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Healthy adipose tissue (AT) contains ST2+ Tregs, ILC2s, and alternatively activated macrophages that are lost in mice or humans on high caloric diet. Understanding how this form of type 2 immunity is regulated could improve treatment of obesity. The STE20 kinase Thousand And One amino acid Kinase-3 (TAOK3) has been linked to obesity in mice and humans, but its precise function is unknown. We found that ST2+ Tregs are upregulated in visceral epididymal white AT (eWAT) of Taok3-/- mice, dependent on IL-33 and the kinase activity of TAOK3. Upon high fat diet feeding, metabolic dysfunction was attenuated in Taok3-/- mice. ST2+ Tregs disappeared from eWAT in obese wild-type mice, but this was not the case in Taok3-/- mice. Mechanistically, AT Taok3-/- Tregs were intrinsically more responsive to IL-33, through higher expression of ST2, and expressed more PPARγ and type 2 cytokines. Thus, TAOK3 inhibits adipose tissue Tregs and regulates immunometabolism under excessive caloric intake., (© 2023 Maes et al.)

Purpose: Recent technical advancements in PET imaging have improved sensitivity and spatial resolution. Consequently, clinical nuclear medicine will be confronted with PET images on a previously unfamiliar resolution. To better understand [ 18 F]FDG distribution at submillimetric scale, a direct correlation of radionuclide-imaging and histopathology is required., Methods: A total of five patients diagnosed with a malignancy of the head and neck were injected with a clinical activity of [ 18 F]FDG before undergoing surgical resection. The resected specimen was imaged using a preclinical high-resolution PET/CT, followed by slicing of the specimen. Multiple slices were rescanned using a micro-PET/CT device, and one of the slices was snap-frozen for frozen sections. Frozen sections were placed on an autoradiographic film, followed by haematoxylin and eosin staining to prepare them for histopathological assessment. The results from both autoradiography and histopathology were co-registered using an iterative co-registration algorithm, and regions of interest were identified to study radiotracer uptake., Results: The co-registration between the autoradiographs and their corresponding histopathology was successful in all specimens. The use of this novel methodology allowed direct comparison of autoradiography and histopathology and enabled the visualisation of uncharted heterogeneity in [ 18 F]FDG uptake in both benign and malignant tissue., Conclusion: We here describe a novel methodology enabling the direct co-registration of [ 18 F]FDG autoradiography with the gold standard of histopathology in human malignant tissue. The future use of the current methodology could further increase our understanding of the distribution of radionuclides in surgically excised malignancies and hence, improve the integration of pathology and molecular imaging in a multiscale perspective., Trial Registration: ClinicalTrials.gov Identifier: NCT05068687., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A wide variety of 18 F-labeled PSMA-targeting PET radiotracers have been developed, including [ 18 F]AlF-PSMA-11. As there is only limited data on the comparison with other 18 F-labeled PSMA PET tracers, a comparative preclinical study between [ 18 F]AlF-PSMA-11 and [ 18 F]PSMA-1007 was conducted. Mice with varying PSMA expressing tumors (C4-2, 22Rv1 and PC-3, each n = 5) underwent two PET/CT scans with both [ 18 F]AlF-PSMA-11 and [ 18 F]PSMA-1007. Ten additional mice bearing C4-2 xenografts were subjected to ex vivo biodistribution with either [ 18 F]AlF-PSMA-11 (n = 5) or [ 18 F]PSMA-1007 (n = 5). Absolute SUV mean and SUV max values were significantly higher for [ 18 F]PSMA-1007 scans in both C4-2 tumors (p < 0.01) and 22Rv1 tumors (p < 0.01). In C4-2 xenograft bearing mice, the tumor-to-organ ratios did not significantly differ between [ 18 F]AlF-PSMA-11 and [ 18 F]PSMA-1007 for liver, muscle, blood and salivary glands (p > 0.05). However, in 22Rv1 xenograft bearing mice, all tumor-to-organ ratios were higher for [ 18 F]AlF-PSMA-11 (p < 0.01). In healthy organs, [ 18 F]PSMA-1007 uptake was higher in the liver, gallbladder, small intestines and glands. Biodistribution data confirmed the increased uptake in the heart, small intestines and liver with [ 18 F]PSMA-1007. Absolute tumor uptake was higher with [ 18 F]PSMA-1007 in all tumors. Tumor-to-organ ratios did not differ significantly in high PSMA expressing tumors, but were higher for [ 18 F]AlF-PSMA-11 in low PSMA expressing tumors. Furthermore, [ 18 F]PSMA-1007 showed higher uptake in healthy organs., (© 2022. The Author(s).)

Introduction: Electrophysiological and neuroimaging studies have demonstrated that large-scale brain networks are affected during the development of epilepsy. These networks can be investigated by using diffusion magnetic resonance imaging (dMRI). The most commonly used model to analyze dMRI is diffusion tensor imaging (DTI). However, DTI metrics are not specific to microstructure or pathology and the DTI model does not take into account crossing fibers, which may lead to erroneous results. To overcome these limitations, a more advanced model based on multi-shell multi-tissue constrained spherical deconvolution was used in this study to perform tractography with more precise fiber orientation estimates and to assess changes in intra-axonal volume by using fixel-based analysis. Methods: dMRI images were acquired before and at several time points after induction of status epilepticus in the intraperitoneal kainic acid (IPKA) rat model of temporal lobe epilepsy. Tractography was performed, and fixel metrics were calculated in several white matter tracts. The tractogram was analyzed by using the graph theory. Results: Global degree, global and local efficiency were decreased in IPKA animals compared with controls during epileptogenesis. Nodal degree was decreased in the limbic system and default-mode network, mainly during early epileptogenesis. Further, fiber density (FD) and fiber-density-and-cross-section (FDC) were decreased in several white matter tracts. Discussion: These results indicate a decrease in overall structural connectivity, integration, and segregation and decreased structural connectivity in the limbic system and default-mode network. Decreased FD and FDC point to a decrease in intra-axonal volume fraction during epileptogenesis, which may be related to neuronal degeneration and gliosis. Impact statement To the best of our knowledge, this is the first longitudinal multi-shell diffusion magnetic resonance imaging study that combines whole-brain tractography and fixel-based analysis to investigate changes in structural brain connectivity and white matter integrity during epileptogenesis in a rat model of temporal lobe epilepsy. Our findings present better insights into how the topology of the structural brain network changes during epileptogenesis and how these changes are related to white matter integrity. This could improve the understanding of the basic mechanisms of epilepsy and aid the rational development of imaging biomarkers and epilepsy therapies.

A rat glioblastoma model to mimic chemo-radiation treatment of human glioblastoma in the clinic was previously established. Similar to the clinical treatment, computed tomography (CT) and magnetic resonance imaging (MRI) were combined during the treatment-planning process. Positron emission tomography (PET) imaging was subsequently added to implement sub-volume boosting using a micro-irradiation system. However, combining three imaging modalities (CT, MRI, and PET) using a micro-irradiation system proved to be labor-intensive because multimodal imaging, treatment planning, and dose delivery have to be completed sequentially in the preclinical setting. This also results in a workflow that is more prone to human error. Therefore, a user-friendly algorithm to further optimize preclinical multimodal imaging-based radiation treatment planning was implemented. This software tool was used to evaluate the accuracy and efficiency of dose painting radiation therapy with micro-irradiation by using an in silico study design. The new methodology for dose painting radiation therapy is superior to the previously described method in terms of accuracy, time efficiency, and intra- and inter-user variability. It is also an important step towards the implementation of inverse treatment planning on micro-irradiators, where forward planning is still commonly used, in contrast to clinical systems.

This two-part preclinical study aims to evaluate prostate specific membrane antigen (PSMA) as a valuable target for expression-based imaging applications and to determine changes in target binding in function of varying apparent molar activities (MA app ) of [ 18 F]AlF-PSMA-11. For the evaluation of PSMA expression levels, male NOD/SCID mice bearing prostate cancer (PCa) xenografts of C4-2 (PSMA+++), 22Rv1 (PSMA+) and PC-3 (PSMA-) were administered [ 18 F]AlF-PSMA-11 with a medium MA app (20.24 ± 3.22 MBq/nmol). SUV mean and SUV max values were respectively 3.22 and 3.17 times higher for the high versus low PSMA expressing tumors (p < 0.0001). To evaluate the effect of varying MA app , C4-2 and 22Rv1 xenograft bearing mice underwent additional [ 18 F]AlF-PSMA-11 imaging with a high (211.2 ± 38.9 MBq/nmol) and/or low MA app (1.92 ± 0.27 MBq/nmol). SUV values showed a significantly increasing trend with higher MA app . Significant changes were found for SUV mean and SUV max between the high versus low MA app and medium versus low MA app (both p < 0.05), but not between the high versus medium MA app (p = 0.055 and 0.25, respectively). The effect of varying MA app was more pronounced in low expressing tumors and PSMA expressing tissues (e.g. salivary glands and kidneys). Overall, administration of a high MA app increases the detection of low expression tumors while also increasing uptake in PSMA expressing tissues, possibly leading to false positive findings. In radioligand therapy, a medium MA app could reduce radiation exposure to dose-limiting organs with only limited effect on radionuclide accumulation in the tumor., (© 2021. The Author(s).)

