The low affinity A2B adenosine receptor enhances migratory and invasive capacity in vitro and angiogenesis in vivo of glioblastoma stem-like cells.

Glioblastoma (GBM) is the most common and deadly malignant brain tumor, with a median survival of 15 to 17 months for a patient. GBM contains a cellular subpopulation known as GBM stem-like cells (GSCs) that persist in hypoxic niches and are capable of infiltrating into healthy brain tissue. For this reason, GSCs are considered one of the main culprits for GBM recurrence. A hypoxic microenvironment increases extracellular adenosine levels, activating the low affinity A2B adenosine receptor (A2BAR). Adenosine, through A2BAR, is capable of modulating invasiveness. However, its role in the invasion/migration of hypoxic-GSCs is still unknown. This study aims to understand the importance of A2BAR in modulating the migratory/invasive capacity of GSCs under hypoxia. Data analysis from The Cancer Genome Atlas (TCGA) program correlates A2BAR expression with high-grade glioma and hypoxic necrotic areas. U87MG and primary culture-derived GSCs under hypoxic conditions (0.5% O2) increased A2BAR mRNA and protein levels. As expected, the migratory and invasive capacity of GSCs increased under hypoxia, which was counteracted by blocking A2BAR, through the downregulation of MMP9 activity and epithelial-mesenchymal transition marker expression. Finally, in a xenograft mouse model, we demonstrate that treatment with MRS1754 did not affect the tumor volume but could decrease blood vessel formation and VEGF expression. Our results suggest that extracellular adenosine, through the activation of A2BAR, enhances the migratory and invasive capacity of GSCs in vitro under hypoxic conditions. Targeting A2BAR can be an effective therapy for GBM recurrence.

Suppressor of fused associates with dissemination patterns in patients with glioma.

Gliomas are the most common brain tumors, which present poor prognosis, due, in part, to tumor cell migration and infiltration into distant brain areas. However, the underlying mechanisms causing such effects are unknown. Hedgehog (HH)-Gli axis is one of the signaling pathways involved, with a high number of molecular mediators. In this study, we investigated the association between HH-Gli intermediates and clinical parameters. We found that high levels of SuFu are associated with high dissemination patterns in patients with glioma. Therefore, we analyzed SuFu expression data in three glioma cohorts of surgical samples (N =1,759) and modified its expression in Glioblastoma Cancer Stem Cells (GB CSC) in vitro models. Our data reveal that SuFu overexpression increases cancer stemness properties together with a migratory phenotype. This work identifies SuFu as a new molecular player in glioma cell migration and a promising target to develop blocking agents to decrease GB dissemination.

Metabolic therapy and bioenergetic analysis: The missing piece of the puzzle.

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.

Clinical Utility of Liquid Biopsy-Based Actionable Mutations Detected via ddPCR.

Cancer is one of the leading causes of death worldwide and remains a major public health challenge. The introduction of more sensitive and powerful technologies has permitted the appearance of new tumor-specific molecular aberrations with a significant cancer management improvement. Therefore, molecular pathology profiling has become fundamental not only to guide tumor diagnosis and prognosis but also to assist with therapeutic decisions in daily practice. Although tumor biopsies continue to be mandatory in cancer diagnosis and classification, several studies have demonstrated that liquid biopsies could be used as a potential tool for the detection of cancer-specific biomarkers. One of the main advantages is that circulating free DNA (cfDNA) provides information about intra-tumoral heterogeneity, reflecting dynamic changes in tumor burden. This minimally invasive tool has become an accurate and reliable instrument for monitoring cancer genetics. However, implementing liquid biopsies across the clinical practice is still ongoing. The main challenge is to detect genomic alterations at low allele fractions. Droplet digital PCR (ddPCR) is a powerful approach that can overcome this issue due to its high sensitivity and specificity. Here we explore the real-world clinical utility of the liquid biopsy ddPCR assays in the most diagnosed cancer subtypes.

Beyond the Warburg Effect: Oxidative and Glycolytic Phenotypes Coexist within the Metabolic Heterogeneity of Glioblastoma.

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.

Ceramide Composition in Exosomes for Characterization of Glioblastoma Stem-Like Cell Phenotypes.

