High-resolution 1H Magnetic Resonance Research Articles

Characterization of lipomatous tumors with high-resolution 1H MRS at 17.6T: Do benign lipomas, atypical lipomatous tumors and liposarcomas have a distinct metabolic signature?

BackgroundDistinguishing between some benign lipomas (BLs), atypical lipomatous tumors (ALTs), and dedifferentiated liposarcomas (DDLs) can be challenging due to overlapping magnetic resonance imaging characteristics, and poorly understood molecular mechanisms underlying the malignant transformation of liposarcomas.PurposeTo identify metabolic biomarkers of the lipomatous tumor spectrum by examining human tissue specimens using high-resolution 1H magnetic resonance spectroscopy (MRS).Materials and methodsIn this prospective study, human tissue specimens were obtained from participants who underwent surgical resection for radiologically-indeterminate lipomatous tumors between November 2016 and May 2019. Tissue specimens were obtained from normal subcutaneous fat (n=9), BLs (n=10), ALTs (n=7) and DDLs (n=8). Extracts from specimens were examined with high-resolution MRS at 17.6T. Computational modeling of pattern recognition-based cluster analysis was utilized to identify significant differences in metabolic signatures between the lipomatous tumor types.ResultsSignificant differences between BLs and ALTs were observed for multiple metabolites, including leucine, valine, branched chain amino acids, alanine, acetate, glutamine, and formate. DDLs were distinguished from ALTs by increased glucose and lactate, and increased phosphatidylcholine. Multivariate principal component analysis showed clear clustering identifying distinct metabolic signatures of the tissue types.ConclusionMetabolic signatures identified in 1H MR spectra of lipomatous tumors provide new insights into malignant progression and metabolic targeting. The metabolic patterns identified provide the foundation of developing noninvasive MRS or PET imaging biomarkers to distinguish between BLs, ALTs, and DDLs.

Abstract 6353: Metabolic changes in the spleen and pancreas induced by PDAC xenografts with or without glutamine transporter downregulation

Abstract Introduction: Our ongoing studies are focused on characterizing metabolic changes induced in the organs of mice with cachexia-inducing Pa04C human pancreatic cancer xenografts. Because pancreatic cancer cells are glutamine dependent [1], we downregulated the glutamine transporter SLC1A5 in Pa04C cells to determine if metabolic changes induced in the spleen and pancreas by Pa04C tumors were normalized when SLC1A5 was downregulated in these tumors. Metabolic patterns were characterized using high-resolution quantitative 1H magnetic resonance spectroscopy (MRS) of spleen and pancreas tissue obtained from normal mice and mice with Pa04C tumors and mice with Pa04C tumors with SLC1A5 downregulated. Method: Patient derived cachexia-inducing Pa04C pancreatic cancer cells were lentivirally transduced to express shRNA to stably downregulate SLC1A5. Mice were euthanized once tumors were ~500 mm3, the spleen and pancreas were excised and snap frozen. Snap frozen spleen (normal n= 5, Pa04C n= 11, Pa04C_SLC1A5 n= 10) and pancreas (normal n= 4, Pa04C n= 16, Pa04C_SLC1A5 n= 10) tissue samples were pulverized for dual phase extraction. The aqueous phase was used for 1H MRS analysis. Topspin 3.5 software was used for data processing and analyses. Results and Discussion: Significant downregulation of SLC1A5 mRNA and protein was confirmed in Pa04C_SLC1A5 cells and tumors. SLC1A5 downregulation resulted in significant growth delay and attenuation of weight loss. A comparison of normal mice vs empty vector/wild type tumor (EV/WT) bearing mice identified significant changes in succinate, aspartate and fumarate in the spleen, lactate, acetate, pyruvate, methionine, asparagine, creatine, choline phosphocholine, uracil, histidine and phenylalanine in the pancreas, with leucine, isoleucine, valine, alanine, glutamate, glutamine, glutathione, glycerophosphocholine, glycine, glucose and tyrosine commonly altered in the spleen and pancreas. A comparison of normal vs Pa04C_ SLC1A5 tumor bearing mice identified similar metabolic changes in the spleen and pancreas but these were reduced. Fumarate did not change in the spleen, and of the metabolic changes common to spleen and pancreas, glutamine did not change when tumor SLC1A5 was downregulated. Metabolite changes induced only in the pancreas were also similar to normal vs EV/WT with the exception of a change in glutamine with SLC1A5 downregulation and no change in lactate. Our data highlight the profound metabolic changes in spleen and pancreas metabolism that occur with growth of a cachexia-inducing pancreatic cancer xenograft, and the impact on these metabolic patterns as a result of downregulating the glutamine transporter in these cancer cells. The metabolic patterns identified in the spleen and pancreas may provide novel targets to reduce the morbidity from cachexia. Reference: 1. Son J et al, Nature. 2013;496(7443):101-5. Citation Format: Raj Kumar Sharma, Santosh Kumar Bharti, Balaji Krishnamachary, Yelena Mironchik, Paul Winnard Jr., Marie-France Penet, Zaver M. Bhujwalla. Metabolic changes in the spleen and pancreas induced by PDAC xenografts with or without glutamine transporter downregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6353.

