Exploring the neuroprotective effects of montelukast on brain inflammation and metabolism in a rat model of quinolinic acid-induced striatal neurotoxicity.

BACKGROUND: One intrastriatal administration of quinolinic acid (QA) in rats induces a lesion with features resembling those observed in Huntington's disease. Our aim is to evaluate the effects of the cysteinyl leukotriene receptor antagonist montelukast (MLK), which exhibited neuroprotection in different preclinical models of neurodegeneration, on QA-induced neuroinflammation and regional metabolic functions. METHODS: The right and left striatum of Sprague Dawley and athymic nude rats were injected with QA and vehicle (VEH), respectively. Starting from the day before QA injection, animals were treated with 1 or 10 mg/kg of MLK or VEH for 14 days. At 14 and 30 days post-lesion, animals were monitored with magnetic resonance imaging (MRI) and positron emission tomography (PET) using [18F]-VC701, a translocator protein (TSPO)-specific radiotracer. Striatal neuroinflammatory response was measured post-mortem in rats treated with 1 mg/kg of MLK by immunofluorescence. Rats treated with 10 mg/kg of MLK also underwent a [18F]-FDG PET study at baseline and 4 months after lesion. [18F]-FDG PET data were then used to assess metabolic connectivity between brain regions by applying a covariance analysis method. RESULTS: MLK treatment was not able to reduce the QA-induced increase in striatal TSPO PET signal and MRI lesion volume, where we only detected a trend towards reduction in animals treated with 10 mg/kg of MLK. Post-mortem immunofluorescence analysis revealed that MLK attenuated the increase in striatal markers of astrogliosis and activated microglia in the lesioned hemisphere. We also found a significant increase in a marker of anti-inflammatory activity (MannR) and a trend towards reduction in a marker of pro-inflammatory activity (iNOS) in the lesioned striatum of MLK-compared to VEH-treated rats. [18F]-FDG uptake was significantly reduced in the striatum and ipsilesional cortical regions of VEH-treated rats at 4 months after lesion. MLK administration preserved glucose metabolism in these cortical regions, but not in the striatum. Finally, MLK was able to counteract changes in metabolic connectivity and measures of network topology induced by QA, in both lesioned and non-lesioned hemispheres. CONCLUSIONS: Overall, MLK treatment produced a significant neuroprotective effect by reducing neuroinflammation assessed by immunofluorescence and preserving regional brain metabolism and metabolic connectivity from QA-induced neurotoxicity in cortical and subcortical regions.

The peritumoral brain zone in glioblastoma: where we are and where we are going.

Glioblastoma (GBM) is the most aggressive and invasive primary brain tumor. Current therapies are not curative, and patients' outcomes remain poor with an overall survival of 20.9 months after surgery. The typical growing pattern of GBM develops by infiltrating the surrounding apparent normal brain tissue within which the recurrence is expected to appear in the majority of cases. Thus, in the last decades, an increased interest has developed to investigate the cellular and molecular interactions between GBM and the peritumoral brain zone (PBZ) bordering the tumor tissue. The aim of this review is to provide up-to-date knowledge about the oncogenic properties of the PBZ to highlight possible druggable targets for more effective treatment of GBM by limiting the formation of recurrence, which is almost inevitable in the majority of patients. Starting from the description of the cellular components, passing through the illustration of the molecular profiles, we finally focused on more clinical aspects, represented by imaging and radiological details. The complete picture that emerges from this review could provide new input for future investigations aimed at identifying new effective strategies to eradicate this still incurable tumor.

Evaluating [18F]FDG and [18F]FLT Radiotracers as Biomarkers of Response for Combined Therapy Outcome in Triple-Negative and Estrogen-Receptor-Positive Breast Cancer Models.