Introduction: [18F]-FDG PET is a widely used imaging modality that visualizes cellular glucose uptake and provides functional information on the metabolic state of different tissues in vivo . Various quantification methods can be used to evaluate glucose metabolism in the brain, including the cerebral metabolic rate of glucose (CMR glc ) and standard uptake values (SUVs). Especially in the brain, these (semi-)quantitative measures can be affected by several physiological factors, such as blood glucose level, age, gender, and stress. Next to this inter- and intra-subject variability, the use of different PET acquisition protocols across studies has created a need for the standardization and harmonization of brain PET evaluation. In this study we present a framework for statistical voxel-based analysis of glucose uptake in the rat brain using histogram-based intensity normalization. Methods: [18F]-FDG PET images of 28 normal rat brains were coregistered and voxel-wisely averaged. Ratio images were generated by voxel-wisely dividing each of these images with the group average. The most prevalent value in the ratio image was used as normalization factor. The normalized PET images were voxel-wisely averaged to generate a normal rat brain atlas. The variability of voxel intensities across the normalized PET images was compared to images that were either normalized by whole brain normalization, or not normalized. To illustrate the added value of this normal rat brain atlas, 9 animals with a striatal hemorrhagic lesion and 9 control animals were intravenously injected with [18F]-FDG and the PET images of these animals were voxel-wisely compared to the normal atlas by group- and individual analyses. Results: The average coefficient of variation of the voxel intensities in the brain across normal [18F]-FDG PET images was 6.7% for the histogram-based normalized images, 11.6% for whole brain normalized images, and 31.2% when no normalization was applied. Statistical voxel-based analysis, using the normal template, indicated regions of significantly decreased glucose uptake at the site of the ICH lesion in the ICH animals, but not in control animals. Conclusion: In summary, histogram-based intensity normalization of [18F]-FDG uptake in the brain is a suitable data-driven approach for standardized voxel-based comparison of brain PET images., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Proesmans, Raedt, Germonpré, Christiaen, Descamps, Boon, De Herdt and Vanhove.)

Magnetic resonance imaging (MRI) is frequently used for preclinical treatment monitoring in glioblastoma (GB). Discriminating between tumors and tumor-associated changes is challenging on in vivo MRI. In this study, we compared in vivo MRI scans with ex vivo MRI and histology to estimate more precisely the abnormal mass on in vivo MRI. Epileptic seizures are a common symptom in GB. Therefore, we used a recently developed GB-associated epilepsy model from our group with the aim of further characterizing the model and making it useful for dedicated epilepsy research. Ten days after GB inoculation in rat entorhinal cortices, in vivo MRI (T2w and mean diffusivity (MD)), ex vivo MRI (T2w) and histology were performed, and tumor volumes were determined on the different modalities. The estimated abnormal mass on ex vivo T2w images was significantly smaller compared to in vivo T2w images, but was more comparable to histological tumor volumes, and might be used to estimate end-stage tumor volumes. In vivo MD images displayed tumors as an outer rim of hyperintense signal with a core of hypointense signal, probably reflecting peritumoral edema and tumor mass, respectively, and might be used in the future to distinguish the tumor mass from peritumoral edema-associated with reactive astrocytes and activated microglia, as indicated by an increased expression of immunohistochemical markers-in preclinical models. In conclusion, this study shows that combining imaging techniques using different structural scales can improve our understanding of the pathophysiology in GB.

Longitudinal in vivo monitoring of transplanted cells is crucial to perform cancer research or to assess the treatment outcome of cell-based therapies. While several bio-imaging techniques can be used, magnetic resonance imaging (MRI) clearly stands out in terms of high spatial resolution and excellent soft-tissue contrast. However, MRI suffers from low sensitivity, requiring cells to be labeled with high concentrations of contrast agents. An interesting option is to label cells with clinically approved gadolinium chelates which generate a hyperintense MR signal. However, spontaneous uptake of the label via pinocytosis results in its endosomal sequestration, leading to quenching of the T 1 -weighted relaxation. To avoid this quenching effect, delivery of gadolinium chelates directly into the cytosol via electroporation or hypotonic cell swelling have been proposed. However, these methods are also accompanied by several drawbacks such as a high cytotoxicity, and changes in gene expression and phenotype. Here, we demonstrate that nanoparticle-sensitized laser induced photoporation forms an attractive alternative to efficiently deliver the contrast agent gadobutrol into the cytosol of both HeLa and SK-OV-3 IP1 cells. After intracellular delivery by photoporation the quenching effect is clearly avoided, leading to a strong increase in the hyperintense T 1 -weighted MR signal. Moreover, when compared to nucleofection as a state-of-the-art electroporation platform, photoporation has much less impact on cell viability, which is extremely important for reliable cell tracking studies. Additional experiments confirm that photoporation does not induce any change in the long-term viability or the migratory capacity of the cells. Finally, we show that gadolinium 'labeled' SK-OV-3 IP1 cells can be imaged in vivo by MRI with high soft-tissue contrast and spatial resolution, revealing indications of potential tumor invasion or angiogenesis.

The histopathological growth patterns (HGPs) of liver metastases of colorectal cancer and of several other tumor types predict outcome of patients in multiple studies. The HGPs of liver metastases have a prognostic but also a predictive value with one of the growth patterns, the replacement growth pattern, related to resistance to systemic treatment. Given that the HGP can only be assessed in a reliable manner when a surgical resection of the metastasis has been performed, this biomarker cannot be exploited to the full. For example, HGPs can at this moment, not be used to decide whether patients with liver metastatic breast or colorectal cancer will benefit or not from locoregional treatment, such as surgery or radiotherapy, and from peri-operative systemic treatment. In this review we highlight studies that suggest that the HGPs of liver metastases can be identified by medical imaging. Although still to be confirmed by a prospective multicenter approach, some studies indeed achieve a high accuracy in predicting the HGPs by applying radiomic algorithms on CT- or MR-images of liver metastases. This is an important step towards a treatment planning of patients with liver metastatic cancer that takes into account the biology and the progression kinetics of the metastases., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: We investigated the potential of [18F]fluorodeoxyglucose ([18F]FDG) and [18F]Fluoromethylcholine ([18F]FCho) PET, compared to contrast-enhanced MRI, for the early detection of treatment response in F98 glioblastoma (GB) rats., Methods: When GB was confirmed on T2- and contrast-enhanced T1-weighted MRI, animals were randomized into a treatment group (n = 5) receiving MRI-guided 3D conformal arc micro-irradiation (20 Gy) with concomitant temozolomide, and a sham group (n = 5). Effect of treatment was evaluated by MRI and [18F]FDG PET on day 2, 5, 9 and 12 post-treatment and [18F]FCho PET on day 1, 6, 8 and 13 post-treatment. The metabolic tumor volume (MTV) was calculated using a semi-automatic thresholding method and the average tracer uptake within the MTV was converted to a standard uptake value (SUV)., Results: To detect treatment response, we found that for [18F]FDG PET (SUVmean x MTV) is superior to MTV only. Using (SUVmean x MTV), [18F]FDG PET detects treatment effect starting as soon as day 5 post-therapy, comparable to contrast-enhanced MRI. Importantly, [18F]FDG PET at delayed time intervals (240 min p.i.) was able to detect the treatment effect earlier, starting at day 2 post-irradiation. No significant differences were found at any time point for both the MTV and (SUVmean x MTV) of [18F]FCho PET., Conclusions: Both MRI and particularly delayed [18F]FDG PET were able to detect early treatment responses in GB rats, whereas, in this study this was not possible using [18F]FCho PET. Further comparative studies should corroborate these results and should also include (different) amino acid PET tracers., Competing Interests: The authors have declared that no competing interests exist.

Recently, a 18 F-labeled derivative of the widely used 68 Ga-PSMA-11 was developed for PET imaging of prostate cancer. Although 18 F-PSMA-11 has already been evaluated in a Phase I and Phase II clinical trial, preclinical evaluation of this radiotracer is important for further understanding its dynamic behavior. Saturation binding experiments were conducted by incubation of LNCaP cells with 18 F-PSMA-11 or 68 Ga-PSMA-11 for 1 h, followed by determination of the specific and aspecific binding. Mice bearing LNCaP or PC-3 xenografts each received ± 3.7 MBq 18 F-PSMA-11 and 68 Ga-PSMA-11 followed by dynamic acquisition of 2.5 h as well as ± 15 MBq 18 F-FDG followed by static acquisition at 1 h post injection (p.i.). Uptake was evaluated by comparison of uptake parameters (SUV mean , SUV max , TBR mean and TBR max ). Mice underwent ex vivo biodistribution where 18 F-PSMA-11 activity was measures in excretory organs (kidneys, bladder and liver) as well as bone fragments (femur, humerus, sternum and skull) to evaluate bone uptake. The dissociation constant (K d ) of 18 F-PSMA-11 and 68 Ga-PSMA-11 was 2.95 ± 0.87 nM and 0.49 ± 0.20 nM, respectively. Uptake parameters were significantly higher in LNCaP compared to PC-3 xenografts for both 18 F-PSMA-11 and 68 Ga-PSMA-11, while no difference was found for 18 F-FDG uptake (except for SUV max ). Tumor uptake of 18 F-PSMA-11 showed a similar trend over time as 68 Ga-PSMA-11, although all uptake parameter curves of the latter were considerably lower. When comparing early (60 min p.i.) to delayed (150 min p.i.) imaging for both radiotracers individually, TBR mean and TBR max were significantly higher at the later timepoint, as well as the SUV max of 68 Ga-PSMA-11. The highest %ID/g was determined in the kidneys (94.0 ± 13.6%ID/g 1 h p.i.) and the bladder (6.48 ± 2.18%ID/g 1 h p.i.). No significant increase in bone uptake was seen between 1 and 2 h p.i. Both radiotracers showed high affinity for the PSMA receptor. Over time, all uptake parameters were higher for 18 F-PSMA-11 compared to 68 Ga-PSMA-11. Delayed imaging with the latter may improve tumor visualization, while no additional benefits could be found for late 18 F-PSMA-11 imaging. Ex vivo biodistribution demonstrated fast renal clearance of 18 F-PSMA-11 as well as no significant increase in bone uptake.