Abstract 2353: Metabolic reprogramming by SLC1A5 downregulation in pancreatic cancer cells

Abstract Introduction: Pancreatic cancers exhibit limited response to chemo- and radiation therapy. Identifying novel targets for pancreatic cancer treatment is important for new treatment strategies. Because pancreatic cancer cells are ‘glutamine avid' [1], targeting glutamine metabolic pathways can provide novel options for treatment. The glutamine transporter, SLC1A5, is being actively investigated as a pharmacological target in cancer [2]. Here we have engineered human pancreatic cells expressing shRNA to downregulate the glutamine transporter SLC1A5. We performed high-resolution 1H magnetic resonance spectroscopy (MRS) to understand the metabolic reprograming that occurs with SLC1A5 downregulation in these two cell lines. Method: Panc1 and Pa04C pancreatic cancer cell lines were genetically engineered to express shRNA against SLC1A5 using lentiviral transduction. Downregulation of SLC1A5 was verified with qRTPCR and immunoblotting. Control cells expressing an empty vector (EV) were also engineered. Dual phase extraction was performed as previously described [3]. High-resolution 1H MRS was performed on the aqueous phase extracts and recorded on a 750 MHz spectrometer. All data acquisition, processing and quantification was performed with TOPSPIN 3.5 software. Spectra were obtained from Panc1EV (n=4), Pa04CEV (n=4), Panc1_SLC1A5_shRNA (n=4) and Pa04C_SLC1A5_shRNA (n=4) cells. Results and Discussion: SLC1A5 downregulation significantly decreased the glutamine/glutamate ratio in Pa04C_SLC1A5_shRNA cells but not in Panc1_SLC1A5_shRNA cells. Instead, in Panc1_SLC1A5_shRNA cells, a significant decrease of the amino acids leucine, isoleucine, valine, tyrosine, histidine, and phenylalanine was observed. Lactate and fumarate also significantly decreased in Panc1_SLC1A5_shRNA cells. Other than the significant decrease of glutamine/glutamate in the Pa04C_SLC1A5_shRNA cells, no other significant metabolic differences were observed in these cells. Our data expand the understanding of the diverse metabolic reprogramming that occurs following SLC1A5 downregulation in two human pancreatic cancer cell lines that may lead to the development of additional metabolic targets. Supported by NIH R01CA193365 and R35CA209960.1. Son J et al., Nature. 2013; 2. Bhutia YD et al., Biochim Biophys Acta. 2016; 3. Winnard PT Jr. et al., J Cachexia Sarcopenia Muscle. 2020. Citation Format: Raj Kumar Sharma, Balalji Krishnamachary, Ishwarya Sivakumar, Yelena Mironchik, Santosh Kumar Bharti, Zaver M. Bhujwalla. Metabolic reprogramming by SLC1A5 downregulation in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2353.

