UNCAN.eu: Toward a European Federated Cancer Research Data Hub.

To enable a collective effort that generates a new level of UNderstanding CANcer (UNCAN.eu) [Cancer Discov (2022) 12 (11): OF1], the European Union supports the creation of a sustainable platform that connects cancer research across Member States. A workshop hosted in Heidelberg gathered European cancer experts to identify ongoing initiatives that may contribute to building this platform and discuss the governance and long-term evolution of a European Federated Cancer Data Hub.

Superfluorinated Extracellular Vesicles for In Vivo Imaging by 19F-MRI.

Extracellular vesicles (EVs) play a crucial role in cell-to-cell communication and have great potential as efficient delivery vectors. However, a better understanding of EV in vivo behavior is hampered by the limitations of current imaging tools. In addition, chemical labels present the risk of altering the EV membrane features and, thus, in vivo behavior. 19F-MRI is a safe bioimaging technique providing selective images of exogenous probes. Here, we present the first example of fluorinated EVs containing PERFECTA, a branched molecule with 36 magnetically equivalent 19F atoms. A PERFECTA emulsion is given to the cells, and PERFECTA-containing EVs are naturally produced. PERFECTA-EVs maintain the physicochemical features, morphology, and biological fingerprint as native EVs but exhibit an intense 19F-NMR signal and excellent 19F relaxation times. In vivo 19F-MRI and tumor-targeting capabilities of stem cell-derived PERFECTA-EVs are also proved. We propose PERFECTA-EVs as promising biohybrids for imaging biodistribution and delivery of EVs throughout the body.

Targeted inducible delivery of immunoactivating cytokines reprograms glioblastoma microenvironment and inhibits growth in mouse models.

Glioblastoma multiforme (GBM) is the most common and lethal brain tumor characterized by a strongly immunosuppressive tumor microenvironment (TME) that represents a barrier also for the development of effective immunotherapies. The possibility to revert this hostile TME by immunoactivating cytokines is hampered by the severe toxicity associated with their systemic administration. Here, we exploited a lentiviral vector-based platform to engineer hematopoietic stem cells ex vivo with the aim of releasing, via their tumor-infiltrating monocyte/macrophage progeny, interferon-α (IFN-α) or interleukin-12 (IL-12) at the tumor site with spatial and temporal selectivity. Taking advantage of a syngeneic GBM mouse model, we showed that inducible release of IFN-α within the TME achieved robust tumor inhibition up to eradication and outperformed systemic treatment with the recombinant protein in terms of efficacy, tolerability, and specificity. Single-cell RNA sequencing of the tumor immune infiltrate revealed reprogramming of the immune microenvironment toward a proinflammatory and antitumoral state associated with loss of a macrophage subpopulation shown to be associated with poor prognosis in human GBM. The spatial and temporal control of IL-12 release was critical to overcome an otherwise lethal hematopoietic toxicity while allowing to fully exploit its antitumor activity. Overall, our findings demonstrate a potential therapeutic approach for GBM and set the bases for a recently launched first-in-human clinical trial in patients with GBM.

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.

In vivo magnetic resonance spectroscopy in the brain of Cdkl5 null mice reveals a metabolic profile indicative of mitochondrial dysfunctions.

Mutations in the X-linked CDKL5 gene cause CDKL5 deficiency disorder (CDD), a severe neurodevelopmental condition mainly characterized by infantile epileptic encephalopathy, intellectual disability, and autistic features. The molecular mechanisms underlying the clinical symptoms remain largely unknown and the identification of reliable biomarkers in animal models will certainly contribute to increase our comprehension of CDD as well as to assess the efficacy of therapeutic strategies. Here, we used different Magnetic Resonance (MR) methods to disclose structural, functional, or metabolic signatures of Cdkl5 deficiency in the brain of adult mice. We found that loss of Cdkl5 does not cause cerebral atrophy but affects distinct brain areas, particularly the hippocampus. By in vivo proton-MR spectroscopy (MRS), we revealed in the Cdkl5 null brain a metabolic dysregulation indicative of mitochondrial dysfunctions. Accordingly, we unveiled a significant reduction in ATP levels and a decrease in the expression of complex IV of mitochondrial electron transport chain. Conversely, the number of mitochondria appeared preserved. Importantly, we reported a significant defect in the activation of one of the major regulators of cellular energy balance, the adenosine monophosphate-activated protein kinase (AMPK), that might contribute to the observed metabolic impairment and become an interesting therapeutic target for future preclinical trials. In conclusion, MRS revealed in the Cdkl5 null brain the presence of a metabolic dysregulation suggestive of a mitochondrial dysfunction that permitted to foster our comprehension of Cdkl5 deficiency and brought our interest towards targeting mitochondria as therapeutic strategy for CDD.

