Search

Glioblastoma (GBM) is the most invasive and deadliest brain cancer in adults. Its inherent heterogeneity has been designated as the main cause of treatment failure. Thus, a deeper understanding of the diversity that shapes GBM pathobiology is of utmost importance. Single-cell RNA sequencing (scRNA-seq) technologies have begun to uncover the hidden composition of complex tumor ecosystems. Herein, a semi-systematic search of reference literature databases provided all existing publications using scRNA-seq for the investigation of human GBM. We compared and discussed findings from these works to build a more robust and unified knowledge base. All aspects ranging from inter-patient heterogeneity to intra-tumoral organization, cancer stem cell diversity, clonal mosaicism, and the tumor microenvironment (TME) are comprehensively covered in this report. Tumor composition not only differs across patients but also involves a great extent of heterogeneity within itself. Spatial and cellular heterogeneity can reveal tumor evolution dynamics. In addition, the discovery of distinct cell phenotypes might lead to the development of targeted treatment approaches. In conclusion, scRNA-seq expands our knowledge of GBM heterogeneity and helps to unravel putative therapeutic targets. post-print 4967 KB

Background: The incidence of community and nosocomial candidiasis has dramatically increased in the last two decades. There are multiple treatments for this infection, but the toxicity of some and the induction of resistant strains require the development of new compounds. Objectives: With the aim of reducing the Candida population in the oropharyngeal cavity, we have formulated a toothpaste with VG-01 agent, composed of a mixture of carnosic acid (CA) and propolis (PP). Methods: We investigated the ability of VG-01 toothpaste to minimize and stabilize fungal presence in 21 patients diagnosed with clinical oropharyngeal candidiasis. Results: Our data indicate that VG-01 toothpaste showed an effect not only against the most frequent species of Candida, C. albicans, but also in the other species analyzed. 82% of patients stated that they would continue using it outside the study. Conclusion: Our data demonstrate that VG-01, composed of CA and PP is a potential antimycotic agent effective against the most common species that cause oropharyngeal candidiasis present in clinical practice.

Nanoparticle (NP)-based applications are becoming increasingly important in the biomedical field. However, understanding the interactions of NPs with biofluids and cells is a major issue in order to develop novel approaches aimed at boosting their internalization and, therefore, their translation into the clinic. To this end, we put forward a transport mathematical model to describe the spatio-temporal dynamics of iron oxide NPs and their interaction with cells under moderate centrifugation. Our numerical simulations allowed us to quantify the relevance of the flux density as one of the unavoidable key features driving NPs interaction with the media as well as for cell internalization processes. These findings will help to increase the efficiency of cell labelling for biomedical applications.

The diagnosis of glioma is mainly based on imaging methods that do not distinguish between stage and subtype prior to histopathological analysis. Patients with gliomas are generally diagnosed in the symptomatic stage of the disease. Additionally, healing scar tissue may be mistakenly identified based on magnetic resonance imaging (MRI) as a false positive tumor recurrence in postoperative patients. Current knowledge of molecular alterations underlying gliomagenesis and identification of tumoral biomarkers allow for their use as discriminators of the state of the organism. Moreover, a multiomics approach provides the greatest spectrum and the ability to track physiological changes and can serve as a minimally invasive method for diagnosing asymptomatic gliomas, preceding surgery and allowing for the initiation of prophylactic treatment. It is important to create a vast biomarker library for adults and pediatric patients due to their metabolic differences. This review focuses on the most promising proteomic, metabolomic and lipidomic glioma biomarkers, their pathways, the interactions, and correlations that can be considered characteristic of tumor grade or specific subtype. post-print 2427 KB

Transcriptional and proteomic profiling of individual cells have revolutionized interpretation of biological phenomena by providing cellular landscapes of healthy and diseased tissues1,2. These approaches, however, do not describe dynamic scenarios in which cells continuously change their biochemical properties and downstream 'behavioural' outputs3-5. Here we used 4D live imaging to record tens to hundreds of morpho-kinetic parameters describing the dynamics of individual leukocytes at sites of active inflammation. By analysing more than 100,000 reconstructions of cell shapes and tracks over time, we obtained behavioural descriptors of individual cells and used these high-dimensional datasets to build behavioural landscapes. These landscapes recognized leukocyte identities in the inflamed skin and trachea, and uncovered a continuum of neutrophil states inside blood vessels, including a large, sessile state that was embraced by the underlying endothelium and associated with pathogenic inflammation. Behavioural screening in 24 mouse mutants identified the kinase Fgr as a driver of this pathogenic state, and interference with Fgr protected mice from inflammatory injury. Thus, behavioural landscapes report distinct properties of dynamic environments at high cellular resolution. This study was supported by RTI2018-095497-B-I00 from Ministerio de Ciencia e Innovación (MCIN), HR17_00527 from Fundación La Caixa, Transatlantic Network of Excellence (TNE-18CVD04) from the Leducq Foundation, and FET-OPEN (no. 861878) from the European Commission to A.H. M.P-S. is supported by a Federation of European Biochemical Societies and the EMBO ALTF (no. 1142-2020) long-term fellowships. J.S. is supported by a fellowship (PRE2019-089130) from MICINN and A.A.-C. is supported by fellowship CF/BQ/ DR19/11740022 from La Caixa Foundation. J.L.Y.L. was supported by A*STAR and a Juan de la Cierva JCI-2017-33136 Fellowship from MICINN. S.D.C. is a recipient of a Marie Sklodowska-Curie fellowship (749731). M.G. is supported by SAF2017-89116R-P from MCIN and HR18_00120 from la Fundación La Caixa. T.R.M. is supported by grant NIH AI163223, L.G.N. is supported by SIgN core funding from A*STAR, and G.F.C. is supported by MCIN/ AEI/10.13039/501100011033 (grant PID2019-110895RB-I00) and by Junta de Comunidades de Castilla-La Mancha (SBPLY/19/180501/000211). F.S.-C. is supported by MCIN (grant RTI2018- 102084-B-I00), O.S. is supported by the Leducq Foundation (TNE-18CVD04), F.D.-d.-M. is supported by MCIN (TEC2017-84395-P), and T.E.S. is supported by the National Cancer Institute, NIH grant CA233576. The CNIC is supported by the MCIN and the Pro-CNIC Foundation. Sí

