Glycolytic activity and in vitro effect of the pyruvate kinase activator AG-946 in red blood cells from low-risk myelodysplastic syndromes patients: A proof-of-concept study.

Glycolytic activity and in vitro effect of the pyruvate kinase activator AG-946 in red blood cells from low-risk myelodysplastic syndromes patients. Data showed decreased glycolytic activity in red blood cells of 2/3 of patients with lower-risk MDS. These results highlight a potential effect of the PK activator in this setting.

Fusion-fission-mitophagy cycling and metabolic reprogramming coordinate nerve growth factor (NGF)-dependent neuronal differentiation.

Neuronal differentiation is regulated by nerve growth factor (NGF) and other neurotrophins. We explored the impact of NGF on mitochondrial dynamics and metabolism through time-lapse imaging, metabolomics profiling, and computer modeling studies. We show that NGF may direct differentiation by stimulating fission, thereby causing selective mitochondrial network fragmentation and mitophagy, ultimately leading to increased mitochondrial quality and respiration. Then, we reconstructed the dynamic fusion-fission-mitophagy cycling of mitochondria in a computer model, integrating these processes into a single network mechanism. Both the computational model and the simulations are able to reproduce the proposed mechanism in terms of mitochondrial dynamics, levels of reactive oxygen species (ROS), mitophagy, and mitochondrial quality, thus providing a computational tool for the interpretation of the experimental data and for future studies aiming to detail further the action of NGF on mitochondrial processes. We also show that changes in these mitochondrial processes are intertwined with a metabolic function of NGF in differentiation: NGF directs a profound metabolic rearrangement involving glycolysis, TCA cycle, and the pentose phosphate pathway, altering the redox balance. This metabolic rewiring may ensure: (a) supply of both energy and building blocks for the anabolic processes needed for morphological reorganization, as well as (b) redox homeostasis.

Production of recombinant human xCT (SLC7A11) and reconstitution in proteoliposomes for functional studies.

The plasma membrane transporter xCT belongs to the SLC7 family and has the physiological role of mediating the exchange of glutamate and cystine across the cell plasma membrane, being crucial for redox control. The xCT protein forms a heterodimer with the ancillary protein CD98. Over the years, xCT became a hot pharmacological target due to the documented over-expression in virtually all human cancers, which rely on cystine availability for their progression. Notwithstanding, several unknown aspects of xCT biology still exist that require a suitable single protein experimental model, to be addressed. To this aim, the recombinant host Escherichia coli has been exploited to over-express the human isoform of xCT. In this widely used and low-cost system, the optimization for growth and protein production has been achieved by acting on the metabolic needs of the bacterial strains. Then, the His-tagged protein has been purified by Ni2+-chelating chromatography and reconstituted in proteoliposomes for transport activity assays. The expressed protein was in a folded/active state allowing functional and kinetic characterization. Interestingly, the features of the recombinant protein meet those of the native one extracted from intact cells, further confirming the suitability of E. coli as a host for the expression of human proteins. This study opens perspectives for elucidating other molecular aspects of xCT, as well as for studying the interaction with endogenous and exogenous compounds, relevant to human health.

Polystyrene micro and nano-particles induce metabolic rewiring in normal human colon cells: A risk factor for human health.

Polystyrene is a thermoplastic polymer widely used in commercial products. Like all plastics, polystyrene can be degraded into microplastic and nanoplastic particles and ingested via food chain contamination. Although the ecological impact due to plastic contamination is well known, there are no studies indicating a carcinogenic potential of polystyrene microplastics (MPs) and nanoplastics (NPs). Here, we evaluated the effects of the MPs and NPs on normal human intestinal CCD-18Co cells. Our results show that internalization of NPs and MPs induces metabolic changes under both acute and chronic exposure by inducing oxidative stress, increasing glycolysis via lactate to sustain energy metabolism and glutamine metabolism to sustain anabolic processes. We also show that this decoupling of nutrients mirrors the effect of the potent carcinogenic agent azoxymethane and HCT15 colon cancer cells, carrying out the typical strategy of cancer cells to optimize nutrients utilization and allowing metabolic adaptation to environmental stress conditions. Taken together our data provide new evidence that chronic NPs and MPs exposure could act as cancer risk factor for human health.

INTEGRATE: Model-based multi-omics data integration to characterize multi-level metabolic regulation.