Breast cancer (BC) is the most frequent cancer and the second leading cause of death in women. A typical feature of BC cells is the metabolic shift toward increased glycolysis, which has become an interesting therapeutic target for metabolic drugs such as metformin (MET). Recently, the administration of the antihypertensive syrosingopine (SYRO) in combination with MET has shown a synergistic effect toward a variety of cancers. However, a fundamental need remains, which is the development of in vivo biomarkers that are able to detect early clinical response. In this study, we exploited a triple-negative murine BC cell line (4T1) and a metastatic ER+ murine BC cell line (TS/A) in order to investigate, in vivo, the early response to treatment, based on MET and/or SYRO administration, evaluating [18F]FDG and [18F]FLT as potential biomarkers via PET/CT. The study provides evidence that SYRO plus MET has a synergistic effect on tumor growth inhibition in both 4T1 and TS/A experimental models and has showed the highest efficacy on the TNBC xenograft mice (4T1) via the expression reduction in the lactate transporter MCT4 and in the epithelial-mesenchymal transition biomarker Snail, promoting its potential application in therapy settings. In addition, the selective reduction in the [18F]FLT tumor uptake (at 7 dd), observed in the SYRO plus MET treated mice in comparison with the vehicle group, suggests that this radiotracer could be potentially used as a biomarker for the early detection of therapy response, in both evaluated xenografts models.

In vivo susceptibility to energy failure parkinsonism and LRRK2 kinase activity.

The G2019S mutation of LRRK2 represents a risk factor for idiopathic Parkinson's disease. Here, we investigate whether LRRK2 kinase activity regulates susceptibility to the environmental toxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP). G2019S knock-in mice (bearing enhanced kinase activity) showed greater nigro-striatal degeneration compared to LRRK2 knock-out, LRRK2 kinase-dead and wild-type mice following subacute MPTP treatment. LRRK2 kinase inhibitors PF-06447475 and MLi-2, tested under preventive or therapeutic treatments, protected against nigral dopamine cell loss in G2019S knock-in mice. MLi-2 also rescued striatal dopaminergic terminal degeneration in both G2019S knock-in and wild-type mice. Immunoblot analysis of LRRK2 Serine935 phosphorylation levels confirmed target engagement of LRRK2 inhibitors. However, MLi-2 abolished phosphoSerine935 levels in the striatum and midbrain of both wild-type and G2019S knock-in mice whereas PF-06447475 partly reduced phosphoSerine935 levels in the midbrain of both genotypes. In vivo and ex vivo uptake of the 18-kDa translocator protein (TSPO) ligand [18F]-VC701 revealed a similar TSPO binding in MPTP-treated wild-type and G2019S knock-in mice which was consistent with an increased GFAP striatal expression as revealed by Real Time PCR. We conclude that LRRK2 G2019S, likely through enhanced kinase activity, confers greater susceptibility to mitochondrial toxin-induced parkinsonism. LRRK2 kinase inhibitors are neuroprotective in this model.

RAB39B-mediated trafficking of the GluA2-AMPAR subunit controls dendritic spine maturation and intellectual disability-related behaviour.

Mutations in the RAB39B gene cause X-linked intellectual disability (XLID), comorbid with autism spectrum disorders or early Parkinson's disease. One of the functions of the neuronal small GTPase RAB39B is to drive GluA2/GluA3 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) maturation and trafficking, determining AMPAR subunit composition at glutamatergic postsynaptic neuronal terminals. Taking advantage of the Rab39b knockout murine model, we show that a lack of RAB39B affects neuronal dendritic spine refinement, prompting a more Ca2+-permeable and excitable synaptic network, which correlates with an immature spine arrangement and behavioural and cognitive alterations in adult mice. The persistence of immature circuits is triggered by increased hypermobility of the spine, which is restored by the Ca2+-permeable AMPAR antagonist NASPM. Together, these data confirm that RAB39B controls AMPAR trafficking, which in turn plays a pivotal role in neuronal dendritic spine remodelling and that targeting Ca2+-permeable AMPARs may highlight future pharmaceutical interventions for RAB39B-associated disease conditions.

Skeletal Muscle Proteomic Profile Revealed Gender-Related Metabolic Responses in a Diet-Induced Obesity Animal Model.