Background Aberrant metabolism is recognized as a hallmark of cancer, a pillar necessary for cellular proliferation. Regarding bioenergetics (ATP generation), most cancers display a preference not only toward aerobic glycolysis (“Warburg effect”) and glutaminolysis (mitochondrial substrate level-phosphorylation) but also toward other metabolites such as lactate, pyruvate, and fat-derived sources. These secondary metabolites can assist in proliferation but cannot fully cover ATP demands. Scope of review The concept of a static metabolic profile is challenged by instances of heterogeneity and flexibility to meet fuel/anaplerotic demands. Although metabolic therapies are a promising tool to improve therapeutic outcomes, either via pharmacological targets or press-pulse interventions, metabolic plasticity is rarely considered. Lack of bioenergetic analysis in vitro and patient-derived models is hindering translational potential. Here, we review the bioenergetics of cancer and propose a simple analysis of major metabolic pathways, encompassing both affordable and advanced techniques. A comprehensive compendium of Seahorse XF bioenergetic measurements is presented for the first time. Major conclusions Standardization of principal readouts might help researchers to collect a complete metabolic picture of cancer using the most appropriate methods depending on the sample of interest., Highlights • Normal and cancer cells differ in substrate level phosphorylation and mitochondrial rewiring of ATP-generating pathways. • Glucose and glutamine are two major fuels for tumor growth. Their allocation is dependent on mitochondrial function. • Secondary metabolites assist in proliferation but cannot fully cover ATP demands. • Metabolic therapy is a promising cancer management strategy, but standardization and patient stratification is required. • Bioenergetics can be assessed in vitro using bench top and integrated solutions and in vivo via metabolic imaging.

Interferon (IFN), the first ever-described cytokine, has a potent activity against viruses. Soon since its discovery, quantification of IFN has been an important issue. Most of the traditional methods to measure IFN biological activity rely on indirect methods that quantify dyes retained by IFN-protected cells against a lytic virus, or by techniques that indirectly quantify viral replication by measuring the expression level of viral-encoded reporter proteins such as the green fluorescent protein (GFP). In both cases, the IFN units are determined by the quantification of an effective dose 50, defined as the IFN dose that prevents 50% cell death of 50% reduction of the maximal amount of GFP intensity. In this study we propose the use of an alternative approach to measure IFN activity by calculating the minimal IFN dose 50 as the amount of IFN able to completely protect 50% of the cells from infection measured by the total absence of virus-dependent GFP signal in a cell culture plate. This sensitive approach could be used to easily quantify the Z value to determine IFN bioassay robustness. We believe that this approximation could be interesting to be considered by the IFN community.

Objective The aim of our study was to evaluate the usefulness of cortical-subcortical intraoperative brain mapping (ioBM) in resective awake surgery of low-grade gliomas (LGG) of the right non-dominant hemisphere (RndH). It was estimated how ioBM may affect both the extent of resection and postoperative outcome of language, spatial cognition, social cognition, and executive functions including attention and working memory. Patients and Methods : Fifteen patients that underwent ioBM in resective awake surgery of LGG located on the RndH, were included. A cohort of 15 patients with the same tumour location operated under general anaesthesia without brain mapping was used as control. Specific intraoperative tasks for each location were carried out and results registered. Neuropsychological assessment was performed preoperatively and at 6 months after surgery. Results In the group of patients operated by using ioBM in awake surgery, an 86.66 % mean of resection was obtained compared to 60.33 % in the control group. Speech arrest and incorrect naming responses were elicited in higher proportion in frontal and insular locations. Parietal stimulation associated higher number of incorrect responses in social cognition task. Parietal and temporal stimulation were more frequently associated with incorrect performance of spatial cognition task. Parietal stimulation associated with higher frequency incorrect execution of attention and working memory tasks. After comparing clinical and neuropsychological results in both cohorts, worst outcome at 6 months was observed in the group of patients operated under general anaesthesia without brain mapping, especially in parietal and insular locations. Conclusions Intraoperative identification of language, cognitive functions, and social cognition of RndH by means of ioBM, can be of paramount importance in improving the extent of resection of low-grade gliomas and positively affects clinical and neuropsychological outcome at six months.

Iron oxide nanoparticles (IONPs) are suitable materials for contrast enhancement in magnetic resonance imaging (MRI). Their potential clinical applications range from diagnosis to therapy and follow‐up treatments. However, a deeper understanding of the interaction between IONPs, culture media and cells is necessary for expanding the application of this technology to different types of cancer therapies. To achieve new insights of these interactions, a set of IONPs were prepared with the same inorganic core and five distinct coatings, to study their aggregation and interactions in different physiological media, as well as their cell labelling efficiency. Then, a second set of IONPs, with six different core sizes and the same coating, were used to study how the core size affects cell labelling and MRI in vitro. Here, IONPs suspended in biological media experience a partial removal of the coating and adhesion of molecules. The FBS concentration alters the labelling of all types of IONPs and hydrodynamic sizes ≥ 300 nm provide the greatest labelling using the centrifugation‐mediated internalization (CMI). The best contrast for MRI results requires a core size range between 12–14 nm coated with dimercaptosuccinic acid (DMSA) producing R2* values of 393.7 s−1 and 428.3 s−1, respectively. These findings will help to bring IONPs as negative contrast agents into clinical settings. post-print 2.788 KB

Background Well-designed studies assessing the treatment outcome of brain arteriovenous malformations (AVMs) are infrequent and have not consistently included all of the available treatment modalities, making their results not completely generalizable. Moreover, the predictors of poor outcome are not well defined. Methods We performed an observational retrospective study of AVM patients. We included patients with clinical, radiologic, and outcome data, with a minimum follow-up of 1 year. Neurologic outcome was documented using the modified Rankin Scale (mRS) at the AVM diagnosis and 30 days after the treatment. Results There were 117 patients, with equal male/female proportion. The mean follow-up time was 51 months. Treatment distribution in the Spetzler–Martin grades I–III was as follows: 52 (54.6%) surgery, 31 (32.35%) radiosurgery, 2 (0.02%) embolization, and 11 (12%) conservative follow-up. Treatment distribution in Spetzler–Martin grades IV and V was as follows: 4 (20%) surgery, 7 (35%) radiosurgery, and 10 (45%) conservative follow-up. Poor neurologic outcome (mRS ≥ 3) was significantly associated with poor clinical status at diagnosis (Glasgow Coma Scale [GCS] score< 14; odds ratio [OR]: 0.20; 95% confidence interval [CI]: 0.001–0.396; p = 0.010). The rupture of the AVM was associated with poor neurologic outcome. The Lawton–Young Supplementary scale (LYSS) proved to be the most effective in predicting poor outcome. The existence of seizures, treatment-related complications, and conservative treatment was associated with the worsening of the mRS score, whereas the existence of hemorrhage was associated with the likelihood of disability. Conclusion Our results suggest that poor neurologic status at diagnosis, AVM rupture, and conservative treatment were associated with worse outcome. Hemorrhage as initial presentation is related to disability, not with mRS worsening. The LYSS appeared to be the best method to predict outcome.