Metabolism is directly and indirectly fine-tuned by a complex web of interacting regulatory mechanisms that fall into two major classes. On the one hand, the expression level of the catalyzing enzyme sets the maximal theoretical flux level (i.e., the net rate of the reaction) for each enzyme-controlled reaction. On the other hand, metabolic regulation controls the metabolic flux through the interactions of metabolites (substrates, cofactors, allosteric modulators) with the responsible enzyme. High-throughput data, such as metabolomics and transcriptomics data, if analyzed separately, do not accurately characterize the hierarchical regulation of metabolism outlined above. They must be integrated to disassemble the interdependence between different regulatory layers controlling metabolism. To this aim, we propose INTEGRATE, a computational pipeline that integrates metabolomics and transcriptomics data, using constraint-based stoichiometric metabolic models as a scaffold. We compute differential reaction expression from transcriptomics data and use constraint-based modeling to predict if the differential expression of metabolic enzymes directly originates differences in metabolic fluxes. In parallel, we use metabolomics to predict how differences in substrate availability translate into differences in metabolic fluxes. We discriminate fluxes regulated at the metabolic and/or gene expression level by intersecting these two output datasets. We demonstrate the pipeline using a set of immortalized normal and cancer breast cell lines. In a clinical setting, knowing the regulatory level at which a given metabolic reaction is controlled will be valuable to inform targeted, truly personalized therapies in cancer patients.

Fundamental Role of Pentose Phosphate Pathway within the Endoplasmic Reticulum in Glutamine Addiction of Triple-Negative Breast Cancer Cells.

Cancer utilization of large glutamine equivalents contributes to diverging glucose-6-P flux toward the pentose phosphate shunt (PPP) to feed the building blocks and the antioxidant responses of rapidly proliferating cells. In addition to the well-acknowledged cytosolic pathway, cancer cells also run a largely independent PPP, triggered by hexose-6P-dehydrogenase within the endoplasmic reticulum (ER), whose activity is mandatory for the integrity of ER-mitochondria networking. To verify whether this reticular metabolism is dependent on glutamine levels, we complemented the metabolomic characterization of intermediates of the glucose metabolism and tricarboxylic acid cycle with the estimation of proliferating activity, energy metabolism, redox damage, and mitochondrial function in two breast cancer cell lines. ER-PPP activity and its determinants were estimated by the ER accumulation of glucose analogs. Glutamine shortage decreased the proliferation rate despite increased ATP and NADH levels. It depleted NADPH reductive power and increased malondialdehyde content despite a marked increase in glucose-6P-dehydrogenase. This paradox was explained by the deceleration of ER-PPP favored by the decrease in hexose-6P-dehydrogenase expression coupled with the opposite response of its competitor enzyme glucose-6P-phosphatase. The decreased ER-PPP activity eventually hampered mitochondrial function and calcium exchanges. These data configure the ER-PPP as a powerful, unrecognized regulator of cancer cell metabolism and proliferation.

Transcriptomics and Metabolomics Integration Reveals Redox-Dependent Metabolic Rewiring in Breast Cancer Cells.

Rewiring glucose metabolism toward aerobic glycolysis provides cancer cells with a rapid generation of pyruvate, ATP, and NADH, while pyruvate oxidation to lactate guarantees refueling of oxidized NAD+ to sustain glycolysis. CtPB2, an NADH-dependent transcriptional co-regulator, has been proposed to work as an NADH sensor, linking metabolism to epigenetic transcriptional reprogramming. By integrating metabolomics and transcriptomics in a triple-negative human breast cancer cell line, we show that genetic and pharmacological down-regulation of CtBP2 strongly reduces cell proliferation by modulating the redox balance, nucleotide synthesis, ROS generation, and scavenging. Our data highlight the critical role of NADH in controlling the oncogene-dependent crosstalk between metabolism and the epigenetically mediated transcriptional program that sustains energetic and anabolic demands in cancer cells.

Gold nanoparticles coated with polyvinylpyrrolidone and sea urchin extracellular molecules induce transient immune activation.

