Tissue Metabolite Profiles Research Articles

Human umbilical cord mesenchymal stem cells restore the ovarian metabolome and rescue premature ovarian insufficiency in mice

BackgroundPremature ovarian insufficiency (POI) is an ovarian dysfunction that seriously affects a woman’s physiological health and reproduction. Mesenchymal stem cell (MSC) transplantation offers a promising treatment option for ovarian restoration in rodent POI models. However, the efficacy and mechanism of it remain unclear.MethodsPOI mice model was generated by cyclophosphamide and busulfan, followed with the treatment of tail-vein injection of the human umbilical cord mesenchymal stem cells (hUCMSCs). Maternal physiological changes and offspring behavior were detected. To reveal the pathogenesis and therapeutic mechanisms of POI, we first compared the metabolite profiles of healthy and POI ovarian tissues using untargeted metabolomics analyses. After stem cell therapy, we then collected the ovaries from control, POI, and hUCMSC-treated POI groups for lipid metabolomics and pseudotargeted metabolomics analysis.ResultsOur results revealed remarkable changes of multiple metabolites, especially lipids, in ovarian tissues after POI generation. Following the transplantation of clinical-grade hUCMSCs, POI mice exhibited significant improvements in body weight, sex hormone levels, estrous cycles, and reproductive capacity. Lipid metabolomics and pseudotargeted metabolomics analyses for the ovaries showed that the metabolite levels in the POI group, mainly lipids, glycerophospholipids, steroids, and amino acids changed significantly compared with the controls’, and most of them returned to near-healthy levels after receiving hUCMSC treatment. Meanwhile, we also observed an increase of monosaccharide levels in the ovaries from POI mice and a decrease after stem cell treatment.ConclusionshUCMSCs restore ovarian function through activating the PI3K pathway by promoting the level of free amino acids, consequently improving lipid metabolism and reducing the concentration of monosaccharides. These findings provide potential targets for the clinical diagnosis and treatment of POI.

Bioactive diterpenoid metabolism and cytotoxic activities of genetically transformed Euphorbia lathyris roots

Plants in the genus Euphorbia produce a wide variety of pharmacologically active diterpenoids with anticancer, multidrug resistance reversal, and antiviral properties. Some are the primary industrial source of ingenol mebutate, which is approved for treatment of the precancerous skin condition actinic keratosis. Similar to other high value phytochemicals, Euphorbia diterpenoids accumulate at low concentrations in planta and chemical synthesis produces similarly low yields. We established genetically transformed root cultures of Euphorbia lathryis as a strategy to gain greater access to diterpenoids from this genus. Transformed roots produced via stem explant infection with Agrobacterium rhizogenes strain 15834 recapitulated the metabolite profiles of field-grown plant roots and aerial tissues. Several putative diterpenoids were present in transformed roots, including ingenol and closely related structures, indicating that root cultures are a promising approach to Euphorbia-specific diterpenoid production. Treatment with methyl jasmonate led to a significant, albeit transient increase in mRNA levels of early diterpenoid biosynthetic enzymes (farnesyl pyrophosphate synthase, geranylgeranyl pyrophosphate synthase, and casbene synthase), suggesting that elicitation could prove useful in future pathway characterization and metabolic engineering efforts. We also show the potential of transformed E. lathyris root cultures for natural product drug discovery applications by measuring their cytotoxic activities using a panel of human carcinoma cell lines derived from prostate, cervix, breast, and lung.