Simpson grading of resection has been used as a predictor of intracranial meningioma (IM) recurrence. Histopathological findings, like the Ki-67/MIB-1 labeling index, may be useful in the assessment risk of recurrence. Our objective was to analyze the predictive value of meningioma recurrence using both parameters. We retrospectively studied 322 consecutive patients with histopathological diagnosis of IM WHO grade I and 43 patients with IM WHO grade II in a 13-year period. Multivariate survival analysis was performed. In the WHO grade I IM group, recurrence was observed in 28 patients (8.69%). The Cox regression model for WHO grade I IM, provided a significative hazard ratio (HR) for Ki-67/MIB-1 index ≥3 (HR = 36.35, p 0.001) and Simpson's grading resection, grade II (HR = 2.03, p = 0.045), grade III (HR = 3.41, p = 0.034) and grade IV (HR = 19.75, p ≥ 0.001). In the WHO grade II IM group, recurrence was observed in 10 patients (23.25%). The Cox regression model for WHO grade II IM, provided a significative hazard ratio (HR) for Ki-67/MIB-1 index ≥3% (HR = 1.66, p 0.001) and Simpson's grading resection grade III (HR = 3.96, p = 0.027). The Kaplan-Meier survival curve showed a similar distribution of survival between WHO grade I IM with Ki-67/MIB-1 ≥3% and WHO grade II IM. In WHO grade I meningiomas, the Ki-67/MIB-1 index and Simpson grading were both independent predictors of recurrence. A similar management protocol should be advisable for WHO grade I with Ki-67/MIB-1 ≥3% and WHO grade II meningiomas.

Glioblastoma multiforme is one of the most malignant types of cancer. This is mainly due to a cell subpopulation with an extremely aggressive potential, called glioblastoma stem-like cells (GSCs). These cells produce high levels of extracellular adenosine which has been associated with increased chemoresistance, migration, and invasion in glioblastoma. In this study, we attempted to elucidate the mechanisms that control extracellular adenosine levels in GSC subtypes. By using primary and U87MG-derived GSCs, we associated increased extracellular adenosine with the mesenchymal phenotype. [3H]-adenosine uptake occurred mainly through the equilibrative nucleoside transporters (ENTs) in GSCs, but mesenchymal GSCs have lower expression and ENT1-mediated uptake activity than proneural GSCs. By analyzing expression and enzymatic activity, we determined that ecto-5&prime, nucleotidase (CD73) is predominantly expressed in proneural GSCs, driving AMPase activity. While in mesenchymal GSCs, both CD73 and Prostatic Acid Phosphatase (PAP) contribute to the AMP (adenosine monophosphate) hydrolysis. We did not observe significant differences between the expression of proteins involved in the metabolization of adenosine among the GCSs subtypes. In conclusion, the lower expression and activity of the ENT1 transporter in mesenchymal GSCs contributes to the high level of extracellular adenosine that these GSCs present.

Background Complete resection of symptomatic supratentorial cavernoma (SCA) and removal of the surrounding gliotic area is recommended to minimize the risk of persistent seizures or (re)bleeding. Surgery of SCA located in an eloquent area, can carry out severe postoperative neurological morbidity. We report a study aimed to assess feasibility, extent of resection and outcome after surgical removal of CA by cortico-subcortical intraoperative brain stimulation (ioBS) in the awake patient. Methods Six patients diagnosed of symptomatic SCA located on an eloquent area and operated on while awake under local anaesthesia ioBS, were included. Preoperative planning included neuropsychologic assessment of language-related functions, sociocognitive functions and executive functions. Intraoperatively, we recorded the results achieved in the planned neuropsychological tasks when stimulation was applied (cortical and subcortical). Postoperative control 3D MRI was scheduled at 1 month after surgery to calculate extent of resection. Neuropsychological assessment at 6 months after surgery was performed in all cases. Results Six patients (5 females, 1 male) aged 24–48 years were included in our study. Locations of the lesions were right insular (n = 1), left insular (n = 1), left temporo-insular (n = 1), left temporal (n = 2) and left frontal (n = 1). In all patients, positive findings were obtained during ioBS. In 5 patients, complete surgical resection was achieved. Two patients had postoperative transient neurological deficits, one case of hemiparesis, one case of dysnomia, both cleared over a 6-month period. Clinical follow-up revealed that all patients experienced complete recovery from preoperative symptoms within a year and five patients with seizures showed marked improvement and eventually quit antiepileptic drugs. Neuropsychological assessment at 6 months provided normal results compared to preoperative baseline in all domains. Conclusions Our study suggests that ioBS in the awake surgery of symptomatic SCA located in eloquent areas, allows to increase the rate of complete resection, minimizing postoperative neurological and neuropsychological deficit, and improving postoperative seizures control.

