Alessandra Palma

Metabolic / Proteomic Signature Defines Two Glioblastoma Subtypes With Different Clinical Outcome

Glioblastoma (GBM) is one of the deadliest human cancers. Because of the extremely unfavorable prognosis of GBM, it is important to develop more effective diagnostic and therapeutic strategies based on biologically and clinically relevant subclassification systems. Analyzing a collection of seventeen patient-derived glioblastoma stem-like cells (GSCs) by gene expression profiling, NMR spectroscopy and signal transduction pathway activation, we identified two GSC clusters, one characterized by a pro-neural-like phenotype and the other showing a mesenchymal-like phenotype. Evaluating the levels of proteins differentially expressed by the two GSC clusters in the TCGA GBM sample collection, we found that SRC activation is associated with a GBM subgroup showing better prognosis whereas activation of RPS6, an effector of mTOR pathway, identifies a subgroup with a worse prognosis. The two clusters are also differentiated by NMR spectroscopy profiles suggesting a potential prognostic stratification based on metabolic evaluation. Our data show that the metabolic/proteomic profile of GSCs is informative of the genomic/proteomic GBM landscape, which differs among tumor subtypes and is associated with clinical outcome.

1H NMR detects different metabolic profiles in glioblastoma stem-like cells

The metabolic profiles of glioblastoma stem‐like cells (GSCs) growing in neurospheres were examined by 1H NMR spectroscopy. Spectra of two GSC lines, labelled 1 and 83, from tumours close to the subventricular zone of the temporal lobe were studied in detail and compared with those of neural stem/progenitor cells from the adult olfactory bulb (OB‐NPCs) and of the T98G glioblastoma cell line. In both GSCs, signals from myoinositol (Myo‐I), UDP‐hexosamines (UDP‐Hex) and glycine indicated an astrocyte/glioma metabolism. For line 1, the presence of signals from N‐acetyl aspartate, GABA and creatine pointed to a neuronal fingerprint. These metabolites were almost absent from line 83 spectra, whereas lipid signals, absent from normal neural lineages, were intense in line 83 spectra and remained low in those of line 1, irrespective of apoptotic fate. Spectra of OB‐NPC cells displayed strong similarities with those from line 1, with low lipid signals and clearly detectable neuronal signals. In contrast, the spectral profile of line 83 was more similar to that of T98G, displaying high lipids and nearly complete absence of the neuronal markers. A mixed neural–astrocyte metabolic phenotype with a strong neuronal fingerprint was therefore found in line 1, while an astrocytic/glioma‐like metabolism prevailed in line 83. We found a signal assigned to the amide proton of N‐acetyl galactosamine in GSC lines and in OB‐NPC spectra, whereas it was absent from those of T98G cells. This signal may be related to a stem‐cell‐specific protein glycosylation pattern and is therefore suggested as a marker of cell multipotency.

Characterization of 1H NMR detectable mobile lipids in cells from human adenocarcinomas

Magnetic resonance spectroscopy studies are often carried out to provide metabolic information on tumour cell metabolism, aiming for increased knowledge for use in anti‐cancer treatments. Accordingly, the presence of intense lipid signals in tumour cells has been the subject of many studies aiming to obtain further insight on the reaction of cancer cells to external agents that eventually cause cell death. The present study explored the relationship between changes in neutral lipid signals during cell growth and after irradiation with gamma rays to provide arrest in cell cycle and cell death. Two cell lines from human tumours were used that were differently prone to apoptosis following irradiation. A sub‐G1 peak was present only in the radiosensitive HeLa cells. Different patterns of neutral lipids changes were observed in spectra from intact cells, either during unperturbed cell growth in culture or after radiation‐induced growth arrest. The intensities of triglyceride signals in the spectra from extracted total lipids changed concurrently. The increase in lipid peak intensities did not correlate with the apoptotic fate. Modelling to fit the experimental data revealed a dynamic equilibrium between the production and depletion of neutral lipids. This is observed for the first time in cells that are different from adipocytes.