Abstract 2354: Downregulating the glutamine transporter, SLC1A5, significantly reduces cachexia in a PDAC xenograft

Abstract Cachexia occurs with high frequency and severity in pancreatic ductal adenocarcinoma (PDAC) patients [1]. Cachectic patients experience a wide range of symptoms affecting the function of organs such as muscle, liver, brain, and heart, causing significant morbidity [2]. In high-resolution 1H magnetic resonance spectroscopy (MRS) studies of extracts, we previously observed significant perturbation of glutamine in the brain and plasma of mice with a cachexia-inducing PDAC xenograft [3] that prompted us to evaluate the effects of modifying tumor glutamine metabolism on cachexia. We investigated tumors derived from cachexia-inducing Pa04C cells engineered to express shRNA against the glutamine transporter, SLC1A5, glutaminase (GLS) 1, and GLS 2. Patient-derived cachexia-inducing Pa04C cells were stably transduced with virions that expressed shRNA against GLS1 or GLS2 or SLC1A5. Pooled clones were obtained with puromycin selection. Empty vector (EV) cells were also established. Downregulation of target genes was confirmed with mRNA and protein expression characterization. The effects of gene knockdown on tumor growth and weight-loss were determined following subcutaneous inoculation of engineered cells or wild type cells in SCID mice. Longitudinal tumor growth, weights and percent weight changes were determined in 7 wild type (WT), 7 EV, 9 GLS1 downregulated, 9 GLS2 downregulated, and 9 SLC1A5 downregulated tumor bearing mice. Once tumors were ~500 mm3 in volume, tumors were harvested from euthanized mice, and snap frozen for molecular analysis. Protein and mRNA obtained from tumors was validated for downregulation of target genes. Efficient downregulation of SLC1A5, GLS1 and GLS2 mRNA and protein was confirmed in tumors. Downregulating SLC1A5 significantly reduced tumor growth. But, downregulating GLS1 or GLS2 did not reduce tumor growth and, in fact, GLS1 downregulated tumors grew significantly faster than WT or EV tumors. Importantly, for comparable tumor volumes, we found that body weight loss was markedly reduced in mice with SLC1A5 downregulated tumors. Although GLS1 downregulated tumors grew faster than WT tumors, weight loss was attenuated at comparable tumor volumes in these tumors although not to the same extent as in SLC1A5 downregulated tumors. These data highlight potential role of SLC1A5 in PDAC tumor treatment and in the treatment of PDAC-induced cachexia, and support targeting the glutamine/glutamate axis in PDAC to reduce or reverse cachexia.Supported by NIH R35 CA209960 and R01 CA193365. 1. Fearon KC, Baracos VE. Cachexia in pancreatic cancer: new treatment options and measures of success. 2010; 2. Inui A. Cancer anorexia-cachexia syndrome: current issues in research and management. CA Cancer J Clin. 2002; 3. Winnard PT, Jr., et al., Brain metabolites in cholinergic and glutamatergic pathways are altered by pancreatic cancer cachexia. J Cachexia Sarcopenia Muscle. 2020. Citation Format: Balaji Krishnamachary, Ishwarya Sivakumar, Yelena Mironchik, Raj Kumar Sharma, Santosh Kumar Bharti, Marie-France Penet, Paul Winnard, Flonne Wildes, Eibhlin Goggins, Anirban Maitra, Michael G. Goggins, Zaver M. Bhujwalla. Downregulating the glutamine transporter, SLC1A5, significantly reduces cachexia in a PDAC xenograft [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2354.

Early Noninvasive Metabolic Biomarkers of Mutant IDH Inhibition in Glioma

Approximately 80% of low-grade glioma (LGGs) harbor mutant isocitrate dehydrogenase 1/2 (IDH1/2) driver mutations leading to accumulation of the oncometabolite 2-hydroxyglutarate (2-HG). Thus, inhibition of mutant IDH is considered a potential therapeutic target. Several mutant IDH inhibitors are currently in clinical trials, including AG-881 and BAY-1436032. However, to date, early detection of response remains a challenge. In this study we used high resolution 1H magnetic resonance spectroscopy (1H-MRS) to identify early noninvasive MR (Magnetic Resonance)-detectable metabolic biomarkers of response to mutant IDH inhibition. In vivo 1H-MRS was performed on mice orthotopically-implanted with either genetically engineered (U87IDHmut) or patient-derived (BT257 and SF10417) mutant IDH1 cells. Treatment with either AG-881 or BAY-1436032 induced a significant reduction in 2-HG. Moreover, both inhibitors led to a significant early and sustained increase in glutamate and the sum of glutamate and glutamine (GLX) in all three models. A transient early increase in N-acetylaspartate (NAA) was also observed. Importantly, all models demonstrated enhanced animal survival following both treatments and the metabolic alterations were observed prior to any detectable differences in tumor volume between control and treated tumors. Our study therefore identifies potential translatable early metabolic biomarkers of drug delivery, mutant IDH inhibition and glioma response to treatment with emerging clinically relevant therapies.

