Search

Supplementary Video 2 shows sonographic visualization of the axial position of the heart of a NOD/SCID mouse injected with 1000 ug K16ApoE intravenously.

Supplementary Video 4 shows a confocal time-lapse series, showing MDCK cells treated with 20 ug of K16ApoE, immediately after labeling with Calcein-AM (green), which indicates live cells, and Ethidium homodimer-1 (red), indicating dead cells.

Supplementary Video 1 shows sonographic visualization of the axial position of the heart of a NOD/SCID mouse injected with 9 mg/mL NaCl intravenously.

Supplementary Video 3 shows a confocal time-lapse series, showing a control sample of MDCK cells labeled with Calcein-AM (green), which indicates live cells, and Ethidium homodimer-1 (red), indicating dead cells.

Supplementary Video 6 shows a confocal time-lapse series, showing MDCK cells treated with 80 ug of K16ApoE, immediately after labeling with Calcein-AM (green), which indicates live cells, and Ethidium homodimer-1 (red), indicating dead cells.

Supplementary Video 5 shows a confocal time-lapse series, showing MDCK cells treated with 40 ug of K16ApoE, immediately after labeling with Calcein-AM (green), which indicates live cells, and Ethidium homodimer-1 (red), indicating dead cells.

Patients with melanoma have a high risk of developing brain metastasis, which is associated with a dismal prognosis. During early stages of metastasis development, the blood–brain barrier (BBB) is likely intact, which inhibits sufficient drug delivery into the metastatic lesions. We investigated the ability of the peptide, K16ApoE, to permeabilize the BBB for improved treatment with targeted therapies preclinically. Dynamic contrast enhanced MRI (DCE-MRI) was carried out on NOD/SCID mice to study the therapeutic window of peptide-mediated BBB permeabilization. Further, both in vivo and in vitro assays were used to determine K16ApoE toxicity and to obtain mechanistic insight into its action on the BBB. The therapeutic impact of K16ApoE on metastases was evaluated combined with the mitogen-activated protein kinase pathway inhibitor dabrafenib, targeting BRAF mutated melanoma cells, which is otherwise known not to cross the intact BBB. Our results from the DCE-MRI experiments showed effective K16ApoE-mediated BBB permeabilization lasting for up to 1 hour. Mechanistic studies showed a dose-dependent effect of K16ApoE caused by induction of endocytosis. At concentrations above IC50, the peptide additionally showed nonspecific disturbances on plasma membranes. Combined treatment with K16ApoE and dabrafenib reduced the brain metastatic burden in mice and increased animal survival, and PET/CT showed that the peptide also facilitated the delivery of compounds with molecular weights as large as 150 kDa into the brain. To conclude, we demonstrate a transient permeabilization of the BBB, caused by K16ApoE, that facilitates enhanced drug delivery into the brain. This improves the efficacy of drugs that otherwise do not cross the intact BBB.

Supplementary Materials shows supplementary methods, Supplementary Figures S1-S9 with legends and Supplementary Table 1. Supplementary Table 1: Mean Ve, Kep and AUC after DCE-MRI; Supplementary Figure S1: In vivo study design; Supplementary Figure S2: Blood smear from mice subjected to 1,000 µg K16ApoE; Supplementary Figure S3: In vitro cell viability: Supplementary Figure S4: Mechanistic studies of the in vitro effects of K16ApoE; Supplementary Figure S5: In vitro BBB model; Supplementary Figure S6: Biodistribution of 125I-K16ApoE; Supplementary Figure S7: Histopathological evaluation of tissue samples after long- and short-term treatment of NOD/SCID mice with the peptide; Supplementary Figure S8: In vivo treatment control experiment; Supplementary Figure S9: Mass spectrometry of treated and untreated brain tissue

An orthotopically xenografted mouse GL26 glioma model (Ccr2RFP/wt-Cx3cr1GFP/wt) was used to evaluate the effect of transient, focal opening of the Blood Brain Barrier (BBB) on the composition of tumor-associated macrophages and microglia (TAMs). Opening of the BBB was induced by Magnetic Resonance Imaging (MRI)-guided focused ultrasound (MRgFUS) combined with systemically-administered microbubbles. CX3CR1-GFP cells and CCR2-RFP cells in brain tumors and in peritumoral tissue were quantified utilizing cell counting in fluroscent, microscopic images. Tumors in animals treated with a single session of MRgFUS did not show significant changes in CX3CR1-GFP or CCR2-RFP cell numbers when compared to tumors in animals not receiving FUS. However, tumors that received two or three sessions of MRgFUS showed significantly increased amounts of both CX3CR1-GFP and CCR2-RFP cells.The effect of MRgFUS on immune cell compositon of tumors and the brain parenchyma was also characterized and quantified utilizing flow cytometry. Glioma implantation resulted in increased amounts of monocytes, blood-derived macrophages, and microglia-derived macrophages in the brain parenchyma. Tumors administered MRgFUS showed increased numbers of monocytes and blood-derived macrophages. In addition, MRgFUS-treated tumors exhibited more CD80+CD206- cells in monocytes, blood-derived macrophages, microglia, and microglia-derived macrophages, and fewer CD80-CD206+ cells in monocytes and microglia. This signature is indicative of a shift toward a more pro-inflammatory polarization. In summary, transient, focal opening of the BBB using MRgFUS combined with microbubbles can activate the homing and differentiation of monocytes, and induce a shift towards a more proinflammatory status of the immune environment in glioblastoma.

