Exploration of oxidized phosphocholine profile in non-small-cell lung cancer.

Introduction: Lung cancer is one of the most frequently studied types of cancer and represents the most common and lethal neoplasm. Our previous research on non-small cell lung cancer (NSCLC) has revealed deep lipid profile reprogramming and redox status disruption in cancer patients. Lung cell membranes are rich in phospholipids that are susceptible to oxidation, leading to the formation of bioactive oxidized phosphatidylcholines (oxPCs). Persistent and elevated levels of oxPCs have been shown to induce chronic inflammation, leading to detrimental effects. However, recent reports suggest that certain oxPCs possess anti-inflammatory, pro-survival, and endothelial barrier-protective properties. Thus, we aimed to measure the levels of oxPCs in NSCLC patients and investigate their potential role in lung cancer. Methods: To explore the oxPCs profiles in lung cancer, we performed in-depth, multi-level metabolomic analyses of nearly 350 plasma and lung tissue samples from 200 patients with NSCLC, including adenocarcinoma (ADC) and squamous cell carcinoma (SCC), the two most prevalent NSCLC subtypes and COPD patients as a control group. First, we performed oxPC profiling of plasma samples. Second, we analyzed tumor and non-cancerous lung tissues collected during the surgical removal of NSCLC tumors. Because of tumor tissue heterogeneity, subsequent analyses covered the surrounding healthy tissue and peripheral and central tumors. To assess whether the observed phenotypic changes in the patients were associated with measured oxPC levels, metabolomics data were augmented with data from medical records. Results: We observed a predominance of long-chain oxPCs in plasma samples and of short-chain oxPCs in tissue samples from patients with NSCLC. The highest concentration of oxPCs was observed in the central tumor region. ADC patients showed higher levels of oxPCs compared to the control group, than patients with SCC. Conclusion: The detrimental effects associated with the accumulation of short-chain oxPCs suggest that these molecules may have greater therapeutic utility than diagnostic value, especially given that elevated oxPC levels are a hallmark of multiple types of cancer.

Optimization of capillary electrophoresis coupled to negative mode electrospray ionization-mass spectrometry using polyvinyl alcohol coated capillaries. Application to a study on non-small cell lung cancer.

Despite recent developments in separation techniques, the analysis of relatively small highly polar negatively charged analytes (e.g. small organic acids, phosphorylated sugars, and underivatized amino acids) remains challenging. Capillary electrophoresis coupled to mass spectrometry (CE-MS) has been included in the untargeted metabolomics toolbox, although mostly in positive polarity. The aim of this study was to assess the use of CE-MS to analyze highly polar and negatively charged metabolites at physiological levels without the need for derivatization. After a preliminary selection, conditions regarding CE (buffers, applied potential, injection time and applied pressure), electrospray parameters (sheath liquid flow, temperature and drying gas flow, nebulizer, and capillary voltage), and fragmentor voltage were optimized using a capillary coated with polyvinyl alcohol (PVA) for the metabolic profiling of anionic compounds compared to fused silica as the reference capillary. In addition, a database of 240 metabolites with two relative migration times (RMT) obtained against methionine sulfone and 2-morpholinoethanesulfonic acid (MES) as internal standards (IS) has been compiled. Finally, the optimized method has been used to characterize the metabolic profile of blood plasma in patients with non-small cell lung cancer (NSCLC). The identified compounds are mostly amino acids and their derivatives, carboxylic acids and organic compounds from the TCA cycle, and sugars and their phosphoderivates. In addition, we performed a comparative study to find significant differences between non-small cell lung cancer (NSCLC) vs non-cancer individuals, and squamous cell carcinoma (SCC) and adenocarcinoma (ADC) vs non-cancer individuals, respectively, searching for differences between the various types of NSCLC.

New molecular mechanisms in cholangiocarcinoma: signals triggering interleukin-6 production in tumor cells and KRAS co-opted epigenetic mediators driving metabolic reprogramming.