Super-Resolution Hyperpolarized 13C Imaging of Human Brain Using Patch-Based Algorithm.

Spatial resolution of metabolic imaging with hyperpolarized 13C-labeled substrates is limited owing to the multidimensional nature of spectroscopic imaging and the transient characteristics of dissolution dynamic nuclear polarization. In this study, a patch-based algorithm (PA) is proposed to enhance spatial resolution of hyperpolarized 13C human brain images by exploiting compartmental information from the corresponding high-resolution 1H images. PA was validated in simulation and phantom studies. Effects of signal-to-noise ratio, upsampling factor, segmentation, and slice thickness on reconstructing 13C images were evaluated in simulation. PA was further applied to low-resolution human brain metabolite maps of hyperpolarized [1-13C] pyruvate and [1-13C] lactate with 3 compartment segmentations (gray matter, white matter, and cerebrospinal fluid). The performance of PA was compared with other conventional interpolation methods (sinc, nearest-neighbor, bilinear, and spline interpolations). The simulation and the phantom tests showed that PA improved spatial resolution by up to 8 times and enhanced the image contrast without compromising quantification accuracy or losing the intracompartment signal inhomogeneity, even in the case of low signal-to-noise ratio or inaccurate segmentation. PA also improved spatial resolution and image contrast of human 13C brain images. Dynamic analysis showed consistent performance of the proposed method even with the signal decay along time. In conclusion, PA can enhance low-resolution hyperpolarized 13C images in terms of spatial resolution and contrast by using a priori knowledge from high-resolution 1H magnetic resonance imaging while preserving quantification accuracy and intracompartment signal inhomogeneity.

Abstract 2546: The immune checkpoint PD-L1 alters choline kinase expression and metabolism in triple negative breast cancer cells

Abstract Introduction Cancer immunotherapy is designed to activate the immune system against cancer cells. This field has been revitalized with the discovery of immune checkpoints such as programmed cell death protein-1 (PD-1) and its ligand (PD-L1). PD-L1 has been exploited for immune therapy with varying success depending on the tumor type, with several studies pointing towards cancer metabolism as one of the possible causes of this variability. One of the most common tumor associated metabolic alteration is increased expression of choline kinase-α (Chk-α), characterized by increased phosphocholine (PC) and total choline-containing compounds. Since the presence of PC on proteins has been related to escape from immune surveillance, here, we investigated the relationship between Chk-α, PC and PD-L1. Methods Experiments were performed using triple negative breast MDA-MB-231 cells transiently transfected with small interfering RNA (siRNA) against Chk-α or PD-L1 following standard protocols. Total RNA was isolated, complementary cDNA synthesised and quantitative real-time PCR performed using IQ SYBR Green supermix and gene specific primers. For high resolution 1H Magnetic Resonance Spectroscopy (MRS), cell extracts were obtained using a dual-phase extraction method. 1H MRS was recorded on a Bruker Biospin Avance-III 750 MHz spectrometer. Integrals of the metabolites of interest were determined and normalized to the TSP reference and the number of cells. Metabolites were estimated from at least three experimental samples. Statistical significance was evaluated using the Student t-test. Results and Discussion We observed that silencing Chk-α resulted in a significant increase of PD-L1 expression and silencing PD-L1 resulted in a significant increase of Chk-α expression. Consistent with the molecular changes, a significant increase of PC was observed in cells transfected with PD-L1 siRNA. Metabolic changes, however, extended well beyond the choline containing compounds. We identified changes in amino acids, organic acids, nucleotides and other compounds. Furthermore, we found that PD-L1 downregulation profoundly altered the lipid profile of MDA-MB-231 cells. Metabolic characterization showed that cells with downregulated Chk-α levels shifted towards a more immunosuppressive profile through metabolic reprogramming, increasing the production of metabolites that have been linked to decreased effectiveness of T-cells. Our data also suggest that low levels of PD-L1 can skew cancer cell metabolism towards a more immunosuppressive profile by a significant increase of lipid production and changes in the lipid profile. These results provide new insights in the role of PD-L1 in the cancer metabolome that may be exploited to improve the outcome of treatment with immune checkpoint inhibitors. Acknowledgement: This work was supported by NIH R35CA209960 and R01CA82337. JPT was funded by Fundación Alonso Martín Escudero and MSCA. Citation Format: Jesus Pacheco-Torres, Marie-France Penet, Flonne Wildes, Yelena Mironchik, Balaji Krishnamachary, Zaver M. Bhujwalla. The immune checkpoint PD-L1 alters choline kinase expression and metabolism in triple negative breast cancer cells [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 2546.