Molecular classification of glioblastoma has enabled a deeper understanding of the disease. The four-subtype model (including Proneural, Classical, Mesenchymal and Neural) has been replaced by a model that discards the Neural subtype, found to be associated with samples with a high content of normal tissue. These samples can be misclassified preventing biological and clinical insights into the different tumor subtypes from coming to light. In this work, we present a model that tackles both the molecular classification of samples and discrimination of those with a high content of normal cells. We performed a transcriptomic in silico analysis on glioblastoma (GBM) samples (n = 810) and tested different criteria to optimize the number of genes needed for molecular classification. We used gene expression of normal brain samples (n = 555) to design an additional gene signature to detect samples with a high normal tissue content. Microdissection samples of different structures within GBM (n = 122) have been used to validate the final model. Finally, the model was tested in a cohort of 43 patients and confirmed by histology. Based on the expression of 20 genes, our model is able to discriminate samples with a high content of normal tissue and to classify the remaining ones. We have shown that taking into consideration normal cells can prevent errors in the classification and the subsequent misinterpretation of the results. Moreover, considering only samples with a low content of normal cells, we found an association between the complexity of the samples and survival for the three molecular subtypes., The Fondo de Investigaciones Sanitarias (FIS) (PI17-01489); the Miguel Servet Program (CP11/00147); del Instituto de Salud Carlos III (AAS) and the Ministerio de Economía y Competitividad–FEDERER (RTC-2016-4990-1); Convocatoria de ayudas para la contratación de investigadores predoctorales e investigadores postdoctorales cofinanciadas por Fondo Social Europeo a través del Programa Operativo de Empleo Juvenil y la Iniciativa de Empleo Juvenil (YEI) to R.M; Instituto de Salud Carlos III, Ministerio de Innovación y Ciencia de España (PT17/0015/0042) to the HGM BioBank, integrated in National Network Biobanks.

Glioblastoma (GBM) is the most aggressive and frequent primary brain tumor in adults with a median overall survival of 15 months. Tumor recurrence and poor prognosis are related to cancer stem cells (CSCs), which drive resistance to therapies. A common characteristic in GBM is CDKN2A gene loss, located close to the cluster of type I IFN genes at Ch9p21. Newcastle disease virus (NDV) is an avian paramyxovirus with oncolytic and immunostimulatory properties that has been proposed for the treatment of GBM. We have analyzed the CDKN2A-IFN I gene cluster in 1018 glioma tumors and evaluated the NDV oncolytic effect in six GBM CSCs ex vivo and in a mouse model. Our results indicate that more than 50% of GBM patients have some IFN deletion. Moreover, GBM susceptibility to NDV is dependent on the loss of the type I IFN. Infection of GBM with an NDV-expressing influenza virus NS1 protein can overcome the resistance to oncolysis by NDV of type I-competent cells. These results highlight the potential of using NDV vectors in antitumor therapies. This research was funded by grants from the “Fondo de Investigaciones Sanitarias” (FIS) (PI17-01489), the Miguel Servet Program (CP11/00147) del Instituto de Salud Carlos III (AAS), the Ministerio de Economía y Competitividad–FEDERER (RTC-2016-4990-1). This work was also partly supported by NIH grant R01CA229818 to AG-S.I.P.-A., was supported by “Ayudas destinadas a la realización de doctorados industriales CAM (IND2019/BMD17222)”. The authors acknowledge the FPI fellowship by Universidad San Pablo CEU to S.E.-R. and S.R.-R. This work has been supported by Proyectos Fundación CEU–San Pablo Banco Santander Grant PPC16/2015 and Consejería de Educación e Investigación de la Comunidad de Madrid, Project NIETO-CM B2017/BMD-3731 (E.N.-V.). Sí

Glioblastoma (GBM) is one of the most aggressive and vascularized brain tumors in adults, with a median survival of 20.9 months. In newly diagnosed and recurrent GBM, bevacizumab demonstrated an increase in progression-free survival, but not in overall survival. We conducted an in silico analysis of VEGF expression, in a cohort of 1082 glioma patients. Then, to determine whether appropriate bevacizumab dose adjustment could increase the anti-angiogenic response, we used in vitro and in vivo GBM models. Additionally, we analyzed VEGFA expression in tissue, serum, and plasma in a cohort of GBM patients before and during bevacizumab treatment. We identified that 20% of primary GBM did not express VEGFA suggesting that these patients would probably not respond to bevacizumab therapy as we proved in vitro and in vivo. We found that a specific dose of bevacizumab calculated based on VEGFA expression levels increases the response to treatment in cell culture and serum samples from mice bearing GBM tumors. Additionally, in a cohort of GBM patients, we observed a correlation of VEGFA levels in serum, but not in plasma, with bevacizumab treatment performance. Our data suggest that bevacizumab dose adjustment could improve clinical outcomes in Glioblastoma treatment. This work was supported by grants from the “Fondo de Investigaciones Sanitarias” (FIS) (PI17-01489), the Miguel Servet Program (CP11/00147) del Instituto de Salud Carlos III (AAS), and the Ministerio de Economía y Competitividad–FEDERER (RTC-2016-4990-1). IPA was supported by “Ayudas para la contratación de ayudantes de investigación cofinanciadas por el Fondo Social Europeo a través del Programa Operativo de Empleo Juvenil y la Iniciativa de Empleo Juvenil (YEI),” and SER was supported by FPI-CEU predoctoral fellowship. Sí

Gliomas that express the mutated isoforms of isocitrate dehydrogenase 1/2 (IDH1/2) have better prognosis than wild-type (wt) IDH1/2 gliomas. However, how these mutant (mut) proteins affect the tumor microenvironment is still a pending question. Here, we describe that the transcription of microtubule-associated protein TAU (MAPT), a gene that has been classically associated with neurodegenerative diseases, is epigenetically controlled by the balance between wt and mut IDH1/2 in mouse and human gliomas. In IDH1/2 mut tumors, we found high expression of TAU that decreased with tumor progression. Furthermore, MAPT was almost absent from tumors with epidermal growth factor receptor (EGFR) mutations, whereas its trancription negatively correlated with overall survival in gliomas carrying wt or amplified (amp) EGFR. We demonstrated that the overexpression of TAU, through the stabilization of microtubules, impaired the mesenchymal/pericyte-like transformation of glioma cells by blocking EGFR, nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) and the transcriptional coactivator with PDZ-binding motif (TAZ). Our data also showed that mut EGFR induced a constitutive activation of this pathway, which was no longer sensitive to TAU. By inhibiting the transdifferentiation capacity of EGFRamp/wt tumor cells, TAU protein inhibited angiogenesis and favored vascular normalization, decreasing glioma aggressiveness and increasing their sensitivity to chemotherapy., This work was supported by NIH/ NINDS grants R01-NS105556 and R37-ND094804 to M.G.C.; by Ministerio de Economía y Competitividad (Acción Estratégica en Salud) grants PI13/01258 to A.H.-L., PI17/01489 and CP11/00147 to A.A.-S., PI18/00263 to R.G.-E., and PI16/01 278 to J.S.; by “Asociación Española contra el Cancer” grants Investigador Junior to R.G. and GCTRA16015SEDA to J.M.S.-S. and J.S.; and by Ministerio de Economía y Competitividad SAF-2014-53040-P to J.A., RTC-2015-3771-1 to J.S., and SAF2015-65175-R/FEDER to P.S.-G