We report that the immunogenicity of colloidal gold nanoparticles coated with polyvinylpyrrolidone (PVP-AuNPs) in a model organism, the sea urchin Paracentrotus lividus, can function as a proxy for humans for in vitro immunological studies. To profile the immune recognition and interaction from exposure to PVP-AuNPs (1 and 10 µg mL-1), we applied an extensive nano-scale approach, including particle physicochemical characterisation involving immunology, cellular biology, and metabolomics. The interaction between PVP-AuNPs and soluble proteins of the sea urchin physiological coelomic fluid (blood equivalent) results in the formation of a protein "corona" surrounding the NPs from three major proteins that influence the hydrodynamic size and colloidal stability of the particle. At the lower concentration of PVP-AuNPs, the P. lividus phagocytes show a broad metabolic plasticity based on the biosynthesis of metabolites mediating inflammation and phagocytosis. At the higher concentration of PVP-AuNPs, phagocytes activate an immunological response involving Toll-like receptor 4 (TLR4) signalling pathway at 24 hours of exposure. These results emphasise that exposure to PVP-AuNPs drives inflammatory signalling by the phagocytes and the resolution at both the low and high concentrations of the PVP-AuNPs and provides more details regarding the immunogenicity of these NPs.

Two high-rate pentose-phosphate pathways in cancer cells.

The relevant role of pentose phosphate pathway (PPP) in cancer metabolic reprogramming has been usually outlined by studying glucose-6-phosphate dehydrogenase (G6PD). However, recent evidence suggests an unexpected role for a less characterized PPP, triggered by hexose-6-phosphate dehydrogenase (H6PD) within the endoplasmic reticulum (ER). Studying H6PD biological role in breast and lung cancer, here we show that gene silencing of this reticular enzyme decreases cell content of PPP intermediates and D-ribose, to a similar extent as G6PD silencing. Decrease in overall NADPH content and increase in cell oxidative status are also comparable. Finally, either gene silencing impairs at a similar degree cell proliferating activity. This unexpected response occurs despite the absence of any cross-interference between the expression of both G6PD and H6PD. Thus, overall cancer PPP reflects the contribution of two different pathways located in the cytosol and ER, respectively. Disregarding the reticular pathway might hamper our comprehension of PPP role in cancer cell biology.

Methionine Supplementation Affects Metabolism and Reduces Tumor Aggressiveness in Liver Cancer Cells.

Liver cancer is one of the most common cancer worldwide with a high mortality. Methionine is an essential amino acid required for normal development and cell growth, is mainly metabolized in the liver, and its role as an anti-cancer supplement is still controversial. Here, we evaluate the effects of methionine supplementation in liver cancer cells. An integrative proteomic and metabolomic analysis indicates a rewiring of the central carbon metabolism, with an upregulation of the tricarboxylic acid (TCA) cycle and mitochondrial adenosine triphosphate (ATP) production in the presence of high methionine and AMP-activated protein kinase (AMPK) inhibition. Methionine supplementation also reduces growth rate in liver cancer cells and induces the activation of both the AMPK and mTOR pathways. Interestingly, in high methionine concentration, inhibition of AMPK strongly impairs cell growth, cell migration, and colony formation, indicating the main role of AMPK in the control of liver cancer phenotypes. Therefore, regulation of methionine in the diet combined with AMPK inhibition could reduce liver cancer progression.

Disruption of redox homeostasis for combinatorial drug efficacy in K-Ras tumors as revealed by metabolic connectivity profiling.

BACKGROUND: Rewiring of metabolism induced by oncogenic K-Ras in cancer cells involves both glucose and glutamine utilization sustaining enhanced, unrestricted growth. The development of effective anti-cancer treatments targeting metabolism may be facilitated by the identification and rational combinatorial targeting of metabolic pathways. METHODS: We performed mass spectrometric metabolomics analysis in vitro and in vivo experiments to evaluate the efficacy of drugs and identify metabolic connectivity. RESULTS: We show that K-Ras-mutant lung and colon cancer cells exhibit a distinct metabolic rewiring, the latter being more dependent on respiration. Combined treatment with the glutaminase inhibitor CB-839 and the PI3K/aldolase inhibitor NVP-BKM120 more consistently reduces cell growth of tumor xenografts. Maximal growth inhibition correlates with the disruption of redox homeostasis, involving loss of reduced glutathione regeneration, redox cofactors, and a decreased connectivity among metabolites primarily involved in nucleic acid metabolism. CONCLUSIONS: Our findings open the way to develop metabolic connectivity profiling as a tool for a selective strategy of combined drug repositioning in precision oncology.

Systems metabolomics: from metabolomic snapshots to design principles.

