Publications by authors named "Christel Vangestel"

36 Publications

Targeting fibroblast activation protein (FAP): next generation PET radiotracers using squaramide coupled bifunctional DOTA and DATA chelators.

EJNMMI Radiopharm Chem 2020 Jul 29;5(1):19. Epub 2020 Jul 29.

Department of Chemistry - TRIGA Site, Johannes Gutenberg University Mainz, 55128, Mainz, Germany.

Background: Fibroblast activation protein (FAP) is a proline selective serine protease that is overexpressed in tumor stroma and in lesions of many other diseases that are characterized by tissue remodeling. In 2014, a most potent FAP-inhibitor (referred to as UAMC1110) with low nanomolar FAP-affinity and high selectivity toward related enzymes such as prolyl oligopeptidase (PREP) and the dipeptidyl-peptidases (DPPs): DPP4, DPP8/9 and DPP2 were developed. This inhibitor has been adopted recently by other groups to create radiopharmaceuticals by coupling bifunctional chelator-linker systems. Here, we report squaric acid (SA) containing bifunctional DATA and DOTA chelators based on UAMC1110 as pharmacophor. The novel radiopharmaceuticals DOTA.SA.FAPi and DATA.SA.FAPi with their non-radioactive derivatives were characterized for in vitro inhibitory efficiency to FAP and PREP, respectively and radiochemical investigated with gallium-68. Further, first proof-of-concept in vivo animal study followed by ex vivo biodistribution were determined with [Ga]Ga-DOTA.SA.FAPi.

Results: [Ga]Ga-DOTA.SA.FAPi and [Ga]Ga-DATA.SA.FAPi showed high complexation > 97% radiochemical yields after already 10 min and high stability over a period of 2 h. Affinity to FAP of DOTA.SA.FAPi and DATA.SA.FAPi and its Ga and Lu-labeled derivatives were excellent resulting in low nanomolar IC values of 0.7-1.4 nM. Additionally, all five compounds showed low affinity for the related protease PREP (high IC with 1.7-8.7 μM). First proof-of-principle in vivo PET-imaging animal studies of the [Ga]Ga-DOTA.SA.FAPi precursor in a HT-29 human colorectal cancer xenograft mouse model indicated promising results with high accumulation in tumor (SUV of 0.75) and low background signal. Ex vivo biodistribution showed highest uptake in tumor (5.2%ID/g) at 60 min post injection with overall low uptake in healthy tissues.

Conclusion: In this work, novel PET radiotracers targeting fibroblast activation protein were synthesized and biochemically investigated. Critical substructures of the novel compounds are a squaramide linker unit derived from the basic motif of squaric acid, DOTA and DATA bifunctional chelators and a FAP-targeting moiety. In conclusion, these new FAP-ligands appear promising, both for further research and development as well as for first human application.
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http://dx.doi.org/10.1186/s41181-020-00102-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7391456PMC
July 2020

Development of Antibody Immuno-PET/SPECT Radiopharmaceuticals for Imaging of Oncological Disorders-An Update.

Cancers (Basel) 2020 Jul 11;12(7). Epub 2020 Jul 11.

Molecular Imaging Center Antwerp, Faculty of Medicine and Health Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium.

Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) are molecular imaging strategies that typically use radioactively labeled ligands to selectively visualize molecular targets. The nanomolar sensitivity of PET and SPECT combined with the high specificity and affinity of monoclonal antibodies have shown great potential in oncology imaging. Over the past decades a wide range of radio-isotopes have been developed into immuno-SPECT/PET imaging agents, made possible by novel conjugation strategies (e.g., site-specific labeling, click chemistry) and optimization and development of novel radiochemistry procedures. In addition, new strategies such as pretargeting and the use of antibody fragments have entered the field of immuno-PET/SPECT expanding the range of imaging applications. Non-invasive imaging techniques revealing tumor antigen biodistribution, expression and heterogeneity have the potential to contribute to disease diagnosis, therapy selection, patient stratification and therapy response prediction achieving personalized treatments for each patient and therefore assisting in clinical decision making.
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http://dx.doi.org/10.3390/cancers12071868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7408676PMC
July 2020

Preclinical Evaluation of a Novel F-Labeled dTCO-Amide Derivative for Bioorthogonal Pretargeted Positron Emission Tomography Imaging.

ACS Omega 2020 Mar 26;5(9):4449-4456. Epub 2020 Feb 26.

Laboratory of Medicinal Chemistry, University of Antwerp, Antwerp, Wilrijk 2610, Belgium.

Pretargeted positron emission tomography (PET) imaging based on the bioorthogonal inverse-electron-demand Diels-Alder reaction between tetrazines (Tz) and -cyclooctenes (TCO) has emerged as a promising tool for solid tumor imaging, allowing the use of short-lived radionuclides in immune-PET applications. With this strategy, it became possible to achieve desirable target-to-background ratios and at the same time to decrease the radiation burden to nontargeted tissues because of the fast clearance of small PET probes. Here, we show the synthesis of novel F-labeled dTCO-amide probes for pretargeted immuno-PET imaging. The PET probes were evaluated regarding their stability, reactivity toward tetrazine, and pharmacokinetic profile. showed an extremely fast kinetic rate (10,553 M s in 50:50 MeOH/water), good stability in saline and plasma up to 4 h at 37 °C with no isomerization observed, and the biodistribution in healthy mice revealed a mixed hepatobiliary and renal clearance with no defluorination and low background in other tissues. was further used for in vivo pretargeted immune-PET imaging carried out in nude mice bearing LS174T colorectal tumors that were previously treated with a tetrazine-modified anti-TAG-72 monoclonal antibody (CC49). Pretargeted μPET imaging results showed clear visualization of the tumor tissue with a significantly higher uptake when compared to the control.
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http://dx.doi.org/10.1021/acsomega.9b03584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7066555PMC
March 2020

Improved stability of a novel fluorine-18 labeled TCO analogue for pretargeted PET imaging.

Nucl Med Biol 2019 Sep - Oct;76-77:36-42. Epub 2019 Nov 5.

Laboratory of Medicinal Chemistry, University of Antwerp, Antwerp, Belgium.

Introduction: Biorthogonal pretargeted imaging using the inverse electron demand Diels Alder (IEDDA) reaction between tetrazine (Tz) and trans-cyclooctene (TCO) is one of the most attractive strategies in molecular imaging. It allows the use of short-lived radioisotopes such as fluorine-18 for imaging of long circulating vectors with improved imaging contrast and reduced radiation dose. Here we aim to develop a novel F-labeled trans-cyclooctene (TCO) with improved metabolic stability and assess its potential usefulness in a pretargeted PET imaging approach.

Methods: We have synthetized a new TCO-analogue containing a 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) chelator, allowing radiolabeling by chelation with aluminum fluoride (Al[F]F). Stability and pharmacokinetic profile of Al[F]F-NOTA-TCO ([F]MICA-205) were evaluated in healthy animals at different timepoints after injection of the radiotracer. To assess the potential use of this new PET tracer for tumor targeting, in vivo pretargeted PET imaging was performed in LS174T tumor-bearing mice pre-treated with a tetrazine-modified anti-TAG-72 monoclonal antibody (CC49).

Results: The radiotracer was obtained with a radiochemical yield (RCY) of 12.8 ± 2.8% and a radiochemical purity (RCP) of ≥95%. It also showed a promising in vivo stability with 51.9 ± 5.16% of radiotracer remaining intact after 1 h. The biodistribution in healthy mice demonstrated mixed hepatobiliary and renal clearance, with a rapid blood clearance and low uptake in other tissues. The low bone uptake indicated lack of tracer defluorination. Interestingly, a pretargeted PET imaging experiment showed a significantly increased radiotracer uptake (0.67 ± 0.16%ID/g, p < 0.001) in the tumors of mice pre-treated with CC49-tetrazine compared to the CC49 alone (0.16 ± 0.08%ID/g).