Pediatric Central Nervous System (CNS) tumors are the most fatal cancer diseases in childhood. Due to their localization and infiltrative nature, some tumor resections or biopsies are not feasible. In those cases, the use of minimally invasive methods as diagnostic, molecular marker detection, prognostic or monitoring therapies are emerging. The analysis of liquid biopsies which contain genetic information from the tumor has been much more widely explored in adults than in children. We compare the detection of BRAF V600E targetable mutation by digital-PCR from cell-free-DNA and EV-derived DNA (ctDNA) in serum, plasma and cerebrospinal fluid (CSF) isolated from a cohort of 29 CNS pediatric patients. Here we demonstrate that ctDNA isolated from serum and plasma could be successfully analyzed to obtain tumor genetic information which could be used to guide critical treatment decisions.

Liquid biopsy is becoming a new source of biomarkers that complement and resolve some of the most important limitations of surgical biopsy, which are the accessibility to the diseased tissue and its heterogeneity, especially relevant for tumors. The diseased tissues release their molecule content to the bloodstream in free form, inside a cell or within extracellular vesicles (EVs). While the identification of molecular alterations in total DNA isolated from peripheral blood is already in use for some tumors that secrete large amounts of DNA, it is challenging to assay those secreting lower amounts of molecules as well as for many other non-tumoral pathologies like immunological and cardiovascular diseases. In this scenery, the compartment of diseased tissue-derived EVs will be one of the best alternatives for the detection and identification of current and new biomarkers and targets in the clinical management of these diseases. Here, we review the mechanisms of molecular internalization as well as the correlation of EV's cargo with clinical parameters in tumor and non-tumor diseases, with special emphasis in clinical application.

Posterior third ventricle ependymomas with intraaqueductal extension are relatively infrequent lesions. Its surgical management represents a formidable technical challenge and includes a wide variety of approaches. Minimally invasive surgery including the endoscopic management can play a crucial role to obtain an optimal clinical outcome. We report the clinical outcome of an 11-year-old female patient with a 6-year history of recurrent episodes of headache and vomiting. On brain MRI a posterior third ventricle lesion with extension to the aqueduct of Sylvius and fourth ventricle, and associated hydrocephalus was observed. Our management of the lesion included a two-step endoscopic surgery: first an anterior third ventriculostomy and biopsy of the lesion that was reported to be a low-grade ependymoma, and posteriorly an endoscopic-assisted resection of the lesion. Clinical outcome was optimal without neurological sequelae. The postoperative MRI showed a thickened ependymal area on the tumor base of implantation. It was considered to be a remnant of the lesion and subsequently treated with radiotherapy. Posterior third ventricle ependymomas with intraaqueductal extension can be endoscopically managed to obtain a successful outcome.

SUMMARYClassification of gliomas as wild-type or mutantIDH1/2tumors has profound clinical implications. However, how these two groups of gliomas progress, in a microenvironment-dependent manner, is still a pending question. Here we describe that the expression of Tau is epigenetically induced by mutant IDH1/2, whereas is almost absent from tumors withEGFR/PTENmutations. Moreover,Tau (MAPT)expression is inversely correlated with overall survival inEGFR-amplified gliomas. Using orthotopicEGFR-related models, we have observed that Tau overexpression or microtubule stabilizers impair the mesenchymal transformation of glioma cells, with profound changes in tumor vasculature and a significant decrease in tumor burden. However, epithelial-to-mesenchymal transformedEGFR-mutant cells, acting as pericytes, induce neo-vasculogenesis and favor aggressive glioma growth, a process that is no longer sensitive to Tau. Altogether our data indicate that the genomic background controls glioma aggressiveness by modifying the vascular microenvironment.GRAPHICAL ABSTRACT

Recent advances in pharmacological immune modulation against tumor cells has dramatically changed the paradigm of cancer treatment. Checkpoint inhibitor therapy is a form of cancer immunotherapy already in clinical setting but also under active basic and clinical investigation. Nevertheless, some patients are primary unresponsive or develop ulterior resistance to these family of drugs. This review aims to update the basic molecular mechanism of resistance as well as the current strategies for checkpoint inhibitor selection in order to propose new approaches to individualize the use of these novel therapies.

BACKGROUND: Extracellular vesicles (EVs) are small membrane-bound vesicles which play an important role in cell-to-cell communication. Their molecular cargo analysis is presented as a new source for biomarker detection, and it might provide an alternative to traditional solid biopsies. However, the most effective approach for EV isolation is not yet well established. RESULTS: Here, we study the efficiency of the most common EV isolation methods-ultracentrifugation, Polyethlyene glycol and two commercial kits, Exoquick® and PureExo®. We isolated circulating EVs from the bloodstream of healthy donors, characterized the size and yield of EVs and analyzed their protein profiles and concentration. Moreover, we have used for the first time Digital-PCR to identify and detect specific gDNA sequences, which has several implications for diagnostic and monitoring many types of diseases. CONCLUSIONS: Our findings present Polyethylene glycol precipitation as the most feasible and less cost-consuming EV isolation technique. We are grateful for the financial support from the ‘Fondo de Investigaciones Sanitarias’ (FIS) (PI14_00077), the Miguel Servet Program (CP11/00147) del Instituto de Salud Carlos III (AAS), and the Ministerio de Economía y Competi‑tividad–FEDERER (RTC‑2016‑4990‑1, RTC‑2015‑3846‑1). SER was supported by FPI‑CEU predoctoral fellowship. Sí

Glioblastoma (GBM) is the most aggressive primary brain tumor, with a median survival at diagnosis of 16–20 months. Metabolism represents a new attractive therapeutic target, however, due to high intratumoral heterogeneity, the application of metabolic drugs in GBM is challenging. We characterized the basal bioenergetic metabolism and antiproliferative potential of metformin (MF), dichloroacetate (DCA), sodium oxamate (SOD) and diazo-5-oxo-L-norleucine (DON) in three distinct glioma stem cells (GSCs) (GBM18, GBM27, GBM38), as well as U87MG. GBM27, a highly oxidative cell line, was the most resistant to all treatments, except DON. GBM18 and GBM38, Warburg-like GSCs, were sensitive to MF and DCA, respectively. Resistance to DON was not correlated with basal metabolic phenotypes. In combinatory experiments, radiomimetic bleomycin exhibited therapeutically relevant synergistic effects with MF, DCA and DON in GBM27 and DON in all other cell lines. MF and DCA shifted the metabolism of treated cells towards glycolysis or oxidation, respectively. DON consistently decreased total ATP production. Our study highlights the need for a better characterization of GBM from a metabolic perspective. Metabolic therapy should focus on both glycolytic and oxidative subpopulations of GSCs.