Metabolomics is a rapidly expanding technology that finds increasing application in a variety of fields, form metabolic disorders to cancer, from nutrition and wellness to design and optimization of cell factories. The integration of metabolic snapshots with metabolic fluxes, physiological readouts, metabolic models, and knowledge-informed Artificial Intelligence tools, is required to obtain a system-level understanding of metabolism. The emerging power of multi-omic approaches and the development of integrated experimental and computational tools, able to dissect metabolic features at cellular and subcellular resolution, provide unprecedented opportunities for understanding design principles of metabolic (dis)regulation and for the development of precision therapies in multifactorial diseases, such as cancer and neurodegenerative diseases.

Titanium dioxide nanoparticles temporarily influence the sea urchin immunological state suppressing inflammatory-relate gene transcription and boosting antioxidant metabolic activity.

Titanium dioxide nanoparticles (TiO2NPs) are revolutionizing biomedicine due to their potential application as diagnostic and therapeutic agents. However, the TiO2NP immune-compatibility remains an open issue, even for ethical reasons. In this work, we investigated the immunomodulatory effects of TiO2NPs in an emergent proxy to human non-mammalian model for in vitro basic and translational immunology: the sea urchin Paracentrotus lividus. To highlight on the new insights into the evolutionarily conserved intracellular signaling and metabolism pathways involved in immune-TiO2NP recognition/interaction we applied a wide-ranging approach, including electron microscopy, biochemistry, transcriptomics and metabolomics. Findings highlight that TiO2NPs interact with immune cells suppressing the expression of genes encoding for proteins involved in immune response and apoptosis (e.g. NF-κB, FGFR2, JUN, MAPK14, FAS, VEGFR, Casp8), and boosting the immune cell antioxidant metabolic activity (e.g. pentose phosphate, cysteine-methionine, glycine-serine metabolism pathways). TiO2NP uptake was circumscribed to phagosomes/phagolysosomes, depicting harmless vesicular internalization. Our findings underlined that under TiO2NP-exposure sea urchin innate immune system is able to control inflammatory signaling, excite antioxidant metabolic activity and acquire immunological tolerance, providing a new level of understanding of the TiO2NP immune-compatibility that could be useful for the development in Nano medicines.

An Intricate Connection between Alternative Splicing and Phenotypic Plasticity in Development and Cancer.

During tumor progression, hypoxia, nutrient deprivation or changes in the extracellular environment (i.e., induced by anti-cancer drugs) elicit adaptive responses in cancer cells. Cellular plasticity increases the chance that tumor cells may survive in a challenging microenvironment, acquire new mechanisms of resistance to conventional drugs, and spread to distant sites. Re-activation of stem pathways appears as a significant cause of cellular plasticity because it promotes the acquisition of stem-like properties through a profound phenotypic reprogramming of cancer cells. In addition, it is a major contributor to tumor heterogeneity, depending on the coexistence of phenotypically distinct subpopulations in the same tumor bulk. Several cellular mechanisms may drive this fundamental change, in particular, high-throughput sequencing technologies revealed a key role for alternative splicing (AS). Effectively, AS is one of the most important pre-mRNA processes that increases the diversity of transcriptome and proteome in a tissue- and development-dependent manner. Moreover, defective AS has been associated with several human diseases. However, its role in cancer cell plasticity and tumor heterogeneity remains unclear. Therefore, unravelling the intricate relationship between AS and the maintenance of a stem-like phenotype may explain molecular mechanisms underlying cancer cell plasticity and improve cancer diagnosis and treatment.

Differential Modulation of NF-κB in Neurons and Astrocytes Underlies Neuroprotection and Antigliosis Activity of Natural Antioxidant Molecules.