Conclusions: [F]MICA-205 represents a large improvement in in vivo metabolic stability compared to previous reported 18F-labeled TCOs, allowing a clear visualization of tumor tissue in a small-animal pretargeted PET imaging experiment. Despite the favorable in vivo stability and image contrast obtained with [F]MICA-205, the development of next-generation derivatives with increased absolute tumor uptake is warranted for future pretargeting applications.
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http://dx.doi.org/10.1016/j.nucmedbio.2019.11.001DOI Listing
July 2020

Effects of metformin on tumor hypoxia and radiotherapy efficacy: a [F]HX4 PET imaging study in colorectal cancer xenografts.

EJNMMI Res 2019 Aug 2;9(1):74. Epub 2019 Aug 2.

Molecular Imaging Center Antwerp (MICA), University of Antwerp, Universiteitsplein 1, Antwerp, 2610, Belgium.

Background: In a colorectal cancer xenograft model, we investigated the therapeutic effect of metformin on tumor hypoxia with [F]flortanidazole ([F]HX4) small-animal positron emission tomography (μPET). We also assessed the additive effect of metformin on long-term radiotherapy outcome and we studied the potential of [F]HX4 as a predictive and/or prognostic biomarker within this setup.

Methods: Colo205-bearing mice (n = 40) underwent a baseline [F]HX4 hypoxia μPET/computed tomography (CT) scan. The next day, mice received 100 mg/kg metformin or saline intravenously (n = 20/group) and [F]HX4 was administered intravenously 30 min later, whereupon a second μPET/CT scan was performed to assess changes in tumor hypoxia. Two days later, mice were further divided into four therapy groups (n = 10/group): control (1), metformin (2), radiotherapy (3), and metformin + radiotherapy, i.e., combination (4). Then, they received a second dose of metformin (groups 2 and 4) or saline (groups 1 and 3), followed by a single radiotherapy dose of 15 Gy (groups 3 and 4) or sham irradiation (groups 1 and 2) 30 min later. Tumor growth was followed three times a week by caliper measurements to assess the therapeutic outcome.

Results: [F]HX4 uptake decreased in metformin-treated tumors with a mean intratumoral reduction in [F]HX4 tumor-to-background ratio (TBR) from 2.53 ± 0.30 to 2.28 ± 0.26 (p = 0.04), as opposed to saline treatment (2.56 ± 0.39 to 3.08 ± 0.39; p = 0.2). The median tumor doubling time (TDT) was 6, 8, 41, and 43 days in the control, metformin, radiotherapy and combination group, respectively (log-rank p < 0.0001), but no metformin-specific therapy effects could be detected. Baseline [F]HX4 TBR was a negative prognostic biomarker for TDT (hazard ratio, 2.39; p = 0.02).

Conclusions: Metformin decreased [F]HX4 uptake of Colo205-tumors, but had no additive effect on radiotherapy efficacy. Nevertheless, [F]HX4 holds promise as a prognostic imaging biomarker.
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http://dx.doi.org/10.1186/s13550-019-0543-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6677842PMC
August 2019

Bone metastases in the era of targeted treatments: insights from molecular biology.

Q J Nucl Med Mol Imaging 2019 Jun 11;63(2):98-111. Epub 2019 Jul 11.

INSERM Laboratory Pathophysiology, Diagnosis and Treatments of Bone Diseases, Lyon, France.

Bone metastases remain a common feature of advanced cancers and are associated with significant morbidity and mortality. Recent research has identified promising novel treatment targets to improve current treatment strategies for bone metastatic disease. This review summarizes the well-known and recently discovered molecular biology pathways in bone that govern normal physiological remodeling or drive the pathophysiological changes observed when bone metastases are present. In the rapidly changing world of targeted cancer treatments, it is important to recognize the specific treatment effects induced in bone by these agents and the potential impact on common imaging strategies. The osteoclastic targets (bisphosphonates, LGR4, RANKL, mTOR, MET-VEGFR, cathepsin K, Src, Dock 5) and the osteoblastic targets (Wnt and endothelin) are discussed, and the emerging field of osteo-immunity is introduced as potential future therapeutic target. Finally, a summary is provided of available trial data for agents that target these pathways and that have been assessed in patients. The ultimate goal of research into novel pathways and targets involved in the tumor-bone microenvironment is to tackle one of the great remaining unmet needs in oncology, that is finding a cure for bone metastatic disease.
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http://dx.doi.org/10.23736/S1824-4785.19.03203-5DOI Listing
June 2019

How to Modulate Tumor Hypoxia for Preclinical In Vivo Imaging Research.

Contrast Media Mol Imaging 2018 18;2018:4608186. Epub 2018 Oct 18.

Molecular Imaging Center Antwerp (MICA), University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp, Belgium.

Tumor hypoxia is related with tumor aggressiveness, chemo- and radiotherapy resistance, and thus a poor clinical outcome. Therefore, over the past decades, every effort has been made to develop strategies to battle the negative prognostic influence of tumor hypoxia. For appropriate patient selection and follow-up, noninvasive imaging biomarkers such as positron emission tomography (PET) radiolabeled ligands are unprecedentedly needed. Importantly, before being able to implement these new therapies and potential biomarkers into the clinical setting, preclinical in vivo validation in adequate animal models is indispensable. In this review, we provide an overview of the different attempts that have been made to create differential hypoxic in vivo cancer models with a particular focus on their applicability in PET imaging studies.
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http://dx.doi.org/10.1155/2018/4608186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6211155PMC
July 2019

F-Flortanidazole Hypoxia PET Holds Promise as a Prognostic and Predictive Imaging Biomarker in a Lung Cancer Xenograft Model Treated with Metformin and Radiotherapy.

J Nucl Med 2019 01 6;60(1):34-40. Epub 2018 Jul 6.

Molecular Imaging Center Antwerp (MICA), University of Antwerp, Wilrijk, Belgium

Metformin may improve tumor oxygenation and thus radiotherapy response, but imaging biomarkers for selection of suitable patients are still under investigation. First, we assessed the effect of acute metformin administration on non-small cell lung cancer xenograft tumor hypoxia using PET imaging with the hypoxia tracer F-flortanidazole. Second, we verified the effect of a single dose of metformin before radiotherapy on long-term treatment outcome. Third, we examined the potential of baseline F-flortanidazole as a prognostic or predictive biomarker for treatment response. A549 tumor-bearing mice underwent a F-flortanidazole PET/CT scan to determine baseline tumor hypoxia. The next day, mice received a 100 mg/kg intravenous injection of metformin. F-flortanidazole was administered intravenously 30 min later, and a second PET/CT scan was performed to assess changes in tumor hypoxia. Two days later, the mice were divided into 3 therapy groups: controls (group 1), radiotherapy (group 2), and metformin + radiotherapy (group 3). Animals received saline (groups 1-2) or metformin (100 mg/kg; group 3) intravenously, followed by a single radiotherapy dose of 10 Gy (groups 2-3) or sham irradiation (group 1) 30 min later. Tumor growth was monitored triweekly by caliper measurement, and tumor volume relative to baseline was calculated. The tumor doubling time (TDT), that is, the time to reach twice the preirradiation tumor volume, was defined as the endpoint. Thirty minutes after metformin treatment, F-flortanidazole demonstrated a significant change in tumor hypoxia, with a mean intratumoral reduction in F-flortanidazole tumor-to-background ratio (TBR) from 3.21 ± 0.13 to 2.87 ± 0.13 ( = 0.0001). Overall, relative tumor volume over time differed across treatment groups ( < 0.0001). Similarly, the median TDT was 19, 34, and 52 d in controls, the radiotherapy group, and the metformin + radiotherapy group, respectively (log-rank < 0.0001). Both baseline F-flortanidazole TBR (hazard ratio, 2.0; = 0.0004) and change from baseline TBR (hazard ratio, 0.39; = 0.04) were prognostic biomarkers for TDT irrespective of treatment, and baseline TBR predicted metformin-specific treatment effects that were dependent on baseline tumor hypoxia. Using F-flortanidazole PET imaging in a non-small cell lung cancer xenograft model, we showed that metformin may act as a radiosensitizer by increasing tumor oxygenation and that baseline F-flortanidazole shows promise as an imaging biomarker.
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http://dx.doi.org/10.2967/jnumed.118.212225DOI Listing
January 2019

Preclinical data on the combination of cisplatin and anti-CD70 therapy in non-small cell lung cancer as an excellent match in the era of combination therapy.