BACKGROUND Extensive tumor cell invasion within the brain represents a major problem for effective treatment of glioblastomas (GBMs). The invasive processes can be divided into three types: Collective cell invasion, perivascular infiltration, and single-cell invasion into the brain parenchyma. GBM cells can form synapses with neural cells pointing at an extensive communication network between brain and GBM cells which can be mediated via the metabolites Glutamine and Glutamate both needed for GBM cell proliferation. In this context, it has been shown in preclinical models that Perampanel, an antiepileptic agent, functioning as non-competitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor antagonist, has an inhibitory effect on GBM growth. To delineate how Perampanel affects GBM invasion, we utilised a highly characterized 3D GBM-brain organoid invasion model where single-cell invasion was studied in real-time following Perampanel treatment. METHODS A brain coculture model, consisting of rat brain organoids expressing various markers of the human adult brain, where confronted with GFP-labelled tumor cells. By using time-lapse confocal microscopy, we quantified single-cell invasion patterns and speed of invasion using two glioma stem cell models (BG5 and BG7). RESULTS Perampanel treatment significantly reduces tumor cell invasion into the brain organoids with the strongest effect seen in the most invasive GBM (BG5). The single-tumor cell invasion ratio was reduced by 72 % compared to the control (p= 0.0033). In contrast, collective cell invasion was reduced by 19 % (p= 0.028). Statistical analysis was performed using an unpaired sample t-test. CONCLUSION The AMPA glutamate receptor antagonist Perampanel significantly inhibits GBM invasion, suggesting an important role of the glutamate-glutamine cycle between the GBM cells and neurons in the invasion process. Moreover, this communication and exchange of metabolites seem to be more prominent where single GBM cells invade into the brain parenchyma compared to areas where collective invasion take place.

Patients with melanoma have a high risk of developing brain metastasis, which is associated with a dismal prognosis. During early stages of metastasis development, the blood–brain barrier (BBB) is likely intact, which inhibits sufficient drug delivery into the metastatic lesions. We investigated the ability of the peptide, K16ApoE, to permeabilize the BBB for improved treatment with targeted therapies preclinically. Dynamic contrast enhanced MRI (DCE-MRI) was carried out on NOD/SCID mice to study the therapeutic window of peptide-mediated BBB permeabilization. Further, both in vivo and in vitro assays were used to determine K16ApoE toxicity and to obtain mechanistic insight into its action on the BBB. The therapeutic impact of K16ApoE on metastases was evaluated combined with the mitogen-activated protein kinase pathway inhibitor dabrafenib, targeting BRAF mutated melanoma cells, which is otherwise known not to cross the intact BBB. Our results from the DCE-MRI experiments showed effective K16ApoE-mediated BBB permeabilization lasting for up to 1 hour. Mechanistic studies showed a dose-dependent effect of K16ApoE caused by induction of endocytosis. At concentrations above IC50, the peptide additionally showed nonspecific disturbances on plasma membranes. Combined treatment with K16ApoE and dabrafenib reduced the brain metastatic burden in mice and increased animal survival, and PET/CT showed that the peptide also facilitated the delivery of compounds with molecular weights as large as 150 kDa into the brain. To conclude, we demonstrate a transient permeabilization of the BBB, caused by K16ApoE, that facilitates enhanced drug delivery into the brain. This improves the efficacy of drugs that otherwise do not cross the intact BBB.

Purpose The Tofts and the extended Tofts models are the pharmacokinetic models commonly used in dynamic contrast-enhanced MRI (DCE-MRI) perfusion analysis, although they do not provide two important biological markers, namely, the plasma flow and the permeability-surface area product. Estimates of such markers are possible using advanced pharmacokinetic models describing the vascular distribution phase, such as the tissue homogeneity model. However, the disadvantage of the advanced models lies in biased and uncertain estimates, especially when the estimates are computed voxelwise. The goal of this work is to improve the reliability of the estimates by including information from neighboring voxels. Theory and methods Information from the neighboring voxels is incorporated in the estimation process through spatial regularization in the form of total variation. The spatial regularization is applied on five maps of perfusion parameters estimated using the tissue homogeneity model. Since the total variation is not differentiable, two proximal techniques of convex optimization are used to solve the problem numerically. Results The proposed algorithm helps to reduce noise in the estimated perfusion-parameter maps together with improving accuracy of the estimates. These conclusions are proved using a numerical phantom. In addition, experiments on real data show improved spatial consistency and readability of perfusion maps without considerable lowering of the quality of fit. Conclusion The reliability of the DCE-MRI perfusion analysis using the tissue homogeneity model can be improved by employing spatial regularization. The proposed utilization of modern optimization techniques implies only slightly higher computational costs compared to the standard approach without spatial regularization.

Brain metastasis (BM) is a major cause of cancer patient morbidity. Clinical magnetic resonance imaging (MRI) and positron emission tomography (PET) represent important resources to assess tumor progression and treatment responses. In preclinical research, anatomical MRI and to some extent functional MRI have frequently been used to assess tumor progression. In contrast, PET has only to a limited extent been used in animal BM research. A considerable culprit is that results from most preclinical studies have shown little impact on the implementation of new treatment strategies in the clinic. This emphasizes the need for the development of robust, high-quality preclinical imaging strategies with potential for clinical translation. This review focuses on advanced preclinical MRI and PET imaging methods for BM, describing their applications in the context of what has been done in the clinic. The strengths and shortcomings of each technology are presented, and recommendations for future directions in the development of the individual imaging modalities are suggested. Finally, we highlight recent developments in quantitative MRI and PET, the use of radiomics and multimodal imaging, and the need for a standardization of imaging technologies and protocols between preclinical centers.

Summary Tumor organoids and patient-derived orthotopic xenografts (PDOXs) are some of the most valuable pre-clinical tools in cancer research. In this protocol, we describe efficient derivation of organoids and PDOX models from glioma patient tumors. We provide detailed steps for organoid culture, intracranial implantation, and detection of tumors in the brain. We further present technical adjustments for standardized functional assays and drug testing. For complete details on the use and execution of this protocol, please refer to Golebiewska et al. (2020)., Graphical abstract, Highlights • Organoids can be generated from diverse glioma patient tumors • High-grade glioma organoids give rise to patient-derived orthotopic xenografts • Serial transplantation in vivo allows for consistent expansion of human tumor cells • The adapted protocol for reconstitution of uniform organoids for functional assays, Tumor organoids and patient-derived orthotopic xenografts (PDOXs) are some of the most valuable pre-clinical tools in cancer research. In this protocol, we describe efficient derivation of organoids and PDOX models from glioma patient tumors. We provide detailed steps for organoid culture, intracranial implantation, and detection of tumors in the brain. We further present technical adjustments for standardized functional assays and drug testing.