Young triple negative breast cancer (TNBC) patients are at high risk for developing very aggressive brain metastases associated with a poor prognosis and a high mortality rate. Preclinical models that allow follow-up by magnetic resonance imaging (MRI) can contribute to the development of new therapeutic approaches for brain metastasis. To date, preclinical brain tumor research has almost exclusively relied on xenograft mouse models. Yet, rats are an ideal model for imaging of brain metastasis as their larger brain offers better relative spatial resolution compared to a mouse brain. For the development of a clinically relevant rat model for TNBC brain metastasis, the MDA-MB-231br/eGFP cancer cell line can be used. However, as a result of species-dependent extracranial features, the propensity of the MDA-MB-231br/eGFP cancer cell line to metastasize exclusively to the brain needs to be enhanced by in vivo selection. In this study, repeated sequential passages of metastatic cancer cells obtained from brain metastases in nude rats were performed. Brain metastasis formation was evaluated using preclinical MRI, while bone metastasis formation was assessed using high-resolution computed tomography (CT) and 2-deoxy-2-[18F] fluoro-D-glucose ([18F] FDG) positron emission tomography (PET) imaging. Our results demonstrated that the metastatic tumor burden in the rat brain (number and volume) significantly increased with increasing passage, while the metastatic tumor burden in the skeleton (i.e., number of metastasis-affected bones) significantly decreased with increasing passage. However, bone metastasis development was not reduced to a negligible amount. Consequently, despite in vivo selection, our rat model is not recommended for investigating brain metastasis as a single disease. Our findings highlight the importance of well-reasoned selection of both the preclinical model and the cancer cell line in order to obtain reliable and reproducible scientific results., Competing Interests: The authors have declared that no competing interests exist.

Traumatic brain injury (TBI) is a major cause of long-term cognitive deficits, even in mild TBI patients. Computerized cognitive training can help alleviate complaints and improve daily life functioning of TBI patients. However, the underlying biological mechanisms of cognitive training in TBI are not fully understood. In the present study, we utilised for the first time a touchscreen cognitive training system in a rat model of mild TBI. Moreover, we wanted to examine whether the beneficial effects of a cognitive training are task-dependent and selective in their target. Specifically, we examined the effect of two training tasks, i.e. the Paired Associate Learning (PAL) task targeting spatial memory functioning and 5-Choice Continuous Performance (5-CCP) task loading on attention and inhibition control, on the microstructural organization of the hippocampus and cingulum, respectively, using diffusion tensor imaging (DTI). Our findings revealed that the two training protocols induced similar effects on the diffusion MRI metrics. Further, in the TBI groups who received training microstructural organization in the hippocampus and cingulum improved (as denoted by increases in fractional anisotropy), while a worsening (i.e., increases in mean diffusivity and radial diffusivity) was found in the TBI control group. In addition, these alterations in diffusion MRI metrics coincided with improved performance on the training tasks in the TBI groups who received training. Our findings show the potential of DTI metrics as reliable measure to evaluate cognitive training in TBI patients and to facilitate future research investigating further improvement of cognitive training targeting deficits in spatial memory and attention.

Viruses, such as white spot syndrome virus, and bacteria, such as Vibrio species, wreak havoc in shrimp aquaculture [C. M. Escobedo-Bonilla et al., J. Fish. Dis. 31, 1-18 (2008)]. As the main portal of entry for shrimp-related pathogens remain unclear, infectious diseases are difficult to prevent and control. Because the cuticle is a strong pathogen barrier, regions lacking cuticular lining, such as the shrimp's excretory organ, "the antennal gland," are major candidate entry portals [M. Corteel et al., Vet. Microbiol. 137, 209-216 (2009)]. The antennal gland, up until now morphologically underexplored, is studied using several imaging techniques. Using histology-based three-dimensional technology, we demonstrate that the antennal gland resembles a kidney, connected to a urinary bladder with a nephropore (exit opening) and a complex of diverticula, spread throughout the cephalothorax. Micromagnetic resonance imaging of live shrimp not only confirms the histology-based model, but also indicates that the filling of the diverticula is linked to the molting cycle and possibly involved therein. Based on function and complexity, we propose to rename the antennal gland as the "nephrocomplex." By an intrabladder inoculation, we showed high susceptibility of this nephrocomplex to both white spot syndrome virus and Vibrio infection compared to peroral inoculation. An induced drop in salinity allowed the virus to enter the nephrocomplex in a natural way and caused a general infection followed by death; fluorescent beads were used to demonstrate that particles may indeed enter through the nephropore. These findings pave the way for oriented disease control in shrimp., Competing Interests: The authors declare no competing interest.

Seizures are common in patients with high-grade gliomas (30-60%) and approximately 15-30% of glioblastoma (GB) patients develop drug-resistant epilepsy. Reliable animal models are needed to develop adequate treatments for glioma-related epilepsy. Therefore, fifteen rats were inoculated with F98 GB cells (GB group) and four rats with vehicle only (control group) in the right entorhinal cortex. MRI was performed to visualize tumor presence. A subset of seven GB and two control rats were implanted with recording electrodes to determine the occurrence of epileptic seizures with video-EEG recording over multiple days. In a subset of rats, tumor size and expression of tumor markers were investigated with histology or mRNA in situ hybridization. Tumors were visible on MRI six days post-inoculation. Time-dependent changes in tumor morphology and size were visible on MRI. Epileptic seizures were detected in all GB rats monitored with video-EEG. Twenty-one days after inoculation, rats were euthanized based on signs of discomfort and pain. This study describes, for the first time, reproducible tumor growth and spontaneous seizures upon inoculation of F98 cells in the rat entorhinal cortex. The development of this new model of GB-related epilepsy may be valuable to design new therapies against tumor growth and associated epileptic seizures.

The metabolic alterations in tumors make it possible to visualize the latter by means of positron emission tomography, enabling diagnosis and providing metabolic information. The alanine serine cysteine transporter-2 (ASCT-2) is the main transporter of glutamine and is upregulated in several tumors. Therefore, a good positron emission tracer targeting this transport protein would have substantial value. Hence, the aim of this study is to develop a fluorine-18-labeled version of a V-9302 analogue, one of the most potent inhibitors of ASCT-2. The precursor was labeled with fluorine-18 via a nucleophilic substitution of the corresponding benzylic bromide. The cold reference product was subjected to in vitro assays with [ 3 H]glutamine in a PC-3 and F98 cell line to determine the affinity for both the human and rat ASCT-2. To evaluate the tracer potential dynamic μPET, images were acquired in a mouse xenograft model for prostate cancer. The tracer could be synthesized with an overall nondecay corrected yield of 3.66 ± 1.90%. in vitro experiments show inhibitor constants K i of 90 and 125 μM for the PC-3 and F98 cells, respectively. The experiments in the PC-3 xenograft demonstrate a low uptake in the tumor tissue. We have successfully synthesized the radiotracer [ 18 F]2-amino-4-((2-((3-fluorobenzyl)oxy)benzyl)(2-((3-(fluoromethyl)benzyl)oxy)benzyl)amino)butanoic acid. in vitro experiments show a good affinity for both the human and rat ASCT-2. However, the tracer suffers from poor in vivo tumor uptake in the PC-3 model. Briefly, we present the first fluorine-18-labeled derivative of compound V-9302, a promising novel ASCT-2 blocker used for inhibition of tumor growth., (© 2020 John Wiley & Sons, Ltd.)

Introduction: Glucose has been deemed the driving force of tumor growth for decades. However, research has shown that several tumors metabolically shift towards glutaminolysis. The development of radiolabeled glutamine derivatives could be a useful molecular imaging tool for visualizing these tumors. We elaborated on the glutamine-derived PET tracers by developing two novel probes, namely [ 18 F]fluorophenylglutamine and [ 18 F]fluorobiphenylglutamine., Materials and Methods: Both tracers were labelled with fluorine-18 using our recently reported ruthenium-based direct aromatic fluorination method. Their affinity was evaluated with a [ 3 H]glutamine inhibition experiment in a human PC-3 and a rat F98 cell line. The imaging potential of [ 18 F]fluorophenylglutamine and [ 18 F]fluorobiphenylglutamine was tested using a mouse PC-3 and a rat F98 tumor model., Results: The radiosynthesis of both tracers was successful with overall non-decay corrected yields of 18.46 ± 4.18% (n = 10) ([ 18 F]fluorophenylglutamine) and 8.05 ± 3.25% (n = 5) ([ 18 F]fluorobiphenylglutamine). In vitro inhibition experiments showed a moderate and low affinity of fluorophenylglutamine and fluorobiphenylglutamine, respectively, towards the human ASCT-2 transporter. Both compounds had a low affinity towards the rat ASCT-2 transporter. These results were endorsed by the in vivo experiments with low uptake of both tracers in the F98 rat xenograft, low uptake of [ 18 F]FBPG in the mice PC-3 xenograft and a moderate uptake of [ 18 F]FPG in the PC-3 tumors., Conclusion: We investigated the imaging potential of two novel PET radiotracers [ 18 F]FPG and [ 18 F]FBPG. [ 18 F]FPG is the first example of a glutamine radiotracer derivatized with a phenyl group which enables the exploration of further derivatization of the phenyl group to increase the affinity and imaging qualities. We hypothesize that increasing the affinity of [ 18 F]FPG by optimizing the substituents of the arene ring can result in a high-quality glutamine-based PET radiotracer. Advances in Knowledge and Implications for patient care: We hereby report novel glutamine-based PET-tracers. These tracers are tagged on the arene group with fluorine-18, hereby preventing in vivo defluorination, which can occur with alkyl labelled tracers (e.g. (2S,4R)4-[ 18 F]fluoroglutamine). [ 18 F]FPG shows clear tumor uptake in vivo, has no in vivo defluorination and has a straightforward production. We believe this tracer is a good starting point for the development of a high-quality tracer which is useful for the clinical visualization of the glutamine transport., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Intra-abdominal dissemination of peritoneal nodules, a condition known as peritoneal carcinomatosis (PC), is typically diagnosed in ovarian cancer patients at the advanced stages. The current treatment of PC consists of perioperative systemic chemotherapy and cytoreductive surgery, followed by intra-abdominal flushing with solutions of chemotherapeutics such as cisplatin and oxaliplatin. In this study, we developed cisplatin-loaded polyarginine-hyaluronic acid nanoscale particles (Cis-pARG-HA NPs) with high colloidal stability, marked drug loading efficiency, unimpaired biological activity, and tumor-targeting ability. Injected Cis-pARG-HA NPs showed enhanced antitumor activity in a rat model of PC, compared to injection of the free cisplatin drug. The activity of Cis-pARG-HA NPs could even be further improved when administered by an intra-abdominal aerosol therapy, referred to as pressurized intraperitoneal aerosol chemotherapy (PIPAC). PIPAC is hypothesized to ensure a more homogeneous drug distribution together with a deeper drug penetration into peritoneal tumor nodules within the abdominal cavity. Using fluorescent pARG-HA NPs, this enhanced nanoparticle deposit on tumors could indeed be observed in regions opposite the aerosolization nozzle. Therefore, this study demonstrates that nanoparticles carrying chemotherapeutics can be synergistically combined with the PIPAC technique for IP therapy of disseminated advanced ovarian tumors, while this synergistic effect was not observed for the administration of free cisplatin.