Oncotarget 2017 Sep 23;8(43):74058-74067. Epub 2017 May 23.

Center for Oncological Research, University of Antwerp, Antwerp, 2610 Wilrijk, Belgium.

In contrast to the negligible expression of the immunomodulating protein CD70 in normal tissue, we have demonstrated constitutive overexpression of CD70 on tumor cells in a subset of primary non-small cell lung cancer (NSCLC) biopsies. This can be exploited by CD70-targeting antibody-dependent cellular cytotoxicity (ADCC)-inducing antibodies. Early clinical trials of these antibodies have already shown promising results in CD70-positive malignancies. In this study, we explored the potential of cisplatin to induce CD70 expression in NSCLC. Using real-time measurement tools, we also assessed the efficacy of a combination regimen with cisplatin and anti-CD70 therapy under normoxia and hypoxia. We identified an induction of CD70 expression on lung cancer cells upon low doses of cisplatin, independent of oxygen levels. More importantly, the use of cisplatin resulted in an enhanced ADCC-effect of anti-CD70 therapy. As such, this combination regimen led to a significant decrease in lung cancer cell survival cell survival, broadening the applicability the applicability of CD70-targeting therapy. This is the first study that proves the potential of a combination therapy with cisplatin and CD70-targeting drugs in NSCLC. Based on our data, we postulate that this combination strategy is an interesting approach to increase tumor-specific cytotoxicity and reduce drug-related side effects.
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http://dx.doi.org/10.18632/oncotarget.18202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5650323PMC
September 2017

[Tc]duramycin for cell death imaging: Impact of kit formulation, purification and species difference.

Nucl Med Biol 2018 Jan 14;56:1-9. Epub 2017 Sep 14.

Molecular Imaging Center Antwerp, University of Antwerp, Campus Drie Eiken, Universiteitsplein 1, B-2610 Wilrijk, Belgium; Antwerp University Hospital, Department of Nuclear Medicine, Wilrijkstraat 10, B-2650, Edegem, Belgium. Electronic address:

Introduction: [Tc]duramycin is a SPECT tracer for cell death imaging. We evaluated the impact of kit formulation, purification and species difference on the pharmacokinetic profile and cell death targeting properties of [Tc]duramycin in order to define the optimal conditions for (pre-)clinical use.

Methods: Three kits were prepared (A: traditional formulation, B: containing 1/3 of ingredients, C: containing HYNIC-PEG-duramycin). Following labeling, the kits were used without purification, or with SPE or HPLC purification. The pharmacokinetic profile was evaluated in mice and rats at 24 h post tracer injection (p.i.). Non-specific accumulation of [Tc]duramcyin was studied by μSPECT imaging in chemotherapy treated COLO205 tumor bearing mice pre-treated with cold duramycin (0.01-50 μg). Cell death targeting ability of the kits displaying the best pharmacokinetic profile was compared in a treatment response study in COLO205 tumor bearing mice treated with conatumumab (anti-DR5 antibody).

Results: HPLC purification of kit prepared [Tc]duramycin and reducing the amount of kit ingredients resulted in the best pharmacokinetic profile with low accumulation in liver, spleen and kidneys. The use of PEGylated [Tc]duramycin required longer circulation times (> 4 h pi) to obtain good imaging characteristics. Pre-treatment with duramycin significantly decreased tracer uptake in chemotherapy treated tumors in a dose-dependent manner. A blocking dose of 50 μg significantly increased non-specific accumulation in liver and spleen. Non-specific accumulation of [Tc]duramycin was however demonstrated to be species dependent. HPLC purified kit A (5.21±1.71 %ID/cc) and non-purified kit B (1.68±0.46 %ID/cc) demonstrated a significant increase in tumor uptake compared to baseline following conatumumab treatment.

Conclusions: To obtain [Tc]duramycin with favorable imaging characteristics for cell death imaging in mice [Tc]duramycin needs to be prepared with high specific activity by applying HPLC purification. The need for HPLC purification appears to be a species dependent phenomenon and might therefore not be required for clinical translation.
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http://dx.doi.org/10.1016/j.nucmedbio.2017.08.005DOI Listing
January 2018

ATG4B inhibitors with a benzotropolone core structure block autophagy and augment efficiency of chemotherapy in mice.

Biochem Pharmacol 2017 08 19;138:150-162. Epub 2017 Jun 19.

Laboratory of Physiopharmacology, Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium. Electronic address:

Autophagy is a cell survival mechanism hijacked by advanced tumors to endure a rough microenvironment. Late autophagy inhibitors such as (hydroxy)chloroquine have been used clinically to halt tumor progression with modest success. However, given the toxic nature of these compounds and their lack of specificity, novel targets should be considered. We recently identified a benzotropolone derivative that significantly inhibited the essential autophagy protein ATG4B. Therefore, we synthesized and tested additional benzotropolone compounds to identify a promising ATG4B inhibitor that impairs autophagy both in vitro and in vivo. A compound library containing 27 molecules with a benzotropolone backbone was synthesized and screened for inhibition of recombinant ATG4B. Depending on the benzotropolone compound, inhibition of recombinant ATG4B ranged from 3 to 82%. Active compounds were evaluated in cellular assays to confirm inhibition of ATG4B and suppression of autophagy. Seven compounds inhibited processing of the autophagy protein LC3 and autophagosome formation. Compound UAMC-2526 was selected for further in vivo use because of its fair plasma stability. This compound abolished autophagy both in nutrient-deprived GFP-LC3 mice and in CD1 Foxn1nu mice bearing HT29 colorectal tumor xenografts. Moreover, addition of UAMC-2526 to the chemotherapy drug oxaliplatin significantly improved inhibition of tumor growth. Our data indicate that suppression of autophagy via ATG4B inhibition is a feasible strategy to augment existing chemotherapy efficacy and to halt tumor progression.
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http://dx.doi.org/10.1016/j.bcp.2017.06.119DOI Listing
August 2017

Characterization of an Orthotopic Colorectal Cancer Mouse Model and Its Feasibility for Accurate Quantification in Positron Emission Tomography.

Mol Imaging Biol 2017 Oct;19(5):762-771

Molecular Imaging Center Antwerp (MICA), Faculty of Medicine and Health Sciences, University of Antwerp, Universiteitsplein 1, 2610, Wilrijk, Belgium.

Purpose: Quantification in positron emission tomography (PET) imaging of an orthotopic mouse model of colorectal cancer (CRC) is challenging due to difficult tumor delineation. We aimed to establish a reproducible delineation approach, evaluate its feasibility for reliable PET quantification and compare its added translational value with its subcutaneous counterpart.

Procedures: A subcutaneous Colo205-luc2 tumor fragment harvested from a donor mouse was transplanted onto the caecum of nude mice, with (n = 10) or without (n = 10) the addition of an X-ray detectable thread. Animals underwent 2-deoxy-2-[F]fluoro-D-glucose ([F]FDG) PET imaging, complemented with X-ray computed tomography (CT) and magnetic resonance imaging (MRI, 7T). Animals without a thread underwent additional contrast enhanced (Exitron) CT imaging. Tumors were delineated on the MRI, μPET image or contrast enhanced μCT images and correlations between in vivo and ex vivo [F]FDG tumor uptake as well as between image-derived and caliper-measured tumor volume were evaluated. Finally, cancer hallmarks were assessed immunohistochemically for the characterization of both models.