Phenanthridine derivatives as promising new anticancer agents: synthesis, biological evaluation and binding studies.

Aim: Phenanthridines are an essential class of nitrogenous heterocycles with extensive applications in medicinal chemistry. The development of efficient and eco-friendly methods for the preparation of chirally pure dihydropyrrolo[1,2-f]phenanthridines (5a-h), and their in vitro evaluation and modeling studies as potential anticancer, antioxidant and DNA cleavage agents is reported. Methodology & results: Compounds 5a-h were prepared through a facile one-pot synthesis and characterized by infrared, high resolution mass spectrometry, 1H and 13C nuclear magnetic resonance. The molecules were subjected to virtual screening and docking analysis against selected human molecular targets. Compound 5g displayed good binding properties as well as significant anticancer and DNA cleavage activity. Conclusion: Compound 5g has been identified as a potential lead candidate for further testing against additional cancer cell lines and animal models in future.

Cardiac metabolomic profile of the naked mole-rat-glycogen to the rescue.

The African naked mole-rat (Heterocephalus glaber) is unique among mammals, displaying extreme longevity, resistance to cardiovascular disease and an ability to survive long periods of extreme hypoxia. The metabolic adaptations required for resistance to hypoxia are hotly debated and a recent report provides evidence that they are able to switch from glucose to fructose driven glycolysis in the brain. However, other systemic alterations in their metabolism are largely unknown. In the current study, a semi-targeted high resolution 1H magnetic resonance spectroscopy (MRS) metabolomics investigation was performed on cardiac tissue from the naked mole-rat (NMR) and wild-type C57/BL6 mice to better understand these adaptations. A range of metabolic differences was observed in the NMR including increased lactate, consistent with enhanced rates of glycolysis previously reported, increased glutathione, suggesting increased resistance to oxidative stress and decreased succinate/fumarate ratio suggesting reduced oxidative phosphorylation and ROS production. Surprisingly, the most significant difference was an elevation of glycogen stores and glucose-1-phosphate resulting from glycogen turnover, that were completely absent in the mouse heart and above the levels found in the mouse liver. Thus, we identified a range of metabolic adaptations in the NMR heart that are relevant to their ability to survive extreme environmental pressures and metabolic stress. Our study underscores the plasticity of energetic pathways and the need for compensatory strategies to adapt in response to the physiological and pathological stress including ageing and ischaemic heart pathologies.

