Altered metabolic landscape in IDH‐mutant gliomas affects phospholipid, energy, and oxidative stress pathways

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

Increased mitochondrial activity in a novel IDH1-R132H mutant human oligodendroglioma xenograft model: in situ detection of 2-HG and α-KG

Background: Point mutations in genes encoding NADP+-dependent isocitrate dehydrogenases (especially IDH1) are common in lower grade diffuse gliomas and secondary glioblastomas and occur early during tumor development. The contribution of these mutations to gliomagenesis is not completely understood and research is hampered by the lack of relevant tumor models. We previously described the development of the patient-derived high-grade oligodendroglioma xenograft model E478 that carries the commonly occurring IDH1-R132H mutation. We here report on the analyses of E478 xenografts at the genetic, histologic and metabolic level. Results: LC-MS and in situ mass spectrometric imaging by LESA-nano ESI-FTICR revealed high levels of the proposed oncometabolite D-2-hydroxyglutarate (D-2HG), the product of enzymatic conversion of α-ketoglutarate (α-KG) by IDH1-R132H, in the tumor but not in surrounding brain parenchyma. α-KG levels and total NADP+-dependent IDH activity were similar in IDH1-mutant and -wildtype xenografts, demonstrating that IDH1-mutated cancer cells maintain α-KG levels. Interestingly, IDH1-mutant tumor cells in vivo present with high densities of mitochondria and increased levels of mitochondrial activity as compared to IDH1-wildtype xenografts. It is not yet clear whether this altered mitochondrial activity is a driver or a consequence of tumorigenesis. Conclusions: The oligodendroglioma model presented here is a valuable model for further functional elucidation of the effects of IDH1 mutations on tumor metabolism and may aid in the rational development of novel therapeutic strategies for the large subgroup of gliomas carrying IDH1 mutations

Glioma proteomics: status and perspectives.

peer reviewedHigh grade gliomas are the most common brain tumors in adults and their malignant nature makes them the fourth biggest cause of cancer death. Major efforts in neuro-oncology research are needed to reach similar progress in treatment efficacy as that achieved for other cancers in recent years. In addition to the urgent need to identify novel effective drug targets against malignant gliomas, the search for glioma biomarkers and grade specific protein signatures will provide a much needed contribution to diagnosis, prognosis, treatment decision and assessment of treatment response. Over the past years glioma proteomics has been attempted at different levels, including proteome analysis of patient biopsies and bodily fluids, of glioma cell lines and animal models. Here we provide an extensive review of the outcome of these studies in terms of protein identifications (protein numbers and regulated proteins), with an emphasis on the methods used and the limitations of the studies with regard to biomarker discovery. This is followed by a perspective on novel technologies and on the potential future contribution of proteomics in a broad sense to understanding glioma biology

Combined effect of tissue stabilization and protein extraction methods on phosphoprotein analysis.

peer reviewedPreanalytical conditions applied during sample collection and processing can affect the detection or quantification of unstable phosphoprotein biomarkers. We evaluated the consequences of tissue stabilization and protein extraction methods on phosphoprotein analysis. The effects of stabilization techniques (heat stabilization, snap-freezing) and time on the levels of phosphoproteins, including phospho-Akt, p-ERK 1/2, p-IkBα, p-JNK, and p38 MAPK, were evaluated using a BioPlex phosphoprotein assay. Additionally, two different protein extraction protocols, using different extraction buffers (8 M urea buffer, or Bio-Rad buffer without urea) were tested. For snap-frozen samples, protein extraction yields were comparable with the two buffer systems. For heat-stabilized samples, total protein yields were significantly lower following extraction in non-urea buffer. However, the concentrations of specific phosphoproteins were significantly higher in heat-stabilized samples than in the corresponding snap-frozen samples, indicating that this tissue processing method better preserved phosphoproteins. Significant differences were found between the measured phosphoprotein levels in heat-stabilized and snap-frozen tissue, suggesting that alterations occur very rapidly after tissue excision. Our results suggest that heat stabilization can be used as a tissue processing method for subsequent phosphoprotein analyses, but also suggest that the BioPlex phosphoprotein assay could be used as a possible quality control method to assess tissue sample integrity

Faecal DNA typing as a tool for investigating territorial behaviour of badgers

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Measles Virus Genotyping by Nucleotide-Specific Multiplex PCR

A simple genotyping method based on multiplex PCR has been developed to discriminate between all active measles virus (MV) clades and genotypes (A, B3.1, B3.2, C2, D2-D9, G2-G3, and H1-H2). The sequencing reaction was replaced by six multiplex PCRs: one to identify the clade and five to identify the respective genotype. Primers were sensitive to clade- and genotype-specific nucleotides and generated fragments of type-specific sizes that were analyzed by conventional agarose gel electrophoresis. On the basis of all published MV sequences, positive and negative predictive values of 99.2% and 98.6% were calculated. Variability in the primer binding sites, which could potentially reduce sensitivity, was very limited among published sequences. As new genotypes are described, additional specific primers can be included in the multiplex PCR with relative ease. Although sequencing remains the “gold standard,” the present method should facilitate MV genotyping especially in developing countries and will therefore contribute to enhanced MV control and elimination strategies as recommended by the World Health Organization

Colorectal cancer derived organotypic spheroids maintain essential tissue characteristics but adapt their metabolism in culture

Background: Organotypic tumor spheroids, a 3D in vitro model derived from patient tumor material, preserve tissue heterogeneity and retain structural tissue elements, thus replicating the in vivo tumor more closely than commonly used 2D and 3D cell line models. Such structures harbour tumorigenic cells, as revealed by xenograft implantation studies in animal models and maintain the genetic makeup of the original tumor material. The aim of our work was a morphological and proteomic characterization of organotypic spheroids derived from colorectal cancer tissue in order to get insight into their composition and associated biology. Results: Morphological analysis showed that spheroids were of about 250 μm in size and varied in structure, while the spheroid cells differed in shape and size and were tightly packed together by desmosomes and tight junctions. Our proteomic data revealed significant alterations in protein expression in organotypic tumor spheroids cultured as primary explants compared to primary colorectal cancer tissue. Components underlying cellular and tissue architecture were changed; nuclear DNA/ chromatin maintenance systems were up-regulated, whereas various mitochondrial components were down-regulated in spheroids. Most interestingly, the mesenchymal cells appear to be substantial component in such cellular assemblies. Thus the observed changes may partly occur in this cellular compartment. Finally, in the proteomics analysis stem cell-like characteristics were observed within the spheroid cellular assembly, reflected by accumulation of Alcam, Ctnnb1, Aldh1, Gpx2, and CD166. These findings were underlined by IHC analysis of Ctnnb1, CD24 and CD44, therefore warranting closer investigation of the tumorigenic compartment in this 3D culture model for tumor tissue. Conclusions: Our analysis of organotypic CRC tumor spheroids has identified biological processes associated with a mixture of cell types and states, including protein markers for mesenchymal and stem-like cells. This 3D tumor model in which tumor heterogeneity is preserved may represent an advantageous model system to investigate novel therapeutic approaches

Bevacizumab treatment induces metabolic adaptation toward anaerobic metabolism in glioblastomas

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