Results: Our results showed the strongest correlation between both in vivo and ex vivo uptake (r = 0.84, p < 0.0001) and image-derived and caliper-measured tumor volume (r = 0.96, p < 0.0001) when the tumor was delineated on the MR image. Orthotopic tumors displayed an abundance of stroma, higher levels of proliferation (p = 0.0007), apoptosis (p = 0.02), and necrosis (p < 0.0001), a higher number of blood vessels (p < 0.0001); yet lower tumor hypoxia (p < 0.0001) as compared with subcutaneous tumors.

Conclusions: This orthotopic mouse model proved to be a promising tool for the investigation of CRC through preclinical imaging studies provided the availability of anatomical MR images for accurate tumor delineation. Furthermore, the tumor microenvironment of the orthotopic tumor resembled more that of human CRC, increasing its likelihood to advance translational nuclear imaging studies of CRC.
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http://dx.doi.org/10.1007/s11307-017-1051-4DOI Listing
October 2017

Evaluation of [F]CP18 as a Substrate-Based Apoptosis Imaging Agent for the Assessment of Early Treatment Response in Oncology.

Mol Imaging Biol 2017 08;19(4):560-569

Molecular Imaging Center Antwerp (MICA), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium.

Purpose: The substrate-based positron emission tomography (PET) tracer [F]CP18 is capable of detecting the activity of caspase-3/7, two key executioner proteases in the apoptosis pathway, through selective cleavage of the ligand by the activated proteases and subsequent accumulation in apoptotic cells. Using an in vitro and in vivo model of colorectal cancer (CRC), we investigated whether [F]CP18 tracer accumulation provides a measure for apoptosis and reliably reflects early treatment response to chemotherapeutics.

Procedures: [F]CP18 cell uptake was assessed in treated Colo205 cells (saline, 5-fluorouracil (5-FU), irinotecan or their combination) and correlated with caspase-3/7 activity. [F]CP18 imaging was performed in Colo205 xenografts, starting with a baseline μPET/micro X-ray computed tomography (​μCT) scan, followed by a 3-day treatment with saline (n = 5), 5-FU (low sensitivity, n = 4), irinotecan (high sensitivity, n = 5), or a combination of both (n = 7). The study was concluded with a second [F]CP18 scan, 24 h after final treatment administration, followed by tumor removal for gamma counting (%ID/g) and for cleaved caspase-3 immunohistochemistry (apoptotic index/necrosis). Tumors were delineated on μCT images and, using the obtained volumes of interest, average percentage injected dose per cubic centimeter (%ID/cm) was calculated from every μPET image.

Results: In vitro, [F]CP18 cell uptake was positively correlated with caspase-3/7 activity (r = 0.59, p = 0.003). A drug-dependent increase in [F]CP18 tumor uptake compared to baseline was observed in animals treated with 5-FU (+14 ± 25 %), irinotecan (+56 ± 54 %), and their combination (+158 ± 69 %, p = 0.002). %ID/cm showed a positive relationship with both %ID/g (r = 0.83, p < 0.0001) and the apoptotic index (r = 0.60, p = 0.004), but not with tumor necrosis (r = 0.22, p = 0.36).

Conclusion: Both our in vitro and in vivo findings have shown the ability of [F]CP18-PET to visualize therapy-induced cancer cell apoptosis and possibly serve as a biomarker for early therapy response.
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http://dx.doi.org/10.1007/s11307-016-1037-7DOI Listing
August 2017

Evaluation of [ F]BR420 and [ F]BR351 as radiotracers for MMP-9 imaging in colorectal cancer.

J Labelled Comp Radiopharm 2017 01 21;60(1):69-79. Epub 2016 Dec 21.

Molecular Imaging Center Antwerp, University of Antwerp, Antwerp, Belgium.

MMP-9 is a zinc-dependent endopeptidase that is involved in the proteolytic degradation of the extracellular matrix and plays an important role in cancer migration, invasion, and metastasis. The aim of this study was to evaluate the potential of MMP-tracers [ F]BR420 and [ F]BR351 for MMP-9 imaging in a colorectal cancer xenograft model. [ F]BR420 and [ F]BR351 were synthesized using an automated synthesis module. For [ F]BR420, a novel and improved radiosynthesis was developed. Plasma stability and MMP-9-targeting capacity of both radiotracers was compared in the Colo205 colorectal cancer model. MMP-9 and MMP-2 expression levels in the tumors were evaluated by immunohistochemistry and in situ zymography. μPET imaging as well as ex vivo biodistribution revealed a higher tumor uptake for [ F]BR420 (3.15% ± 0.03% ID/g vs 0.94% ± 0.18% ID/g for [ F]BR351 at 2 hours pi) but slower blood clearance compared with [ F]BR351. [ F]BR351 was quickly metabolized in plasma with 20.28% ± 5.41% of intact tracer remaining at 15 minutes postinjection (PI). By contrast, [ F]BR420 displayed a higher metabolic stability with >86% intact tracer remaining at 2 hours PI. Immunohistochemistry revealed the presence of MMP-9 and MMP-2 in the tumor tissue, which was confirmed by in situ zymography. However, an autoradiography analysis of tracer distribution in the tumors did not correlate with MMP-9 expression. [ F]BR420 displayed a higher tumor uptake and higher stability compared with [ F]BR351 but a low tumor-to-blood ratio and discrepancy between tracer distribution and MMP-9 immunohistochemistry. Therefore, both tracers will not be usefulness for MMP-9 imaging in colorectal cancer.
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http://dx.doi.org/10.1002/jlcr.3476DOI Listing
January 2017

Tc-Duramycin SPECT Imaging of Early Tumor Response to Targeted Therapy: A Comparison with F-FDG PET.

J Nucl Med 2017 Apr 22;58(4):665-670. Epub 2016 Nov 22.

Molecular Imaging Center Antwerp, University of Antwerp, Wilrijk, Belgium

Molecular imaging of cell death may provide a detailed readout of the cellular response to novel therapies and prognostic information on tumor treatment efficacy, assisting in the design of individualized therapy. We compared the predictive power of cell death imaging using Tc-duramycin with the current gold standard F-FDG for treatment response evaluation after targeted therapy. Early therapy response evaluation was assessed by Tc-duramycin SPECT and F-FDG PET imaging in treatment-sensitive COLO205 and treatment-resistant HT29 human colorectal cancer xenografts 24 h after a single dose of conatumumab or IgG1 control. The specificity of Tc-duramycin for apoptosis was assessed using Tc-linear duramycin control radiotracer. Radiotracer uptake was validated ex vivo by γ-counting and autoradiography and compared with cleaved caspase-3 (CC3) activation and DNA fragmentation (TdT-mediated dUTP nick-end labeling [TUNEL]). Data were analyzed with the Student test and Pearson correlation. All statistical tests were 2-sided. COLO205 tumor uptake of Tc-duramycin was increased 7-fold from baseline in conatumumab- versus IgG1-treated control mice ( < 0.001), in good correlation with histologic analysis of apoptosis (CC3, = 0.842, and TUNEL, = 0.894; < 0.001). No response was detected in HT29 tumors. No change in Tc-linear duramycin uptake could be detected in COLO205 tumors after treatment, indicating specificity of the Tc-duramycin tumor signal. F-FDG uptake was not significantly increased from baseline in conatumumab- versus IgG1-treated COLO205 and HT29 tumor-bearing mice ( = 0.104 and 0.779, respectively) and did not correlate with immunohistochemical evidence of apoptosis. We have demonstrated that Tc-duramycin specifically accumulates in apoptotic tumors in which F-FDG was not able to differentiate responding from nonresponding tumors early after treatment. Tc-duramycin holds promise as a noninvasive imaging radiotracer for early treatment evaluation in the clinic.
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http://dx.doi.org/10.2967/jnumed.116.182014DOI Listing
April 2017

Coadministration of a Gloriosa superba extract improves the in vivo antitumoural activity of gemcitabine in a murine pancreatic tumour model.

Phytomedicine 2016 Nov 1;23(12):1434-1440. Epub 2016 Aug 1.