Abstract 3510: The metabolomic profile of urine from prostate cancer patients using capillary electrophoresis mass spectrometry (CEMS)

Abstract Background: Prostate cancer (PCa) pathogenesis is influenced by alterations in cellular metabolism. Metabolomics measures these biochemical changes to create global tissue metabolite profiles. Urinary studies are noninvasive and can potentially identify biomarkers for PCa. Objective: We used Capillary Electrophoresis Mass Spectrometry (CE-MS) to analyze urine from men undergoing prostate biopsy to investigate their metabolomic profiles. Methods: We retrospectively analyzed 40 urine samples (20 from bx demonstrating PCa, 20 from benign) that were prospectively collected from men undergoing prostate biopsy. Analysis of charged metabolites done with Agilent CE-MS system (J Proteome Res. 2:488; 2003). Screening of potential biomarkers was performed with statistical protocols and pathway analyses. Metabolites with levels below the detection limit in all samples were excluded. Relative abundances of metabolites were normalized to levels of creatinine. Pathway analysis with MetaboAnalyst (Metabolites 9:57; 2019). Conclusions: CE-MS analysis identified several metabolic pathways that were altered in urine of men with PCa. These metabolites are involved in steroid, aromatic, microorganism and SCFA processes and warrant targeted studies which are underway in our lab. If validated, they have potential to serve as non-invasive biomarkers for PCa diagnosis and therapeutics Citation Format: Alexander Buko, Leo Cheng, Andrew Gustev, Adam Feldman. The metabolomic profile of urine from prostate cancer patients using capillary electrophoresis mass spectrometry (CEMS) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3510.

Undetectable Free Aromatic Amino Acids in Nails of Breast Carcinoma: Biomarker Discovery by a Novel Metabolite Purification VTGE System.

Background: Metabolic reprogramming in breast cancer is depicted as a crucial change in the tumor microenvironment. Besides the molecular understanding of metabolic heterogeneity, appreciable attention is drawn to characterizing metabolite profiles in tumor tissue and derived biological fluids and tissue materials. Several findings reported on the metabolic alterations of free aromatic amino acids (FAAAs) and other metabolites in biological fluids. Furthermore, there is a significant gap in the development of a suitable method for the purification and analysis of metabolite biomarkers in nails of cancer patients.Methods: To address the metabolite alterations, specifically FAAA levels in nails, fingernail clippings of breast cancer patients (N = 10) and healthy subjects (N-12) were used for extraction and purification of metabolites. Here, we reported a novel and specifically designed vertical tube gel electrophoresis (VTGE) system that helped in the purification of metabolites in the range of 100–1,000 Da from nail materials. Here, the VTGE system uses 15% polyacrylamide under non-denaturing and non-reducing conditions, which makes eluted metabolites directly compatible with LC-HRMS and other analytical techniques. Qualitative and quantitative determination of FAAAs in nail lysates was done in positive ESI mode of the Agilent LC-HRMS platform.Results: The analysis on collected data of nail metabolites clearly suggested that FAAAs including tryptophan, tyrosine, phenylalanine, and histidine were undetectable in nail lysates of breast cancer over healthy subjects. This is a first report that showed highly reduced levels of FAAAs in nails of breast cancer patients. Furthermore, the present observation is in consonance with previous findings that showed cancer cachexia and high amino acid catabolism in breast cancer patients that drive metabolite-led cancer growth and proliferation.Conclusion: This paper provides a proof of concept for a novel and specifically developed VTGE process that showed first evidence on the undetectable level of FAAAs in nails of breast cancer patients as metabolite biomarkers. Here, the authors propose the potential use of a VTGE-assisted process to achieve metabolomic discovery in nails of breast cancer and other tumor types.

Immune response in probiotic-fed New Zealand black-footed abalone (Haliotis iris) under Vibrio splendidus challenge

Vibriosis disease is a major constraint for sustainable molluscan aquaculture. Development of strategies to enhance disease resistance during grow out would greatly reduce stock mortality and boost production yields. In this study, New Zealand black-footed abalone (Haliotis iris) were fed a commercial diet enhanced with multi-strain probiotics (Exiguobacterium JHEb1, Vibrio JH1 and Enterococcus JHLDc) for four months, then challenged with an injection of pathogenic Vibrio splendidus. Host immune responses in haemocytes were characterized using flow cytometry by measuring total haemocyte counts (THC) and viability, degree of apoptosis, and production of reactive oxygen species (ROS) 48 h post-challenge. Probiotic-fed abalone had significantly higher survival rates compared to control animals after the bacterial challenge. Infected probiotic-fed abalone also had significantly higher haemocyte viabilities, slightly lower proportions of haemocytes undergoing early apoptosis, and lower proportions of ROS-producing haemocytes compared to infected control-fed abalone. In addition, metabolite profiles of muscle tissues generated via gas chromatography–mass spectrometry (GC-MS) delivered complimentary evidence to support a perturbed ROS-regulatory system in infected abalone through changes in key metabolites associated with glutathione biosynthesis. The results of this study provide valuable information to assist in farm management practices, leading to enhance production and sustainability of the New Zealand abalone aquaculture industry.