Arterial tortuosity syndrome (ATS) is a recessively inherited connective tissue disorder, mainly characterized by tortuosity and aneurysm formation of the major arteries. ATS is caused by loss-of-function mutations in SLC2A10, encoding the facilitative glucose transporter GLUT10. Former studies implicated GLUT10 in the transport of dehydroascorbic acid, the oxidized form of ascorbic acid (AA). Mouse models carrying homozygous Slc2a10 missense mutations did not recapitulate the human phenotype. Since mice, in contrast to humans, are able to intracellularly synthesize AA, we generated a novel ATS mouse model, deficient for Slc2a10 as well as Gulo, which encodes for L-gulonolactone oxidase, an enzyme catalyzing the final step in AA biosynthesis in mouse. Gulo;Slc2a10 double knock-out mice showed mild phenotypic anomalies, which were absent in single knock-out controls. While Gulo;Slc2a10 double knock-out mice did not fully phenocopy human ATS, histological and immunocytochemical analysis revealed compromised extracellular matrix formation. Transforming growth factor beta signaling remained unaltered, while mitochondrial function was compromised in smooth muscle cells derived from Gulo;Slc2a10 double knock-out mice. Altogether, our data add evidence that ATS is an ascorbate compartmentalization disorder, but additional factors underlying the observed phenotype in humans remain to be determined., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Epilepsy is a neurological disorder characterized by recurrent epileptic seizures. The involvement of abnormal functional brain networks in the development of epilepsy and its comorbidities has been demonstrated by electrophysiological and neuroimaging studies in patients with epilepsy. This longitudinal study investigated changes in dynamic functional connectivity (dFC) and network topology during the development of epilepsy using the intraperitoneal kainic acid (IPKA) rat model of temporal lobe epilepsy (TLE). Resting state functional magnetic resonance images (rsfMRI) of 20 IPKA animals and 7 healthy control animals were acquired before and 1, 3, 6, 10 and 16 weeks after status epilepticus (SE) under medetomidine anaesthesia using a 7 T MRI system. Starting from 17 weeks post-SE, hippocampal EEG was recorded to determine the mean daily seizure frequency of each animal. Dynamic FC was assessed by calculating the correlation matrices between fMRI time series of predefined regions of interest within a sliding window of 50 s using a step length of 2 s. The matrices were classified into 6 FC states, each characterized by a correlation matrix, using k-means clustering. In addition, several time-variable graph theoretical network metrics were calculated from the time-varying correlation matrices and classified into 6 states of functional network topology, each characterized by a combination of network metrics. Our results showed that FC states with a lower mean functional connectivity, lower segregation and integration occurred more often in IPKA animals compared to control animals. Functional connectivity also became less variable during epileptogenesis. In addition, average daily seizure frequency was positively correlated with percentage dwell time (i.e. how often a state occurs) in states with high mean functional connectivity, high segregation and integration, and with the number of transitions between states, while negatively correlated with percentage dwell time in states with a low mean functional connectivity, low segregation and low integration. This indicates that animals that dwell in states of higher functional connectivity, higher segregation and higher integration, and that switch more often between states, have more seizures., Competing Interests: Declaration of Competing Interest This research was financially supported by a PhD grant from the Special Research Fund (BOF) of Ghent University. Emma Christiaen and Marie-Gabrielle Goossens are SB PhD fellows at Research Foundation – Flanders (project numbers 1S90218N and 1S30017N). The authors have no competing interests to declare., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Since atherosclerotic plaques are small and sparse, their non-invasive detection via PET imaging requires both highly specific radiotracers as well as imaging systems with high sensitivity and resolution. This study aimed to assess the targeting and biodistribution of a novel fluorine-18 anti-VCAM-1 Nanobody (Nb), and to investigate whether sub-millimetre resolution PET imaging could improve detectability of plaques in mice. The anti-VCAM-1 Nb functionalised with the novel restrained complexing agent (RESCA) chelator was labelled with [ 18 F]AlF with a high radiochemical yield (>75%) and radiochemical purity (>99%). Subsequently, [ 18 F]AlF(RESCA)-cAbVCAM1-5 was injected in ApoE -/- mice, or co-injected with excess of unlabelled Nb (control group). Mice were imaged sequentially using a cross-over design on two different commercially available PET/CT systems and finally sacrificed for ex vivo analysis. Both the PET/CT images and ex vivo data showed specific uptake of [ 18 F]AlF(RESCA)-cAbVCAM1-5 in atherosclerotic lesions. Non-specific bone uptake was also noticeable, most probably due to in vivo defluorination. Image analysis yielded higher target-to-heart and target-to-brain ratios with the β-CUBE (MOLECUBES) PET scanner, demonstrating that preclinical detection of atherosclerotic lesions could be improved using the latest PET technology.

Radiotherapeutic treatment consists of targeted application of radiation beams to a tumor but exposure of surrounding healthy tissue is inevitable. In the brain, ionizing radiation induces breakdown of the blood-brain barrier by effects on brain microvascular endothelial cells. Damage from directly irradiated cells can be transferred to surrounding non-exposed bystander cells, known as the radiation-induced bystander effect. We investigated involvement of connexin channels and paracrine signaling in radiation-induced bystander DNA damage in brain microvascular endothelial cells exposed to focused X-rays. Irradiation caused DNA damage in the directly exposed area, which propagated over several millimeters in the bystander area. DNA damage was significantly reduced by the connexin channel-targeting peptide Gap26 and the Cx43 hemichannel blocker TAT-Gap19. ATP release, dye uptake, and patch clamp experiments showed that hemichannels opened within 5 min post irradiation in both irradiated and bystander areas. Bystander signaling involved cellular Ca 2+ dynamics and IP 3 , ATP, ROS, and NO signaling, with Ca 2+ , IP 3 , and ROS as crucial propagators of DNA damage. We conclude that bystander effects are communicated by a concerted cascade involving connexin channels, and IP 3 /Ca 2+ , ATP, ROS, and NO as major contributors of regenerative signal expansion.