Natural Products & Food Research and Analysis (NatuRA), Department of Pharmaceutical Sciences, University of Antwerp, Antwerp (Wilrijk), Belgium. Electronic address:

Background: Gloriosa superba L. (glory lily, Colchicaceae) contains colchicine, and related alkaloids such as 3-O-demethylcolchicine and its glycoside colchicoside. Previously the in vivo efficacy of a crude extract and a colchicine-poor / colchicoside-rich extract of G. superba seeds was shown in a murine model of pancreatic adenocarcinoma.

Hypothesis/purpose: The efficacy can be improved without obvious signs of toxicity by increasing the treatment dose; the efficacy of gemcitabine can be improved by coadministration of a Gloriosa superba extract.

Study Design: A survival experiment was carried out in a murine model of pancreatic adenocarcinoma and the semi-long-term toxicity of both G. superba extracts was determined; a combination therapy with gemcitabine was evaluated.

Methods: A crude ethanolic extract (GS) and a colchicine-poor / colchicoside-rich extract (GS2B) were prepared, containing 3.22% colchicine, 2.52% colchicoside and 1.52% 3-O-demethylcolchicine (GS), and 0.07%, 2.26% and 0.46% (m/m) (GS2B). They were evaluated in a murine model of pancreatic adenocarcinoma at a dose of 4.5mg/kg (p.o., daily) total content of colchicine and derivatives during 3 weeks, or at 3.0mg/kg (p.o., daily) combined with gemcitabine (60mg/kg, i.p., 3x/week) during 54 days.

Results: A significant effect in tumour growth over time was observed for gemcitabine and the combination therapy compared to the control group. No significant difference was observed for the groups treated with colchicine and both extracts. However, combination therapy was significantly better than the monotherapy with gemcitabine. Moreover, survival analysis showed a significant prolongation of the survival of the groups treated with gemcitabine and the combination therapy. A slight difference in survival was observed between gemcitabine and the combination therapy, the latter one being slightly better. No significant prolongation of survival was observed for the extracts and colchicine compared to the control group.

Conclusion: Although a relevant tumour growth inhibition and a difference of relative tumour volume compared to the control group were observed on day 11, and a slightly longer survival was noticed for GS2B, the most important conclusion from this study is that the crude G. superba extract (GS) might have an added value combined with gemcitabine in the treatment of pancreatic tumours.
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http://dx.doi.org/10.1016/j.phymed.2016.07.012DOI Listing
November 2016

Preclinical evaluation of [ In]MICA-401, an activity-based probe for SPECT imaging of in vivo uPA activity.

Contrast Media Mol Imaging 2016 11 24;11(6):448-458. Epub 2016 Aug 24.

Molecular Imaging Center Antwerp, University of Antwerp, Universiteitsplein 1, B-2610, Antwerp, Belgium.

Urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 are key players in cancer invasion and metastasis. Both uPA and PAI-1 have been described as prognostic biomarkers; however, non-invasive methods measuring uPA activity are lacking. We developed an indium-111 ( In)-labelled activity-based probe to image uPA activity in vivo by single photon emission computed tomography (SPECT). A DOTA-conjugated uPA inhibitor was synthesized and radiolabelled with In ([ In]MICA-401), together with its inactive, hydrolysed form ([ In]MICA-402). A biodistribution study was performed in mice (healthy and tumour-bearing), and tumour-targeting properties were evaluated in two different cancer xenografts (MDA-MB-231 and HT29) with respectively high and low levels of uPA expression in vitro, with either the active or hydrolysed radiotracer. MicroSPECT was performed 95 h post injection followed by ex vivo biodistribution. Tumour uptake was correlated with human and murine uPA expression determined by ELISA and immunohistochemistry (IHC). Biodistribution data with the hydrolysed probe [ In]MICA-402 showed almost complete clearance 95 h post injection. The ex vivo biodistribution and SPECT data with [ In]MICA-401 demonstrated similar tumour uptakes in the two models: ex vivo 5.68 ± 1.41%ID/g versus 5.43 ± 1.29%ID/g and in vivo 4.33 ± 0.80 versus 4.86 ± 1.18 for MDA-MB-231 and HT-29 respectively. Human uPA ELISA and IHC showed significantly higher uPA expression in the MDA-MB-231 tumours, while mouse uPA staining revealed similar staining intensities of the two tumours. Our data demonstrate non-invasive imaging of uPA activity in vivo, although the moderate tumour uptake and hence potential clinical translation of the radiotracer warrants further investigation. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/cmmi.1706DOI Listing
November 2016

Development of a novel antibody-tetrazine conjugate for bioorthogonal pretargeting.

Org Biomol Chem 2016 Aug 19;14(31):7544-51. Epub 2016 Jul 19.

Laboratory of Medicinal Chemistry, University of Antwerp, Belgium.

Recently, bioorthogonal chemistry based on the Inverse Electron-Demand Diels-Alder (IEDDA) cycloaddition between 1,2,4,5-tetrazines and trans-cyclooctene (TCO) analogues added an interesting dimension to molecular imaging. Until now, antibodies (Abs) were tagged with TCO and after pretargeting they were reacted with tetrazines substituted with reporters. However, TCO tags have the tendency to degrade under physiological conditions, and due to their hydrophobic nature are buried within the protein. This results in loss of reactivity and a low Ab functional loading. To circumvent these problems, we report for the first time an approach in which tetrazines are used as tags for antibody (Ab) modification, and TCO as the imaging agent. We developed a new Ab-tetrazine conjugate, which displays a high functional loading, good stability and reactivity. We utilized this immunoconjugate for live-cell imaging together with novel TCO probes, resulting in selective and rapid labeling of SKOV-3 cells. Our approach may be useful for in vivo pretargeted imaging.
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http://dx.doi.org/10.1039/c6ob01411aDOI Listing
August 2016

Evaluation of [F]Fluorothymidine as a Biomarker for Early Therapy Response in a Mouse Model of Colorectal Cancer.

Mol Imaging Biol 2017 02;19(1):109-119

Molecular Imaging Center Antwerp (MICA), Faculty of Medicine and Health Sciences, University of Antwerp, Universiteitsplein 1, 2610, Wilrijk, Belgium.

Purpose: In oncology, positron emission tomography imaging using dedicated tracers as biomarkers may assist in early evaluation of therapy efficacy. Using 3'-deoxy-3'-[F]fluorothymidine ([F]FLT), we investigated the early effects of chemotherapeutic treatment on cancer cell proliferation in a BRAF-mutated colorectal cancer xenograft model.

Procedures: Colo205 subcutaneously inoculated animals underwent 90-min dynamic imaging before and 24 h after treatment with vehicle (control), cetuximab (resistant) or irinotecan (sensitive). Total distribution volume was quantified from dynamic data, and standardized uptake values as well as tumor-to-blood ratios were calculated from static images averaged over the last 20 min. In vivo imaging data was correlated with ex vivo proliferation and thymidine metabolism proteins.

Results: All imaging parameters showed a significant post-treatment decrease from [F]FLT baseline uptake for the irinotecan group (p ≤ 0.001) as compared with the cetuximab and vehicle group and correlated strongly with each other (p ≤ 0.0001). In vivo data were in agreement with Ki67 staining, showing a significantly lower percentage of Ki67-positive cells in the irinotecan group as compared with other groups (p ≤ 0.0001). Tumor expression of thymidine kinase 1 phosphorylated on serine 13, thymidylate synthase, and thymidine phosphorylase remained unaffected, while thymidine kinase 1 expression was, surprisingly, significantly higher in irinotecan-treated animals (p ≤ 0.01). In contrast, tumor ATP levels were lowest in this group.

Conclusions: [F]FLT positron emission tomography was found to be a suitable biomarker of early tumor response to anti-proliferative treatment, with static imaging not being inferior to full compartmental analysis in our xenograft model. The dynamics of thymidine kinase 1 protein expression and protein activity in low ATP environments merits further investigation.
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http://dx.doi.org/10.1007/s11307-016-0974-5DOI Listing
February 2017

Efficacy Screening of Gloriosa Superba Extracts in a Murine Pancreatic Cancer Model Using (18)F-FDG PET/CT for Monitoring Treatment Response.