Combining MALDI mass spectrometry imaging and droplet-base surface sampling analysis for tissue distribution, metabolite profiling, and relative quantification of cyclic peptide melanotan II

Peptides have become a fast-growing segment of the pharmaceutical industry over the past few decades. It is essential to develop cutting edge analytical techniques to support the discovery and development of peptide therapeutics, especially to examine their absorption, distribution, metabolism and excretion (ADME) properties. Herein, we utilized two label-free mass spectrometry (MS) based techniques to investigate representative challenges in developing therapeutic peptides, such as tissue distribution, metabolic stability and clearance. A tool proof-of-concept cyclic peptide, melanotan II, was used in this study. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), which is a well-developed label-free imaging technique, was used to map the detailed molecular distribution of melanotan II and its metabolites. Droplet-based liquid microjunction surface sampling liquid chromatography-high resolution mass spectrometry (LMJ-SSP-LC-HRMS) was used in combination with MALDI-MSI to rapidly profile molecular information and provide structural insights on drug and metabolites. Using both techniques in parallel allowed a more comprehensive and complementary data set than using either technique independently. We envision MALDI-MSI and droplet-based LMJ-SSP-LC-HRMS, which can be used in combination or as standalone techniques, to become valuable tools for assessing the in vivo fate of peptide therapeutics in support of drug discovery and development.

Alterations in Tissue Metabolite Profiles with Amifostine-Prophylaxed Mice Exposed to Gamma Radiation.

Acute exposure to high-dose ionizing irradiation has the potential to severely injure the hematopoietic system and its capacity to produce vital blood cells that innately serve to ward off infections and excessive bleeding. Developing a medical radiation countermeasure that can protect individuals from the damaging effects of irradiation remains a significant, unmet need and an area of great public health interest and concern. Despite significant advancements in the field of radiation countermeasure development to find a nontoxic and effective prophylactic agent for acute radiation syndrome, no such drug has yet been approved by the Food and Drug Administration. This study focuses on examining the metabolic corrections elicited by amifostine, a potent radioprotector, on tissues of vital body organs, such as the heart, spleen, and kidney. Our findings indicate that prophylaxis with this drug offers significant protection against potentially lethal radiation injury, in part, by correction of radiation-induced metabolic pathway perturbations.

Proteomic and Metabolite Profiling Reveals Profound Structural and Metabolic Reorganization of Adipocyte Mitochondria in Obesity.

Previous studies have revealed decreased mitochondrial respiration in adipocytes of obese mice. This study aimed to identify the molecular underpinnings of altered mitochondrial metabolism in adipocytes. Untargeted proteomics of mitochondria isolated from adipocytes and metabolite profiling of adipose tissues were conducted in diet-induced obese (DIO) and lean mice. Subcutaneous and intra-abdominal adipose tissues were studied to depict depot-specific alterations. In subcutaneous adipocytes of DIO mice, changes in proteins related to mitochondrial structure and function were observed. Mitochondrial proteins of the inner and outer membrane were strongly reduced, whereas proteins of key matrix metabolic pathways were increased in the obese versus lean state, as further substantiated by metabolite profiling. A pronounced decrease in the oxidative phosphorylation (OXPHOS) enzymatic equipment and cristae density of the inner membrane was identified. In intra-abdominal adipocytes, similar systematic downregulation of the OXPHOS machinery in obesity occurred, but there was no regulation of outer membrane or matrix proteins. Protein components of the OXPHOS machinery are systematically downregulated in adipose tissues of DIO mice compared with lean mice. Loss of the mitochondrial OXPHOS capacity in adipocytes may aggravate the development of metabolic disease.