P13.08 1H-MRS metabolomics as a predictor of overall survival in glioma patients

Abstract BACKGROUND We assess the efficacy of the metabolomic profile from glioma biopsies, in providing estimates of postsurgical Overall Survival in glioma patients. MATERIAL AND METHODS Tumor biopsies from 46 patients bearing gliomas, obtained neurosurgically in the period 1992–1998, were analyzed by high resolution 1H magnetic resonance spectroscopy (HR- 1H MRS), following retrospectively individual postsurgical Overall Survival up to 720 weeks. RESULTS The Overall Survival profile could be resolved in three groups; Short (shorter than 52 weeks, n=19), Intermediate (between 53 and 364 weeks, n=19) or Long (longer than 365 weeks, n=8), respectively. Classical histopathological analysis assigned WHO grades II-IV to every biopsy but notably, some patients with low grade glioma depicted unexpectedly Short Overall Survival, while some patients with high grade glioma, presented unpredictably Long Overall Survival. To explore reasons underlying this behavior, we analyzed HR- 1H MRS spectra from acid extracts of these biopsies, to identify the metabolite patterns underlying OS predictions. Poor prognosis was found in biopsies with higher contents of alanine, acetate, glutamate, total choline, phosphorylcholine and glycine, while more favorable prognosis was achieved in biopsies with larger contents of total creatine, glycerol-phosphorylcholine and myo-inositol. We implemented then a multivariate analysis approach to identify hierarchically the influence of these metabolomic biomarkers on OS predictions, using Classification Regression Trees (CRTs). Metabolomic CRTs grew up to 3 branches and split into 8 nodes, predicting correctly the outcome of 94.7% of the patients in the Short Overall Survival group, 78.9 % of the patients in the Intermediate Overall Survival group, and 75% of the patients in the Long Overall Survival group, respectively. CONCLUSION Present results suggest that metabolic profiling by HR-1H MRS provides more accurate Overall Survival estimates of glioma patients than classical histopathological grading, thus allowing to implement more accurate therapeutic decisions.

P13.11 Metabolomics and molecular profiling in glioma patients: an interactomic approach

Abstract BACKGROUND A wider view of the interaction between different omic-domains is needed to identify potential biomarkers of low- and high-grade gliomas. Using an interactomic approach, we analyzed the correlation between radiological data, IDH mutation, gene expression profiling and metabolic signature in glioma samples. MATERIAL AND METHODS Tumor biopsies from 25 patients with clinical diagnosis of glioma were surgically collected during 2017–2019 at the senior author’s institution. Metabolomic data was obtained by high resolution 31P and 1H magnetic resonance spectroscopy (MRS, 19 metabolites quantified with LCModel). Gene expression profiling was performed using real-time qPCR of 19 genes related to energy metabolism. IDH1/2 common mutation (IDH1R132H/IDH2R172H) was verified by immunohistochemistry and amplicon sanger sequencing. All data was integrated using the R package mixOmics, and we built correlation network plot graphs and correlation maps to identify the most significant interactions, that were analyzed thereafter. RESULTS Mean age was 48±10 years and 72% were men. The most frequent clinical presentation was intracranial hypertension and focal deficit. Imaging revealed 88% of single lobar tumors, 96% of contrast enhancement, 52% located near eloquent areas, 48% with augmented perfusion (mean values of 300±130%) and 60% showed restricted diffusion. WHO 2016 diagnosis were glioblastoma IDH mutated (IDHmut, 16%), IDH wildtype (IDHw, 56%); anaplastic astrocytoma IDHmut (4%), IDHw (16%); diffuse astrocytoma IDHmut (4%), SEGA (4%). The genetic and metabolic profiles were normalized per sample using the total sum of all the studied variables per case. This step made the interactomic approach possible. We found no differences between the metabolic or genetic profiles of glioma grade III and IV samples. However, there was a statistical significance or near-threshold correlation between some metabolic patterns and IDH-mutation, where Alanine (4.7±1.3% IDHw vs 2.5±0.7 IDHmut, p=0.046), Glycine (2.7±0.5% vs. 1.6±0.4%, p=0.095), Glycerophosphorylcholine (3.9±0.4% vs. 6.4±0.9%, p=0.013) and Myo-inositol (4.9±1.0% vs 11.9±2.1%, p=0.004) were the most important biomarkers. Overexpression of Lactate Dehydrogenase subunit B (LDHB, 19±3% vs. 31±6%, p=0.039) and Aconitase 1 (ACO1, 0.5±0.1% vs 1.2±0.3%, p=0.08) had also a significant or near-threshold relationship with IDH-mutation. These correlations were shown as hot spots in the correlation graphs and maps. CONCLUSION These preliminary results indicate that metabolic patterns by high resolution 31P and 1H MRS could be a useful tool to improve our knowledge about glioma gene expression profiles and to identify potential biomarkers to tackle this pathology.