Cancer Biother Radiopharm 2016 Apr;31(3):99-109

3 Molecular Imaging Center Antwerp (MICA), University of Antwerp , Antwerp, Belgium .

Purpose: In vivo efficacy of two herbal extracts of Gloriosa superba L. (Colchicaceae) was investigated in a murine pancreatic tumor model by tumor volume measurements and Positron Emission Tomography (PET) imaging using 2-deoxy-2-[(18)F]fluoro-d-glucose ((18)F-FDG).

Materials And Methods: A crude extract of G. superba (GS) seeds rich in colchicine and a colchicine-poor extract (GS2B) containing mostly colchicoside as a putative prodrug were prepared. PANC02-bearing C57BL/6 mice were treated with either placebo, gemcitabine, or one of the extracts (three different doses) for 10 days. Tumor volume measurements were performed daily during treatment and additionally (18)F-FDG Positron emission tomography/computed tomography was acquired at baseline and after 7 days of treatment. Ki-67 and cleaved caspase-3 immunostaining was performed on the resected tumors.

Results: After 7 days of treatment, a dose-dependent tumor growth inhibition of both extracts was observed with the highest in vivo response at the highest dose of GS and GS2B and gemcitabine. A positive significant correlation was found between Ki-67 scores and relative tumor volumes (RTV), and a negative significant correlation between caspase-3 staining scores and RTV. A decrease in (18)F-FDG uptake was clearly observed in all treatment groups.

Conclusions: The therapeutic efficacy of the two different herbal extracts was demonstrated in an in vivo pancreatic tumor model. (18)F-FDG PET was able to detect an early response as overall lower (18)F-FDG uptake was measured in the treated groups.
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http://dx.doi.org/10.1089/cbr.2015.1954DOI Listing
April 2016

Long-term acquired everolimus resistance in pancreatic neuroendocrine tumours can be overcome with novel PI3K-AKT-mTOR inhibitors.

Br J Cancer 2016 Mar;114(6):650-8

Section of Endocrinology, Department of Internal Medicine, Erasmus Medical Center, Dr Molewaterplein 40, 3015GD Rotterdam, The Netherlands.

Background: The mTOR-inhibitor everolimus improves progression-free survival in advanced pancreatic neuroendocrine tumours (PNETs). However, adaptive resistance to mTOR inhibition is described.

Methods: QGP-1 and BON-1, two human PNET cell lines, were cultured with increasing concentrations of everolimus up to 22 weeks to reach a dose of 1 μM everolimus, respectively, 1000-fold and 250-fold initial IC50. Using total DNA content as a measure of cell number, growth inhibitory dose-response curves of everolimus were determined at the end of resistance induction and over time after everolimus withdrawal. Response to ATP-competitive mTOR inhibitors OSI-027 and AZD2014, and PI3K-mTOR inhibitor NVP-BEZ235 was studied. Gene expression of 10 PI3K-Akt-mTOR pathway-related genes was evaluated using quantitative real-time PCR (RT-qPCR).

Results: Long-term everolimus-treated BON-1/R and QGP-1/R showed a significant reduction in everolimus sensitivity. During a drug holiday, gradual return of everolimus sensitivity in BON-1/R and QGP-1/R led to complete reversal of resistance after 10-12 weeks. Treatment with AZD2014, OSI-027 and NVP-BEZ235 had an inhibitory effect on cell proliferation in both sensitive and resistant cell lines. Gene expression in BON-1/R revealed downregulation of MTOR, RICTOR, RAPTOR, AKT and HIF1A, whereas 4EBP1 was upregulated. In QGP-1/R, a downregulation of HIF1A and an upregulation of ERK2 were observed.

Conclusions: Long-term everolimus resistance was induced in two human PNET cell lines. Novel PI3K-AKT-mTOR pathway-targeting drugs can overcome everolimus resistance. Differential gene expression profiles suggest different mechanisms of everolimus resistance in BON-1 and QGP-1.
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http://dx.doi.org/10.1038/bjc.2016.25DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4800296PMC
March 2016

Early Prediction of Tumor Response to Treatment: Preclinical Validation of 99mTc-Duramycin.

J Nucl Med 2016 05 2;57(5):805-11. Epub 2016 Feb 2.

Molecular Imaging Center Antwerp, University of Antwerp, Wilrijk, Belgium Department of Nuclear Medicine, University Hospital Antwerp, Edegem, Belgium

Unlabelled: Noninvasive imaging of cell death can provide an early indication of the efficacy of tumor treatment, aiding clinicians in distinguishing responding patients from nonresponding patients early on. (99m)Tc-duramycin is a SPECT tracer for cell death imaging. In this study, our aim was to validate the use of (99m)Tc-duramycin for imaging the early response of tumors to treatment.

Methods: An in vitro binding assay was performed on COLO205 cells treated with 5-fluorouracil (3.1, 31, or 310 μM) and oxaliplatin (0.7 or 7 μM) or radiation (2 or 4.5 Gy). (99m)Tc-duramycin cell binding and the levels of cell death were evaluated after treatment. In vivo imaging was performed on 4 groups of CD1-deficient mice bearing COLO205 human colorectal cancer tumors. Each group included 6 tumors. The first group was given irinotecan (100 mg/kg), the second oxaliplatin (5 mg/kg), the third irinotecan (80 mg/kg) plus oxaliplatin (5 mg/kg), and the fourth vehicle (0.9% NaCl and 5% glucose). For radiotherapy studies, COLO205 tumors received 4.5 Gy, 2 fractions of 4.5 Gy in a 24-h interval, pretreatment with an 80 mg/kg dose of irinotecan combined with 2 fractions of 4.5 Gy in a 24-h interval, or no treatment (n = 5-6/group). Therapy response was evaluated by (99m)Tc-duramycin SPECT 24 h after the last dose of therapy. Blocking was used to confirm tracer specificity. Radiotracer uptake in the tumors was validated ex vivo using γ-counting, cleaved caspase-3, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) histology.

Results: Chemotherapy and radiotherapy increased (99m)Tc-duramycin binding to COLO205 cells in a concentration/dose- and time-dependent manner, which correlated well with cell death levels (P < 0.05) as analyzed by annexin V and caspase 3/7 activity. In vivo, (99m)Tc-duramycin uptake in COLO205 xenografts was increased 2.3- and 2.8-fold (P < 0.001) in mice treated with irinotecan and combination therapy, respectively. Blocking with unlabeled duramycin demonstrated specific binding of the radiotracer. After tumor irradiation with 4.5 Gy, (99m)Tc-duramycin uptake in tumors increased significantly (1.24 ± 0.07 vs. 0.57 ± 0.08 percentage injected dose per gram in the unirradiated tumors; P < 0.001). γ-counting of radioactivity in the tumors positively correlated with cleaved caspase-3 (r = 0.85, P < 0.001) and TUNEL (r = 0.81, P < 0.001) staining.

Conclusion: We demonstrated that (99m)Tc-duramycin can be used to image induction of cell death early after chemotherapy and radiotherapy. It holds potential to be translated into clinical use for early assessment of treatment response.
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http://dx.doi.org/10.2967/jnumed.115.168344DOI Listing
May 2016

Baseline [(18)F]FMISO μPET as a Predictive Biomarker for Response to HIF-1α Inhibition Combined with 5-FU Chemotherapy in a Human Colorectal Cancer Xenograft Model.

Mol Imaging Biol 2016 08;18(4):606-16

Molecular Imaging Center Antwerp (MICA), University of Antwerp, Universiteitsplein 1, Wilrijk, Antwerp, Belgium.

Purpose: The purpose of this study was to characterize imaging biomarkers for the potential benefit of hypoxia-inducible factor-1 (HIF-1)α inhibition (by PX-12) during 5-fluorouracil (5-FU) chemotherapy in the treatment of colorectal cancer (CRC).