Obesity is a chronic, complex pathology associated with a risk of developing secondary pathologies, including cardiovascular diseases, cancer, type 2 diabetes (T2DM) and musculoskeletal disorders. Since skeletal muscle accounts for more than 70% of total glucose disposal, metabolic alterations are strictly associated with the onset of insulin resistance and T2DM. The present study relies on the proteomic analysis of gastrocnemius muscle from 15 male and 15 female C56BL/J mice fed for 14 weeks with standard, 45% or 60% high-fat diets (HFD) adopting a label-free LC-MS/MS approach followed by bioinformatic pathway analysis. Results indicate changes in males due to HFD, with increased muscular stiffness (Col1a1, Col1a2, Actb), fiber-type switch from slow/oxidative to fast/glycolytic (decreased Myh7, Myl2, Myl3 and increased Myh2, Mylpf, Mybpc2, Myl1), increased oxidative stress and mitochondrial dysfunction (decreased respiratory chain complex I and V and increased complex III subunits). At variance, females show few alterations and activation of compensatory mechanisms to counteract the increase of fatty acids. Bioinformatics analysis allows identifying upstream molecules involved in regulating pathways identified at variance in our analysis (Ppargc1a, Pparg, Cpt1b, Clpp, Tp53, Kdm5a, Hif1a). These findings underline the presence of a gender-specific response to be considered when approaching obesity and related comorbidities.

Differentiation of human telencephalic progenitor cells into MSNs by inducible expression of Gsx2 and Ebf1.

Medium spiny neurons (MSNs) are a key population in the basal ganglia network, and their degeneration causes a severe neurodegenerative disorder, Huntington's disease. Understanding how ventral neuroepithelial progenitors differentiate into MSNs is critical for regenerative medicine to develop specific differentiation protocols using human pluripotent stem cells. Studies performed in murine models have identified some transcriptional determinants, including GS Homeobox 2 (Gsx2) and Early B-cell factor 1 (Ebf1). Here, we have generated human embryonic stem (hES) cell lines inducible for these transcription factors, with the aims of (i) studying their biological role in human neural progenitors and (ii) incorporating TF conditional expression in a developmental-based protocol for generating MSNs from hES cells. Using this approach, we found that Gsx2 delays cell-cycle exit and reduces Pax6 expression, whereas Ebf1 promotes neuronal differentiation. Moreover, we found that Gsx2 and Ebf1 combined overexpression in hES cells achieves high yields of MSNs, expressing Darpp32 and Ctip2, in vitro as well in vivo after transplantation. We show that hES-derived striatal progenitors can be transplanted in animal models and can differentiate and integrate into the host, extending fibers over a long distance.

Molecular and Cellular Complexity of Glioma. Focus on Tumour Microenvironment and the Use of Molecular and Imaging Biomarkers to Overcome Treatment Resistance.

This review highlights the importance and the complexity of tumour biology and microenvironment in the progression and therapy resistance of glioma. Specific gene mutations, the possible functions of several non-coding microRNAs and the intra-tumour and inter-tumour heterogeneity of cell types contribute to limit the efficacy of the actual therapeutic options. In this scenario, identification of molecular biomarkers of response and the use of multimodal in vivo imaging and in particular the Positron Emission Tomography (PET) based molecular approach, can help identifying glioma features and the modifications occurring during therapy at a regional level. Indeed, a better understanding of tumor heterogeneity and the development of diagnostic procedures can favor the identification of a cluster of patients for personalized medicine in order to improve the survival and their quality of life.

18F-VC701-PET and MRI in the in vivo neuroinflammation assessment of a mouse model of multiple sclerosis.

BACKGROUND: Positron emission tomography (PET) using translocator protein (TSPO) ligands has been used to detect neuroinflammatory processes in neurological disorders, including multiple sclerosis (MS). The aim of this study was to evaluate neuroinflammation in a mouse MS model (EAE) using TSPO-PET with 18F-VC701, in combination with magnetic resonance imaging (MRI). METHODS: MOG35-55/CFA and pertussis toxin protocol was used to induce EAE in C57BL/6 mice. Disease progression was monitored daily, whereas MRI evaluation was performed at 1, 2, and 4 weeks post-induction. Microglia activation was assessed in vivo by 18F-VC701 PET at the time of maximum disease score and validated by radioligand ex vivo distribution and immunohistochemistry at 2 and 4 weeks post-immunization. RESULTS: In vivo and ex vivo analyses show that 18F-VC701 significantly accumulates within the central nervous system (CNS), particularly in the cortex, striatum, hippocampus, cerebellum, and cervical spinal cord of EAE compared to control mice, at 2 weeks post-immunization. MRI confirmed the presence of focal brain lesions at 2 weeks post-immunization in both T1-weighted and T2 images. Of note, MRI abnormalities attenuated in later post-immunization phase. Neuropathological analysis confirmed the presence of microglial activation in EAE mice, consistent with the in vivo increase of 18F-VC701 uptake. CONCLUSION: Increase of 18F-VC701 uptake in EAE mice is strongly associated with the presence of microglia activation in the acute phase of the disease. The combined use of TSPO-PET and MRI provided complementary evidence on the ongoing disease process, thus representing an attractive new tool to investigate neuronal damage and neuroinflammation at preclinical levels.

