Mass spectrometry imaging of L-[ring-13C6]-labeled phenylalanine and tyrosine kinetics in non-small cell lung carcinoma.

BACKGROUND: Metabolic reprogramming is a common phenomenon in tumorigenesis and tumor progression. Amino acids are important mediators in cancer metabolism, and their kinetics in tumor tissue are far from being understood completely. Mass spectrometry imaging is capable to spatiotemporally trace important endogenous metabolites in biological tissue specimens. In this research, we studied L-[ring-13C6]-labeled phenylalanine and tyrosine kinetics in a human non-small cell lung carcinoma (NSCLC) xenografted mouse model using matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FTICR-MSI). METHODS: We investigated the L-[ring-13C6]-Phenylalanine (13C6-Phe) and L-[ring-13C6]-Tyrosine (13C6-Tyr) kinetics at 10 min (n = 4), 30 min (n = 3), and 60 min (n = 4) after tracer injection and sham-treated group (n = 3) at 10 min in mouse-xenograft lung tumor tissues by MALDI-FTICR-MSI. RESULTS: The dynamic changes in the spatial distributions of 19 out of 20 standard amino acids are observed in the tumor tissue. The highest abundance of 13C6-Phe was detected in tumor tissue at 10 min after tracer injection and decreased progressively over time. The overall enrichment of 13C6-Tyr showed a delayed temporal trend compared to 13C6-Phe in tumor caused by the Phe-to-Tyr conversion process. Specifically, 13C6-Phe and 13C6-Tyr showed higher abundances in viable tumor regions compared to non-viable regions. CONCLUSIONS: We demonstrated the spatiotemporal intra-tumoral distribution of the essential aromatic amino acid 13C6-Phe and its de-novo synthesized metabolite 13C6-Tyr by MALDI-FTICR-MSI. Our results explore for the first time local phenylalanine metabolism in the context of cancer tissue morphology. This opens a new way to understand amino acid metabolism within the tumor and its microenvironment.

Impaired mitochondrial complex I function as a candidate driver in the biological stress response and a concomitant stress-induced brain metabolic reprogramming in male mice.

Mitochondria play a critical role in bioenergetics, enabling stress adaptation, and therefore, are central in biological stress responses and stress-related complex psychopathologies. To investigate the effect of mitochondrial dysfunction on the stress response and the impact on various biological domains linked to the pathobiology of depression, a novel mouse model was created. These mice harbor a gene trap in the first intron of the Ndufs4 gene (Ndufs4GT/GT mice), encoding the NDUFS4 protein, a structural component of complex I (CI), the first enzyme of the mitochondrial electron transport chain. We performed a comprehensive behavioral screening with a broad range of behavioral, physiological, and endocrine markers, high-resolution ex vivo brain imaging, brain immunohistochemistry, and multi-platform targeted mass spectrometry-based metabolomics. Ndufs4GT/GT mice presented with a 25% reduction of CI activity in the hippocampus, resulting in a relatively mild phenotype of reduced body weight, increased physical activity, decreased neurogenesis and neuroinflammation compared to WT littermates. Brain metabolite profiling revealed characteristic biosignatures discriminating Ndufs4GT/GT from WT mice. Specifically, we observed a reversed TCA cycle flux and rewiring of amino acid metabolism in the prefrontal cortex. Next, exposing mice to chronic variable stress (a model for depression-like behavior), we found that Ndufs4GT/GT mice showed altered stress response and coping strategies with a robust stress-associated reprogramming of amino acid metabolism. Our data suggest that impaired mitochondrial CI function is a candidate driver for altered stress reactivity and stress-induced brain metabolic reprogramming. These changes result in unique phenomic and metabolomic signatures distinguishing groups based on their mitochondrial genotype.

Glutamine deprivation counteracts hypoxia-induced chemoresistance.

The microenvironment of solid tumors is a key determinant of therapy efficacy. The co-occurrence of oxygen and nutrient deprivation is a common phenomenon of the tumor microenvironment and associated with treatment resistance. Cholangiocarcinoma (CCA) is characterized by a very poor prognosis and pronounced chemoresistance. A better understanding of the underlying molecular mechanisms is urgently needed to improve therapy strategies against CCA. We sought to investigate the importance of the conditionally essential amino acid glutamine, a centrally important nutrient for a variety of solid tumors, for CCA. Glutamine levels were strongly decreased in CCA samples and the growth of established human CCA cell lines was highly dependent on glutamine. Using gradual reduction of external glutamine, we generated derivatives of CCA cell lines which were able to grow without external glutamine (termed glutamine-depleted (GD)). To analyze the effects of coincident oxygen and glutamine deprivation, GD cells were treated with cisplatin or gemcitabine under normoxia and hypoxia. Strikingly, the well-established phenomenon of hypoxia-induced chemoresistance was completely reversed in GD cells. In order to better understand the underlying mechanisms, we focused on the oncogene c-Myc. The combination of cisplatin and hypoxia led to sustained c-Myc protein expression in wildtype cells. In contrast, c-Myc expression was reduced in response to the combinatorial treatment in GD cells, suggesting a functional importance of c-Myc in the process of hypoxia-induced chemoresistance. In summary, these findings indicate that the mechanisms driving adaption to tumor microenvironmental changes and their relevance for the response to therapy are more complex than expected.