Introduction: Considering the need for rapid change of treatment in recurrent glioblastoma (GB), it is of utmost importance to characterize PET radiopharmaceuticals that allow early discrimination of tumor from therapy-related effects. In this study, we examined the value of 2-[ 18 F]FELP as a LAT1 tumor-specific PET tracer in comparison with [ 18 F]FDG and [ 18 F]FET in a combined orthotopic rat radiation necrosis and glioblastoma model. A second experiment compared 2-[ 18 F]FELP to [ 18 F]FDG in a mouse glioblastoma - inflammation model., Methods: Using the small animal radiation research platform (SARRP), radiation necrosis (RN) was induced in the left frontal lobe of the rat brain. When radiation-induced changes were visible on MRI, F98 rat glioblastoma cells were stereotactically inoculated in the contralateral right frontal lobe. When tumor growth was confirmed on MRI, 2-[ 18 F]FELP, [ 18 F]FET and [ 18 F]FDG PET scans were acquired on three consecutive days. In an inflammation experiment, mice were inoculated in the left thigh with U87 human glioblastoma cells. After heterotopic tumor growth was confirmed macroscopically, inflammation was induced by injection of turpentine subcutaneously in the right thigh. Subsequently, 2-[ 18 F]FELP and [ 18 F]FDG scans were acquired on two consecutive days., Results: The in vivo PET images demonstrated that 2-[ 18 F]FELP could differentiate glioblastoma and radiation necrosis using SUV mean (p = 0.0016) and LNR mean (p = 0.009), while [ 18 F]FET was only able to differentiate both lesions by means of the SUV mean . (p = 0.047) Delayed [ 18 F]FDG late PET (4 h postinjection) was also able to distinguish glioblastoma from radiation necrosis, but smaller lesion-to-normal brain ratios were observed (SUV mean : p = 0.009; LNR mean : p = 0.028). In the inflammation study, 2-[ 18 F]FELP showed no significant uptake in the inflammation lesion when compared to the control group (SUVmean: p = 0.149; LNRmean: p = 0.083). In contrast, both conventional and delayed [ 18 F]FDG displayed significant uptake in the turpentine-invoked lesion (SUVmean: p = 0.021; LNRmean: p = 0.021)., Conclusion: This study suggests that the 2-[ 18 F]FELP PET is able to differentiate glioblastoma from radiation necrosis and that the 2-[ 18 F]FELP uptake is less likely to be contaminated by the presence of inflammation than the [ 18 F]FDG signal., Advances in Knowledge: These results are clinically relevant for the differential diagnosis between tumor and radiation necrosis because radiation necrosis always contains a certain amount of inflammatory cells. Hence, 2-[ 18 F]FELP is preferred to discriminate tumor from radiation necrosis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Although intraperitoneal chemotherapy (IPC) has evolved into an established treatment modality for patients with peritoneal metastasis (PM), drug penetration into tumor nodules remains limited. Drug transport during IPC is a complex process that depends on a large number of different parameters (e.g. drug, dose, tumor size, tumor pressure, tumor vascularization). Mathematical modeling allows for a better understanding of the processes that underlie drug transport and the relative importance of the parameters influencing it. In this work, we expanded our previously developed 3D Computational Fluid Dynamics (CFD) model of the drug mass transport in idealized tumor nodules during IP chemotherapy to include realistic tumor geometries and spatially varying vascular properties. DCE-MRI imaging made it possible to distinguish between tumorous tissues, healthy surrounding tissues and necrotic zones based on differences in the vascular properties. We found that the resulting interstitial pressure profiles within tumors were highly dependent on the irregular geometries and different zones. The tumor-specific cisplatin penetration depths ranged from 0.32 mm to 0.50 mm. In this work, we found that the positive relationship between tumor size and IFP does not longer hold in the presence of zones with different vascular properties, while we did observe a positive relationship between the percentage of viable tumor tissue and the maximal IFP. Our findings highlight the importance of incorporating both the irregular tumor geometries and different vascular zones in CFD models of IPC.

Epilepsy is a neurological disorder characterized by recurrent epileptic seizures. Electrophysiological and neuroimaging studies in patients with epilepsy suggest that abnormal functional brain networks play a role in the development of epilepsy, i.e. epileptogenesis, resulting in the generation of spontaneous seizures and cognitive impairment. In this longitudinal study, we investigated changes in functional brain networks during epileptogenesis in the intraperitoneal kainic acid (IPKA) rat model of temporal lobe epilepsy (TLE) using resting state functional magnetic resonance imaging (rsfMRI) and graph theory. Additionally, we investigated whether these changes are related to the frequency of occurrence of spontaneous epileptic seizures in the chronic phase of epilepsy. Using a 7T MRI system, rsfMRI images were acquired under medetomidine anaesthesia before and 1, 3, 6, 10 and 16 weeks after status epilepticus (SE) induction in 20 IPKA animals and 7 healthy control animals. To obtain a functional network, correlation between fMRI time series of 38 regions of interest (ROIs) was calculated. Then, several graph theoretical network measures were calculated to describe and quantify the network changes. At least 17 weeks post-SE, IPKA animals were implanted with electrodes in the left and right dorsal hippocampus, EEG was measured for 7 consecutive days and spontaneous seizures were counted. Our results show that correlation coefficients of fMRI time series shift to lower values during epileptogenesis, indicating weaker whole brain network connections. Segregation and integration in the functional brain network also decrease, indicating a lower local interconnectivity and a lower overall communication efficiency. Secondly, this study demonstrates that the largest decrease in functional connectivity is observed for the retrosplenial cortex. Finally, post-SE changes in functional connectivity, segregation and integration are correlated with seizure frequency in the IPKA rat model., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Even with an optimal treatment protocol, the median survival of glioblastoma (GB) patients is only 12-15 months. Hence, there is need for novel effective therapies that improve survival outcomes. Recent evidence suggests an important role for connexin (Cx) proteins (especially Cx43) in the microenvironment of malignant glioma. Cx43-mediated gap junctional communication has been observed between tumor cells, between astrocytes and between tumor cells and astrocytes. Therefore, gap junction directed therapy using a pharmacological suppressor or modulator, such as tonabersat, could be a promising target in the treatment of GB. In this preclinical study, we evaluated the possible therapeutic potential of tonabersat in the F98 model., Procedures: Female Fischer rats were inoculated with ± 25.000 F98 tumor cells in the right frontal lobe. Eight days post-inoculation contrast-enhanced T1-weighted (CE-T1w) magnetic resonance (MR) images were acquired to confirm tumor growth in the brain. After tumor confirmation, rats were randomized into a Control Group, a Connexin Modulation Group (CM), a Standard Medical Treatment Group (ST), and a Standard Medical Treatment with adjuvant Connexin Modulation Group (STCM). To evaluate therapy response, T2-weighted (T2w) and CE-T1w sequences were acquired at several time points. Tumor volume analysis was performed on CE-T1w images and statistical analysis was performed using a linear mixed model., Results: Significant differences in estimated geometric mean tumor volumes were found between the ST Group and the Control Group and also between the STCM Group and the Control Group. In addition, significant differences in estimated geometric mean tumor volumes between the ST Group and the STCM Group were demonstrated. No significant differences in estimated geometric mean tumor volumes were found between the Control Group and the CM Group., Conclusion: Our results demonstrate a therapeutic potential of tonabersat for the treatment of GB when used in combination with radiotherapy and temozolomide chemotherapy., Competing Interests: The authors have declared that no competing interests exist.

Patients with advanced ovarian cancer develop recurrence despite initial treatment response to standard treatment of surgery and intravenous/intraperitoneal (IP) chemotherapy, partly due to a limited peritoneal exposure time of chemotherapeutics. Paclitaxel-loaded genipin-crosslinked gelatin microspheres (PTX-GP-MS) are evaluated for the treatment of microscopic peritoneal carcinomatosis and prevention of recurrent disease. The highest drug load (39.2 µg PTX/mg MS) was obtained by immersion of GP-MS in aqueous PTX nanosuspension (PTX nano -GP-MS) instead of ethanolic PTX solution (PTX EtOH -GP-MS). PTX release from PTX-GP-MS was prolonged. PTX nano -GP-MS displayed a more controlled release compared to a biphasic release from PTX EtOH -GP-MS. Anticancer efficacy of IP PTX-GP-MS (PTX EtOH -GP-MS, D = 7.5 mg PTX/kg; PTX nano -GP-MS D = 7.5 and 35 mg PTX/kg), IP nanoparticular albumin-bound PTX (D = 35 mg PTX/kg) and controls (0.9% NaCl, blank GP-MS) was evaluated in a microscopic peritoneal carcinomatosis xenograft mouse model. PTX nano -GP-MS showed superior anticancer efficacy with significant increased survival time, decreased peritoneal carcinomatosis index score and ascites incidence. However, prolonged PTX release over 14 days from PTX nano -GP-MS caused drug-related toxicity in 27% of high-dosed PTX nano -GP-MS-treated mice. Dose simulations for PTX nano -GP-MS demonstrated an optimal survival without drug-induced toxicity in a range of 7.5-15 mg PTX/kg. Low-dosed PTX nano -GP-MS can be a promising IP drug delivery system to prevent recurrent ovarian cancer.

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous variability and heterogeneity (age, gender, type of trauma, other possible pathologies, etc.), animal models play a key role in unraveling factors that are limitations in clinical research. They provide a standardized and controlled setting to investigate the biological mechanisms of injury and repair following TBI. However, not all animal models mimic the diffuse and subtle nature of mTBI effectively. For example, the commonly used controlled cortical impact (CCI) and lateral fluid percussion injury (LFPI) models make use of a craniotomy to expose the brain and induce widespread focal trauma, which are not commonly seen in mTBI. Therefore, these experimental models are not valid to mimic mTBI. Thus, an appropriate model should be used to investigate mTBI. The Marmarou weight drop model for rats induces similar microstructural alterations and cognitive impairments as seen in patients who sustain mild trauma; therefore, this model was selected for this protocol. Conventional computed tomography and magnetic resonance imaging (MRI) scans commonly show no damage following a mild injury, because mTBI induces often only subtle and diffuse injuries. With diffusion weighted MRI, it is possible to investigate microstructural properties of brain tissue, which can provide more insight into the microscopic alterations following mild trauma. Therefore, the goal of this study is to obtain quantitative information of a selected region-of-interest (i.e., hippocampus) to follow up disease progression after obtaining a mild and diffuse brain injury.

Purpose: Pressurized intraperitoneal aerosol chemotherapy (PIPAC) is a novel technique delivering drugs into the abdominal cavity as an aerosol under high pressure. It is hypothesized to have advantages such as enhancing tissue uptake, distributing drugs homogeneously within the closed and expanded abdominal cavity and higher local concentration of drugs in the peritoneal cavity. However, the clinical trials of PIPAC so far are limited to liquid chemotherapeutic solution, and the applicability of biomolecules (such as mRNA, siRNA and oligonucleotide) is not known. We aimed to investigate the feasibility of administrating mRNA lipoplexes to the peritoneal cavity via high pressure nebulization., Methods: We firstly investigated the influences of nebulization on physicochemical properties and in vitro transfection efficiency of mRNA lipoplexes. Then, mRNA lipoplexes were delivered to healthy rats through intravenous injection, intraperitoneal injection and PIPAC, respectively., Results: mRNA lipoplexes can withstand the high pressure applied during the PIPAC procedure in vitro. Bioluminescence localized to the peritoneal cavity of rats after administration by IP injection and nebulization, while intravenous injection mainly induced protein expression in the spleen., Conclusion: This study demonstrated that local nebulization is feasible to apply mRNA complexes in the peritoneal cavity during a PIPAC procedure.