BACKGROUND: Cholangiocarcinoma (CCA) is still a deadly tumour. Histological and molecular aspects of thioacetamide (TAA)-induced intrahepatic CCA (iCCA) in rats mimic those of human iCCA. Carcinogenic changes and therapeutic vulnerabilities in CCA may be captured by molecular investigations in bile, where we performed bile proteomic and metabolomic analyses that help discovery yet unknown pathways relevant to human iCCA. METHODS: Cholangiocarcinogenesis was induced in rats (TAA) and mice (JnkΔhepa + CCl4 + DEN model). We performed proteomic and metabolomic analyses in bile from control and CCA-bearing rats. Differential expression was validated in rat and human CCAs. Mechanisms were addressed in human CCA cells, including Huh28-KRASG12D cells. Cell signaling, growth, gene regulation and [U-13C]-D-glucose-serine fluxomics analyses were performed. In vivo studies were performed in the clinically-relevant iCCA mouse model. RESULTS: Pathways related to inflammation, oxidative stress and glucose metabolism were identified by proteomic analysis. Oxidative stress and high amounts of the oncogenesis-supporting amino acids serine and glycine were discovered by metabolomic studies. Most relevant hits were confirmed in rat and human CCAs (TCGA). Activation of interleukin-6 (IL6) and epidermal growth factor receptor (EGFR) pathways, and key genes in cancer-related glucose metabolic reprogramming, were validated in TAA-CCAs. In TAA-CCAs, G9a, an epigenetic pro-tumorigenic writer, was also increased. We show that EGFR signaling and mutant KRASG12D can both activate IL6 production in CCA cells. Furthermore, phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in serine-glycine pathway, was upregulated in human iCCA correlating with G9a expression. In a G9a activity-dependent manner, KRASG12D promoted PHGDH expression, glucose flow towards serine synthesis, and increased CCA cell viability. KRASG12D CAA cells were more sensitive to PHGDH and G9a inhibition than controls. In mouse iCCA, G9a pharmacological targeting reduced PHGDH expression. CONCLUSIONS: In CCA, we identified new pro-tumorigenic mechanisms: Activation of EGFR signaling or KRAS mutation drives IL6 expression in tumour cells; Glucose metabolism reprogramming in iCCA includes activation of the serine-glycine pathway; Mutant KRAS drives PHGDH expression in a G9a-dependent manner; PHGDH and G9a emerge as therapeutic targets in iCCA.

Comprehensive Plasma Metabolomic Profile of Patients with Advanced Neuroendocrine Tumors (NETs). Diagnostic and Biological Relevance.

PURPOSE: High-throughput "-omic" technologies have enabled the detailed analysis of metabolic networks in several cancers, but NETs have not been explored to date. We aim to assess the metabolomic profile of NET patients to understand metabolic deregulation in these tumors and identify novel biomarkers with clinical potential. METHODS: Plasma samples from 77 NETs and 68 controls were profiled by GC-MS, CE-MS and LC-MS untargeted metabolomics. OPLS-DA was performed to evaluate metabolomic differences. Related pathways were explored using Metaboanalyst 4.0. Finally, ROC and OPLS-DA analyses were performed to select metabolites with biomarker potential. RESULTS: We identified 155 differential compounds between NETs and controls. We have detected an increase of bile acids, sugars, oxidized lipids and oxidized products from arachidonic acid and a decrease of carnitine levels in NETs. MPA/MSEA identified 32 enriched metabolic pathways in NETs related with the TCA cycle and amino acid metabolism. Finally, OPLS-DA and ROC analysis revealed 48 metabolites with diagnostic potential. CONCLUSIONS: This study provides, for the first time, a comprehensive metabolic profile of NET patients and identifies a distinctive metabolic signature in plasma of potential clinical use. A reduced set of metabolites of high diagnostic accuracy has been identified. Additionally, new enriched metabolic pathways annotated may open innovative avenues of clinical research.

In vitro generation of oxidized standards for lipidomics. Application to major membrane lipid components.

Membrane lipids (sphingolipids, glycerophospholipids, cardiolipins, and cholesteryl esters) are critical in cellular functions. Alterations in the levels of oxidized counterparts of some of these lipids have been linked to the onset and development of many pathologies. Unfortunately, the scarce commercial availability of chemically defined oxidized lipids is a limitation for accurate quantitative analysis, characterization of oxidized composition, or testing their biological effects in lipidomic studies. To address this dearth of standards, several approaches rely on in-house prepared mixtures of oxidized species generated under in vitro conditions from different sources - non-oxidized commercial standards, liposomes, micelles, cells, yeasts, and human preparations - and using different oxidant systems - UVA radiation, air exposure, enzymatic or chemical oxidant systems, among others. Moreover, high-throughput analytical techniques such as liquid chromatography coupled to mass spectrometry (LC-MS) have provided evidence of their capabilities to study oxidized lipids both in in vitro models and complex biological samples. In this review, we describe the commercial resources currently available, the in vitro strategies carried out for obtaining oxidized lipids as standards for LC-MS analysis, and their applications in lipidomics studies, specifically for lipids found in cell and mitochondria membranes.

Oxidized lipids in the metabolic profiling of neuroendocrine tumors - Analytical challenges and biological implications.

Untargeted metabolomics can be a great tool for exploring new scientific areas; however, wrong metabolite annotation questions the credibility and puts the success of the entire research at risk. Therefore, an effort should be made to improve the quality and robustness of the annotation despite of the challenges, especially when final identification with standards is not possible. Through non-targeted analysis of human plasma samples, from a large cancer cohort study using RP-LC-ESI-QTOF-MS/MS, we have resolved MS/MS annotation through spectral matching, directed to hydroxyeicosatetraenoic acids (HETEs) and, MS/MS structural elucidation for newly annotated oxidized lyso-phosphatidylcholines (oxLPCs). The annotation of unknowns is supported with structural information from fragmentation spectra as well as the fragmentation mechanisms involved, necessarily including data from both polarity modes and different collision energies. In this work, we present evidences that various oxidation products show significant differences between cancer patients and control individuals and we establish a workflow to help identify such modifications. We report here the upregulation of HETEs and oxLPCs in patients with neuroendocrine tumors (NETs). To our knowledge, this is the first attempt to determine HETEs in NETs and one of very few studies where oxLPCs are annotated. The obtained results provide an important insight regarding lipid oxidation in NETs, although their physiological functions still have to be established and require further research.