Neuroinflammation, a hallmark of chronic neurodegenerative disorders, is characterized by sustained glial activation and the generation of an inflammatory loop, through the release of cytokines and other neurotoxic mediators that cause oxidative stress and limit functional repair of brain parenchyma. Dietary antioxidants may protect against neurodegenerative diseases by counteracting chronic neuroinflammation and reducing oxidative stress. Here, we describe the effects of a number of natural antioxidants (polyphenols, carotenoids, and thiolic molecules) in rescuing astrocytic function and neuronal viability following glial activation by reducing astrocyte proliferation and restoring astrocytic and neuronal survival and basal levels of reactive oxygen species (ROS). All antioxidant molecules are also effective under conditions of oxidative stress and glutamate toxicity, two maladaptive components of neuroinflammatory processes. Moreover, it is remarkable that their antioxidant and anti-inflammatory activity occurs through differential modulation of NF-κB binding activity in neurons and astrocytes. In fact, we show that inflammatory stimuli promote a significant induction of NF-κB binding activity in astrocytes and its concomitant reduction in neurons. These changes are prevented in astrocytes and neurons pretreated with the antioxidant molecules, suggesting that NF-κB plays a key role in the modulation of survival and anti-inflammatory responses. Finally, we newly demonstrate that effective antigliosis and neuroprotective activity is achieved with a defined cocktail of four natural antioxidants at very low concentrations, suggesting a promising strategy to reduce inflammatory and oxidative damage in neurodegenerative diseases with limited side effects.

Radiosensitizing effect of curcumin-loaded lipid nanoparticles in breast cancer cells.

In breast cancer (BC) care, radiotherapy is considered an efficient treatment, prescribed both for controlling localized tumors or as a therapeutic option in case of inoperable, incompletely resected or recurrent tumors. However, approximately 90% of BC-related deaths are due to the metastatic tumor progression. Then, it is strongly desirable to improve tumor radiosensitivity using molecules with synergistic action. The main aim of this study is to develop curcumin-loaded solid nanoparticles (Cur-SLN) in order to increase curcumin bioavailability and to evaluate their radiosensitizing ability in comparison to free curcumin (free-Cur), by using an in vitro approach on BC cell lines. In addition, transcriptomic and metabolomic profiles, induced by Cur-SLN treatments, highlighted networks involved in this radiosensitization ability. The non tumorigenic MCF10A and the tumorigenic MCF7 and MDA-MB-231 BC cell lines were used. Curcumin-loaded solid nanoparticles were prepared using ethanolic precipitation and the loading capacity was evaluated by UV spectrophotometer analysis. Cell survival after treatments was evaluated by clonogenic assay. Dose-response curves were generated testing three concentrations of free-Cur and Cur-SLN in combination with increasing doses of IR (2-9 Gy). IC50 value and Dose Modifying Factor (DMF) was measured to quantify the sensitivity to curcumin and to combined treatments. A multi-"omic" approach was used to explain the Cur-SLN radiosensitizer effect by microarray and metobolomic analysis. We have shown the efficacy of the Cur-SLN formulation as radiosensitizer on three BC cell lines. The DMFs values, calculated at the isoeffect of SF = 50%, showed that the Luminal A MCF7 resulted sensitive to the combined treatments using increasing concentration of vehicled curcumin Cur-SLN (DMF: 1,78 with 10 µM Cur-SLN.) Instead, triple negative MDA-MB-231 cells were more sensitive to free-Cur, although these cells also receive a radiosensitization effect by combination with Cur-SLN (DMF: 1.38 with 10 µM Cur-SLN). The Cur-SLN radiosensitizing function, evaluated by transcriptomic and metabolomic approach, revealed anti-oxidant and anti-tumor effects. Curcumin loaded- SLN can be suggested in future preclinical and clinical studies to test its concomitant use during radiotherapy treatments with the double implications of being a radiosensitizing molecule against cancer cells, with a protective role against IR side effects.

Differentiation by nerve growth factor (NGF) involves mechanisms of crosstalk between energy homeostasis and mitochondrial remodeling.

Neuronal differentiation involves extensive modification of biochemical and morphological properties to meet novel functional requirements. Reorganization of the mitochondrial network to match the higher energy demand plays a pivotal role in this process. Mechanisms of neuronal differentiation in response to nerve growth factor (NGF) have been largely characterized in terms of signaling, however, little is known about its impact on mitochondrial remodeling and metabolic function. In this work, we show that NGF-induced differentiation requires the activation of autophagy mediated by Atg9b and Ambra1, as it is disrupted by their genetic knockdown and by autophagy blockers. NGF differentiation involves the induction of P-AMPK and P-CaMK, and is prevented by their pharmacological inhibition. These molecular events correlate with modifications of energy and redox homeostasis, as determined by ATP and NADPH changes, higher oxygen consumption (OCR) and ROS production. Our data indicate that autophagy aims to clear out exhausted mitochondria, as determined by enhanced localization of p62 and Lysotracker-red to mitochondria. In addition, we newly demonstrate that NGF differentiation is accompanied by increased mitochondrial remodeling involving higher levels of fission (P-Drp1) and fusion proteins (Opa1 and Mfn2), as well as induction of Sirt3 and the transcription factors mtTFA and PPARγ, which regulate mitochondria biogenesis and metabolism to sustain increased mitochondrial mass, potential, and bioenergetics. Overall, our data indicate a new NGF-dependent mechanism involving mitophagy and extensive mitochondrial remodeling, which plays a key role in both neurogenesis and nerve regeneration.