The elucidation of mechanisms involved in resistance to therapies is essential to improve the survival of patients with malignant gliomas. A major feature possessed by glioma cells that may aid their ability to survive therapy and reconstitute tumors is the capacity for self-renewal. We show here that glioma stem cells (GSCs) express low levels of MKP1, a dual-specificity phosphatase, which acts as a negative inhibitor of JNK, ERK1/2, and p38 MAPK, while induction of high levels of MKP1 expression are associated with differentiation of GSC. Notably, we find that high levels of MKP1 correlate with a subset of glioblastoma patients with better prognosis and overall increased survival. Gain of expression studies demonstrated that elevated MKP1 impairs self-renewal and induces differentiation of GSCs while reducing tumorigenesis in vivo. Moreover, we identified that MKP1 is epigenetically regulated and that it mediates the anti-tumor activity of histone deacetylase inhibitors (HDACIs) alone or in combination with temozolomide. In summary, this study identifies MKP1 as a key modulator of the interplay between GSC self-renewal and differentiation and provides evidence that the activation of MKP1, through epigenetic regulation, might be a novel therapeutic strategy to overcome therapy resistance in glioblastoma., O.A. (BFI_2011_195), L.G.-R. (PRE_2013_1_760), L.M.-C. (PRE_2014_1_92), and J.A.-I. (PRE_2016_1_0375) were each recipient of a predoctoral fellowship from the Department of Education, University and Research of the Basque Government, and P.A. from the Spanish Association Against Cancer (AECC Gipuzkoa). This work was supported by grants from the Instituto de Salud Carlos III and FEDER Funds (PI13/02277, PI14/01495, PI15/00186, CP16/00039, PI16/01580, DTS16/084), European Union (Marie Curie CIG 2012/712404, REFBIO13/BIOD/009, and 011), and AICR/WCR [13–1270].

Extracellular vesicles (EVs) have been increasingly recognized as a potential source of disease biomarkers, since they contain a multitude of biologically active protein, DNA and RNA species, and they can be retrieved from circulating blood of patients. Here, we describe a protocol for DNA extraction from exosomes, shedding microvesicles and apoptotic bodies isolated from peripheral blood in a mouse xenograft model of solid tumor. In this model, human DNA isolated from tumor-derived EVs can be readily distinguished from the one of the hosts, which is of particular interest for studies aimed at molecular characterization of tumor biomarkers.

// Noemi Garcia-Romero 1, 2, * , Josefa Carrion-Navarro 1, 3, * , Susana Esteban-Rubio 1, 3, * , Elisa Lazaro-Ibanez 4 , Maria Peris-Celda 5 , Marta M. Alonso 6 , Juan Guzman-De-Villoria 7 , Carlos Fernandez-Carballal 8 , Ana Ortiz de Mendivil 1 , Sara Garcia-Duque 1 , Carmen Escobedo-Lucea 4 , Ricardo Prat-Acin 9 , Cristobal Belda-Iniesta 1, 3 , Angel Ayuso-Sacido 1, 2, 3 1 Fundacion de Investigacion HM Hospitales, HM Hospitales, Madrid, Spain 2 IMDEA Nanoscience, Madrid, Spain 3 Facultad de Medicina (IMMA), Universidad San Pablo-CEU, Madrid, Spain 4 Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland 5 Department of Neurological Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA 6 Clinica Universidad de Navarra, CIMA, Pamplona, Spain 7 Servicio de Radiodiagnostico, Hospital General Universitario Gregorio Maranon, Madrid, Spain 8 Servicio de Neurocirugia, Hospital General Universitario Gregorio Maranon, Madrid, Spain 9 Departamento de Neurocirugia, Hospital Universitario la Fe, Valencia, Spain * These authors have contributed equally to this work Correspondence to: Angel Ayuso-Sacido, email: ayusosacido@gmail.com Keywords: extracellular vesicles, brain tumors, blood-brain barrier, biomarkers Received: September 20, 2016 Accepted: November 07, 2016 Published: November 26, 2016 ABSTRACT Tumor-cell-secreted extracellular vesicles (EVs) can cross the disrupted blood-brain barrier (BBB) into the bloodstream. However, in certain gliomas, the BBB remains intact, which might limit EVs release. To evaluate the ability of tumor-derived EVs to cross the BBB, we used an orthotopic xenotransplant mouse model of human glioma-cancer stem cells featuring an intact BBB. We demonstrated that all types of tumor cells-derived EVs−apoptotic bodies, shedding microvesicles and exosomes−cross the intact BBB and can be detected in the peripheral blood, which provides a minimally invasive method for their detection compared to liquid biopsies obtained from cerebrospinal fluid (CSF). Furthermore, these EVs can be readily distinguished from total murine EVs, since they carry human-specific DNA sequences relevant for GBM biology. In a small cohort of glioma patients, we finally demonstrated that peripheral blood EVs cargo can be successfully used to detect the presence of IDH1 G395A , an essential biomarker in the current management of human glioma