Metabolite profile comparisons between ascending and descending colon tissue in healthy adults.

BACKGROUNDObesity is a risk factor for colorectal cancer, yet metabolic distinctions between healthy right and left colon tissue, before cancer is diagnosed, remains largely unknown. This study compared right-ascending and left-descending colon tissue metabolomes to identify differences from the stool metabolome in normal weight, overweight, and obese adults.AIMTo examine right and left colon tissue metabolites according to body mass index that may serve as mechanistic targets for interventions and biomarkers for colon cancer risk.METHODSGlobal, non-targeted metabolomics was applied to assess right-ascending and left-descending colon tissue collected from healthy adults undergoing screening colonoscopies to test the hypothesis that BMI differentially impacts colon tissue metabolite profiles. The colon tissue and stool metabolome of healthy adults (n = 24) was analyzed for metabolite signatures and metabolic pathway networks implicated in progression of colorectal cancer.RESULTSAscending and descending colon contained 504 host, food, and microbiota-derived metabolites from normal weight, overweight and obese adults grouped according to body mass index. Amino acids, lipids, and nucleotides were among the chemical types that further differentiated from the stool metabolite profiles. Normal weight adults had 46 significantly different metabolites between ascending and descending colon tissue locations, whereas there were 37 metabolite differences in overweight and 28 metabolite differences for obese adults (P < 0.05). Obese adults had trimethylamine N-oxide, endocannabinoids and monoacylglycerols with different relative abundances identified between ascending and descending colon. Primary and secondary bile acids, vitamins, and fatty acids also showed marked relative abundance differences in colon tissue from overweight/obese adults.CONCLUSIONThere were metabolite profile differences between right-ascending and left-descending colon tissue in healthy adults. Colon lipids and other metabolites in obese and overweight adults were distinguished from normal weight participants and associated with gut inflammation, nutrient absorption, and products of microbiota metabolism.

The Effect of Tryptophan 2,3-Dioxygenase Inhibition on Kynurenine Metabolism and Cognitive Function in the APP23 Mouse Model of Alzheimer's Disease.

Alzheimer’s disease (AD) is associated with progressive endogenous neurotoxicity and hampered inflammatory regulation. The kynurenine (Kyn) pathway, which is controlled by tryptophan 2,3-dioxygenase (TDO), produces neuroactive and anti-inflammatory metabolites. Age-related Kyn pathway activation might contribute to AD pathology in humans, and inhibition of TDO was found to reduce AD-related cellular toxicity and behavioral deficits in animal models. To further explore the effect of aging on the Kyn pathway in the context of AD, we analyzed Kyn metabolite profiles in serum and brain tissue of the APP23 amyloidosis mouse model. We found that aging had genotype-independent effects on Kyn metabolite profiles in serum, cortex, hippocampus and cerebellum, whereas serum concentrations of many Kyn metabolites were reduced in APP23 mice. Next, to further establish the role of TDO in AD-related behavioral deficits, we investigated the effect of long-term pharmacological TDO inhibition on cognitive performance in APP23 mice. Our results indicated that TDO inhibition reversed recognition memory deficits without producing measurable changes in cerebral Kyn metabolites. TDO inhibition did not affect spatial learning and memory or anxiety-related behavior. These data indicate that age-related Kyn pathway activation is not specific for humans and could represent a cross-species phenotype of aging. These data warrant further investigation on the role of peripheral Kyn pathway disturbances and cerebral TDO activity in AD pathophysiology.