Background: Glioblastoma (GB) is the most common primary malignant brain tumor. Standard medical treatment consists of a maximal safe surgical resection, subsequently radiation therapy (RT) and chemotherapy with temozolomide (TMZ). An accurate definition of the tumor volume is of utmost importance for guiding RT. In this project we investigated the feasibility and treatment response of subvolume boosting to a PET-defined tumor part., Method: F98 GB cells inoculated in the rat brain were imaged using T2- and contrast-enhanced T1-weighted (T1w) MRI. A dose of 20 Gy (5 × 5 mm 2 ) was delivered to the target volume delineated based on T1w MRI for three treatment groups. Two of those treatment groups received an additional radiation boost of 5 Gy (1 × 1 mm 2 ) delivered to the region either with maximum [ 18 F]FET or [ 18 F]FAZA PET tracer uptake, respectively. All therapy groups received intraperitoneal (IP) injections of TMZ. Finally, a control group received no RT and only control IP injections. The average, minimum and maximum dose, as well as the D 90 -, D 50 - and D 2 - values were calculated for nine rats using both RT plans. To evaluate response to therapy, follow-up tumor volumes were delineated based on T1w MRI., Results: When comparing the dose volume histograms, a significant difference was found exclusively between the D 2 -values. A significant difference in tumor growth was only found between active therapy and sham therapy respectively, while no significant differences were found when comparing the three treatment groups., Conclusion: In this study we showed the feasibility of PET guided subvolume boosting of F98 glioblastoma in rats. No evidence was found for a beneficial effect regarding tumor response. However, improvements for dose targeting in rodents and studies investigating new targeted drugs for GB treatment are mandatory.

Purpose: To compare the Ghent and Graz magnetic resonance imaging (MRI) protocols for third molars, focusing on the assessment of apical closure. To study the influence of (1) voxel size and (2) head fixation using a bite bar. To compare both protocols with a ground truth of apical development., Materials and Methods: In 11 healthy volunteers, 3T MRI was conducted, including four Ghent sequences and two Graz sequences, with and without bite bar. After removal, 39 third molars were scanned with 7T μMRI and μCT to establish the ground truth of apical development. Three observers in consensus evaluated assessability and allocated developmental stages., Results: The Ghent T2 FSE sequence (0.33 × 0.33 × 2 mm 3 ) was more assessable than the Graz T1 3D FSE sequence (0.59 × 0.59 × 1 mm 3 ). Comparing assessability in both sequences with bite bar rendered P = 0.02, whereas comparing those without bite bar rendered P < 0.001. Within the same sequence, the bite bar increased assessability, with P = 0.03 for the Ghent T2 FSE and P = 0.07 for the Graz T1 3D FSE. Considering μCT as ground truth for staging, allocated stages on MRI were most frequently equal or higher. Among in vivo protocols, the allocated stages did not differ significantly., Conclusion: Imaging modality-specific and MRI sequence-specific reference data are needed in age estimation. A higher in-plane resolution and a bite bar increase assessability of apical closure, whereas they do not affect stage allocation of assessable apices.

The use of O-(2-[ 18 F]fluoroethyl)-L-tyrosine ([ 18 F]FET) as a positron emission tomography (PET) tracer for brain tumor imaging might have some limitations because of the relatively low affinity for the L-type amino acid transporter 1 (LAT1). To assess the stereospecificity and evaluate the influence of aromatic ring modification of phenylalanine LAT1 targeting tracers, six different fluoroalkylated phenylalanine analogues were synthesized. After in vitro K i determination, the most promising compound, 2-[ 18 F]-2-fluoroethyl-L-phenylalanine (2-[ 18 F]FELP), was selected for further evaluation and in vitro comparison with [ 18 F]FET. Subsequently, 2-[ 18 F]FELP was assessed in vivo and compared with [ 18 F]FET and [ 18 F]FDG in a F98 glioblastoma rat model. 2-[ 18 F]FELP showed improved in vitro characteristics over [ 18 F]FET, especially when the affinity and specificity for system L is concerned. Based on our results, 2-[ 18 F]FELP is a promising new PET tracer for brain tumor imaging.

Glioblastoma is the most aggressive and malignant primary brain tumor in adults. Despite the current state-of-the-art treatment, which consists of maximal surgical resection followed by radiation therapy, concomitant, and adjuvant chemotherapy, progression remains rapid due to aggressive tumor characteristics. Several new therapeutic targets have been investigated using chemotherapeutics and targeted molecular drugs, however, the intrinsic resistance to induced cell death of brain cells impede the effectiveness of systemic therapies. Also, the unique immune environment of the central nervous system imposes challenges for immune-based therapeutics. Therefore, it is important to consider other approaches to treat these tumors. There is a well-known dose-response relationship for glioblastoma with increased survival with increasing doses, but this effect seems to cap around 60 Gy, due to increased toxicity to the normal brain. Currently, radiation treatment planning of glioblastoma patients relies on CT and MRI that does not visualize the heterogeneous nature of the tumor, and consequently, a homogenous dose is delivered to the entire tumor. Metabolic imaging, such as positron-emission tomography, allows to visualize the heterogeneous tumor environment. Using these metabolic imaging techniques, an approach called dose painting can be used to deliver a higher dose to the tumor regions with high malignancy and/or radiation resistance. Preclinical studies are required for evaluating the benefits of novel radiation treatment strategies, such as PET-based dose painting . The aim of this review is to give a brief overview of promising PET tracers that can be evaluated in laboratory animals to bridge the gap between PET-based dose painting in glioblastoma patients.

Diffusion magnetic resonance imaging biomarkers can provide quantifiable information of the brain tissue after a mild traumatic brain injury (mTBI). However, the commonly applied diffusion tensor imaging (DTI) model is not very specific to changes in the underlying cellular structures. To overcome these limitations, other diffusion models have recently emerged to provide a more complete view on the damage profile following TBI. In this study, we investigated longitudinal changes in advanced diffusion metrics following experimental mTBI, utilising three different diffusion models in a rat model of mTBI, including DTI, diffusion kurtosis imaging and a white matter model. Moreover, we investigated the association between the diffusion metrics with histological markers, including glial fibrillary acidic protein (GFAP), neurofilaments and synaptophysin in order to investigate specificity. Our results revealed significant decreases in mean diffusivity in the hippocampus and radial diffusivity and radial extra axonal diffusivity (RadEAD) in the cingulum one week post injury. Furthermore, correlation analysis showed that increased values of fractional anisotropy one day post injury in the hippocampus was highly correlated with GFAP reactivity three months post injury. Additionally, we observed a positive correlation between GFAP on one hand and the kurtosis parameters in the hippocampus on the other hand three months post injury. This result indicated that prolonged glial activation three months post injury is related to higher kurtosis values at later time points. In conclusion, our findings point out to the possible role of kurtosis metrics as well as metrics from the white matter model as prognostic biomarker to monitor prolonged glial reactivity and inflammatory responses after a mTBI not only at early timepoints but also several months after injury., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Metastatic brain tumors pose a severe problem in the treatment of patients with breast carcinoma. Preclinical models have been shown to play an important role in unraveling the underlying mechanisms behind the metastatic process and evaluation of new therapeutic approaches. As the size of the rat brain allows improved in vivo imaging, we attempted to establish a rat model for breast cancer brain metastasis that allows follow-up by 7 tesla (7T) preclinical Magnetic Resonance Imaging (MRI)., Procedures: Green fluorescent protein-transduced (eGFP) MDA-MB-231br breast cancer cells were labeled with micron-sized particles of iron oxide (MPIOs) and intracardially injected in the left ventricle of female nude rats and mice. 7T preclinical MRI was performed to show the initial distribution of MPIO-labeled cancer cells and to visualize metastasis in the brain. Occurrence of potential metastasis outside the brain was evaluated by 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) positron emission tomography (PET)/computed tomography (CT) and potential bone lesions were assessed using [18F]sodium fluoride ([18F]NaF) PET/CT., Results: The first signs of brain metastasis development were visible as hyperintensities on T2-weighted (T2w) MR images acquired 3 weeks after intracardiac injection in rats and mice. Early formation of unexpected bone metastasis in rats was clinically observed and assessed using PET/CT. Almost no bone metastasis development was observed in mice after PET/CT evaluation., Conclusions: Our results suggest that the metastatic propensity of the MDA-MB-231br/eGFP cancer cell line outside the brain is species-dependent. Because of early and abundant formation of bone metastasis with the MDA-MB-231br/eGFP cancer cell line, this rat model is currently not suitable for investigating brain metastasis as a single disease model nor for evaluation of novel brain metastasis treatment strategies., Competing Interests: Sara Neyt, Bert Vandeghinste and Pieter Mollet are employees at MOLECUBES NV. The other authors have declared that no competing interests exist. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Objective- The NTs (neurotrophins), BDNF (brain-derived neurotrophic factor) and NT-3 promote vascular development and angiogenesis. This study investigated the contribution of endogenous NTs in embryonic stem cell (ESC) vascular differentiation and the potential of exogenous BDNF to improve the process of ESC differentiation to endothelial cells (ECs). Approach and Results- Mouse ESCs were differentiated into vascular cells using a 2-dimensional embryoid body (EB) model. Supplementation of either BDNF or NT-3 increased EC progenitors' abundance at day 7 and enlarged the peripheral vascular plexus with ECs and SM22α + (smooth muscle 22 alpha-positive) smooth muscle cells by day 13. Conversely, inhibition of either BDNF or NT-3 receptor signaling reduced ECs, without affecting smooth muscle cells spread. This suggests that during vascular development, endogenous NTs are especially relevant for endothelial differentiation. At mechanistic level, we have identified that BDNF-driven ESC-endothelial differentiation is mediated by a pathway encompassing the transcriptional repressor EZH2 (enhancer of zeste homolog 2), microRNA-214 (miR-214), and eNOS (endothelial nitric oxide synthase). It was known that eNOS, which is needed for endothelial differentiation, can be transcriptionally repressed by EZH2. In turn, miR-214 targets EZH2 for inhibition. We newly found that in ESC-ECs, BDNF increases miR-214 expression, reduces EZH2 occupancy of the eNOS promoter, and increases eNOS expression. Moreover, we found that NRP-1 (neuropilin 1), KDR (kinase insert domain receptor), and pCas 130 (p130 Crk-associated substrate kinase), which reportedly induce definitive endothelial differentiation of pluripotent cells, were increased in BDNF-conditioned ESC-EC. Mechanistically, miR-214 mediated the BDNF-induced expressional changes, contributing to BDNF-driven endothelial differentiation. Finally, BDNF-conditioned ESC-ECs promoted angiogenesis in vitro and in vivo. Conclusions- BDNF promotes ESC-endothelial differentiation acting via miR-214.