Poor outcome in hypoxic endometrial carcinoma is related to vascular density.

BACKGROUND: Identification of endometrial carcinoma (EC) patients at high risk of recurrence is lacking. In this study, the prognostic role of hypoxia and angiogenesis was investigated in EC patients. METHODS: Tumour slides from EC patients were stained by immunofluorescence for carbonic anhydrase IX (CAIX) as hypoxic marker and CD34 for assessment of microvessel density (MVD). CAIX expression was determined in epithelial tumour cells, with a cut-off of 1%. MVD was assessed according to the Weidner method. Correlations with disease-specific survival (DSS), disease-free survival (DFS) and distant disease-free survival (DDFS) were calculated using Kaplan-Meier curves and Cox regression analysis. RESULTS: Sixty-three (16.4%) of 385 ECs showed positive CAIX expression with high vascular density. These ECs had a reduced DSS compared to tumours with either hypoxia or high vascular density (log-rank p = 0.002). Multivariable analysis showed that hypoxic tumours with high vascular density had a reduced DSS (hazard ratio [HR] 3.71, p = 0.002), DDFS (HR 2.68, p = 0.009) and a trend for reduced DFS (HR 1.87, p = 0.054). CONCLUSIONS: This study has shown that adverse outcome in hypoxic ECs is seen in the presence of high vascular density, suggesting an important role of angiogenesis in the metastatic process of hypoxic EC. Differential adjuvant treatment might be indicated for these patients.

Detection of Localized Hepatocellular Amino Acid Kinetics by using Mass Spectrometry Imaging of Stable Isotopes.

Mass spectrometry imaging (MSI) simultaneously detects and identifies the spatial distribution of numerous molecules throughout tissues. Currently, MSI is limited to providing a static and ex vivo snapshot of highly dynamic systems in which molecules are constantly synthesized and consumed. Herein, we demonstrate an innovative MSI methodology to study dynamic molecular changes of amino acids within biological tissues by measuring the dilution and conversion of stable isotopes in a mouse model. We evaluate the method specifically on hepatocellular metabolism of the essential amino acid l-phenylalanine, associated with liver diseases. Crucially, the method reveals the localized dynamics of l-phenylalanine metabolism, including its in vivo hydroxylation to l-tyrosine and co-localization with other liver metabolites in a time course of samples from different animals. This method thus enables the dynamics of localized biochemical synthesis to be studied directly from biological tissues.

Interdependent Conductances Drive Infraslow Intrinsic Rhythmogenesis in a Subset of Accessory Olfactory Bulb Projection Neurons.

The accessory olfactory system controls social and sexual behavior. However, key aspects of sensory signaling along the accessory olfactory pathway remain largely unknown. Here, we investigate patterns of spontaneous neuronal activity in mouse accessory olfactory bulb mitral cells, the direct neural link between vomeronasal sensory input and limbic output. Both in vitro and in vivo, we identify a subpopulation of mitral cells that exhibit slow stereotypical rhythmic discharge. In intrinsically rhythmogenic neurons, these periodic activity patterns are maintained in absence of fast synaptic drive. The physiological mechanism underlying mitral cell autorhythmicity involves cyclic activation of three interdependent ionic conductances: subthreshold persistent Na(+) current, R-type Ca(2+) current, and Ca(2+)-activated big conductance K(+) current. Together, the interplay of these distinct conductances triggers infraslow intrinsic oscillations with remarkable periodicity, a default output state likely to affect sensory processing in limbic circuits. SIGNIFICANCE STATEMENT: We show for the first time that some rodent accessory olfactory bulb mitral cells-the direct link between vomeronasal sensory input and limbic output-are intrinsically rhythmogenic. Driven by ≥ 3 distinct interdependent ionic conductances, infraslow intrinsic oscillations show remarkable periodicity both in vitro and in vivo. As a novel default state, infraslow autorhythmicity is likely to affect limbic processing of pheromonal information.

A full digital magnetic induction measurement device for non-contact vital parameter monitoring (MONTOS).

Magnetic induction measurements enable contactless monitoring of breathing and heart activity. Since this technique is in the scope of many research groups, there are several research devices available. Most of these devices are suitable for tomography approaches, e.g. edema detection or for monitoring technical processes, such as fluid in tubes or metal blocks. However, these devices are less useable for vital parameter monitoring. In this article, we present an new modular magnetic induction measurement system called MONTOS (Monitoring System) for this scenario. Since the implementation is fully digital, each module can easily be applied to several measurement conditions in vital parameter monitoring, i.e. Multi-Frequency measurement modes, Single-Excitation and Multiple-Measurements or Multiple-Excitation and Single-Measurement. Data output is realized via local area networks (LAN), thereby streaming the data to a monitoring computer. Finally, it will be demonstrated that impedance changes due to breathing of a human adult can be detected.