Acute and short-term administrations of delta-9-tetrahydrocannabinol modulate major gut metabolomic regulatory pathways in C57BL/6 mice

Delta-9-tetrahydrocannabinol (THC) is the primary psychoactive compound in Cannabis, which is studied extensively for its medicinal value. A central gap in the science is the underlying mechanisms surrounding THC’s therapeutic effects and the role of gut metabolite profiles. Using a mass-spectrometry based metabolomics, we show here that intraperitoneal injection of THC in C57BL/6 mice modulates metabolic profiles that have previously been identified as integral to health. Specifically, we investigated the effects of acute (single THC injection denoted here as ‘1X’) and short -term (five THC injections on alternate days denoted as ‘5X’) THC administration on fecal and intestinal tissue metabolite profiles. Results are consistent with the hypothesis that THC administration alters host metabolism by targeting two prominent lipid metabolism pathways: glycerophospholipid metabolism and fatty acid biosynthesis.

NMR for metabolomics studies of Crataegus rhipidophylla Gand.

Crataegus products for food, nutritional, and pharmaceutical uses are available over the counter in the global market. The aerial parts are enriched with biochemicals and are used to treat cardiovascular, respiratory, and liver disorders and cancer. Detailed study to understand the chemical composition and different levels within medicinally important plant tissues needs hours, such as plant response for metabolite flux and quality control purposes. Therefore, comprehensive metabolite profiling (qualitatively and quantitatively) of different tissues [leaves, flowers, stems, fruits (without seeds), and seeds; n = 5] of Crataegus rhipidophylla Gand. was performed by NMR techniques in one session. Fifty-eight compounds of different classes were unambiguously identified with theuse of 1D and 2D NMR techniques (1H, 13C, distortionless enhancement by polarization transfer with 135° angle, correlation spectroscopy, heteronuclear single quantum coherence, heteronuclear multiple bond correlation) and quantified by a relative quantification method. Chemical compounds such as amygdalin (seeds), rutin (flowers), isovitexin (fruits), shikimic acid (leaves), and epicatechin (stems) were identified as authentication as well as discrimination markers for the respective C. rhipidophylla tissues. The fruits contain a significant mixture of important metabolites, followed by flowers and leaves. Trends of metabolite levels, upregulation, and downregulation in plant tissues were found, which highlights the responses of plant tissues in a specific time. NMR-based comprehensive metabolite information was used to develop the chemical signature, quality control chemical markers, and strategies to understand the chemical diversity of C. rhipidophylla. This chemical information from different aerial tissues will provide guidance for the extensive utilization of Crataegus.

A Combined Metabolomic and Proteomic Study Revealed the Difference in Metabolite and Protein Expression Profiles in Ruminal Tissue From Goats Fed Hay or High-Grain Diets.

Currently, knowledge about the impact of high-grain (HG) feeding on metabolite and protein expression profiles in ruminal tissue is limited. In this study, a combination of proteomic and metabolomic approaches was applied to evaluate metabolic and proteomic changes of the rumen epithelium in goats fed a hay diet (Hay) or HG diet. At the metabolome level, results from principal component analysis (PCA) and PLS-DA revealed clear differences in the biochemical composition of ruminal tissue of the control (Hay) and the grain-fed groups, demonstrating the evident impact of HG feeding on metabolite profile of ruminal epithelial tissues. As compared with the Hay group, HG feeding increased the levels of eight metabolites and decreased the concentrations of seven metabolites in ruminal epithelial tissues. HG feeding mainly altered starch and sucrose metabolism, purine metabolism, glyoxylate and dicarboxylate metabolism, glycerolipid metabolism, pyruvate metabolism, glycolysis or gluconeogenesis, galactose metabolism, glycine, serine and threonine metabolism, and arginine and proline metabolism in ruminal epithelium. At the proteome level, 35 differentially expressed proteins were found in the rumen epithelium between the Hay and HG groups, with 12 upregulated and 23 downregulated proteins. The downregulated proteins were related to fatty acid metabolism, carbohydrate metabolic processes and nucleoside metabolic processes, while most of upregulated proteins were involved in oxidative stress and detoxification. In general, our findings revealed that HG feeding resulted in differential proteomic and metabolomic profiles in the rumen epithelia of goats, which may contribute to better understanding how rumen epithelium adapt to HG feeding.

Effect of Thermal Stress on Tissue Ultrastructure and Metabolite Profiles During Initiation of Radiata Pine Somatic Embryogenesis.