A review of validated biomarkers obtained through metabolomics.

INTRODUCTION: Studying changes in the whole set of small molecules, final products of biochemical reactions in living systems or metabolites, is extremely appealing because they represent the best approach to identifying what occurs in an organism when samples are collected. However, their usefulness as potential biomarkers is limited by discoveries obtained in small groups without proper validation or even confirmation of the chemical structure. Areas covered: During the past 5 years, more than 900 papers have been published on metabolomics for biomarker discovery, but the numbers are much lower when some criteria of validation are applied. In total, 102 papers have been included in this review. The most frequent disease areas in which these markers have been discovered include the following: cancer, diabetes, and related diseases and neurodegenerative, cardiovascular, autoimmune, liver, and kidney diseases. Expert commentary: Metabolomics has been demonstrated as rapidly growing due to the improvements in instrumentation, mainly mass spectrometry, and data mining software. For application in the clinic, the results should be validated in different stages, from analytical validation to validation in independent sets of samples, using thousands of samples from different sources.

Disfonía como presentación precoz de perforación colónica / Dysphonia as early presentation symptom in colonic perforation

Se presentan dos casos clínicos, en los que la sintomatología principal fueron disfonía y enfisema subcutáneo cervical, encontrándose neumomediastino, neumoperitoneo y retroneumoperitoneo causados por perforación colónica, ambos pacientes fueron intervenidos en el Hospital Campo Arañuelo de Navalmoral de la Mata. Rara vez la disfonía y el enfisema subcutáneo cervical pueden ser la primera manifestación de una perforación oculta del tracto gastrointestinal o de otro proceso retroperitoneal. El enfisema subcutáneo causado por perforaciones colónicas no traumáticas es extremadamente raro, pero debe considerarse cuando el origen del enfisema subcutáneo no ha sido encontrado. Los enfisemas subcutáneos no iatrogénicos son escasos en la bibliografía, y constituyen signos de diverticulitis sigmoidea perforada o carcinoma asociado como casos mas frecuentes. Generalmente el pronóstico de estos enfermos es malo, siendo el enfisema subcutáneo un signo poco frecuente pero ominoso de perforación de víscera hueca abdominal. Ambos pacientes fueron intervenidos y requirieron tratamiento antibiótico de amplio espectro, uno de ellos necesito ser trasladado por necesidad de Unidad de Cuidados Intensivos para soporte ventilatorio, siendo posteriormente reintervenido en nuestro hospital por un absceso residual y dado de alta.

We report two cases with similar main complaints: dysphonia and subcutaneous cervical emphysema. After diagnosis procedures we identified pneumomediastinum, pneumoperitoneum and pneumoretroperitoneum caused by colonic perforation. Both patients underwent surgical treatment at Hospital Campo Arañuelo, Navalmoral de la Mata. Dysphonia and subcutaneous cervical emphysema are infrequent symptoms of presentation for occult gastrointestinal tract perforation or any retroperitoneal condition. Subcutaneous emphysema due to colonic perforation is extremely uncommon but it must to be considerateas a cause when its etiology remains unknown. Non iatrogenic subcutaneous emphysema is very rare in publications and is mostly associated with perforated sigmoid diverticulitis or carcinoma and the prognosis is poor Both patients underwent surgical treatment and also a wide spectrum antibiotic course. One of them needed respiratory support in the intensive care unit, and, in the outcome, he required a second surgical procedure to evacuate a residual abscess before discharge.

[Dysphonia as early presentation symptom in colonic perforation]. / Disfonía como Presentación Precoz de Perforación Colonica.

We report two cases with similar main complaints: dysphonia and subcutaneous cervical emphysema. After diagnosis procedures we identified pneumomediastinum, pneumoperitoneum and pneumoretroperitoneum caused by colonic perforation. Both patients underwent surgical treatment at Hospital Campo Arañuelo, Navalmoral de la Mata. Dysphonia and subcutaneous cervical emphysema are infrequent symptoms of presentation for occult gastrointestinal tract perforation or any retroperitoneal condition. Subcutaneous emphysema due to colonic perforation is extremely uncommon but it must to be considerate as a cause when its etiology remains unknown. Non iatrogenic subcutaneous emphysema is very rare in publications and is mostly associated with perforated sigmoid diverticulitis or carcinoma and the prognosis is poor Both patients underwent surgical treatment and also a wide spectrum antibiotic course. One of them needed respiratory support in the intensive care unit, and, in the outcome, he required a second surgical procedure to evacuate a residual abscess before discharge.