Glioblastoma (GBM) is one of the most malignant central nervous system tumor types. Comparative analysis of GBM tissues has rendered four major molecular subtypes. From them, two molecular subtypes are mainly found in their glioblastoma cancer stem-like cells (GSCs) derived in vitro: proneural (PN) and mesenchymal (MES) with nodular (MES-N) and semi-nodular (MES-SN) disseminations, which exhibit different metabolic, growth, and malignancy properties. Many studies suggest that cancer cells communicate between them, and the surrounding microenvironment, via exosomes. Identifying molecular markers that allow the specific isolation of GSC-derived exosomes is key in the development of new therapies. However, the differential exosome composition produced by main GSCs remains unknown. The aim of this study was to determine ceramide (Cer) composition, one of the critical lipids in both cells and their cell-derived exosomes, from the main three GSC phenotypes using mass spectrometry-based lipidomics. GSCs from human tissue samples and their cell-derived exosomes were measured using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS) in an untargeted analysis. Complete characterization of the ceramide profile, in both cells and cell-derived exosomes from GSC phenotypes, showed differential distributions among them. Results indicate that such differences of ceramide are chain-length dependent. Significant changes for the C16 Cer and C24:1 Cer and their ratio were observed among GSC phenotypes, being different for cells and their cell-derived exosomes.

Newcastle Disease Virus (NDV) Oncolytic Activity in Human Glioma Tumors Is Dependent on CDKN2A-Type I IFN Gene Cluster Codeletion.

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.

BRAF V600E Detection in Liquid Biopsies from Pediatric Central Nervous System Tumors.

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.

Vesicle-Mediated Control of Cell Function: The Role of Extracellular Matrix and Microenvironment.

Extracellular vesicles (EVs) - including exosomes, microvesicles and apoptotic bodies - have received much scientific attention last decade as mediators of a newly discovered cell-to-cell communication system, acting at short and long distances. EVs carry biologically active molecules, thus providing signals that influence a spectrum of functions in recipient cells during various physiological and pathological processes. Recent findings point to EVs as very attractive immunomodulatory therapeutic agents, vehicles for drug delivery and diagnostic and prognostic biomarkers in liquid biopsies. In addition, EVs interact with and regulate the synthesis of extracellular matrix (ECM) components, which is crucial for organ development and wound healing, as well as bone and cardiovascular calcification. EVs carrying matrix metalloproteinases (MMPs) are involved in ECM remodeling, thus modifying tumor microenvironment and contributing to premetastatic niche formation and angiogenesis. Here we review the role of EVs in control of cell function, with emphasis on their interaction with ECM and microenvironment in health and disease.

Extracellular vesicles compartment in liquid biopsies: Clinical application.

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.

DNA sequences within glioma-derived extracellular vesicles can cross the intact blood-brain barrier and be detected in peripheral blood of patients.

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 IDH1G395A, an essential biomarker in the current management of human glioma.

Cancer stem cells from human glioblastoma resemble but do not mimic original tumors after in vitro passaging in serum-free media.

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.

SOX2+ cell population from normal human brain white matter is able to generate mature oligodendrocytes.

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.

Cancer-initiating enriched cell lines from human glioblastoma: preparing for drug discovery assays.

Glioblastoma multiforme (GBM) is the most lethal type of brain tumour in the adult humans. The cancer-initiating cell (CIC) hypothesis supports the notion that failures in current approaches to GBM treatment might be attributed to the survival of the CIC subpopulation. Recent evidence shows the idea that using CIC-enriched cell lines derived from human GBM as new targets for drug discovery programs, may improve the chance of successfully translating the basic research findings into clinical trials. Although this approach appears promising, many important biological and technical issues (characterization of functional CIC markers, inter- and intra-tumoral CIC heterogeneity, and isolation and maintenance inconsistency) need to be resolved.

Genomic instability of surgical sample and cancer-initiating cell lines from human glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor in the adult human, with an average survival of 16 months. A small population of cells within the GBM termed cancer-initiating cells is responsible for the initiation and maintenance of the tumor mass. The traditional glioblastoma cancer cells, grown with serum containing media, display increased rate of genomic instability events, which in turn renders the cell cultures with little resembling to the original tumor, making doubtful their use as preclinical models for screening therapeutic agents. On the contrary, the cancer-initiating cells grown in serum-free media seems to show lower rate of genomic instability processes. However, considering the diversity of genetic and/or epigenetic background, we will need to evaluate the possibility of using different culture conditions to allow for the isolation and culture of such cancer-initiating cells diversity, keeping, at the same time, the genomic instability rate as the original tumor. We summarized the main genetic and epigenetic mechanisms that are driving genomic instability in cancer-initiating cells from human glioblastoma.