Role of Glutathione in Apoptosis Induced by Radiation as Determined by 1H MR Spectra of Cultured Tumor Cells

Abstract Rosi, A., Grande, S., Luciani, A. M., Palma, A., Giovannini, C., Guidoni, L., Sapora, O. and Viti, V. Role of Glutathione in Apoptosis Induced by Radiation as Determined by 1H MR Spectra of Cultured Tumor Cells. Radiat. Res. 167, 268–282 (2007). The relationship between apoptosis induced by γ radiation and glutathione in cells of two human cancer cell lines, HeLa from cervix carcinoma and MCF-7 from mammary carcinoma, was examined. MCF-7 cells appeared to be more radioresistant than HeLa cells, and radiation-induced apoptosis, which was monitored by assessing phosphatidylserine externalization, was observed in HeLa cells but not in MCF-7 cells. Glutathione levels monitored by 1H MRS were higher in MCF-7 cells than in HeLa cells, while the opposite was true for the free glu signals. MCF-7 cells became more radiosensitive when treated with 0.1 mM buthionine sulfoximine, which inhibits GSH synthesis through inactivation of γ-glutamylcysteine synthetase, with the concomitant appearance of radiation-induced apoptosis. We can thus reasonably associate, at least in part, the resistance of MCF-7 cells to apoptosis with a high level of glutathione and probably with a high activity of γ-glutamylcysteine synthetase. A late decrease in glutathione concentration after irradiation was observed in MCF-7 cells, but not in HeLa cells and to a lesser degree in buthionine sulfoximine-treated MCF-7 cells. This would indicate that the radiation-induced decrease in glutathione concentration is not related to the onset of apoptosis, but it is more likely related to glutathione consumption as a result of detoxification reactions.

Glycosidic intermediates identified in 1H MR spectra of intact tumour cells may contribute to the clarification of aspects of glycosylation pathways.

The glycosylation process, through the addition of carbohydrates, is a major post‐translational modification of proteins and glycolipids. Proteins may be glycosylated in either the secretory pathway leading to N‐linked or O‐linked glycoproteins or as nucleocytoplasmic glycosylation that targets only single proteins involving a single β‐linked N‐acetylglucosamine. In both cases, the key precursors are the uridine diphospho‐N‐acetylhexosamines synthesised by the hexosamine biosynthetic pathway. Furthermore, uridine diphospho‐N‐acetylglucosamine participates in the biosynthesis of sialic acid. In this work, we propose MRS for the detection of uridine diphospho‐N‐acetylhexosamines visible in high‐resolution MR spectra of intact cells from different human tumours. Signals from the nucleotide and amino sugar moieties, including amide signals observed for the first time in whole cells, are assigned, also taking advantage of spectral changes that follow cell treatment with ammonium chloride. Finally, parallel changes in uridine diphospho‐N‐acetylhexosamines and glutamine pools, observed after pH changes induced by ammonium chloride in the different tumour cell lines, may provide more details on the glycosylation processes. Copyright

Metabolism of glutathione in tumour cells as evidenced by 1H MRS

1H MRS signals of glutathione and of free glutamate were examined in samples from cultured tumour cells, namely MCF‐7 from mammary carcinoma and TG98 from malignant glioma, with the aim of relating signal intensities to aspects of GSH metabolism. Spectra of cells harvested at different cell densities suggest that GSH and glu signal intensities are related to cell density and proliferation and their ratio is dependent on the activity of the γ‐glutamyl cysteine synthetase. The hypothesis is confirmed by experiments performed on cells treated with buthionine sulfoximine that inhibits the enzyme activity.

1H NMR spectroscopy of glioblastoma stem-like cells identifies alpha-aminoadipate as a marker of tumor aggressiveness

Patients suffering from glioblastoma multiforme (GBM) face a poor prognosis with median survival of about 14 months. High recurrence rate and failure of conventional treatments are attributed to the presence of GBM cells with stem‐like properties (GSCs). Metabolite profiles of 42 GSC lines established from the tumor tissue of adult GBM patients were screened with 1H NMR spectroscopy and compared with human neural progenitor cells from human adult olfactory bulb (OB‐NPCs) and from the developing human brain (HNPCs).