Preclinical research has been indispensable in the exploration of the neurological basis of major depressive disorder (MDD). The present study aimed to examine effects on regional brain activity of two frequently used depression models, the chronic unpredictable mild stress (CUMS)- and the chronic corticosterone (CORT) depression model. The CUMS and CORT depression model were induced by exposing male Long-Evans rats to a 4-week procedure of unpredictable mild stressors or a 3-week procedure of chronic corticosterone, respectively. Positron emission tomography with [ 18 F]FDG was performed to determine alterations in regional brain activity. In addition, depressive- and anxiety-like behaviour was assessed via the forced swim test and the open field test, respectively. The chronic CORT administration, but not the CUMS model, significantly induced depressive-like behaviour and elevated plasma corticosterone levels. Compared to control, induction of the CORT depression model resulted in a significantly reduced glucose consumption in the insular cortex and the striatum, and a significantly elevated consumption in the cerebellum and the midbrain. Induction of the CUMS model replicated the findings with respect to the activity in the striatum region, and cerebellum, but missed significance in the insular cortex and the midbrain. Based on the alterations in behaviour and regional [ 18 F]FDG uptake, a superior face validity and construct validity can be observed after induction of depression via chronic CORT injections, compared to the used CUMS paradigm.

The performances of an intra-operative optical imaging system for Cerenkov luminescence imaging of resected tumor specimens were evaluated with phantom studies. The spatial resolution, the linearity of the measured signal with the activity concentration and the minimum detectable activity concentration were considered. A high linearity was observed over a broad range of activity concentration (R 2 ⩾0.99 down to ∼40 kBq/ml of 18 F-FDG). For 18 F-FDG activity distributions 2 mm deep in biological tissue, the measured detection limit was 8 kBq/ml and a spatial resolution of 2.5 mm was obtained. The detection limit of the imaging system is comparable with clinical activity concentrations in tumor specimens, and the spatial resolution is compatible with clinical requirements., (Copyright © 2018 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.)

Background: Esophageal cancer is an aggressive disease with poor survival rates. A more patient-tailored approach based on predictive biomarkers could improve outcome. We aimed to predict radiotherapy (RT) response by imaging tumor hypoxia with 18 F-FAZA PET/CT in an esophageal adenocarcinoma (EAC) mouse model. Additionally, we investigated the radiosensitizing effect of the hypoxia modifier nimorazole in vitro and in vivo., Methods: In vitro MTS cell proliferation assays (OACM5 1.C SC1, human EAC cell line) were performed under normoxic and hypoxic (< 1%) conditions: control (100 μL PBS), nimorazole, irradiation (5, 10 or 20 Gy) with or without nimorazole. In vivo, subcutaneous xenografts were induced in nude mice (OACM5 1.C SC1). Treatment was given daily for 5 consecutive days: (A) control (600 μl NaCl 0.9% intraperitoneally (IP)) (N = 5, n = 7), (B) RT (5 Gy/d) (N = 11, n = 20), (C) combination (nimorazole (200 mg/kg/d IP) 30 min before RT) (N = 13, n = 21). N = number of mice, n = number of tumors. 18 F-FAZA PET/CT was performed before treatment and tumor to background (T/B) ratios were calculated. Relative tumor growth was calculated and tumor sections were examined histologically (hypoxia, proliferation)., Results: A T/B ≥ 3.59 on pre-treatment 18 F-FAZA PET/CT was predictive for worse RT response (sensitivity 92.3%, specificity 71.4%). Radiation was less effective in hypoxic tumors (T/B ≥ 3.59) compared to normoxic tumors (T/B < 3.59) (P = 0.0025). In vitro, pre-treatment with nimorazole significantly decreased hypoxic radioresistance (P < 0.01) while in vivo, nimorazole enhanced the efficacy of RT to suppress cancer cell proliferation in hypoxic tumor areas (Ki67, P = 0.064), but did not affect macroscopic tumor growth., Conclusions: Tumor tissue hypoxia as measured with 18 F-FAZA PET/CT is predictive for RT response in an EAC xenograft model. The radiosensitizing effect of nimorazole was questionable and requires further investigation.

Preoperative radiotherapy (RT) is a mainstay in the management of rectal cancer, a tumor characterized by desmoplastic stroma containing cancer-associated fibroblasts (CAF). Although CAFs are abundantly present, the effects of RT to CAF and its impact on cancer cells are unknown. We evaluated the damage responses of CAF to RT and investigated changes in colorectal cancer cell growth, transcriptome, metabolome, and kinome in response to paracrine signals emerging from irradiated CAF. RT to CAF induced DNA damage, p53 activation, cell-cycle arrest, and secretion of paracrine mediators, including insulin-like growth factor-1 (IGF1). Subsequently, RT-activated CAFs promoted survival of colorectal cancer cells, as well as a metabolic switch favoring glutamine consumption through IGF1 receptor (IGF1R) activation. RT followed by IGF1R neutralization in orthotopic colorectal cancer models reduced the number of mice with organ metastases. Activation of the downstream IGF1R mediator mTOR was significantly higher in matched (intrapatient) samples and in unmatched (interpatient) samples from rectal cancer patients after neoadjuvant chemoradiotherapy. Taken together, our data support the notion that paracrine IGF1/IGF1R signaling initiated by RT-activated CAF worsens colorectal cancer progression, establishing a preclinical rationale to target this activation loop to further improve clinical responses and patient survival. Significance: These findings reveal that paracrine IGF1/IGF1R signaling promotes colorectal cancer progression, establishing a preclinical rationale to target this activation loop. Cancer Res; 78(3); 659-70. ©2017 AACR ., (©2017 American Association for Cancer Research.)

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without the ability to target a specific or well-delineated tumor volume. The delivery of radiation was achieved using fixed radiation sources or linear accelerators producing megavoltage (MV) X-rays. These devices are unable to achieve sub-millimeter precision required for small animals. Furthermore, the high doses delivered to healthy surrounding tissue hamper response assessment. To increase the translation between small animal studies and humans, our goal was to mimic the treatment of human glioblastoma in a rat model. To enable a more accurate irradiation in a preclinical setting, recently, precision image-guided small animal radiation research platforms were developed. Similar to human planning systems, treatment planning on these micro-irradiators is based on computed tomography (CT). However, low soft-tissue contrast on CT makes it very challenging to localize targets in certain tissues, such as the brain. Therefore, incorporating magnetic resonance imaging (MRI), which has excellent soft-tissue contrast compared to CT, would enable a more precise delineation of the target for irradiation. In the last decade also biological imaging techniques, such as positron emission tomography (PET) gained interest for radiation therapy treatment guidance. PET enables the visualization of e.g., glucose consumption, amino-acid transport, or hypoxia, present in the tumor. Targeting those highly proliferative or radio-resistant parts of the tumor with a higher dose could give a survival benefit. This hypothesis led to the introduction of the biological tumor volume (BTV), besides the conventional gross target volume (GTV), clinical target volume (CTV), and planned target volume (PTV). At the preclinical imaging lab of Ghent University, a micro-irradiator, a small animal PET, and a 7 T small animal MRI are available. The goal was to incorporate MRI-guided irradiation and PET-guided sub-volume boosting in a glioblastoma rat model.

