radiolabeled with 57Co and 55Co with high yields and demonstrated high stability. The radiopeptide showed retained binding specificity to GRPR in vitro and in vivo. 57Co-NOTA-PEG2-RM26 biodistribution in mice was characterized by rapid clearance of radioactivity from blood and normal non-GRPR-expressing organs and low hepatic uptake. The clearance was predominantly renal with a low degree of radioactivity reabsorption. Tumor-to-blood ratios were approximately 200 (3 h pi) and 1000 (24 h pi). The favorable biodistribution of cobalt-labeled NOTA-PEG2-RM26 translated into high contrast preclinical PET/CT (using 55Co) and SPECT/CT (using 57Co) images of PC-3 xenografts. The initial biological results suggest that 55Co-NOTA-PEG2-RM26 is a promising tracer for PET visualization of GRPR-expressing tumors.

Chelation, formulation, encapsulation, retention, and in vivo biodistribution of hydrophobic nanoparticles labelled with 57Co-porphyrin: Oleylamine ensures stable chelation of cobalt in nanoparticles that accumulate in tumors

Background and motivation: While small molecules can be used in cancer diagnosis there is a need for imageable diagnostic NanoParticles (NPs) that act as surrogates for the therapeutic NPs. Many NPs are composed of hydrophobic materials so the challenge is to formulate hydrophobic imaging agents. To develop individualized medical treatments based on NP, a first step should be the selection of patients who are likely responders to the treatment as judged by imaging tumor accumulation of NPs. This requires NPs with the same size and structure as the subsequent therapeutic NPs but labelled with a long‐lived radionuclide. Cobalt isotopes are good candidates for NP labelling since 55Co has half‐life of 17.5h and positron energy of 570keV while 57Co (t1/2 271.6 d) is an isotope suited for preclinical single photon emission tomography (SPECT) to visualize biodistribution and pharmacokinetics of NPs. We used the hydrophobic octaethyl porphyrin (OEP) to chelate cobalt and to encapsulate it inside hydrophobic liquid NPs (LNPs). We hypothesized that at least two additional hydrophobic axial ligands (oleylamine, OA) must be provided to the OEP‐Co complex in order to encapsulate and retain Co inside LNP. Results: 1. Cobalt chelation by OEP and OA. The association constant of cobalt to OEP was 2.49×105 M−1 and the formation of the hexacoordinate complex OEP‐Co‐4OA was measured by spectroscopy. 2. NP formulation and characterization: LNPs were prepared by the fast ethanol injection method and were composed of a liquid core (triolein) surrounded by a lipid monolayer (DSPC:Cholesterol:DSPE‐PEG2000). The size of the LNPs loaded with the cobalt complex was 40±5nm, 3. Encapsulation of OEP‐Co‐OA: The loading capacity of OEP‐Co‐OA in LNP was 5mol%. 4. Retention of OEP‐57Co‐4OA complex in the LNPs: the positive effect of the OA ligands was demonstrated on the stability of the OEP‐57Co‐4OA complex, providing a half‐life for retention in PBS of 170h (7days) while in the absence of the axial OA ligands was only 22h. 5 Biodistribution Study: the in vivo biodistribution of LNP was studied in AR42J pancreatic tumor‐bearing mice. The estimated half‐life of LNPs in blood was about 7.2h. Remarkably, the accumulation of LNPs in the tumor was as high as 9.4% ID/g 24h after injection with a doubling time for tumor accumulation of 3.22h. The most important result was that the nanoparticles could indeed accumulate in the AR42J tumors up to levels greater than those of other NPs previously measured in the same tumor model, and at about half the values reported for the molecular agent 57Co‐DOTATATE. Conclusions: The additional hydrophobic chelator OA was indeed needed to obtain a stable octahedral OEP‐Co‐4OA. Cobalt was actually well‐retained inside LNP in the OEP‐Co‐4OA complex. The method described in the present work for the core‐labelling of LNPs with cobalt is now ready for labeling of NPs with 55Co, or indeed other hexadentate radionuclides of interest for preclinical in vivo PET‐imaging and radio‐therapeutics.

Formulation and in vivo biodistribution of 57Co-porphyrin-labelled hydrophobic liquid nanoparticles

European Journal of Nuclear Medicine and Molecular Imaging Volume 44, Supplement 2 10.1007/s00259-017-3822-1 This supplement was not sponsored by outside commercial interests. It was funded entirely by the association’s own resources DOI 10.1007/s00259-017-3822-1 S119 Eur J Nucl Med Mol Imaging (2017) 44 (Suppl 2):S119–S956