Procedures: Therapy response to 5-FU ± PX-12 was assessed with baseline [(18)F]fluoromisonidazole ([(18)F]FMISO) and longitudinal 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) positron emission computed tomography (μPET/CT) in CRC xenograft model (n = 36) during breathing of a hypoxic (10 % O2) or normoxic (21 % O2) atmosphere. Ex vivo, immunohistochemistry was performed.

Results: Baseline [(18)F]FMISO uptake and relative tumor volume (RTV) 2 days after 5-FU or 5-FU + PX-12 administration correlated significantly (p ≤ 0.01). Under hypoxic breathing conditions, [(18)F]FDG uptake (-53.1 ± 8.4 %) and Ki67 expression (-16 %) decreased and RTV stagnated in the 5-FU + PX-12 treatment group, but not in 5-FU alone-treated tumors. Under normoxic breathing, [(18)F]FDG uptake (-23.5 ± 15.2 % and -72.8 ± 7.1 %) and Ki67 expression (-5 % and -19 %) decreased and RTV stagnated in both the 5-FU and the combination treatment group, respectively.

Conclusion: Baseline [(18)F]FMISO μPET may predict the beneficial effect of HIF-1α inhibition during 5-FU chemotherapy in CRC.
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http://dx.doi.org/10.1007/s11307-015-0926-5DOI Listing
August 2016

Characterization of [(99m)Tc]Duramycin as a SPECT Imaging Agent for Early Assessment of Tumor Apoptosis.

Mol Imaging Biol 2015 Dec;17(6):838-47

Molecular Imaging Center Antwerp, University of Antwerp, Antwerp, Belgium.

Purpose: We investigated the usefulness of [(99m)Tc]duramycin for monitoring early response to cancer therapy in mice, with an eye towards clinical translation.

Procedures: [(99m)Tc]Duramycin was injected in healthy CD1-/- mice to estimate human [(99m)Tc]duramycin radiation dose. [(99m)Tc]Duramycin single-photon emission computed tomography (SPECT) imaging of apoptosis was evaluated in a mouse model of colorectal cancer treated with irinotecan and validated ex vivo using autoradiography, cleaved caspase-3, and TdT-mediated dUTP nick-end labeling (TUNEL) histology of the tumors.

Results: The mean effective dose was estimated to be 3.74 × 10(-3) ± 3.43 × 10(-4) mSv/MBq for non-purified and 3.19 × 10(-3) ± 2.16 × 10(-4) mSv/MBq for purified [(99m)Tc]duramycin. [(99m)Tc]Duramycin uptake in vivo following therapy increased significantly in apoptotic irinotecan-treated tumors (p = 0.008). Radioactivity in the tumors positively correlated with cleaved caspase-3 (r = 0.85, p < 0.001) and TUNEL (r = 0.92, p < 0.001) staining.

Conclusion: [(99m)Tc]Duramycin can be used to detect early chemotherapy-induced tumor cell death, and thus, may be a prospective candidate for clinical SPECT imaging of tumor response to therapy.
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http://dx.doi.org/10.1007/s11307-015-0852-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4641155PMC
December 2015

Synthesis and in vivo preclinical evaluation of an (18)F labeled uPA inhibitor as a potential PET imaging agent.

Nucl Med Biol 2014 Jul 29;41(6):477-87. Epub 2014 Mar 29.

Molecular Imaging Center of Antwerp (MICA), University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium. Electronic address:

Introduction: The urokinase plasminogen activator (uPA) system is a proteolytic cascade involved in tumor invasion and metastasis. uPA and its inhibitor PAI-1 are described as biomarkers for breast cancer with the highest level of evidence. The present study describes the synthesis and first in vivo application of an activity based uPA PET probe.

Methods: Based on the design of a small irreversible and selective uPA inhibitor we developed an (18)F-labeled activity based probe for uPA imaging. Human uPA expressing MDA-MB-231-luc2-GFP cells were inoculated in the mammary fat pads of nude mice and treated with the probe once tumors reached a volume of 150mm(3). Scans were performed at 0.25, 0.75, 1.5, 4 and 6h post injection. To evaluate tumor uptake in vivo and ex vivo data were gathered. Biodistribution data of the organs and tissues of interest were collected at all time points. Due to a relatively low tumor uptake, probe stability was further evaluated.

Results: The uPA targeting PET tracer was produced in high purity and with good specific radioactivity. In vivo PET data showed a maximum tumor uptake of 2,51±0,32 %ID/g at 4h p.i. A significant correlation between in vivo and ex vivo tumor uptake calculation was found (R=0.75; p<0.01). Due to a high blood signal at all time points, probe stability was further examined revealing high plasma protein binding and low plasma stability.

Conclusions: In vivo and ex vivo results clearly demonstrate that uPA expressing tumors can be detected with non-invasive PET imaging. Stability tests suggest that further optimization is needed to provide a better tumor-to-background contrast.
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http://dx.doi.org/10.1016/j.nucmedbio.2014.03.019DOI Listing
July 2014

Pharmacologic activation of tumor hypoxia: a means to increase tumor 2-deoxy-2-[18F]fluoro-D-glucose uptake?

Mol Imaging 2013 Jan-Feb;12(1):49-58

Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.

Tumor hypoxia and tumor metabolism are linked through the activation of metabolic genes following hypoxia-inducible factor 1 (HIF-1) activation. This raises the question of whether this relationship can be exploited to improve 2-deoxy-2-[(18)F]fluoro-D-glucose positron emission tomography ([(18)F]FDG-PET). To do this, [(18)F]FDG uptake was investigated after chemical induction of hypoxia and chemical activation of HIF-1 in an in vitro and an in vivo model of a human colorectal carcinoma. [(18)F]FDG uptake, HIF-1α protein levels, and messenger ribonucleic acid expression of glucose transporter 1 (GLUT1), hexokinase 2, HIF-1α, and carbonic anhydrase IX (CA IX) were determined in HT29 cells after treatment with 200 μM CoCl(2) and 500 μM dimethyloxalylglycine (DMOG). In an HT29 xenograft, the distribution of endogenous and exogenous markers of hypoxia was investigated using immunohistochemistry, and tumor [(18)F]FDG uptake was determined after treatment with a single dose of 5 mg/kg hydralazine and 8 mg DMOG. Treatment of HT29 cells with CoCl(2) and DMOG induced functional HIF-1 and resulted in increased [(18)F]FDG uptake. In an HT29 xenograft, a similar spatial distribution of pimonidazole, CA IX, and GLUT1 was found, and treatment with DMOG resulted in significant increases in maximum and mean standardized uptake values using [(18)F]FDG-PET. Chemical activation of HIF-1 can increase in vitro and in vivo [(18)F]FDG uptake. Imaging after pharmacologic HIF-1 activation might increase tumor [(18)F]FDG uptake when using [(18)F]FDG-PET.
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July 2013

Single-photon emission computed tomographic imaging of the early time course of therapy-induced cell death using technetium 99m tricarbonyl His-annexin A5 in a colorectal cancer xenograft model.

Mol Imaging 2012 Apr;11(2):135-47

Department of Gastroenterology, Ghent University Hospital, De Pintelaan 185, 9000-B Ghent, Belgium.