A metabolic core model elucidates how enhanced utilization of glucose and glutamine, with enhanced glutamine-dependent lactate production, promotes cancer cell growth: The WarburQ effect.

Cancer cells share several metabolic traits, including aerobic production of lactate from glucose (Warburg effect), extensive glutamine utilization and impaired mitochondrial electron flow. It is still unclear how these metabolic rearrangements, which may involve different molecular events in different cells, contribute to a selective advantage for cancer cell proliferation. To ascertain which metabolic pathways are used to convert glucose and glutamine to balanced energy and biomass production, we performed systematic constraint-based simulations of a model of human central metabolism. Sampling of the feasible flux space allowed us to obtain a large number of randomly mutated cells simulated at different glutamine and glucose uptake rates. We observed that, in the limited subset of proliferating cells, most displayed fermentation of glucose to lactate in the presence of oxygen. At high utilization rates of glutamine, oxidative utilization of glucose was decreased, while the production of lactate from glutamine was enhanced. This emergent phenotype was observed only when the available carbon exceeded the amount that could be fully oxidized by the available oxygen. Under the latter conditions, standard Flux Balance Analysis indicated that: this metabolic pattern is optimal to maximize biomass and ATP production; it requires the activity of a branched TCA cycle, in which glutamine-dependent reductive carboxylation cooperates to the production of lipids and proteins; it is sustained by a variety of redox-controlled metabolic reactions. In a K-ras transformed cell line we experimentally assessed glutamine-induced metabolic changes. We validated computational results through an extension of Flux Balance Analysis that allows prediction of metabolite variations. Taken together these findings offer new understanding of the logic of the metabolic reprogramming that underlies cancer cell growth.

Metformin and temozolomide, a synergic option to overcome resistance in glioblastoma multiforme models.

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with poor survival. Cytoreduction in association with radiotherapy and temozolomide (TMZ) is the standard therapy, but response is heterogeneous and life expectancy is limited. The combined use of chemotherapeutic agents with drugs targeting cell metabolism is becoming an interesting therapeutic option for cancer treatment. Here, we found that metformin (MET) enhances TMZ effect on TMZ-sensitive cell line (U251) and overcomes TMZ-resistance in T98G GBM cell line. In particular, combined-treatment modulated apoptosis by increasing Bax/Bcl-2 ratio, and reduced Reactive Oxygen Species (ROS) production. We also observed that MET associated with TMZ was able to reduce the expression of glioma stem cells (GSC) marker CD90 particularly in T98G cells but not that of CD133. In vivo experiments showed that combined treatment with TMZ and MET significantly slowed down growth of TMZ-resistant tumors but did not affect overall survival of TMZ-sensitive tumor bearing mice. In conclusion, our results showed that metformin is able to enhance TMZ effect in TMZ-resistant cell line suggesting its potential use in TMZ refractory GBM patients. However, the lack of effect on a GBM malignancy marker like CD133 requires further evaluation since it might influence response duration.

Divergent in vitro/in vivo responses to drug treatments of highly aggressive NIH-Ras cancer cells: a PET imaging and metabolomics-mass-spectrometry study.

Oncogenic K-ras is capable to control tumor growth and progression by rewiring cancer metabolism. In vitro NIH-Ras cells convert glucose to lactate and use glutamine to sustain anabolic processes, but their in vivo environmental adaptation and multiple metabolic pathways activation ability is poorly understood. Here, we show that NIH-Ras cancer cells and tumors are able to coordinate nutrient utilization to support aggressive cell proliferation and survival. Using PET imaging and metabolomics-mass spectrometry, we identified the activation of multiple metabolic pathways such as: glycolysis, autophagy recycling mechanism, glutamine and serine/glycine metabolism, both under physiological and under stress conditions. Finally, differential responses between in vitro and in vivo systems emphasize the advantageous and uncontrolled nature of the in vivo environment, which has a pivotal role in controlling the responses to therapy.