Regional Differences in Cerebral Glucose Metabolism After Cardiac Arrest and Resuscitation in Rats Using [18F]FDG Positron Emission Tomography and Autoradiography.

BACKGROUND: Cardiac arrest is an important cause of morbidity and mortality. Brain injury severity and prognosis of cardiac arrest patients are related to the cerebral areas affected. To this aim, we evaluated the variability and the distribution of brain glucose metabolism after cardiac arrest and resuscitation in an adult rat model. METHODS: Ten rats underwent 8-min cardiac arrest, induced with a mixture of potassium and esmolol, and resuscitation, performed with chest compressions and epinephrine. Eight sham animals received anesthesia and experimental procedures identical to the ischemic group except cardiac arrest induction. Brain metabolism was assessed using [18F]FDG autoradiography and small animal-dedicated positron emission tomography. RESULTS: The absolute glucose metabolism measured with [18F]FDG autoradiography 2 h after cardiac arrest and resuscitation was lower in the frontal, parietal, occipital, and temporal cortices of cardiac arrest animals, showing, respectively, a 36% (p = 0.006), 32% (p = 0.016), 36% (p = 0.009), and 32% (p = 0.013) decrease compared to sham group. Striatum, hippocampus, thalamus, brainstem, and cerebellum showed no significant changes. Relative regional metabolism indicated a redistribution of metabolism from cortical area to brainstem and cerebellum. CONCLUSIONS: Our data suggest that cerebral regions have different susceptibility to moderate global ischemia in terms of glucose metabolism. The neocortex showed a higher sensibility to hypoxia-ischemia than other regions. Other subcortical regions, in particular brainstem and cerebellum, showed no significant change compared to non-ischemic rats.

Glycine N-methylation in NGR-Tagged Nanocarriers Prevents Isoaspartate formation and Integrin Binding without Impairing CD13 Recognition and Tumor Homing.

NGR (asparagine-glycine-arginine) is a tumor vasculature-homing peptide motif widely used for the functionalization of drugs, nanomaterials and imaging compounds for cancer treatment and diagnosis. Unfortunately, this motif has a strong propensity to undergo rapid deamidation. This reaction, which converts NGR into isoDGR, is associated with receptor switching from CD13 to integrins, with potentially important manufacturing, pharmacological and toxicological implications. It is found that glycine N-methylation of NGR-tagged nanocarriers completely prevents asparagine deamidation without impairing CD13 recognition. Studies in animal models have shown that the methylated NGR motif can be exploited for delivering radiolabeled compounds and nanocarriers, such as tumor necrosis factor-α (TNF)-bearing nanogold and liposomal doxorubicin, to tumors with improved selectivity. These findings suggest that this NGR derivative is a stable and efficient tumor-homing ligand that can be used for delivering functional nanomaterials to tumor vasculature.

Radiosynthesis and Preliminary Biological Evaluation of [18F]VC701, a Radioligand for Translocator Protein.