Surgery can be highly effective for treating certain cases of drug resistant epilepsy. The current study tested a novel, non-invasive, surgical strategy for treating seizures in a rat model of temporal lobe epilepsy. The surgical approach uses magnetic resonance-guided, low-intensity focused ultrasound (MRgFUS) in combination with intravenous microbubbles to open the blood-brain barrier (BBB) in a transient and focal manner. During the period of BBB opening, a systemically administered neurotoxin (Quinolinic Acid: QA) that is normally impermeable to the BBB gains access to a targeted area in the brain, destroying neurons where the BBB has been opened. This strategy is termed Precise Intracerebral Non-invasive Guided Surgery (PING). Spontaneous recurrent seizures induced by pilocarpine were monitored behaviorally prior to and after PING or under control conditions. Seizure frequency in untreated animals or animals treated with MRgFUS without QA exhibited expected seizure rate fluctuations frequencies between the monitoring periods. In contrast, animals treated with PING targeting the intermediate-temporal aspect of the hippocampus exhibited substantial reductions in seizure frequency, with convulsive seizures being eliminated entirely in two animals. These findings suggest that PING could provide a useful alternative to invasive surgical interventions for treating drug resistant epilepsy, and perhaps for treating other neurological disorders in which aberrant neural circuitries play a role.

Patient-derived cancer models are essential tools for studying tumor biology and preclinical interventions. Here, we show that glioma patient-derived orthotopic xenografts (PDOXs) enable long-term propagation of patient tumors and represent clinically relevant patient avatars. We created a large collection of PDOXs from primary and recurrent gliomas with and without mutations in IDH1, which retained histopathological, genetic, epigenetic and transcriptomic features of patient tumors with no mouse-specific clonal evolution. Longitudinal PDOX models recapitulate the limited genetic evolution of gliomas observed in patient tumors following treatment. PDOX-derived standardized tumor organoid cultures enabled assessment of drug responses, which were validated in mice. PDOXs showed clinically relevant responses to Temozolomide and to targeted treatments such as EGFR and CDK4/6 inhibitors in (epi)genetically defined groups, according to MGMT promoter and EGFR/CDK status respectively. Dianhydrogalactitol, a bifunctional alkylating agent, showed promising potential against glioblastoma. Our study underlines the clinical relevance of glioma PDOX models for translational research and personalized treatment studies.

BACKGROUND: High-grade gliomas likely remodel the metabolic machinery to meet the increased demands for amino acids and nucleotides during rapid cell proliferation. Glycine, a non-essential amino acid and intermediate of nucleotide biosynthesis, may increase with proliferation. Non-invasive measurement of glycine by magnetic resonance spectroscopy (MRS) was evaluated as an imaging biomarker for assessment of tumor aggressiveness. METHODS: We measured glycine, 2-hydroxyglutarate (2HG), and other tumor-related metabolites in 35 glioma patients using an MRS sequence tailored for co-detection of glycine and 2HG in gadolinium-enhancing and non-enhancing tumor regions on 3T MRI. Glycine and 2HG concentrations as measured by MRS were correlated with tumor cell proliferation (MIB-1 labeling index), expression of mitochondrial serine hydroxymethyltransferase (SHMT2), and glycine decarboxylase (GLDC) enzymes, and patient overall survival. RESULTS: Elevated glycine was strongly associated with presence of gadolinium enhancement, indicating more rapidly proliferative disease. Glycine concentration was positively correlated with MIB-1, and levels higher than 2.5 mM showed significant association with shorter patient survival, irrespective of isocitrate dehydrogenase status. Concentration of 2HG did not correlate with MIB-1 index. A high glycine/2HG concentration ratio, >2.5, was strongly associated with shorter survival (P

Patient-based cancer models are essential tools for studying tumor biology and for the assessment of drug responses in a translational context. We report the establishment a large cohort of unique organoids and patient-derived orthotopic xenografts (PDOX) of various glioma subtypes, including gliomas with mutations in IDH1, and paired longitudinal PDOX from primary and recurrent tumors of the same patient. We show that glioma PDOXs enable long-term propagation of patient tumors and represent clinically relevant patient avatars that retain histopathological, genetic, epigenetic, and transcriptomic features of parental tumors. We find no evidence of mouse-specific clonal evolution in glioma PDOXs. Our cohort captures individual molecular genotypes for precision medicine including mutations in IDH1, ATRX, TP53, MDM2/4, amplification of EGFR, PDGFRA, MET, CDK4/6, MDM2/4, and deletion of CDKN2A/B, PTCH, and PTEN. Matched longitudinal PDOX recapitulate the limited genetic evolution of gliomas observed in patients following treatment. At the histological level, we observe increased vascularization in the rat host as compared to mice. PDOX-derived standardized glioma organoids are amenable to high-throughput drug screens that can be validated in mice. We show clinically relevant responses to temozolomide (TMZ) and to targeted treatments, such as EGFR and CDK4/6 inhibitors in (epi)genetically defined subgroups, according to MGMT promoter and EGFR/CDK status, respectively. Dianhydrogalactitol (VAL-083), a promising bifunctional alkylating agent in the current clinical trial, displayed high therapeutic efficacy, and was able to overcome TMZ resistance in glioblastoma. Our work underscores the clinical relevance of glioma organoids and PDOX models for translational research and personalized treatment studies and represents a unique publicly available resource for precision oncology.

