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.

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.

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.

Slc2a10 knock-out mice deficient in ascorbic acid synthesis recapitulate aspects of arterial tortuosity syndrome and display mitochondrial respiration defects.

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.

Dynamic functional connectivity and graph theory metrics in a rat model of temporal lobe epilepsy reveal a preference for brain states with a lower functional connectivity, segregation and integration.

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.

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.

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.

Improved Detection of Molecular Markers of Atherosclerotic Plaques Using Sub-Millimeter PET Imaging.

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 [18F]AlF with a high radiochemical yield (>75%) and radiochemical purity (>99%). Subsequently, [18F]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 [18F]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 ß-CUBE (MOLECUBES) PET scanner, demonstrating that preclinical detection of atherosclerotic lesions could be improved using the latest PET technology.

Alterations in the functional brain network in a rat model of epileptogenesis: A longitudinal resting state fMRI study.

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.

High Pressure Nebulization (PIPAC) Versus Injection for the Intraperitoneal Administration of mRNA Complexes.

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.

Magnetic resonance imaging of third molars in forensic age estimation: comparison of the Ghent and Graz protocols focusing on apical closure.

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 mm3) was more assessable than the Graz T1 3D FSE sequence (0.59 × 0.59 × 1 mm3). 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.

Regional alterations of cerebral [18F]FDG metabolism in the chronic unpredictable mild stress- and the repeated corticosterone depression model in rats.

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 [18F]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 [18F]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.

Haematopoietic prolyl hydroxylase-1 deficiency promotes M2 macrophage polarization and is both necessary and sufficient to protect against experimental colitis.

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 (Phd1f/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 Phd2f/+ Tie2:cre and Phd3f/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-1ß 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.

The VEGFR Inhibitor Cediranib Improves the Efficacy of Fractionated Radiotherapy in a Colorectal Cancer Xenograft Model.

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.

Vulnerable plaque detection and quantification with gold particle-enhanced computed tomography in atherosclerotic mouse models.

Recently, an apolipoprotein E-deficient (ApoE-/-) mouse model with a mutation (C1039G+/-) in the fibrillin-1 (Fbn1) gene (ApoE-/-Fbn1C1039G+/- mouse model) was developed showing vulnerable atherosclerotic plaques, prone to rupture, in contrast to the ApoE-/- mouse model, where mainly stable plaques are present. One indicator of plaque vulnerability is the level of macrophage infiltration. Therefore, this study aimed to measure and quantify in vivo the macrophage infiltration related to plaque development and progression. For this purpose, 5-weekly consecutive gold nanoparticle-enhanced micro-computed tomography (microCT) scans were acquired. Histology confirmed that the presence of contrast agent coincided with the presence of macrophages. Based on the microCT scans, regions of the artery wall with contrast agent present were calculated and visualized in three dimensions. From this information, the contrast-enhanced area and contrast-enhanced centerline length were calculated for the branches of the carotid bifurcation (common, external, and internal carotid arteries). Statistical analysis showed a more rapid development and a larger extent of plaques in the ApoE-/-Fbn1C1039G+/- compared to the ApoE-/- mice. Regional differences between the branches were also observable and quantifiable. We developed and applied a methodology based on gold particle-enhanced microCT to visualize the presence of macrophages in atherosclerotic plaques in vivo.

MRI-guided 3D conformal arc micro-irradiation of a F98 glioblastoma rat model using the Small Animal Radiation Research Platform (SARRP).

Current glioblastoma (GB) small animal models for cranial radiation therapy (RT) use simple single beam technologies, which differ from the advanced conformal image-guided radiation techniques used in clinical practice. This technological disparity presents a major disadvantage for the development of new therapeutic approaches. Hence, we established a F98 GB rat model using magnetic resonance imaging (MRI)-guided three-dimensional (3D)-conformal arc RT with the Small Animal Radiation Research Platform (SARRP). Ten Fischer rats were inoculated with F98 tumor cells. When the tumor reached a volume of approximately 27 mm(3) on T2-weighted MR images, the animals were randomized into a treatment group (n = 5) receiving RT and concomitant temozolomide, and a sham group (n = 5) receiving control injections. For the treated animals, contrast-enhanced T1-weighted MR images were acquired followed by a cone-beam computed tomography (CBCT) on the SARRP system. Both scans were co-registered; MRI was used to define the target whereas CBCT was used for calculating a dose plan (20 Gy, three non-coplanar arc beams, 3 × 3 mm collimator). Tumor volumes were evaluated on follow-up contrast-enhanced T1-weighted MR images. Verification of treatment accuracy with γH2AX immunohistochemical staining was performed. Tumors in the control animals showed rapid proliferation during follow-up, encompassing almost the entire right cerebral hemisphere at day 12-15. Treated animals showed no significant tumor growth from 2 to 9 days post RT. γH2AX results confirmed the accuracy of dose delivery. This model, which is quite similar to the approach in the clinic, is valid for combined RT and chemotherapy of GB in rats.

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.

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.

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.

Quantifying hemodynamic refractory bold effects in normal subjects at the single-subject level using an inverse logit fitting procedure.

PURPOSE: To evaluate whether hemodynamic refractory effects provoked by repeated visual stimulation can be detected and quantified at the single-subject level using a recently described hemodynamic response function (HRF) fitting algorithm. MATERIALS AND METHODS: Hemodynamic refractory effects were induced with an easily applicable functional MRI (fMRI) paradigm. A fitting method with inverse logit (IL) functions was applied to quantify net HRFs at the single-subject level with three interstimulus intervals (ISI; 1, 2, and 6 s). The model yielded amplitude, latencies, and width for each HRF. RESULTS: HRF fitting was possible in 44 of 51 healthy volunteers, with excellent goodness-of-fit (R(2) = 0.9745 ± 0.0241). Refractory effects were most pronounced for the 1-s ISI (P < 0.001) and had nearly disappeared for the 6-s ISI. CONCLUSION: Quantifying refractory effects in individuals was possible in 86.3% of normal subjects using the IL fitting algorithm. This setup may be suitable to explore such effects in individual patients.