Detrimental and protective action of microglial extracellular vesicles on myelin lesions: astrocyte involvement in remyelination failure.

Microglia are highly plastic immune cells which exist in a continuum of activation states. By shaping the function of oligodendrocyte precursor cells (OPCs), the brain cells which differentiate to myelin-forming cells, microglia participate in both myelin injury and remyelination during multiple sclerosis. However, the mode(s) of action of microglia in supporting or inhibiting myelin repair is still largely unclear. Here, we analysed the effects of extracellular vesicles (EVs) produced in vitro by either pro-inflammatory or pro-regenerative microglia on OPCs at demyelinated lesions caused by lysolecithin injection in the mouse corpus callosum. Immunolabelling for myelin proteins and electron microscopy showed that EVs released by pro-inflammatory microglia blocked remyelination, whereas EVs produced by microglia co-cultured with immunosuppressive mesenchymal stem cells promoted OPC recruitment and myelin repair. The molecular mechanisms responsible for the harmful and beneficial EV actions were dissected in primary OPC cultures. By exposing OPCs, cultured either alone or with astrocytes, to inflammatory EVs, we observed a blockade of OPC maturation only in the presence of astrocytes, implicating these cells in remyelination failure. Biochemical fractionation revealed that astrocytes may be converted into harmful cells by the inflammatory EV cargo, as indicated by immunohistochemical and qPCR analyses, whereas surface lipid components of EVs promote OPC migration and/or differentiation, linking EV lipids to myelin repair. Although the mechanisms through which the lipid species enhance OPC maturation still remain to be fully defined, we provide the first demonstration that vesicular sphingosine 1 phosphate stimulates OPC migration, the first fundamental step in myelin repair. From this study, microglial EVs emerge as multimodal and multitarget signalling mediators able to influence both OPCs and astrocytes around myelin lesions, which may be exploited to develop novel approaches for myelin repair not only in multiple sclerosis, but also in neurological and neuropsychiatric diseases characterized by demyelination.

The Danger Signal Extracellular ATP Is Involved in the Immunomediated Damage of α-Sarcoglycan-Deficient Muscular Dystrophy.

In muscular dystrophies, muscle membrane fragility results in a tissue-specific increase of danger-associated molecular pattern molecules (DAMPs) and infiltration of inflammatory cells. The DAMP extracellular ATP (eATP) released by dying myofibers steadily activates muscle and immune purinergic receptors exerting dual negative effects: a direct damage linked to altered intracellular calcium homeostasis in muscle cells and an indirect toxicity through the triggering of the immune response and inhibition of regulatory T cells. Accordingly, pharmacologic and genetic inhibition of eATP signaling improves the phenotype in models of chronic inflammatory diseases. In α-sarcoglycanopathy, eATP effects may be further amplified because α-sarcoglycan extracellular domain binds eATP and displays an ecto-ATPase activity, thus controlling eATP concentration at the cell surface and attenuating the magnitude and/or the duration of eATP-induced signals. Herein, we show that in vivo blockade of the eATP/P2X purinergic pathway by a broad-spectrum P2X receptor-antagonist delayed the progression of the dystrophic phenotype in α-sarcoglycan-null mice. eATP blockade dampened the muscular inflammatory response and enhanced the recruitment of forkhead box protein P3-positive immunosuppressive regulatory CD4+ T cells. The improvement of the inflammatory features was associated with increased strength, reduced necrosis, and limited expression of profibrotic factors, suggesting that pharmacologic purinergic antagonism, altering the innate and adaptive immune component in muscle infiltrates, might provide a therapeutic approach to slow disease progression in α-sarcoglycanopathy.

Ocular surface injury induces inflammation in the brain: in vivo and ex vivo evidence of a corneal-trigeminal axis.

