Generation of post-surgical minimal residual disease models to investigate metastasis in soft tissue sarcoma patient-derived orthotopic xenografts.

Despite optimal multimodal treatment including surgical resection, 50%-80% of high-grade soft tissue sarcoma (STS) patients metastasize. Here, we present a protocol for the generation and use of post-surgical minimal residual disease models to investigate metastatic relapse in STS patient-derived xenografts. We describe steps for orthotopic engraftment of high-grade STS patient-derived tumor tissue. We then detail procedures for primary tumor resection with broad, negative resection margins and follow-up until metastases using MRI. For complete details on the use and execution of this protocol, please refer to Fischer et al. (2023).1.

A preclinical platform for assessing long-term drug efficacy exploiting mechanically tunable scaffolds colonized by a three-dimensional tumor microenvironment.

BACKGROUND: Long-term drug evaluation heavily relies upon rodent models. Drug discovery methods to reduce animal models in oncology may include three-dimensional (3D) cellular systems that take into account tumor microenvironment (TME) cell types and biomechanical properties. METHODS: In this study we reconstructed a 3D tumor using an elastic polymer (acrylate-endcapped urethane-based poly(ethylene glycol) (AUPPEG)) with clinical relevant stiffness. Single cell suspensions from low-grade serous ovarian cancer (LGSOC) patient-derived early passage cultures of cancer cells and cancer-associated fibroblasts (CAF) embedded in a collagen gel were introduced to the AUPPEG scaffold. After self-organization in to a 3D tumor, this model was evaluated by a long-term (> 40 days) exposure to a drug combination of MEK and HSP90 inhibitors. The drug-response results from this long-term in vitro model are compared with drug responses in an orthotopic LGSOC xenograft mouse model. RESULTS: The in vitro 3D scaffold LGSOC model mimics the growth ratio and spatial organization of the LGSOC. The AUPPEG scaffold approach allows to test new targeted treatments and monitor long-term drug responses. The results correlate with those of the orthotopic LGSOC xenograft mouse model. CONCLUSIONS: The mechanically-tunable scaffolds colonized by a three-dimensional LGSOC allow long-term drug evaluation and can be considered as a valid alternative to reduce, replace and refine animal models in drug discovery.

STE20 kinase TAOK3 regulates type 2 immunity and metabolism in obesity.

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.

Direct co-registration of [18F]FDG uptake and histopathology in surgically excised malignancies of the head and neck: a feasibility study.

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 [18F]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 [18F]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 [18F]FDG uptake in both benign and malignant tissue. CONCLUSION: We here describe a novel methodology enabling the direct co-registration of [18F]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.

Preclinical comparative study of [18F]AlF-PSMA-11 and [18F]PSMA-1007 in varying PSMA expressing tumors.

A wide variety of 18F-labeled PSMA-targeting PET radiotracers have been developed, including [18F]AlF-PSMA-11. As there is only limited data on the comparison with other 18F-labeled PSMA PET tracers, a comparative preclinical study between [18F]AlF-PSMA-11 and [18F]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 [18F]AlF-PSMA-11 and [18F]PSMA-1007. Ten additional mice bearing C4-2 xenografts were subjected to ex vivo biodistribution with either [18F]AlF-PSMA-11 (n = 5) or [18F]PSMA-1007 (n = 5). Absolute SUVmean and SUVmax values were significantly higher for [18F]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 [18F]AlF-PSMA-11 and [18F]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 [18F]AlF-PSMA-11 (p < 0.01). In healthy organs, [18F]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 [18F]PSMA-1007. Absolute tumor uptake was higher with [18F]PSMA-1007 in all tumors. Tumor-to-organ ratios did not differ significantly in high PSMA expressing tumors, but were higher for [18F]AlF-PSMA-11 in low PSMA expressing tumors. Furthermore, [18F]PSMA-1007 showed higher uptake in healthy organs.

Positron Emission Tomography-based Dose Painting Radiation Therapy in a Glioblastoma Rat Model using the Small Animal Radiation Research Platform.

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.

White Matter Integrity in a Rat Model of Epileptogenesis: Structural Connectomics and Fixel-Based Analysis.

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.

Impact of the molar activity and PSMA expression level on [18F]AlF-PSMA-11 uptake in prostate cancer.