BACKGROUND Extracellular vesicles (EVs) are cell-derived membrane vesicles. EVs contain several RNAs such as mRNA, microRNAs, and ncRNAs, but less is known of their genomic DNA (gDNA) content. It is also unknown whether the DNA cargo is randomly sorted or if it is systematically packed into specific EV subpopulations. The aim of this study was to analyze whether different prostate cancer (PCa) cell-derived EV subpopulations (apoptotic bodies, microvesicles, and exosomes) carry different gDNA fragments. METHODS EV subpopulations were isolated from three PCa cell lines (LNCaP, PC-3, and RC92a/hTERT) and the plasma of PCa patients and healthy donors, and characterized by transmission electron microscopy, nanoparticle tracking analysis and total protein content. gDNA fragments of different genes were detected by real time quantitative PCR and confirmed by DNA sequencing. RESULTS We report that the concentration of EVs was higher in the cancer patients than in the healthy controls. EV subpopulations differed from each other in terms of total protein and DNA content. Analysis of gDNA fragments of MLH1, PTEN, and TP53 genes from the PCa cell-derived EV subpopulations showed that different EVs carried different gDNA content, which could even harbor specific mutations. Altogether, these results suggest that both nucleic acids and proteins are selectively and cell-dependently packed into the EV subtypes. CONCLUSIONS EVs derived from PCa cell lines and human plasma samples contain double-stranded gDNA fragments which could be used to detect specific mutations, making EVs potential biomarkers for cancer diagnostics and prognostics. Prostate 74:1379–1390, 2014. © 2014 The Authors. The Prostate published by Wiley Periodicals, Inc.

Object To determine the effectiveness of fluorescence-guided resection of fronto-basal high grade gliomas by using the supraorbital trans-eyebrow craniotomy. Methods We present a single-institution experience of 6 consecutive patients presenting high grade brain glioma located on the fronto-basal area that were operated through a supraorbital trans-eyebrow craniotomy. Previous to surgery all patients were administered 20 mg/kg of 5 aminolevulic acid so microscopic fluorescence-guided resection could be accomplished. Tumors were located on gyrus rectus (3 patients), medial orbital gyrus (2 patients), and anterior orbital gyrus (1 patient). Results Despite the narrow surgical corridor, fluorescence was useful in all cases. Fluorescence-guided resection allowed inclusion into the margins of resection of areas previously considered as normal under white light. Complete resection was obtained in 5 patients. No neurological postoperative new deficit was observed in this series. All six cases corresponded to glioblastoma. Only one case of superficial infection with delayed wound healing was reported as complication. All patients expressed a high level of satisfaction related to cosmetic result. Conclusions Fluorescence-guided resection of fronto-basal high grade gliomas can be successfully achieved through supraorbital trans-eyebrow craniotomy. Benefits of supraorbital craniotomy in the management of fronto-basal high grade gliomas as well as usefulness of fluorescence-guided resection through a very narrow corridor are exposed.

Nanotechnology plays an increasingly important role in the biomedical arena. Iron oxide nanoparticles (IONPs)-labelled cells is one of the most promising approaches for a fast and reliable evaluation of grafted cells in both preclinical studies and clinical trials. Current procedures to label living cells with IONPs are based on direct incubation or physical approaches based on magnetic or electrical fields, which always display very low cellular uptake efficiencies. Here we show that centrifugation-mediated internalization (CMI) promotes a high uptake of IONPs in glioblastoma tumour cells, just in a few minutes, and via clathrin-independent endocytosis pathway. CMI results in controllable cellular uptake efficiencies at least three orders of magnitude larger than current procedures. Similar trends are found in human mesenchymal stem cells, thereby demonstrating the general feasibility of the methodology, which is easily transferable to any laboratory with great potential for the development of improved biomedical applications.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., Human gliomas harbour cancer stem cells (CSCs) that evolve along the course of the disease, forming highly heterogeneous subpopulations within the tumour mass. These cells possess self-renewal properties and appear to contribute to tumour initiation, metastasis and resistance to therapy. CSC cultures isolated from surgical samples are considered the best preclinical in vitro model for primary human gliomas. However, it is not yet well characterized to which extent their biological and functional properties change during in vitro passaging in the serum-free culture conditions. Here, we demonstrate that our CSC-enriched cultures harboured from one to several CSC clones from the human glioma sample. When xenotransplanted into mouse brain, these cells generated tumours that reproduced at least three different dissemination patterns found in original tumours. Along the passages in culture, CSCs displayed increased expression of stem cell markers, different ratios of chromosomal instability events, and a varied response to drug treatment. Our findings highlight the need for better characterization of CSC-enriched cultures in the context of their evolution in vitro, in order to uncover their full potential as preclinical models in the studies aimed at identifying molecular biomarkers and developing new therapeutic approaches of human gliomas., We are grateful for the financial support from PI10/01069, PI14/00077, and the ‘Miguel Servet Program’ CP11/00147 (AAS), PI-01495 (RP), and PI12/00775) (PSG) from Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spain, supported by FEDER funds, and Ministerio de Economía y Competitividad, Red Temática de Investigación Cooperativa en Cáncer (RTICC) (RD12/0036/0027) (PSG).

We have studied the influence of anisotropic nanopatterns (ripples) on the adhesion and morphology of mouse neural stem cells (C17.2) on glass substrates using cell viability assay, optical microscopy and atomic force microscopy. The ripples were produced by defocused ion beam sputtering with inert Ar ions, which physically remove atoms from the surface at the energy of 800 eV. The ripple periodicity (∼200 nm) is comparable to the thickness of the cytoplasmatic microspikes (filopodia) which link the stem cells to the substrate. All methods show that the cell adhesion is significantly lowered compared to the same type of cells on flat glass surfaces. Furthermore, the AFM analysis reveals that the filopodia tend to be trapped parallel or perpendicular to the ripples, which limits the spreading of the stem cell on the rippled substrate. This opens the perspective of controlling the micro-adhesion of stem cells and the orientation of their filopodia by tuning the anisotropic substrate morphology without chemical reactions occurring at the surface.

Accumulating evidence suggests that a better understanding of normal human brain stem cells and tumor stem cells (TSCs) will have profound implications for treating central nervous system disease during the next decade. Neurosurgeons routinely resect excess surgical tissue containing either normal brain stem cells or TSCs. These cells are immediately available for expansion and use in basic biological assays, animal implantation, and comparative analysis studies. Although normal stem cells have much slower kinetics of expansion than TSCs, they are easily expandable and can be frozen for future use in stem cell banks. This nearly limitless resource holds promise for understanding the basic biology of normal brain stem cells and TSCs, which will likely direct the next major shift in therapeutics for brain tumors, brain and spinal cord injury, and neurodegenerative disease. This report reviews the progress that has been made in harvesting and expanding both normal and tumor-derived stem cells and emphasizes the integral role neurosurgeons will play in moving the neural stem cell field forward.