PURPOSE: To test whether a corneal injury can stimulate inflammation in the trigeminal ganglion (TG), a structure located in the brain. METHODS: At 4 and 8 days after alkali burn induced in the right eyes of mice, in vivo magnetic resonance imaging (MRI) of the brain was done before and after ultrasmall superparamagnetic iron oxide nanoparticle (USPIO) contrast to track macrophages. Trigeminal ganglia were stained for Prussian Blue and inflammatory cell markers. Interleukin-1ß, TNF-α, and VEGF-A transcripts were quantified on days 1, 4, and 8, and 4 days after corneal topical anti-inflammatory treatment with 0.2% dexamethasone. The expression of Substance P and its receptor NK-1R was also measured in the TG on day 4. RESULTS: Corneal alkali burn induced leukocyte infiltration, including T cells, in the right TG at 4 and 8 days. In vivo MRI showed an increased contrast uptake in the right TG, which peaked at day 8. Prussian Blue(+) USPIO(+) macrophages were observed in the right TG and exhibited an M2 phenotype. The M2-macrophage infiltration was preponderant in the TG after damage. The proinflammatory cytokines Substance P and NK-1R were significantly increased in both the TGs. The expression of IL-1ß and VEGF-A was significantly reduced in the right TG with dexamethasone treatment. CONCLUSIONS: We suggest, for the first time, inflammatory involvement of brain structures following ocular surface damage. Our findings support the hypothesis that the neuropeptide Substance P may be involved in the propagation of inflammation from the cornea to the TG through corneal nerves.

Novel Gd(III)-based probes for MR molecular imaging of matrix metalloproteinases.

Two novel Gd-based contrast agents (CAs) for the molecular imaging of matrix metalloproteinases (MMPs) were synthetized and characterized in vitro and in vivo. These probes were based on the PLG*LWAR peptide sequence, known to be hydrolyzed between Gly and Leu by a broad panel of MMPs. A Gd-DOTA chelate was conjugated to the N-terminal position through an amide bond, either directly to proline (compd Gd-K11) or through a hydrophilic spacer (compd Gd-K11N). Both CA were made strongly amphiphilic by conjugating an alkyl chain at the C-terminus of the peptide sequence. Gd-K11 and Gd-K11N have a good affinity for ß-cyclodextrins (K(D) 310 and 670 µ m respectively) and for serum albumin (K(D) 350 and 90 µ m respectively), and can be efficiently cleaved in vitro at the expected site by MMP-2 and MMP-12. Upon MMP-dependent cleavage, the CAs lose the C-terminal tetrapeptide and the alkyl chain, thus undergoing to an amphiphilic-to-hydrophilic transformation that is expected to alter tissue pharmacokinetics. To prove this, Gd-K11 was systemically administered to mice bearing a subcutaneous B16.F10 melanoma, either pre-treated or not with the broad spectrum MMP inhibitor GM6001 (Ilomastat). The washout of the Gd-contrast enhancement in MR images was significantly faster for untreated subjects (displaying MMP activity) with respect to treated ones (MMP activity inhibited). The washout kinetics of Gd-contrast enhancement from the tumor microenvironment could be then interpreted in terms of the local activity of MMPs.

Novel MRI and fluorescent probes responsive to the Factor XIII transglutaminase activity.

Transglutaminases, including factor XIII and tissue transglutaminase, participate in multiple extracellular processes associated with remodeling of the extracellular matrix during wound repair, blood clotting, tumor progression and fibrosis of ischemic injuries. The aim of this work was to evaluate a novel substrate analog for transglutaminase optimized by molecular modeling calculations (DCCP16), which can serve for molecular imaging of transglutaminase activity by magnetic resonance imaging and by near-infrared imaging. Experimental data showed covalent binding of Gd-DCCP16 and DCCP16-IRIS Blue to human clots, to basement membrane components and to casein in purified systems as well as in three-dimensional multicellular spheroids. In vivo, DCCP16 showed enhancement with a prolonged retention in clots and tumors, demonstrating the ability to detect both factor XIII and tissue transglutaminase mediated covalent binding of the contrast material.

Evidence for in vivo macrophage mediated tumor uptake of paramagnetic/fluorescent liposomes.