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 (MAapp) of [18F]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 [18F]AlF-PSMA-11 with a medium MAapp (20.24 ± 3.22 MBq/nmol). SUVmean and SUVmax 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 MAapp, C4-2 and 22Rv1 xenograft bearing mice underwent additional [18F]AlF-PSMA-11 imaging with a high (211.2 ± 38.9 MBq/nmol) and/or low MAapp (1.92 ± 0.27 MBq/nmol). SUV values showed a significantly increasing trend with higher MAapp. Significant changes were found for SUVmean and SUVmax between the high versus low MAapp and medium versus low MAapp (both p < 0.05), but not between the high versus medium MAapp (p = 0.055 and 0.25, respectively). The effect of varying MAapp was more pronounced in low expressing tumors and PSMA expressing tissues (e.g. salivary glands and kidneys). Overall, administration of a high MAapp 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 MAapp could reduce radiation exposure to dose-limiting organs with only limited effect on radionuclide accumulation in the tumor.

Voxel-Based Analysis of [18F]-FDG Brain PET in Rats Using Data-Driven Normalization.

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 (CMRglc) 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.

Comparison of In Vivo and Ex Vivo Magnetic Resonance Imaging in a Rat Model for Glioblastoma-Associated Epilepsy.

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.

Cytosolic delivery of gadolinium via photoporation enables improved in vivo magnetic resonance imaging of cancer cells.

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 T1-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 T1-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.

Assessment of the effect of therapy in a rat model of glioblastoma using [18F]FDG and [18F]FCho PET compared to contrast-enhanced MRI.

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.

Can medical imaging identify the histopathological growth patterns of liver metastases?

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.

In vivo selection of the MDA-MB-231br/eGFP cancer cell line to obtain a clinically relevant rat model for triple negative breast cancer brain metastasis.

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.

Intra-individual dynamic comparison of 18F-PSMA-11 and 68Ga-PSMA-11 in LNCaP xenograft bearing mice.

Recently, a 18F-labeled derivative of the widely used 68Ga-PSMA-11 was developed for PET imaging of prostate cancer. Although 18F-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 18F-PSMA-11 or 68Ga-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 18F-PSMA-11 and 68Ga-PSMA-11 followed by dynamic acquisition of 2.5 h as well as ± 15 MBq 18F-FDG followed by static acquisition at 1 h post injection (p.i.). Uptake was evaluated by comparison of uptake parameters (SUVmean, SUVmax, TBRmean and TBRmax). Mice underwent ex vivo biodistribution where 18F-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 (Kd) of 18F-PSMA-11 and 68Ga-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 18F-PSMA-11 and 68Ga-PSMA-11, while no difference was found for 18F-FDG uptake (except for SUVmax). Tumor uptake of 18F-PSMA-11 showed a similar trend over time as 68Ga-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, TBRmean and TBRmax were significantly higher at the later timepoint, as well as the SUVmax of 68Ga-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 18F-PSMA-11 compared to 68Ga-PSMA-11. Delayed imaging with the latter may improve tumor visualization, while no additional benefits could be found for late 18F-PSMA-11 imaging. Ex vivo biodistribution demonstrated fast renal clearance of 18F-PSMA-11 as well as no significant increase in bone uptake.

The shrimp nephrocomplex serves as a major portal of pathogen entry and is involved in the molting process.

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.

Development of a Rat Model for Glioma-Related Epilepsy.

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.

Synergy between Intraperitoneal Aerosolization (PIPAC) and Cancer Nanomedicine: Cisplatin-Loaded Polyarginine-Hyaluronic Acid Nanocarriers Efficiently Eradicate Peritoneal Metastasis of Advanced Human Ovarian Cancer.

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.

Radiosynthesis, in vitro and preliminary biological evaluation of [18 F]2-amino-4-((2-((3-fluorobenzyl)oxy)benzyl)(2-((3-(fluoromethyl)benzyl)oxy)benzyl)amino)butanoic acid, a novel alanine serine cysteine transporter 2 inhibitor-based positron emission tomography tracer.

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 Ki 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.

Radiosynthesis, in vitro and preliminary in vivo evaluation of the novel glutamine derived PET tracers [18F]fluorophenylglutamine and [18F]fluorobiphenylglutamine.

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 [18F]fluorophenylglutamine and [18F]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 [3H]glutamine inhibition experiment in a human PC-3 and a rat F98 cell line. The imaging potential of [18F]fluorophenylglutamine and [18F]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) ([18F]fluorophenylglutamine) and 8.05 ± 3.25% (n = 5) ([18F]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 [18F]FBPG in the mice PC-3 xenograft and a moderate uptake of [18F]FPG in the PC-3 tumors. CONCLUSION: We investigated the imaging potential of two novel PET radiotracers [18F]FPG and [18F]FBPG. [18F]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 [18F]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-[18F]fluoroglutamine). [18F]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.