Climate change will inevitably lead to environmental variations, thus plant drought tolerance will be a determinant factor in the success of plantations and natural forestry recovery. Some metabolites, such as soluble carbohydrates and amino acids, have been described as being the key to both embryogenesis efficiency and abiotic stress response, contributing to phenotypic plasticity and the adaptive capacity of plants. For this reason, our main objectives were to evaluate if the temperature during embryonal mass initiation in radiata pine was critical to the success of somatic embryogenesis, to alter the morphological and ultrastructural organization of embryonal masses at cellular level and to modify the carbohydrate, protein, or amino acid contents. The first SE initiation experiments were carried out at moderate and high temperatures for periods of different durations prior to transfer to the control temperature of 23°C. Cultures initiated at moderate temperatures (30°C, 4 weeks and 40°C, 4 days) showed significantly lower initiation and proliferation rates than those at the control temperature or pulse treatment at high temperatures (50°C, 5 min). No significant differences were observed either for the percentage of embryogenic cell lines that produced somatic embryos, or for the number of somatic embryos per gram of embryonal mass. Based on the results from the first experiments, initiation was carried out at 40°C 4 h; 50°C, 30 min; and a pulse treatment of 60°C, 5 min. No significant differences were found for the initiation or number of established lines or for the maturation of somatic embryos. However, large morphological differences were observed in the mature somatic embryos. At the same time, changes observed at cellular level suggested that strong heat shock treatments may trigger the programmed cell death of embryogenic cells, leading to an early loss of embryogenic potential, and the formation of supernumerary suspensor cells. Finally, among all the differences observed in the metabolic profile, it is worth highlighting the accumulation of tyrosine and isoleucine, both amino acids involved in the synthesis of abiotic stress response-related secondary metabolites.

Depot-Specific Adipose Tissue Metabolite Profiles and Corresponding Changes Following Aerobic Exercise.

Objectives: Total, visceral, and abdominal subcutaneous adipose tissue (AT) depots have distinct associations with cardiometabolic health; however, the metabolite profiles that characterize each AT depot and its reduction following exercise are poorly understood. Our objectives were to (1) assess the independent associations between identified metabolites and total, visceral and abdominal subcutaneous AT; and (2) examine whether changes in metabolite concentrations and AT mass following aerobic exercise are associated.Methods: A secondary analysis was performed in 103 middle-aged abdominally obese men and women {[mean (SD)], 52.4 (8.0) years} randomized into one of four groups varying in exercise amount and intensity for 6 months duration: high amount high intensity, high amount low intensity, low amount low intensity, and control. One hundred and forty seven metabolites were profiled by liquid chromatography-tandem mass spectrometry. AT mass was measured by magnetic resonance imaging (MRI).Results: Individual metabolite associations with AT depots confirmed several established cross-sectional relationships between the obesity phenotype and metabolic pathways. Collapsed across exercise groups, reduction in visceral AT predicted increases in pyroglutamic acid (B = −0.41) and TCA cycle intermediates [succinic (B = −0.41) and fumaric acid (B = −0.20)], independent of change in total AT. Changes in UDP-GlcNAc (B = 0.43), pyroglutamic acid (B = −0.35), histidine (B = 0.20), citric acid/isocitric acid (B = −0.20), and creatine (B = 0.27) were significantly associated with changes in total AT (false discovery rate = 0.1).Conclusions: Our findings point to potential biomarkers of depot-specific AT reduction that may play a direct role in mediating cardiometabolic improvements.