Nanotechnology plays an increasingly important role in the biomedical arena. Iron oxide nanoparticles (IONPs)-labelled cells is one of the most promising approaches for a fast and reliable evaluation of grafted cells in both preclinical studies and clinical trials. Current procedures to label living cells with IONPs are based on direct incubation or physical approaches based on magnetic or electrical fields, which always display very low cellular uptake efficiencies. Here we show that centrifugation-mediated internalization (CMI) promotes a high uptake of IONPs in glioblastoma tumour cells, just in a few minutes and via clathrin-independent endocytosis pathway. CMI results in controllable cellular uptake efficiencies at least three orders of magnitude larger than current procedures. Similar trends are found in human mesenchymal stem cells, thereby demonstrating the general feasibility of the methodology, which is easily transferable to any laboratory with great potential for the development of improved biomedical applications.

Extracellular vesicles (EVs), including exosomes and microvesicles, carry a variety of bio-macromolecules, including mRNA, microRNA, other non-coding RNAs, proteins and lipids. EVs have emerged as a promising, minimally invasive (liquid biopsies) and novel source of material for molecular diagnostics, and may provide a surrogate to tissue biopsy-based biomarkers for a variety of diseases. Although EVs can be easily identified and collected from biological fluids using commercial kits, further research and proper validation is needed in order for them to be useful in the clinical setting. Currently, several EV-based research and diagnostic companies have developed research-based kits and are in the process of working with clinical laboratories to develop and validate EV-based assays for a variety of diseases. The successful clinical application of EV-based diagnostic assays will require close collaboration between industry, academia, regulatory agencies and access to patient samples. We expect that international, integrative and interdisciplinary translational research teams, along with the emergence of FDA-approved platforms, will set the framework for EV-based diagnostics. We recognize that the EV field offers new promise for personalized/precision medicine and targeted treatment in a variety of diseases. A short course was held as a four-session webinar series in September and October 2014, presented by pioneers and experts in the EV domain, covering a broad range of topics from an overview of the field to its applications, and the current state and challenges of the commercialization of EVs for research and an introduction to the clinic. It was concluded with a panel discussion on the regulatory aspects and funding opportunities in this field. A summary of the short course is presented as a meeting dispatch.

Actinomycetes, one of the major communities of the microbial population present in soil, can also be found inhabiting a wide diversity of ecological sources. We have explored the use of lichens as an alternative source for the isolation of novel actinomycetes. Lichens are symbiotic mixtures of fungi, green algae and/or cyanobacteria and whereas these symbiotic components have been extensively described, the microbial community inhabiting this niche has not been well characterized. We studied the diversity of the actinomycete population isolated from lichens collected in tropical areas from the Hawaii and Reunion islands and in cold areas from Alaska. The diversity of the microbial population was evaluated using fatty acid analysis and molecular fingerprinting. A PCR approach to screen the isolates for genes associated with secondary metabolite production was applied to evaluate the biosynthetic potential of these strains; profiles obtained for each isolate were compared to the antimicrobial activity exhibited by these isolates in laboratory conditions. Our results demonstrate that lichens represent an extremely rich reservoir for the isolation of a wide diversity of actinomycetes many of them representing still today a rich untapped source of secondary metabolites.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., [Objectives]: A number of neurodegenerative diseases progress with a loss of myelin, which makes them candidate diseases for the development of cell-replacement therapies based on mobilisation or isolation of the endogenous neural/glial progenitor cells, in vitro expansion, and further implantation. Cells expressing A2B5 or PDGFRA/CNP have been isolated within the pool of glial progenitor cells in the subcortical white matter of the normal adult human brain, all of which demonstrate glial progenitor features. However, the heterogeneity and differentiation potential of this pool of cells is not yet well established. [Methods]: We used diffusion tensor images, histopathology, and immunostaining analysis to demonstrate normal cytoarchitecture and the absence of abnormalities in human temporal lobe samples from patients with mesial temporal sclerosis. These samples were used to isolate and enrich glial progenitor cells in vitro, and later to detect such cells in vivo. [Results]: We have identified a subpopulation of SOX2+ cells, most of them co-localising with OLIG2, in the white matter of the normal adult human brain in vivo. These cells can be isolated and enriched in vitro, where they proliferate and generate immature (O4+) and mature (MBP+) oligodendrocytes and, to a lesser extent, astrocytes (GFAP+). [Conclusion]: Our results demonstrate the existence of a new glial progenitor cell subpopulation that expresses SOX2 in the white matter of the normal adult human brain. These cells might be of use for tissue regeneration procedures., JOC is a recipient of a predoctoral fellowship from the FPU program (AP2008/02823), Ministry of Education, Spain. This work was supported in part by grants from the Alicia Koplowitz Foundation (AAS, JOC, CEL), the Gent x Gent Foundation (AAS) and Health Research Fund (Fondo de Investigaciones Sanitarias, FIS) from the Carlos III Health Institute (PI10/01069 and CP11/00147) (AAS).

Human adipose stem cells (hASCs) play a crucial role in the fields of regenerative medicine and tissue engineering for different reasons: the abundance of adipose tissue, their easy harvesting, the ability to multipotent differentiation and the fact that they do not trigger allogeneic blood response or secrete cytokines that act as immunosuppressants. The vast majority of protocols use animal origin reagents, with the underlying risk of transmitting infections by non-human pathogens. We have designed a protocol to isolate and maintain the properties of hASCs avoiding xenogeneic reagents. These changes not only preserve hASCs morphology, but also increase cell proliferation and maintain their stem cell marker profile. On the other hand, human serum albumin (HSA), Tryple® and human Serum (HS), do not affect hASCs multipotent differentiation ability. The amendments introduced do not trigger modifications in the transcriptional profile of hASCs, alterations in key biochemical pathways or malignization. Thus, we have proven that it is possible to isolate and maintain hASCs avoiding animal reagents and, at the same time, preserving crucial culture parameters during long term culture. Thereby we have revealed a novel and effective tool for the improvement of clinical, cell-based therapies.