Dysprosium (Dy)-loaded liposomes act as excellent T(2)-susceptibility agents at high magnetic field strength. The R(2)-enhancement increases with the size of the liposomes and the concentration of entrapped paramagnetic metal complexes. Neuro-2a tumor cells are readily labeled when Dy-loaded liposomes, suitably functionalized with glutamine residues (Gln), are added to the culture medium as glutamine receptors are highly expressed in such proliferating tumor cells. By using fluorescent liposomes doped with fluorescent dyes (either incorporated in the membrane or included in the inner cavity), confocal microscopy experiments showed that targeted liposomes are taken up much more avidly than non-targeted vesicles. In vivo studies showed that glutamine-functionalized and non-functionalized liposomes accumulate in the tumor region to a similar extent. Confocal images of the excised tumor showed extensive co-localization of liposomes and macrophages in both cases. It is suggested that the loss of tumor specificity, shown by Gln-functionalized liposomes in vivo, has to be associated with the efficient removal of liposomes operated by the RES (reticulo endoplasmatic system) or tumor associated macrophages.

Tumor microvasculature observed using different contrast agents: a comparison between Gd-DTPA-Albumin and B-22956/1 in an experimental model of mammary carcinoma.

OBJECTIVE: The aim of this study was to compare a pure macromolecular contrast agent (Gd-DTPA-albumin) with a new protein-binding blood pool contrast agent (B22956/1) in terms of their capacity to investigate the microvasculature in an experimental model of mammary carcinoma. MATERIALS AND METHODS: Tumors were induced by subcutaneous injection of 5 x 10(5) BB1 cells into the backs of 5-7 week-old female FVB/neuNT233 mice. The animals were observed using DCE-MRI when the longest diameter of the tumor was 10.2+/-2.0 mm. DCE-MRI experiments were carried out using B22956/1 and (24 h later) Gd-DTPA-albumin. RESULTS: DCE-MRI data showed that vasculature in the tumor rim was characterized by greater fractional plasma volume and transendothelial permeability than vasculature in the tumor core as measured by both contrast agents. Permeability to Gd-DTPA-albumin in the tumor core was hardly measurable while permeability to B22956/1 was substantial. Histologically the tumor core showed areas of well vascularized, viable tissue surrounded by necrotic regions. CONCLUSIONS: DCE-MRI experiments performed with B22956/1 are useful in the investigation of vasculature in those tumor regions that are characterized by low permeability to macromolecules.

Dose-related effects of repeated ETC-216 (recombinant apolipoprotein A-I Milano/1-palmitoyl-2-oleoyl phosphatidylcholine complexes) administrations on rabbit lipid-rich soft plaques: in vivo assessment by intravascular ultrasound and magnetic resonance imaging.

OBJECTIVES: This study sought to evaluate in vivo the minimal dose of apolipoprotein (apo) A-I(Milano) phospholipid complex (recombinant apoA-I(Milano) and 1-palmitoyl-2-oleoyl phosphatidylcholine complexes [ETC-216]) able to induce atherosclerosis regression in a rabbit model of lipid-rich plaques. BACKGROUND: A single high dose of recombinant apoA-I(Milano) has promoted atherosclerosis regression in animal models. More recently, regression of atherosclerosis was achieved in coronary patients by repeated infusions of ETC-216. METHODS: Thirty-six rabbits underwent perivascular injury at both carotid arteries, followed by a 1.5% cholesterol diet. After 90 days, rabbits were randomly divided into 6 groups and treated 5 times with vehicle or ETC-216 at 5, 10, 20, 40, or 150 mg/kg dose every 4 days. Carotid plaque changes were evaluated in vivo by intravascular ultrasound (IVUS) and magnetic resonance imaging (MRI), performed before and at the end of treatments. Magnetic resonance imaging scans were also recorded after administration of the second dose for rabbits infused with vehicle 40 or 150 mg/kg. RESULTS: Atheroma volume in vehicle-treated rabbits increased dramatically between the first and the second IVUS analyses (+26.53%), whereas in ETC-216-treated animals, a reduced progression at the lower doses and a significant regression at the higher doses, up to -6.83%, was detected. Results obtained by MRI analysis correlated significantly with those at IVUS (r = 0.706; p < 0.0001). The MRI evaluations after the second infusion established that a significant regression was achieved with only 2 administrations of the highest dose. CONCLUSIONS: These results confirm the efficacy of ETC-216 for atherosclerosis treatment and provide guidance for dose selection and frequency to obtain a significant reduction of plaque volume.