BACKGROUND Extensive tumor cell invasion within the brain represents a major problem for effective treatment of glioblastomas (GBMs). The invasive processes can be divided into three types: Collective cell invasion, perivascular infiltration and single-cell invasion into the brain parenchyma. It has recently been shown that GBM cells have the ability to form synapses with neural cells pointing at an extensive communication network between brain cells GBM cells. This communication network can be mediated via the metabolites glutamine and glutamate both needed for GBM cell proliferation. In this context, it has been shown in preclinical models that Perampanel, an antiepileptic agent, functioning as non-competitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor antagonist, has an inhibitory effect on GBM growth. In order to delineate how Perampanel affects GBM invasion, we here utilised a highly characterized 3D GBM-brain organoid invasion model where single-cell invasion was studied in real-time following Perampanel treatment. MATERIAL AND METHODS A brain coculture model, consisting of rat brain organoids expressing various markers of the human adult brain, where confronted with GFP-labelled tumor cells. By using time-lapse confocal microscopy, we quantified single-cell invasion patterns and speed of invasion using two glioma stem cell models (GSCs; BG5 and BG7). RESULTS Perampanel treatment significantly reduces tumor cell invasion into the brain organoids with the strongest effect seen in the most invasive GBM (BG5). Here, the single-tumor cell invasion ratio was reduced by 72 % compared to the control group (p=0.0033). In contrast, collective cell invasion was reduced by 19 % (p=0.028). Statistical analysis was performed using an unpaired sample t-test. CONCLUSION The AMPA glutamate receptor antagonist Perampanel significantly inhibits GBM invasion, suggesting an important role of the glutamate-glutamine cycle between the GBM cells and neurons in the invasion process. Moreover, this communication and exchange of metabolites seems to be more prominent where single GBM cells invade into the brain parenchyma compared to areas where collective invasion take place.

Neo-angiogenesis represents an important factor for the delivery of oxygen and nutrients to a growing tumour, and is considered to be one of the main pathodiagnostic features of glioblastomas (GBM). Anti-angiogenic therapy by vascular endothelial growth factor (VEGF) blocking agents has been shown to lead to morphological vascular normalisation resulting in a reduction of contrast enhancement as seen by magnetic resonance imaging (MRI). Yet the functional consequences of this normalisation and its potential for improved delivery of cytotoxic agents to the tumour are not known. The presented study aimed at determining the early physiologic changes following bevacizumab treatment. A time series of perfusion MRI and hypoxia positron emission tomography (PET) scans were acquired during the first week of treatment, in two human GBM xenograft models treated with either high or low doses of bevacizumab. We show that vascular morphology was normalised over the time period investigated, but vascular function was not improved, resulting in poor tumoural blood flow and increased hypoxia.

Heterozygous mutations in NADP‐dependent isocitrate dehydrogenases (IDH) define the large majority of diffuse gliomas and are associated with hypermethylation of DNA and chromatin. The metabolic dysregulations imposed by these mutations, whether dependent or not on the oncometabolite D‐2‐hydroxyglutarate (D2HG), are less well understood. Here, we applied mass spectrometry imaging on intracranial patient‐derived xenografts of IDH‐mutant versus IDH wild‐type glioma to profile the distribution of metabolites at high anatomical resolution in situ . This approach was complemented by in vivo tracing of labeled nutrients followed by liquid chromatography–mass spectrometry (LC‐MS) analysis. Selected metabolites were verified on clinical specimen. Our data identify remarkable differences in the phospholipid composition of gliomas harboring the IDH1 mutation. Moreover, we show that these tumors are characterized by reduced glucose turnover and a lower energy potential, correlating with their reduced aggressivity. Despite these differences, our data also show that D2HG overproduction does not result in a global aberration of the central carbon metabolism, indicating strong adaptive mechanisms at hand. Intriguingly, D2HG shows no quantitatively important glucose‐derived label in IDH‐mutant tumors, which suggests that the synthesis of this oncometabolite may rely on alternative carbon sources. Despite a reduction in NADPH, glutathione levels are maintained. We found that genes coding for key enzymes in de novo glutathione synthesis are highly expressed in IDH‐mutant gliomas and the expression of cystathionine‐β‐synthase ( CBS ) correlates with patient survival in the oligodendroglial subtype. This study provides a detailed and clinically relevant insight into the in vivo metabolism of IDH1‐mutant gliomas and points to novel metabolic vulnerabilities in these tumors.

Background: The efficiency of traditional anthropometric measurements such as body mass index (BMI) or waist circumference (Waist C) used to replace biomedical imaging for assessing visceral adipose tissue (VAT) is still highly controversial in youth. Hypothesis and Objectives: We evaluated the most accurate model predicting VAT in overweight/obese youth, using various anthropometric measurements and their correlation with different body fat compartments, especially by testing, for the first time in youth, the hypothesis that subtracting the anthropometric measurement the most highly correlated with subcutaneous abdominal adipose tissue (SAAT) and less correlated possible with VAT from an anthropometric abdominal measurement highly correlated with visceral and total abdominal adipose tissue (TAAT), predicts VAT with higher accuracy. Subjects and Methods: VAT and SAAT data resulted from magnetic resonance imaging (MRI) analysis performed on 181 boys and girls (7-17 y) from Diabetes & Endocrinology Care Paediatrics Clinic in Luxembourg. Height, weight, abdominal diameters, waist, hip, and thigh circumferences were measured with a view to developing the anthropometric VAT predictive algorithms. Results: In girls, subtracting proximal thigh circumference (Proximal Thigh C), the most closely correlated anthropometric measurement with SAAT, from Waist C, the most closely correlated anthropometric measurement with VAT was instrumental in improving VAT prediction, in comparison with the most accurate single VAT anthropometric surrogate. VAT =1:111 × Waist C – 0:675 × Proximal Thigh C + 0:26 age – 46:761 (R =55:7%, P < 10 ) Residual analysis showed a negligible estimation error (5 cm ). In boys, Waist C was the best VAT predictor. Conclusions: Subtraction of abdominal subcutaneous fat is important to predict VAT in overweight/obese girls. Girls 2 −4 2