Positron emission tomography (PET) can be used to monitor in vivo translocator protein (TSPO) expression by using specific radioligands. Recently, several [11C]PK11195 analogues have been synthesized to improve binding stability and brain availability. [18F]VC701 was synthesized and validated in CD healthy rats by biodistribution and inhibition analysis. Imaging studies were also conducted on animals injected unilaterally in the striatum with quinolinic acid (QA) to evaluate the TSPO ligand uptake in a neuroinflammation/neurodegenerative model. [18F]VC701 was synthesized with a good chemical and radiochemical purity and specific activity higher than 37 GBq/µmol. Kinetic studies performed on healthy animals showed the highest tracer biodistribution in TSPO-rich organs, and preadministration of cold PK11195 caused an overall radioactivity reduction. Metabolism studies showed the absence of radiometabolites in the rat brain of QA lesioned rats, and biodistribution analysis revealed a progressive increase in radioactivity ratios (lesioned to nonlesioned striatum) during time, reaching an approximate value of 5 4 hours after tracer injection. These results encourage further evaluation of this TSPO radioligand in other models of central and peripheral diseases.

A novel AMPK activator reduces glucose uptake and inhibits tumor progression in a mouse xenograft model of colorectal cancer.

The anticancer activity of a novel pure 1,4-Diaryl-2-azetidinone (1), endowed with a higher solubility than the well known Combretastatin A4, is tested in mice. We previously reported that Compound (1) showed specific antiproliferative activity against duodenal and colon cancer cells, inducing activation of AMP-activated protein kinase and apoptosis. Here we estimate that the maximum tolerated dose in a mouse model is 40 mg/kg; the drug is well tolerated both in single dose and in repeated administration schedules. The drug displays a significant antitumor activity and a tumor growth delay when administered at the MTD both in single and fractionated i.v. administration in a mouse xenograft model of colorectal cancer. Arrest of tumor growth and relapse after drug suspension are parallel to modification in glucose demand as shown by PET studies with [(18)F] FDG. These data strongly support Compound (1) as a promising molecule for in vivo treatment of colorectal cancer.

Positive relation between dopamine neuron degeneration and metabolic connectivity disruption in the MPTP plus probenecid mouse model of Parkinson's disease.

The clinical manifestation of Parkinson's disease (PD) appears when neurodegeneration is already advanced, compromising the efficacy of disease-modifying treatment approaches. Biomarkers to identify the early stages of PD are therefore of paramount importance for the advancement of the therapy of PD. In the present study, by using a mouse model of PD obtained by subchronic treatment with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the clearance inhibitor probenecid (MPTPp), we identified prodromal markers of PD by combining in vivo positron emission tomography (PET) imaging and ex vivo immunohistochemistry. Longitudinal PET imaging of the dopamine transporter (DAT) by [18F]-N-(3-fluoropropyl)-2ß-carboxymethoxy-3ß-(4-iodophenyl) nortropane ([18F]-FP-CIT), and brain glucose metabolism by 2-deoxy-2-[18F]-fluoroglucose ([18F]-FDG) were performed before MPTPp treatment and after 1, 3, and 10 MPTPp administrations, in order to assess relation between dopamine neuron integrity and brain connectivity. The results show that in vivo [18F]-FP-CIT in the dorsal striatum was not modified after the first administration of MPTPp, tended to decrease after 3 administrations, and significantly decreased after 10 MPTPp administrations. Post-mortem immunohistochemical analyses of DAT and tyrosine hydroxylase (TH) in the striatum showed a positive correlation with [18F]-FP-CIT, confirming the validity of repeated MPTPp-treated mice as a model that can reproduce the progressive pathological changes in the early phases of PD. Analysis of [18F]-FDG uptake in several brain areas connected to the striatum showed that metabolic connectivity was progressively disrupted, starting from the first MPTPp administration, and that significant connections between cortical and subcortical regions were lost after 10 MPTPp administrations, suggesting an association between dopamine neuron degeneration and connectivity disruption in this PD model. The results of this study provide a relevant model, where new drugs that can alleviate neurodegeneration in PD could be evaluated preclinically.

Automated radiosynthesis and preclinical evaluation of two new PSMA-617 derivatives radiolabelled via [18F]AlF2+ method.