A three-microRNA signature identifies two subtypes of glioblastoma patients with different clinical outcomes

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor in adults, characterized by aggressive growth, limited response to therapy, and inexorable recurrence. Because of the extremely unfavorable prognosis of GBM, it is important to develop more effective diagnostic and therapeutic strategies based on biologically and clinically relevant patient stratification systems. Analyzing a collection of patient‐derived GBM stem‐like cells (GSCs) by gene expression profiling, nuclear magnetic resonance spectroscopy, and signal transduction pathway activation, we identified two GSC clusters characterized by different clinical features. Due to the widely documented role played by microRNAs (miRNAs) in the tumorigenesis process, in this study we explored whether these two GBM patient subtypes could also be discriminated by different miRNA signatures. Global miRNA expression pattern was analyzed by oblique principal component analysis and principal component analysis. By a combined inferential strategy on PCA results, we identified a reduced set of three miRNAs – miR‐23a, miR‐27a, and miR‐9* (miR‐9‐3p) – able to discriminate the proneural‐ and mesenchymal‐like GSC phenotypes as well as mesenchymal and proneural subtypes of primary GBM included in The Cancer Genome Atlas (TCGA) data set. Kaplan–Meier analysis showed a significant correlation between the selected miRNAs and overall survival in 429 GBM specimens from TCGA‐identifying patients who had an unfavorable outcome. The survival prognostic capability of the three‐miRNA signatures could have important implications for the understanding of the biology of GBM subtypes and could be useful in patient stratification to facilitate interpretation of results from clinical trials.

1H-MRS can detect aberrant glycosylation in tumour cells: a study of the HeLa cell line

Glycosylation is the most abundant and diverse form of post‐translational modification of proteins. Two types of glycans exist in glycoproteins: N‐glycans and O‐glycans often coexisting in the same protein. O‐glycosylation is frequently found on secreted or membrane‐bound mucins whose overexpression and structure alterations are associated with many types of cancer. Mucins have several cancer‐associated structures, including high levels of Lewis antigens characterized by the presence of terminal fucose. The present study deals with the identification of MR signals from N‐acetylgalactosamine and from fucose in HeLa cells by detecting a low‐field signal in one‐dimensional (1D) spectra assigned to the NH of N‐acetylgalactosamine and some cross peaks assigned to fucose in two‐dimensional (2D) spectra. The increase of Golgi pH by treatment with ammonium chloride allowed the N‐acetylgalactosamine signal assignment to be confirmed. Behaviour of MR peak during cell growth and comparison with studies from literature taken together made it possible to have more insight into the relationship between aberrantly processed mucin and the presence of non‐processed N‐acetylgalactosamine residues in HeLa cells. Fucose signals, tentatively ascribed to residues bound to galactose and to N‐acetylglucosamine, are visible in both intact cell and perchloric acid spectra. Signals assigned to fucose bound to galactose are more evident in ammonium chloride‐treated cells where structural changes of mucin‐related Lewis antigens are expected as a result of the higher Golgi pH. A common origin for the N‐acetylgalactosamine and fucose resonances attributing them to aberrantly processed mucin can be inferred from the present results. Copyright

Metabolic Study of Breast MCF-7 Tumor Spheroids after Gamma Irradiation by 1H NMR Spectroscopy and Microimaging

Multicellular tumor spheroids are an important model system to investigate the response of tumor cells to radio- and chemotherapy. They share more properties with the original tumor than cells cultured as 2D monolayers do, which helps distinguish the intrinsic properties of monolayer cells from those induced during cell aggregation in 3D spheroids. The paper investigates some metabolic aspects of small tumor spheroids of breast cancer and their originating MCF-7 cells, grown as monolayer, by means of high–resolution (HR) 1H NMR spectroscopy and MR microimaging before and after gamma irradiation. The spectra of spheroids were characterized by higher intensity of mobile lipids, mostly neutral lipids, and glutamine (Gln) signals with respect to their monolayer cells counterpart, mainly owing to the lower oxygen supply in spheroids. Morphological changes of small spheroids after gamma-ray irradiation, such as loss of their regular shape, were observed by MR microimaging. Lipid signal intensity increased after irradiation, as evidenced in both MR localized spectra of the single spheroid and in HR NMR spectra of spheroid suspensions. Furthermore, the intense Gln signal from spectra of irradiated spheroids remained unchanged, while the low Gln signal observed in monolayer cells increased after irradiation. Similar results were observed in cells grown in hypoxic conditions. The different behavior of Gln in 2D monolayers and in 3D spheroids supports the hypothesis that a lower oxygen supply induces both an upregulation of Gln synthetase and a downregulation of glutaminases with the consequent increase in Gln content, as already observed under hypoxic conditions. The data herein indicate that 1H NMR spectroscopy can be a useful tool for monitoring cell response to different constraints. The use of spheroid suspensions seems to be a feasible alternative to localized spectroscopy since similar effects were found after radiation treatment.