Individuals with brain metastasis usually await a poor prognosis. Even with the ongoing development of novel targeted therapies, a major impediment in the treatment of brain metastasis is delivering of drugs across the blood-brain/tumor barrier. Different strategies to accomplish temporary opening of the heterogenous blood-brain barrier have been described. Here, we report on a synthetic peptide, K16ApoE, which is able to open the blood-brain barrier for improved drug-delivery in a non-permanent manner. We carried out extensive dynamic contrast enhanced magnetic resonance imaging experiments to study the ability of the peptide to open the blood-brain barrier in healthy mice. We further performed dynamic positron emission/computer tomography, with F18-labeled albumin and IgG to validate an open blood-brain barrier after co-injecting with K16ApoE. Further, we studied the effects of the peptide on cells and tissue in vitro and in vivo. The biodistribution of the peptide was studied in healthy mice by labeling I-125 to the imidazole ring of histidine in K16ApoE and tracking the peptide in plasma and tissue. We also conducted a treatment study on BRAF(V600E)mutant brain metastases, where the treatment groups received K16ApoE before a BRAF inhibitor was administered, BRAF inhibitor only, K16ApoE only or vehicle. We found a peptide-induced therapeutic window at approximately 30 minutes using dynamic contrast enhanced magnetic resonance imaging. The dynamic positron emission/computer tomography showed cerebral uptake of albumin (∼67 kDa) and IgG (∼150 kDa) in healthy mice with a presumed intact blood-brain barrier. Further, we identified lysis of cells treated with K16ApoE in addition to in part endocytosis-mediated peptide uptake. In vitro blood-brain barrier modeling demonstrated endothelial and astrocyte cell layer regrowth within approximately three and 15 hours, respectively, after treatment with the peptide. We observed a significant treatment effect of co-injecting the peptide with a BRAF inhibitor on BRAF mutated brain metastases.

Glioblastoma (GBM) is a typically lethal type of brain tumor with a median survival of 15 months postdiagnosis. This negative prognosis prompted the exploration of alternative treatment options. In particular, the reliance of GBM on angiogenesis triggered the development of anti‐VEGF (vascular endothelial growth factor) blocking antibodies such as bevacizumab. Although its application in human GBM only increased progression‐free periods but did not improve overall survival, physicians and researchers still utilize this treatment option due to the lack of adequate alternatives. In an attempt to improve the efficacy of anti‐VEGF treatment, we explored the role of the egfl7 gene in malignant glioma. We found that the encoded extracellular matrix protein epidermal growth factor‐like protein 7 (EGFL7) was secreted by glioma blood vessels but not glioma cells themselves, while no major role could be assigned to the parasitic miRNAs miR‐126/126*. EGFL7 expression promoted glioma growth in experimental glioma models in vivo and stimulated tumor vascularization. Mechanistically, this was mediated by an upregulation of integrin α 5 β 1 on the cellular surface of endothelial cells, which enhanced fibronectin‐induced angiogenic sprouting. Glioma blood vessels that formed in vivo were more mature as determined by pericyte and smooth muscle cell coverage. Furthermore, these vessels were less leaky as measured by magnetic resonance imaging of extravasating contrast agent. EGFL7‐inhibition using a specific blocking antibody reduced the vascularization of experimental gliomas and increased the life span of treated animals, in particular in combination with anti‐VEGF and the chemotherapeutic agent temozolomide. Data allow for the conclusion that this combinatorial regimen may serve as a novel treatment option for GBM.

This paper introduces new variational formulation for reconstruction from subsampled dynamic contrast-enhanced DCE-MRI data, that combines a data-driven approach using estimated temporal basis and total variation regularization (PCA TV). We also experimentally compares the performance of such model with two other state-of-the-art formulations. One models the shape of perfusion curves in time as a sum of a curve belonging to a low-dimensional space and a function sparse in a suitable domain (L + S model). The other possibility is to regularize both spatial and time domains (ICTGV). We are dealing with the specific situation of the DCE-MRI acquisition with a 9.4T small animal scanner, working with noisier signals than human scanners and with a smaller number of coil elements that can be used for parallel acquisition and small voxels. Evaluation of the selected methods is done through subsampled reconstruction of radially-sampled DCE-MRI data. Our analysis shows that compressed sensed MRI in the form of regularization can be used to increase the temporal resolution of acquisition while keeping a sufficient signal-to-noise ratio.

L-Glutamine (Gln) functions physiologically to balance the carbon and nitrogen requirements of tissues. It has been proposed that in cancer cells undergoing aerobic glycolysis, accelerated anabolism is sustained by Gln-derived carbons, which replenish the tricarboxylic acid (TCA) cycle (anaplerosis). However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation. Moreover, Gln-starved cells are not rescued by TCA cycle replenishment. Instead, the conversion of Glu to Gln by glutamine synthetase (GS; cataplerosis) confers Gln prototrophy, and fuels de novo purine biosynthesis. In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons. Finally, the Gln required for the growth of GBM tumours is contributed only marginally by the circulation, and is mainly either autonomously synthesized by GS-positive glioma cells, or supplied by astrocytes.