Metabolic analyses reveal growth characteristics of young tea shoots in spring

Tea plant (Camellia sinensis L.) is one of the three leading crop sources of non-alcoholic commercial beverages around the world. It is harvested for its young shoots and steeped into tea infusion. To reveal the metabolic characteristics of young tea shoots growing in early spring, we conducted metabolite profiling of different shoot tissues using gas chromatography–mass spectrometry and ultra-high performance liquid chromatography–tandem mass spectrometry. Metabolic profiling indicated that different tissues varied widely mainly in levels of organic acids, flavonoids, amino acids, polyols and sugars. The results suggested that the buds needed more energy and amino acids for chloroplasts’ biogenesis and expansion. The leaves accumulated large amounts of tryptophan and indoleacrylic acid, which contributed to leaf growth. They also accumulated sugars, sugar alcohols and flavonoids that contributed to tea quality. The stems accumulated gibberellin A14, l-theanine, pyroglutamic acid, 4-aminobutyric acid and glutamine, which may promote stem elongation; and stem accumulation of quinic and shikimic acids contributed to stem lignification. This study showed metabolic characteristics of tea shoot growth in spring, and revealed that different parts of the young shoot may specifically rely on tissue-specific metabolites for their growth.

Tissue and serum metabolite profiling reveals potential biomarkers of human hepatocellular carcinoma

Background and aimsMetabolomics serves as an important tool in distinguishing changes in metabolic pathways and the diagnosis of human disease. Hepatocellular carcinoma (HCC) is a malignance present of heterogeneous metabolic disorder and lack of effective biomarker for surveillance and diagnosis. In this study, we searched for potential metabolite biomarkers of HCC using tissue and serum metabolomics approach. MethodsA total of 30 pairs of matched liver tissue samples from HCC patients and 90 serum samples (30 HCC patients, 30 liver cirrhosis patients, and 30 healthy individuals) were assessed. Metabolomics was performed through ultra performance liquid chromatography-mass spectrometry in conjunction with multivariate and univariate statistical analyses. ResultsA total of six differential metabolites including chenodeoxycholic acid (CDCA), glycocholic acid (GCA), LPC20:5, LPE18:0, succinyladenosine and uridine were present in HCC tissue and serum samples. CDCA, LPC20:5, succinyladenosine and uridine were used to construct a diagnostic model based on logistic regression. The four-metabolite panel discriminated HCC from liver cirrhosis with an AUC score of 0.938, sensitivity of 93.3% and specificity of 86.7%. For all HCC and cirrhosis patients, the diagnostic accuracy increased to 96.7% and 90.0%, respectively. ConclusionThe combination of CDCA, LPC20:5, succinyladenosine and uridine can be used as a biomarker panel to improve HCC sensitivity and specificity. This panel significantly benefits HCC diagnostics and reveals new insight into HCC pathogenesis.

Terpene profiling, transcriptome analysis and characterization of cis-β-terpineol synthase from Ocimum

Ocimum species produces a varied mix of different metabolites that imparts immense medicinal properties. To explore this chemo-diversity, we initially carried out metabolite profiling of different tissues of five Ocimum species and identified the major terpenes. This analysis broadly classified these five Ocimum species into two distinct chemotypes namely, phenylpropanoid-rich and terpene-rich. In particular, β-caryophyllene, myrcene, limonene, camphor, borneol and selinene were major terpenes present in these Ocimum species. Subsequently, transcriptomic analysis of pooled RNA samples from different tissues of Ocimum gratissimum, O. tenuiflorum and O. kilimandscharicum identified 38 unique transcripts of terpene synthase (TPS) gene family. Full-length gene cloning, followed by sequencing and phylogenetic analysis of three TPS transcripts were carried out along with their expression in various tissues. Terpenoid metabolite and expression profiling of candidate TPS genes in various tissues of Ocimum species revealed spatial variances. Further, putative TPS contig 19414 (TPS1) was selected to corroborate its role in terpene biosynthesis. Agrobacterium-mediated transient over-expression assay of TPS1 in the leaves of O. kilimandscharicum and subsequent metabolic and gene expression analyses indicated it as a cis-β-terpineol synthase. Overall, present study provided deeper understanding of terpene diversity in Ocimum species and might help in the enhancement of their terpene content through advanced biotechnological approaches.