BACKGROUND: In the last decade the development of new PSMA-ligand based radiopharmaceuticals for the imaging and therapy of prostate cancer has been a highly active and important area of research. The most promising derivative in terms of interaction with the antigen and clinical properties has been found to be "PSMA-617", and its lutetium-177 radiolabelled version has recently been approved by EU and USA regulatory agencies for therapeutic purposes. For the above reasons, the development of new derivatives of PSMA-617 radiolabelled with fluorine-18 may still be of great interest. This paper proposes the comparison of two different PSMA-617 derivatives functionalized with NODA and RESCA chelators, respectively, radiolabelled via [18F]AlF2+ complexation. RESULTS: The organic synthesis of two PSMA-617 derivatives and their radiolabelling via [18F]AlF2+ complexation resulted to proceed efficiently and successfully. Moreover, stability in solution and in plasma has been evaluated. The whole radiosynthesis procedure has been fully automated, and the final products have been obtained with radiochemical yield and purity potentially suitable for clinical studies. The biodistribution of the two derivatives was performed both in prostate cancer and glioma tumour models. Compared with the reference [18F]F-PSMA-1007 and [18F]F-PSMA-617-RESCA, [18F]F-PSMA-617-NODA derivative showed a higher uptake in both tumors, faster clearance in non-target organs, and lower uptake in salivary glands. CONCLUSION: PSMA-617 NODA and RESCA derivatives were radiolabelled successfully via [18F]AlF2+ chelation, the former being more stable in solution and human plasma. Moreover, preclinical biodistribution studies showed that [18F]F-PSMA-617-NODA might be of potential interest for clinical applications.

New orphan disease therapies from the proteome of industrial plasma processing waste- a treatment for aceruloplasminemia.

Plasma-derived therapeutic proteins are produced through an industrial fractionation process where proteins are purified from individual intermediates, some of which remain unused and are discarded. Relatively few plasma-derived proteins are exploited clinically, with most of available plasma being directed towards the manufacture of immunoglobulin and albumin. Although the plasma proteome provides opportunities to develop novel protein replacement therapies, particularly for rare diseases, the high cost of plasma together with small patient populations impact negatively on the development of plasma-derived orphan drugs. Enabling therapeutics development from unused plasma fractionation intermediates would therefore constitute a substantial innovation. To this objective, we characterized the proteome of unused plasma fractionation intermediates and prioritized proteins for their potential as new candidate therapies for human disease. We selected ceruloplasmin, a plasma ferroxidase, as a potential therapy for aceruloplasminemia, an adult-onset ultra-rare neurological disease caused by iron accumulation as a result of ceruloplasmin mutations. Intraperitoneally administered ceruloplasmin, purified from an unused plasma fractionation intermediate, was able to prevent neurological, hepatic and hematological phenotypes in ceruloplasmin-deficient mice. These data demonstrate the feasibility of transforming industrial waste plasma fraction into a raw material for manufacturing of new candidate proteins for replacement therapies, optimizing plasma use and reducing waste generation.

Ibuprofen plus isosorbide dinitrate treatment in the mdx mice ameliorates dystrophic heart structure.

BACKGROUND: Co-administration of ibuprofen (IBU) and isosorbide dinitrate (ISDN) provides synergistic beneficial effects on dystrophic skeletal muscle. Whether this treatment has also cardioprotective effects in this disease was still unknown. AIMS: To evaluate the effects of co-administration of IBU and ISDN (a) on left ventricular (LV) structure and function, and (b) on cardiac inflammatory response and fibrosis in mdx mice. METHODS: Three groups of mice were studied: mdx mice treated with IBU (50 mg kg⁻¹)+ISDN (30 mg kg⁻¹) administered daily in the diet, mdx mice that received standard diet without drugs and wild type aged-matched mice. Animals were analysed after 10-11 months of treatment. Structural and functional parameters were evaluated by echocardiography while histological analyses were performed to evaluate inflammatory response, collagen deposition, cardiomyocyte number and area. RESULTS: Treatment for 10-11 months with IBU+ISDN preserved LV wall thickness and LV mass. Drug treatment also preserved the total number of cardiomyocytes in the LV and attenuated the increase in cardiomyocyte size, when compared to untreated mdx mice. Moreover, a trend towards a decreased number of inflammatory cells, a reduced LV myocardial interstitial fibrosis and an enhanced global LV function response to stress was observed in treated mdx mice. CONCLUSIONS: Treatment for 10-11 months with IBU+ISDN is effective in preventing the alterations in LV morphology of mdx mice while not reaching statistical significance on LV function and cardiac inflammation.