Background Childhood obesity is associated with early cardiometabolic risk (CMR), increased risk of adulthood obesity, and worse health outcomes. Leg fat mass (LFM) is protective beyond total fat mass (TFM) in adults. However, the limited evidence in children remains controversial. Objective We investigated the relationship between LFM and CMR factors in youth. Subjects A total of 203 overweight/obese children, 7–17-yr-old, followed in the Pediatric Clinic, Luxembourg. Methods TFM and LFM by dual energy x-ray absorptiometry and a detailed set of CMR markers were analyzed. Results After TFM, age, sex, body mass index (BMI) Z-score, sexual maturity status, and physical activity adjustments, negative significant partial correlations were shown between LFM and homeostasis model assessment of insulin resistance (HOMA) (variance explained: 6.05% by LFM*; 7.18% by TFM**), fasting insulin (variance explained: 5.71% by LFM*; 6.97% by TFM**), triglycerides (variance explained: 3.96% by LFM*; 2.76% by TFM*), systolic blood pressure (variance explained: 2.68% by LFM*; 4.33% by TFM*), C-reactive protein (variance explained: 2.31% by LFM*; 4.28% by TFM*), and resistin (variance explained: 2.16% by LFM*; 3.57% by TFM*). Significant positive partial correlations were observed between LFM and high-density lipoprotein (HDL) cholesterol (variance explained: 4.16% by LFM*) and adiponectin (variance explained: 3.09% by LFM*) (*p-value

BACKGROUND: Melanoma patients have a high risk of developing brain metastases, which is associated with a poor prognosis. The blood-brain barrier (BBB) inhibits sufficient drug delivery into metastatic lesions. We investigated the ability of a synthetic peptide (K16ApoE) to permeabilize the BBB for more effective drug treatment. METHODS: DCE-MRI was performed to study the therapeutic window of BBB opening facilitated by K16ApoE. In vivoand in vitroassays were used to determine K16ApoE toxicity and also to obtain mechanistic insight into its action on the BBB. The therapeutic impact of K16ApoE on melanoma metastases was determined together with dabrafenib, which is otherwise known not to cross an intact BBB. RESULTS: DCE-MRI exhibited an effective K16ApoE-mediated BBB opening for up to 1h. Mechanistic studies displayed a dose-dependent effect of K16ApoE caused by induction of endocytosis. At higher concentrations, the peptide also showed unspecific disturbances on plasma membranes. Combined treatment with K16ApoE and dabrafenib reduced the brain metastatic burden in mice compared to dabrafenib. We also showed by PET/CT that the peptide facilitated the delivery of compounds up to 150 kDa into the brain. CONCLUSIONS: We demonstrate a transient opening of the BBB, caused by K16ApoE, that facilitates improved drug-delivery into the brain. This improves the efficacy of drugs that otherwise do not cross the intact BBB.

Background Deregulated growth factor signaling is a major driving force in the initiation and progression of glioblastoma. The tumor suppressor and stem cell marker Lrig1 is a negative regulator of the epidermal growth factor receptor (EGFR) family. Here, we addressed the therapeutic potential of the soluble form of Lrig1 (sLrig1) in glioblastoma treatment and the mechanism of sLrig1-induced growth inhibition. Methods With use of encapsulated cells, recombinant sLrig1 was locally delivered in orthotopic glioblastoma xenografts generated from freshly isolated patient tumors. Tumor growth and mouse survival were evaluated. The efficacy of sLrig1 and the affected downstream signaling was studied in vitro and in vivo in glioma cells displaying variable expression of wild-type and/or a constitutively active EGFR mutant (EGFRvIII). Results Continuous interstitial delivery of sLrig1 in genetically diverse patient-derived glioma xenografts led to strong tumor growth inhibition. Glioma cell proliferation in vitro and tumor growth in vivo were potently inhibited by sLrig1, irrespective of EGFR expression levels. Of importance, tumor growth was also suppressed in EGFRvIII-driven glioma. sLrig1 induced cell cycle arrest without changing total receptor level or phosphorylation. Affected downstream effectors included MAP kinase but not AKT signaling. Of importance, local delivery of sLrig1 into established tumors led to a 32% survival advantage in treated mice. Conclusions To our knowledge, this is the first report demonstrating that sLrig1 is a potent inhibitor of glioblastoma growth in clinically relevant experimental glioma models and that this effect is largely independent of EGFR status. The potent anti-tumor effect of sLrig1, in combination with cell encapsulation technology for in situ delivery, holds promise for future treatment of glioblastoma.

Angiogenesis is regarded as a hallmark of cancer progression and it has been postulated that solid tumor growth depends on angiogenesis. At present, however, it is clear that tumor cell invasion can occur without angiogenesis, a phenomenon that is particularly evident by the infiltrative growth of malignant brain tumors, such as glioblastomas (GBMs). In these tumors, amplification or overexpression of wild-type (wt) or truncated and constitutively activated epidermal growth factor receptor (EGFR) are regarded as important events in GBM development, where the complex downstream signaling events have been implicated in tumor cell invasion, angiogenesis and proliferation. Here, we show that amplification and in particular activation of wild-type EGFR represents an underlying mechanism for non-angiogenic, invasive tumor growth. Using a clinically relevant human GBM xenograft model, we show that tumor cells with EGFR gene amplification and activation diffusely infiltrate normal brain tissue independent of angiogenesis and that transient inhibition of EGFR activity by cetuximab inhibits the invasive tumor growth. Moreover, stable, long-term expression of a dominant-negative EGFR leads to a mesenchymal to epithelial-like transition and induction of angiogenic tumor growth. Analysis of human GBM biopsies confirmed that EGFR activation correlated with invasive/non-angiogenic tumor growth. In conclusion, our results indicate that activation of wild-type EGFR promotes invasion and glioblastoma development independent of angiogenesis, whereas loss of its activity results in angiogenic tumor growth. Electronic supplementary material The online version of this article (doi:10.1007/s00401-013-1101-1) contains supplementary material, which is available to authorized users.