As apoptosis occurs over an interval of time after administration of apoptosis-inducing therapy in tumors, the changes in technetium 99m ((99m)Tc)-tricarbonyl (CO)₃ His-annexin A5 (His-ann A5) accumulation over time were examined. Colo205-bearing mice were divided into six treatment groups: (1) control, (2) 5-fluorouracil (5-FU; 250 mg/kg), (3) irinotecan (100 mg/kg), (4) oxaliplatin (30 mg/kg), (5) bevacizumab (5 mg/kg), and (6) panitumumab (6 mg/kg). (99m)Tc-(CO)₃ His-ann A5 was injected 4, 8, 12, 24, and 48 hours posttreatment, and micro-single-photon emission computed tomography was performed. Immunostaining of caspase-3 (apoptosis), survivin (antiapoptosis), and LC3-II (autophagy marker) was also performed. Different dynamics of (99m)Tc-(CO)₃ His-ann A5 uptake were observed in this colorectal cancer xenograft model, in response to a single dose of three different chemotherapeutics (5-FU, irinotecan, and oxaliplatin). Bevacizumab-treated mice showed no increased uptake of the radiotracer, and a peak of (99m)Tc-(CO)₃ His-ann A5 uptake in panitumumab-treated mice was observed 24 hours posttreatment, as confirmed by caspase-3 immunostaining. For irinotecan-, oxaliplatin-, and bevacizumab-treated tumors, a significant correlation was established between the radiotracer uptake and caspase-3 immunostaining (r  =  .8, p < .05; r  =  .9, p < .001; r  =  .9, p < .001, respectively). For 5-FU- and panitumumab-treated mice, the correlation coefficients were r  =  .7 (p  =  .18) and r  =  .7 (p  =  .19), respectively. Optimal timing of annexin A5 imaging after the start of different treatments in the Colo205 model was determined.
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April 2012

(99)mTc-(CO)(3) His-annexin A5 micro-SPECT demonstrates increased cell death by irinotecan during the vascular normalization window caused by bevacizumab.

J Nucl Med 2011 Nov;52(11):1786-94

Department of Gastroenterology, Ghent University Hospital, Ghent, Belgium.

Unlabelled: Colorectal tumors are dependent on angiogenesis for growth, and vascular endothelial growth factor (VEGF) is a key mediator of tumor angiogenesis. Antiangiogenic drugs can induce a transient normalization of the tumor vasculature with improved delivery of coadministered chemotherapy. The efficacy of antihuman VEGF antibody (bevacizumab) with or without irinotecan was evaluated in a colorectal cancer xenograft using (99m)Tc-(CO)(3) His-annexin A5.

Methods: Colo205-bearing mice were treated with a single dose of bevacizumab (5 mg/kg) during 2, 4, or 6 d. Microvessel density, pericyte coverage (α-smooth-muscle actin immunostaining), collagen-covered tumor vessels (Masson trichrome staining), and tumor hypoxic fraction (pimonidazole staining) were determined at the 3 different time points after treatment with bevacizumab. To investigate the possible synergistic effects of combination therapy with bevacizumab and irinotecan, Colo205-bearing mice were treated with a single dose of bevacizumab 2, 4, or 6 d before administration of a single dose of irinotecan (100 mg/kg) or 0.9% NaCl. The apoptosis-detecting radiotracer (99m)Tc-(CO)(3) His-annexin A5 was injected (18.5 MBq) in mice 12, 24, and 48 h after the start of the irinotecan or NaCl treatment, and micro-SPECT was subsequently performed 3.5 h after injection of the radiotracer. Results were correlated to histologic analysis for apoptosis (caspase-3 activation).

Results: Four days after bevacizumab administration, microvessel density decreased significantly, and α-smooth-muscle actin and collagen-covered vessels, compared with control tumors, were increased, suggesting normalization of the tumor vasculature. Hypoxic fraction was slightly reduced 4 d after treatment with bevacizumab. SPECT analyses demonstrated a significant increase in tumoral (99m)Tc-(CO)(3) His-annexin A5 uptake 4 d after bevacizumab treatment and 24 h after irinotecan administration (232.78 ± 24.82 percentage injected dose/tumor weight [g]/body weight [kg], P < 0.05), compared with each monotherapy, indicating a synergistic effect of both therapies.

Conclusion: (99m)Tc-(CO)(3) His-annexin A5 micro-SPECT demonstrates increased antitumor activity of irinotecan during the transient vascular normalization period caused by bevacizumab. Our data outline the importance of timing of combined anti-VEGF treatment with chemotherapy.
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http://dx.doi.org/10.2967/jnumed.111.092650DOI Listing
November 2011

Expression and prognostic value of glucose transporters and hexokinases in tonsil and mobile tongue squamous cell carcinoma.

Histol Histopathol 2011 09;26(9):1165-72

Department of Head and Neck Surgery, University Hospital Ghent, Gent, Belgium.

The aim of this study was to assess the expression pattern and prognostic value of the high affinity glucose transporters GLUT-1, 3, 4, 8 and 9, SGLT-1 and of hexokinases (HK) I, II and III in squamous cell carcinoma of the tonsil and mobile tongue (TTSCC) by means of immunohistochemistry. Seventy-one consecutive patients suffering from TTSCC were included. The intensity and amount of positive tumour cells in the immunoreaction (histology score (H-score)) for GLUT-1, 3, 4, 8 and 9 as well as for HK-I, II and III were assessed independently by two experienced observers, blinded to the clinical results. H-scores as well as clinical variables were related to patient outcome. Median follow-up time was 49 months (range 1-123 months). Mean H-scores for GLUT expression in decreasing order of magnitude were respectively 10.99 for GLUT-1 (sd 3.9), 5.7 for GLUT-8 (sd 4.0), 5.4 for GLUT-3 (sd 3.7), 1.0 for GLUT-4 (sd 2.0), 1.1 (sd 1.3) for SGLT-1, and 0.4 for GLUT-9 (sd 0.6); GLUT-1 > GLUT-8 = GLUT-3 > GLUT-4 = GLUT-9 = SGLT-1 (with > meaning significantly (p<0.05 on ANOVA + posthoc Bonferroni correction) higher than and =, meaning not significantly different from). Mean H-scores for hexokinase expression were respectively 5.8 for HK-I (sd 3.5), 4.6 for HK-II (sd 3.0) and 2.0 for HK-III (sd 2.0); HK-I > HK-II > HK-III. Finally high H-scores for GLUT-4 were favourably related to disease-free and overall survival on multivariate analysis. To conclude, TTSCC expresses a wide variety of glucose transporter systems and hexokinase enzymes with the "housekeeping" GLUT-1 and HK-I being the most intensely expressed. GLUT-4 over-expression appears to confer a favourable prognosis in squamous cell carcinoma of the tonsil and mobile tongue. Additional studies confirming this finding in larger cohorts of patients are mandatory.
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http://dx.doi.org/10.14670/HH-26.1165DOI Listing
September 2011

In vivo imaging of apoptosis in oncology: an update.

Mol Imaging 2011 Oct 26;10(5):340-58. Epub 2011 Apr 26.

Department ofGastroenterology, University Hospital Ghent, Belgium.

In this review, data on noninvasive imaging of apoptosis in oncology are reviewed. Imaging data available are presented in order of occurrence in time of enzymatic and morphologic events occurring during apoptosis. Available studies suggest that various radiopharmaceutical probes bear great potential for apoptosis imaging by means of positron emission tomography and single-photon emission computed tomography (SPECT). However, for several of these probes, thorough toxicologic studies are required before they can be applied in clinical studies. Both preclinical and clinical studies support the notion that 99mTc-hydrazinonicotinamide-annexin A5 and SPECT allow for noninvasive, repetitive, quantitative apoptosis imaging and for assessing tumor response as early as 24 hours following treatment instigation. Bioluminescence imaging and near-infrared fluorescence imaging have shown great potential in small-animal imaging, but their usefulness for in vivo imaging in humans is limited to structures superficially located in the human body. Although preclinical tumor-based data using high-frequency-ultrasonography (US) are promising, whether or not US will become a routinely clinically useful tool in the assessment of therapy response in oncology remains to be proven. The potential of magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) for imaging late apoptotic processes is currently unclear. Neither 31P MRS nor 1H MRS signals seems to be a unique identifier for apoptosis. Although MRI-measured apparent diffusion coefficients are altered in response to therapies that induce apoptosis, they are also altered by nonapoptotic cell death, including necrosis and mitotic catastrophe. In the future, rapid progress in the field of apoptosis imaging in oncology is expected.
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http://dx.doi.org/10.2310/7290.2010.00058DOI Listing
October 2011