A stapled chromogranin A-derived peptide homes in on tumors that express αvß6 or αvß8 integrins.

Rationale: The αvß6- and αvß8-integrins, two cell-adhesion receptors upregulated in many tumors and involved in the activation of the latency associated peptide (LAP)/TGFß complex, represent potential targets for tumor imaging and therapy. We investigated the tumor-homing properties of a chromogranin A-derived peptide containing an RGDL motif followed by a chemically stapled alpha-helix (called "5a"), which selectively recognizes the LAP/TGFß complex-binding site of αvß6 and αvß8. Methods: Peptide 5a was labeled with IRDye 800CW (a near-infrared fluorescent dye) or with 18F-NOTA (a label for positron emission tomography (PET)); the integrin-binding properties of free peptide and conjugates were then investigated using purified αvß6/αvß8 integrins and various αvß6/αvß8 single - or double-positive cancer cells; tumor-homing, biodistribution and imaging properties of the conjugates were investigated in subcutaneous and orthotopic αvß6-positive carcinomas of the pancreas, and in mice bearing subcutaneous αvß8-positive prostate tumors. Results: In vitro studies showed that 5a can bind both integrins with high affinity and inhibits cell-mediated TGFß activation. The 5a-IRDye and 5a-NOTA conjugates could bind purified αvß6/αvß8 integrins with no loss of affinity compared to free peptide, and selectively recognized various αvß6/αvß8 single- or double-positive cancer cells, including cells from pancreatic carcinoma, melanoma, oral mucosa, bladder and prostate cancer. In vivo static and dynamic optical near-infrared and PET/CT imaging and biodistribution studies, performed in mice with subcutaneous and orthotopic αvß6-positive carcinomas of the pancreas, showed high target-specific uptake of fluorescence- and radio-labeled peptide by tumors and low non-specific uptake in other organs and tissues, except for excretory organs. Significant target-specific uptake of fluorescence-labeled peptide was also observed in mice bearing αvß8-positive prostate tumors. Conclusions: The results indicate that 5a can home to αvß6- and/or αvß8-positive tumors, suggesting that this peptide can be exploited as a ligand for delivering imaging or anticancer agents to αvß6/αvß8 single- or double-positive tumors, or as a tumor-homing inhibitor of these TGFß activators.

Aberrant L-Fucose Accumulation and Increased Core Fucosylation Are Metabolic Liabilities in Mesenchymal Glioblastoma.

Glioblastoma (GBM) is a common and deadly form of brain tumor in adults. Dysregulated metabolism in GBM offers an opportunity to deploy metabolic interventions as precise therapeutic strategies. To identify the molecular drivers and the modalities by which different molecular subgroups of GBM exploit metabolic rewiring to sustain tumor progression, we interrogated the transcriptome, the metabolome, and the glycoproteome of human subgroup-specific GBM sphere-forming cells (GSC). L-fucose abundance and core fucosylation activation were elevated in mesenchymal (MES) compared with proneural GSCs; this pattern was retained in subgroup-specific xenografts and in subgroup-affiliated human patient samples. Genetic and pharmacological inhibition of core fucosylation significantly reduced tumor growth in MES GBM preclinical models. Liquid chromatography-mass spectrometry (LC-MS)-based glycoproteomic screening indicated that most MES-restricted core-fucosylated proteins are involved in therapeutically relevant GBM pathological processes, such as extracellular matrix interaction, cell adhesion, and integrin-mediated signaling. Selective L-fucose accumulation in MES GBMs was observed using preclinical minimally invasive PET, implicating this metabolite as a potential subgroup-restricted biomarker.Overall, these findings indicate that L-fucose pathway activation in MES GBM is a subgroup-specific dependency that could provide diagnostic markers and actionable therapeutic targets. SIGNIFICANCE: Metabolic characterization of subgroup-specific glioblastoma (GBM) sphere-forming cells identifies the L-fucose pathway as a vulnerability restricted to mesenchymal GBM, disclosing a potential precision medicine strategy for targeting cancer metabolism.