Recent results from 2 double-blind, placebo-controlled phase III trials (RTOG 0825) and (AVAglio) for first-line treatment of glioblastoma patients with the VEGF antibody bevacizumab, showed similar results, related to overall and progression-free survival. The RTOG 0825 trial indicated, opposed to the AVAglio trial, that patients treated with bevacizumab showed a decline in global neurocognitive function compared to untreated patients, -a decline that was most obvious after prolonged treatment. At present, there is a considerably controversy related to these observations. In the present work we point at the possibility that bevacizumab treatment of the normal brain can reduce synaptic plasticity in the hippocampus. We believe that such a phenomenon may partly explain the reduced cognitive function observed in patients in the RTOG 0825 trial. Since the same effects were not clearly defined in the AVAglio trial, further studies on putative neurocognitive effects after bevacizumab treatment are warranted.

Patients with brain metastases usually have a poor prognosis. Regardless of the constant progress in novel drug establishments, a crucial obstacle is the delivery of drugs across the blood brain barrier (BBB) and into the metastatic neoplasms. Different study designs to achieve temporary BBB opening have previously been studied. Here, we describe a synthetic peptide termed K16ApoE, which is able to transiently open the BBB for drug-delivery into experimental brain metastases.We conducted a systemic study of the ability of the peptide to open the BBB by dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) in nonobese diabetic/severe combined (nod/scid) mice. Further, we studied the mechanisms of the peptide using flow cytometry, electron microscopy and dynamic positron emission tomography (PET). The biodistribution of the peptide was studied in blood plasma and organs using I-125 labeled K16ApoE. Finally, a treatment study using the peptide in combination with the B-RAF inhibitor dabrafenib was conducted.An opening of the BBB for up to 60 mins was clearly demonstrated by DCE-MRI. Microscopy showed that the peptide disrupted brain endothelial cell monolayers by reducing the barrier properties of the cells. The treatment study demonstrated that the group of animals receiving K16ApoE followed by dabrafenib had smaller tumor volumes than the other two animal groups.In conclusion, the synthetic K16ApoE peptide in combination with dabrafenib decreased the number of experimental brain metastases. Thus, the current strategy could have the potential to improve the treatment of patients with brain metastatic disease. Citation Format: Synnøve Nymark Aasen, Heidi Espedal, Christopher Florian Holte, Olivier Keunen, Tine Veronica Karlsen, Olav Tenstad, Habib Baghirov, Dag Erlend Olberg, Rolf Bjerkvig, Gobinda Sarkar, Frits Thorsen. The good drug, the bad barrier and the handy peptide: Improved treatment of experimental melanoma brain metastases using a synthetic peptide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-314.

The histopathological and molecular heterogeneity of glioblastomas represents a major obstacle for effective therapies. Glioblastomas do not develop autonomously, but evolve in a unique environment that adapts to the growing tumour mass and contributes to the malignancy of these neoplasms. Here, we show that patient-derived glioblastoma xenografts generated in the mouse brain from organotypic spheroids reproducibly give rise to three different histological phenotypes: (i) a highly invasive phenotype with an apparent normal brain vasculature, (ii) a highly angiogenic phenotype displaying microvascular proliferation and necrosis and (iii) an intermediate phenotype combining features of invasion and vessel abnormalities. These phenotypic differences were visible during early phases of tumour development suggesting an early instructive role of tumour cells on the brain parenchyma. Conversely, we found that tumour-instructed stromal cells differentially influenced tumour cell proliferation and migration in vitro, indicating a reciprocal crosstalk between neoplastic and non-neoplastic cells. We did not detect any transdifferentiation of tumour cells into endothelial cells. Cell type-specific transcriptomic analysis of tumour and endothelial cells revealed a strong phenotype-specific molecular conversion between the two cell types, suggesting co-evolution of tumour and endothelial cells. Integrative bioinformatic analysis confirmed the reciprocal crosstalk between tumour and microenvironment and suggested a key role for TGFβ1 and extracellular matrix proteins as major interaction modules that shape glioblastoma progression. These data provide novel insight into tumour-host interactions and identify novel stroma-specific targets that may play a role in combinatorial treatment strategies against glioblastoma.

Epigenetic modifications play a major role in the development of multiple myeloma. We have previously reported that the PPARγ agonist pioglitazone (PIO) enhances, in-vitro, the cytotoxic effect of the Histone deacetylase inhibitor (HDACi), valproic acid (VPA), on multiple myeloma cells. Here, we described the development of a new multiple myeloma mouse model using MOLP8 cells, in order to evaluate the effect of VPA/PIO combination on the progression of myeloma cells, by analyzing the proliferation of bone marrow plasma cells. We showed that VPA/PIO delays the progression of the disease and the invasion of myeloma cells in the bone marrow. Mechanistically, we demonstrated that VPA/PIO increases the cleavage of caspase 3 and PARP, and induces the acetylation of Histone 3 (H3). Furthermore, we provided evidence that PPARγ agonist is able to enhance the action of other HDACi such as Vorinostat or Mocetinostat. Using PPARγ antagonist or siPPARγ, we strongly suggest that, as described during adipogenesis, PIO behaves as an epigenetic regulator by improving the activity of HDACi. This study highlights the therapeutic benefit of PIO/VPA combination, compared to VPA treatment as a single-arm therapy on multiple myeloma and further highlights that such combination may constitute a new promising treatment strategy which should be supported by clinical trials.