Purpose: In this study, the potential of semiquantitative and quantitative analysis of dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) was investigated to differentiate glioblastoma (GB) from radiation necrosis (RN) in rats., Procedures: F98 GB growth was seen on MRI 8-23 days post-inoculation (n = 15). RN lesions developed 6-8 months post-irradiation (n = 10). DCE-MRI was acquired using a fast low-angle shot (FLASH) sequence. Regions of interest (ROIs) encompassed peripheral contrast enhancement in GB (n = 15) and RN (n = 10) as well as central necrosis within these lesions (GB (n = 4), RN (n = 3)). Dynamic contrast-enhanced time series, obtained from the DCE-MRI data, were fitted to determine four function variables (amplitude A, offset from zero C, wash-in rate k, and wash-out rate D) as well as maximal intensity (Imax F ) and time to peak (TTP F ). Secondly, maps of semiquantitative and quantitative parameters (extended Tofts model) were created using Olea Sphere ( O ). Semiquantitative DCE-MRI parameters included wash-in O , wash-out O , area under the curve (AUC O ), maximal intensity (Imax O ), and time to peak (TTP O ). Quantitative parameters included the rate constant plasma to extravascular-extracellular space (EES) (K trans ), the rate constant EES to plasma (K ep ), plasma volume (V p ), and EES volume (V e ). All (semi)quantitative parameters were compared between GB and RN using the Mann-Whitney U test. ROC analysis was performed., Results: Wash-in rate (k) and wash-out rate (D) were significantly higher in GB compared to RN using curve fitting (p = 0.016 and p = 0.014). TTP F and TTP O were significantly lower in GB compared to RN (p = 0.001 and p = 0.005, respectively). The highest sensitivity (87 %) and specificity (80 %) were obtained for TTP F by applying a threshold of 581 s. K trans , K ep , and V e were not significantly different between GB and RN. A trend towards higher V p values was found in GB compared to RN, indicating angiogenesis in GB (p = 0.075)., Conclusions: Based on our results, in a rat model of GB and RN, wash-in rate, wash-out rate, and the time to peak extracted from DCE-MRI time series data may be useful to discriminate GB from RN.

This study reports on the design of mRNA and adjuvant-loaded lipid nanoparticles for therapeutic cancer vaccination. The use of nucleoside-modified mRNA has previously been shown to improve the translational capacity and safety of mRNA-therapeutics, as it prevents the induction of type I interferons (IFNs). However, type I IFNs were identified as the key molecules that trigger the activation of antigen presenting cells, and as such drive T cell immunity. We demonstrate that nucleoside-modified mRNA can be co-delivered with the clinically approved TLR agonist monophosphoryl lipid A (MPLA). As such, we simultaneously allow high antigen expression in vivo while substituting the type I IFN response by a more controllable adjuvant. This strategy shows promise to induce effective antigen-specific T cell immunity and may be useful to enhance the safety of mRNA vaccines., (Copyright © 2017. Published by Elsevier B.V.)

The present study aimed to investigate the orthotopic growth potential of two generally available esophageal adenocarcinoma cell lines, OE33 and OACM5 1.C, and a third in vivo selected subpopulation, OACM5 1.C SC1. One group of mice was subcutaneously injected in the hind legs. Tumor growth was measured with calipers. Another group was injected orthotopically in the distal esophageal wall through median laparotomy. Tumor development was evaluated macroscopically and confirmed microscopically. A subset of mice was evaluated with magnetic resonance imaging (MRI) to follow tumor progression. Additionally, functional cell line characteristics were evaluated in vitro (clonogenic, collagen invasion and sphere formation assays, and protein analysis of cell-cell adhesion and cytoskeletal proteins) to better understand xenograft behavior. OE33 cells were shown to be epithelial‑like, whereas OACM5 1.C and OACM5 1.C SC1 were more mesenchymal-like. The three cell lines were non‑invasive into native type I collagen gels. In vivo, OE33 cells led to 63.6 and 100% tumor nodules after orthotopic (n=12) and subcutaneous (n=8) injection, respectively. Adversely, OACM5 1.C cells did not lead to tumor formation after orthotopic injection (n=6) and only 50% of subcutaneous injections led to tumor nodules (n=8). However, the newly established cell line OACM5 1.C SC1 resulted in 33% tumor formation when orthotopically injected (n=6) and in 100% tumors when injected subcutaneously (n=8). The higher xenograft rate of OACM5 1.C SC1 (P<0.05) corresponded with a higher clonogenic potential compared to its parental cell line (P<0.0001). All models showed local tumor growth without metastasis formation. In conclusion, OACM5 1.C has a poor tumor take rate at an orthotopic and ectopic site. A subpopulation obtained through in vivo selection, OACM5 1.C SC1, gives a significant higher take rate, ectopically. Furthermore, OE33 establishes orthotopic (and subcutaneous) xenografts in mice. These models can be of interest for future studies, and their slow growth rates are a challenge for therapeutic intervention.

Prolyl hydroxylase domain-containing proteins (PHDs) regulate the adaptation of cells to hypoxia. Pan-hydroxylase inhibition is protective in experimental colitis, in which PHD1 plays a prominent role. However, it is currently unknown how PHD1 targeting regulates this protection and which cell type(s) are involved. Here, we demonstrated that Phd1 deletion in endothelial and haematopoietic cells (Phd1 f/ f Tie2:cre) protected mice from dextran sulphate sodium (DSS)-induced colitis, with reduced epithelial erosions, immune cell infiltration, and colonic microvascular dysfunction, whereas the response of Phd2 f/+ Tie2:cre and Phd3 f/ f Tie2:cre mice to DSS was similar to that of their littermate controls. Using bone marrow chimeras and cell-specific cre mice, we demonstrated that ablation of Phd1 in haematopoietic cells but not in endothelial cells was both necessary and sufficient to inhibit experimental colitis. This effect relied, at least in part, on skewing of Phd1-deficient bone marrow-derived macrophages towards an anti-inflammatory M2 phenotype. These cells showed an attenuated nuclear factor-κB-dependent response to lipopolysaccharide (LPS), which in turn diminished endothelial chemokine expression. In addition, Phd1 deficiency in dendritic cells significantly reduced interleukin-1β production in response to LPS. Taken together, our results further support the development of selective PHD1 inhibitors for ulcerative colitis, and identify haematopoietic cells as their primary target. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Objective: The aim of this work was to investigate whether quantitative dual-energy CT (DECT) imaging is feasible for small animal irradiators with an integrated cone-beam CT (CBCT) system., Methods: The optimal imaging protocols were determined by analyzing different energy combinations and dose levels. The influence of beam hardening effects and the performance of a beam hardening correction (BHC) were investigated. In addition, two systems from different manufacturers were compared in terms of errors in the extracted effective atomic numbers (Z eff ) and relative electron densities (ρ e ) for phantom inserts with known elemental compositions and relative electron densities., Results: The optimal energy combination was determined to be 50 and 90 kVp. For this combination, Z eff and ρ e can be extracted with a mean error of 0.11 and 0.010, respectively, at a dose level of 60 cGy., Conclusion: Quantitative DECT imaging is feasible for small animal irradiators with an integrated CBCT system. To obtain the best results, optimizing the imaging protocols is required. Well-separated X-ray spectra and a sufficient dose level should be used to minimize the error and noise for Z eff and ρ e . When no BHC is applied in the image reconstruction, the size of the calibration phantom should match the size of the imaged object to limit the influence of beam hardening effects. No significant differences in Z eff and ρ e errors are observed between the two systems from different manufacturers. Advances in knowledge: This is the first study that investigates quantitative DECT imaging for small animal irradiators with an integrated CBCT system.

Background/purpose: Radiotherapy (RT) increases local tumor control in locally advanced rectal cancer, but complete histological response is seen in only a minority of cases. Antiangiogenic therapy has been proposed to improve RT efficacy by "normalizing" the tumor microvasculature. Here, we examined whether cediranib, a pan-vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor, improves microvascular function and tumor control in combination with RT in a mouse colorectal cancer (CRC) model., Methods: CRC xenografts (HT29) were grown subcutaneously in mice. Animals were treated for 5 consecutive days with vehicle, RT (1.8 Gy daily), cediranib (6 mg/kg po), or combined therapy (cediranib 2 h prior to radiation). Tumor volume was measured with calipers. Vascular changes were analyzed by dynamic contrast-enhanced MRI, oxygenation and interstitial fluid pressure probes and histology. To investigate vascular changes more in detail, a second set of mice were fitted with titanium dorsal skinfold window chambers, wherein a HT29 tumor cell suspension was injected. In vivo fluorescence microscopy was performed before and after treatment (same treatment protocol)., Results: In vivo microscopy analyses showed that VEGFR inhibition with cediranib led to a "normalization" of the vessel wall, with decreased microvessel permeability (p < 0.0001) and tortuosity (p < 0.01), and a trend to decreased vessel diameters. This seemed to lead to lower tumor hypoxia rates in the cediranib and combination groups compared to the control and RT groups. This led to an increased tumor control in the combination group compared to controls or monotherapy (p < 0.0001)., Conclusions: The combination of RT with cediranib enhances tumor control in a CRC xenograft mouse model. Microvascular analyses suggest that cediranib leads to vascular normalization and improved oxygenation., (© 2016 S. Karger AG, Basel.)

A generic site-specific conjugation method that generates a homogeneous product is of utmost importance in tracer development for molecular imaging and therapy. We explored the protein-ligation capacity of the enzyme Sortase A to label camelid single-domain antibody-fragments, also known as nanobodies. The versatility of the approach was demonstrated by conjugating independently three different imaging probes: the chelating agents CHX-A"-DTPA and NOTA for single-photon emission computed tomography (SPECT) with indium-111 and positron emission tomography (PET) with gallium-68, respectively, and the fluorescent dye Cy5 for fluorescence reflectance imaging (FRI). After a straightforward purification process, homogeneous single-conjugated tracer populations were obtained in high yield (30-50%). The enzymatic conjugation did not affect the affinity of the tracers, nor the radiolabeling efficiency or spectral characteristics. In vivo, the tracers enabled the visualization of human epidermal growth factor receptor 2 (HER2) expressing BT474M1-tumors with high contrast and specificity as soon as 1 h post injection in all three imaging modalities. These data demonstrate Sortase A-mediated conjugation as a valuable strategy for the development of site-specifically labeled camelid single-domain antibody-fragments for use in multiple molecular imaging modalities. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)