Tissue metabolite profiles for the characterisation of paediatric cerebellar tumours

Paediatric brain tumors are becoming well characterized due to large genomic and epigenomic studies. Metabolomics is a powerful analytical approach aiding in the characterization of tumors. This study shows that common cerebellar tumors have metabolite profiles sufficiently different to build accurate, robust diagnostic classifiers, and that the metabolite profiles can be used to assess differences in metabolism between the tumors. Tissue metabolite profiles were obtained from cerebellar ependymoma (n = 18), medulloblastoma (n = 36), pilocytic astrocytoma (n = 24) and atypical teratoid/rhabdoid tumors (n = 5) samples using HR-MAS. Quantified metabolites accurately discriminated the tumors; classification accuracies were 94% for ependymoma and medulloblastoma and 92% for pilocytic astrocytoma. Using current intraoperative examination the diagnostic accuracy was 72% for ependymoma, 90% for medulloblastoma and 89% for pilocytic astrocytoma. Elevated myo-inositol was characteristic of ependymoma whilst high taurine, phosphocholine and glycine distinguished medulloblastoma. Glutamine, hypotaurine and N-acetylaspartate (NAA) were increased in pilocytic astrocytoma. High lipids, phosphocholine and glutathione were important for separating ATRTs from medulloblastomas. This study demonstrates the ability of metabolic profiling by HR-MAS on small biopsy tissue samples to characterize these tumors. Analysis of tissue metabolite profiles has advantages in terms of minimal tissue pre-processing, short data acquisition time giving the potential to be used as part of a rapid diagnostic work-up.

Abstract 628: Immunometabolic signature of papillary thyroid carcinoma

Abstract Introduction: Papillary thyroid carcinoma (PTC) is the most common subtype of thyroid cancer with rapidly rising incidence. The interface between PTC tumor metabolic reprogramming and metabolic immunomodulation remains poorly understood. Experimental Procedures: We sought to investigate the immunometabolic interaction patterns of PTC tumors and to determine whether a correlative relationship exists between tumor-infiltrating immune cell phenotypes and the expression of tumor metabolic enzymes. We performed a computational immunogenomic analysis to calculate leukocyte fractions within 496 PTC tumors from The Cancer Genome Atlas (TCGA). Immunogenomic analysis was performed using CIBERSORT (Newman AM et al., Nature Methods2015), a deconvolution method to calculate leukocyte fractions using RNA gene expression signatures. The PTC immune cell fractions were then compared to the RNA expression of tumor metabolic enzymes within the TCGA data using regression analysis. Matching targeted metabolomic analysis (Metabolon, Inc.) of 20 human PTC tumors was performed to determine tumor metabolite profiles associated with perturbed tumor cellular metabolic pathways based on the TCGA data analysis. Results: The immunogenomic and PTC metabolic pathway enzyme expression analysis demonstrated significant correlation between immune cell types and major metabolic pathway enzymes. Specifically, tumor glycolytic enzymes upstream of the TCA cycle correlated with CD8 (hexokinase-1, p&amp;lt;0.0001) and T regulatory (enolase, p&amp;lt;0.0001) cell infiltrates. Enhanced pyruvate cycling into the TCA cycle correlated with the presence of several immune cell types (malic enzyme 2 vs. follicular helper T cells, p&amp;lt;0.0001; pyruvate carboxylase vs CD8 T cells, p&amp;lt;0.0001). The expression of TCA cycle enzymes correlated with the fraction of macrophages (aconitase, p&amp;lt;0.0001; IDH1, p&amp;lt;0.05). Significant differences in PTC tumor and normal thyroid tissue metabolite profiles were observed in glycolysis and TCA cycle, further supporting our findings of immunometabolic plasticity it PTC. Together, these findings highlight the central role TCA cycle activity plays in PTC tumor immunometabolism. Conclusion: We demonstrate, for the first time, a distinct immunometabolic tumor signature supported by tumor metabolic reprogramming in PTC tumors. This novel characterization approach provides an important foundation for future functional metabolic studies within the tumor and immunologic microenvironment. A mechanistic understanding of the complex integration of immune networks and cellular metabolism can be harnessed for future therapeutic immunometabolic modulation in PTC. Citation Format: Vivian L. Weiss, Naira Baregamian. Immunometabolic signature of papillary thyroid carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 628.