Anti-angiogenic therapy in glioblastoma (GBM) has unfortunately not led to the anticipated improvement in patient prognosis. We here describe how human GBM adapts to bevacizumab treatment at the metabolic level. By performing 13C6-glucose metabolic flux analysis, we show for the first time that the tumors undergo metabolic re-programming toward anaerobic metabolism, thereby uncoupling glycolysis from oxidative phosphorylation. Following treatment, an increased influx of 13C6-glucose was observed into the tumors, concomitant to increased lactate levels and a reduction of metabolites associated with the tricarboxylic acid cycle. This was confirmed by increased expression of glycolytic enzymes including pyruvate dehydrogenase kinase in the treated tumors. Interestingly, l-glutamine levels were also reduced. These results were further confirmed by the assessment of in vivo metabolic data obtained by magnetic resonance spectroscopy and positron emission tomography. Moreover, bevacizumab led to a depletion in glutathione levels indicating that the treatment caused oxidative stress in the tumors. Confirming the metabolic flux results, immunohistochemical analysis showed an up-regulation of lactate dehydrogenase in the bevacizumab-treated tumor core as well as in single tumor cells infiltrating the brain, which may explain the increased invasion observed after bevacizumab treatment. These observations were further validated in a panel of eight human GBM patients in which paired biopsy samples were obtained before and after bevacizumab treatment. Importantly, we show that the GBM adaptation to bevacizumab therapy is not mediated by clonal selection mechanisms, but represents an adaptive response to therapy. Electronic supplementary material The online version of this article (doi:10.1007/s00401-014-1352-5) contains supplementary material, which is available to authorized users.

Introduction: Patients with brain metastases await a dismal prognosis. Regardless of the continuous progress in drug development, a major problem is the delivery of drugs across the blood brain barrier (BBB) and into the metastatic neoplasms. The BBB excludes almost all compounds, in particular highly charged, hydrophilic or large compounds, and most of the current chemotherapeutic agents are thus unable to penetrate the BBB. Varying strategies to transiently open the BBB have been studied previously. Here, we describe a peptide transporter comprising 16 lysine residues and 20 amino acid residues corresponding to the low density lipoprotein receptor (LDLR) binding domain of apolipoprotein E (ApoE). We show that the peptide (K16ApoE) is able to transiently open the BBB for drug-delivery into experimental brain metastases. Experimental procedures: A systemic study of the ability of the peptide to open the BBB was conducted by dynamic contrast enhanced magnetic resonance imaging (DCE MRI) in nonobese diabetic/severe combined (nod/scid) mice. The BBB permeability was studied after administering 200 μg of the peptide intravenously. Further, cellular effects after treatment with the peptide was investigated in vitro using confocal microscopy, flow cytometry and impedance experiments. The biodistribution of the peptide was studied in blood plasma and several organs using 125I labeled K16ApoE. Finally, a treatment study was initiated, treating the animals with the peptide in combination with the B-RAF inhibitor Dabrafenib, only Dabrafenib or vehicle. Summary: After injecting the K16ApoE peptide into the mice, a transient opening of the BBB for up to 4 hours was clearly demonstrated by DCE-MRI. Microscopy showed that the peptide disrupted brain endothelial cell monolayers, reducing the barrier properties of the cells. The impedance experiments displayed that the permeability through endothelial cell barriers was increased after treatment with K16ApoE, and a dose-dependent cell death pattern was observed at higher concentrations of K16ApoE.The peptide did not affect endothelial cell tight junctions. The biodistribution study showed that the peptide was eliminated from blood plasma in less than five minutes through the kidneys. The treatment study displayed that the group of animals receiving K16ApoE followed by Dabrafenib had smaller tumor volumes than the other two animal groups. Conclusions: We have shown that the peptide opens the BBB and facilitates a therapeutic window of 4 hours. The peptide did in combination with Dabrafenib decrease the number of experimental brain metastases in our studies. Thus, the current strategy could also have the potential to improve the treatment of patients with brain metastatic disease. Citation Format: Synnøve Nymark Aasen, Heidi Espedal, Olivier Keunen, Christopher Florian Holte, Habib Baghirov, Rolf Bjerkvig, Tine Veronica Karlsen, Olav Tenstad, Dag Erlend Olberg, Gobinda Sarkar, Robert B Jenkins, Frits Thorsen. The peptide transporter K16ApoE increases drug delivery across the blood brain barrier in an experimental animal model of melanoma brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2865. doi:10.1158/1538-7445.AM2017-2865

Individuals with metastatic disease within the brain usually await a poor prognosis. Regardless of the ongoing development of novel drugs, a significant challenge is the delivery of drugs across the blood brain barrier (BBB) and into the metastatic neoplasms. The BBB excludes almost all therapeutic compounds, as highly charged, hydrophilic or large compounds are unable to penetrate the BBB. Different strategies to temporarily open the BBB have been studied previously. Here, we describe a peptide transporter encompassing 16 lysine residues and 20 amino acid residues corresponding to the low density lipoprotein receptor (LDLR) binding domain of apolipoprotein E (ApoE). We show that this peptide, K16ApoE, is able to transiently open the BBB for drug-delivery into experimental brain metastases. The ability of the peptide to open the BBB was studied in vivo using dynamic contrast enhanced magnetic resonance imaging (DCE MRI) in nonobese diabetic/severe combined (nod/scid) mice. Further, cellular effects after treatment with the peptide was investigated in vitro using confocal microscopy, flow cytometry and impedance experiments. The biodistribution of the peptide was studied in blood plasma and several organs using isotope labeled K16ApoE. Finally, we treated animals with the peptide in combination with the B-RAF inhibitor Dabrafenib, only Dabrafenib or vehicle. After intravenously administering the K16ApoE peptide into the mice, a transient opening of the BBB for up to 4 hours was clearly demonstrated by DCE-MRI. Microscopy showed that the peptide disrupted brain endothelial cell monolayers by reducing the barrier properties of the cells. The impedance experiments displayed that the permeability through endothelial cell barriers was increased after treatment with K16ApoE, and dose-dependent cell death was seen. The peptide did not affect endothelial cell tight junctions. The biodistribution study showed that the peptide was eliminated from blood plasma in less than five minutes through the kidneys. In the treatment study it was observed that the group of animals receiving K16ApoE followed by Dabrafenib had smaller tumor volumes than the other two experimental groups. K16ApoE did in combination with Dabrafenib decrease the number of experimental brain metastases, as a therapeutic window up to 4 hours was seen when using the peptide. The described strategy could thus have the potential to improve the treatment of patients with brain metastatic disease.