Abstract 5265: Spleen metabolism altered by human pancreatic cancer xenografts

Abstract Cancer-induced cachexia accounts for approximately 20% of all cancer deaths [1] and affects nearly 80% of pancreatic cancer patients [2, 3]. Cachectic patients experience a wide range of symptoms affecting several organ functions such as muscle, liver, brain, and heart that decrease quality of life and worsen prognosis. A major characteristic of cachexia is the accelerated skeletal muscle and fat storage wasting causing nutrient mobilization both directly as lipid and amino acids, and indirectly as glucose derived from the exploitation of liver gluconeogenesis that reaches the tumor through the bloodstream [4]. Here, for the first time, we have performed high-resolution quantitative 1H magnetic resonance spectroscopy (MRS) of spleen tissue obtained from normal mice and mice bearing PDAC that are cachectic (Pa04C) and non-cachectic (Panc1). The Panc1 (non-cachectic), and Pa04C (cachectic) PDAC cell lines were used [5]. Eight-week-old male severe combined immunodeficient mice were inoculated in the right flank with cancer cells (5×106). Mice were euthanized once tumors were ~ 300 mm3. Control, cachectic and non-cachectic groups consisted of 9, 10 and 9 mice per group respectively. Spleen water soluble metabolites were extracted, freeze dried, reconstituted in D2O PBS and transferred to an NMR tube for spectral acquisition with a 750 MHz (17.6T) MR spectrometer [6]. As anticipated, mice with cachexia-inducing Pa04C tumors showed significant weight loss with time. We observed, for the first time, that spleens from cachectic Pa04C tumor bearing mice showed a profound weight loss compared to normal mice and mice with Panc1 tumors. However, an increase of liver and spleen size has been previously reported in terminal cachectic human patients from colorectal cancer measured by CT scan 2-11 months prior to death [7]. A significant decrease in almost all amino acids was observed in cachectic (Pa04C) mouse spleens compared to normal and non-cachectic (Panc1) mouse spleens. Differences in choline metabolites, creatine, glutamine, glutamate, glutathione and aspartate were observed in cachectic mouse spleens compared to non-cachectic mouse spleens and spleens from healthy control mice. The significant decrease of amino acids in the cachectic spleens may reflect increased utilization of amino acids by the tumor or other organs during the cachexia muscle/protein wasting [4]. These results provide new insights into changes in spleen metabolism during cachexia, and support investigating metabolic targets to reduce cachexia associated morbidity. Supported by NIH R01CA193365 and R35CA209960. Reference: 1. Argiles et al. Nat. Rev. Cancer 2014, 14 (11), 754; 2. Fearon et al. HPB (Oxford) 2010, 12 (5), 323; 3. Ozola et al. Pancreatology 2015, 15 (1), 19; 4. Porporato et al. Oncogenesis 2016, 5, e200; 5. Winnard et al. Can. Res. 2016, 76(6): 1441; 6. Penet et al. Clin. Can. Res. 2015, 21 (2), 386; 7. Lieffers et al. Am J Clin Nutr. 2009, 89 (4), 1173 Citation Format: Santosh Kumar Bharti, Paul T. Winnard, Raj Kumar Sharma, Yelena Mironchik, Marie-France Penet, Zaver M. Bhujwalla. Spleen metabolism altered by human pancreatic cancer xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5265.

Abstract 5258: 1 H MRS analysis of pancreas metabolites altered by cachexia

Abstract Cachexia is a multifactorial syndrome characterized by skeletal muscle weight loss and reduced physical activity. The extreme weight loss due to cachexia results in a particularly poor quality of life causing profound weakness, listlessness, and inability to function [1, 2]. Severe weight loss decreases the tolerance to treatments and anticancer therapies, and leads to the reduced survival of patients. In pancreatic cancer, the syndrome affects nearly 80% of patients [3]. Cancers can stimulate cachexia through the dysfunction of multiple organs [4]. Metabolic abnormalities may interfere with the regular functioning of these organs to increase the severity of the disease. Here we determined metabolic changes in the pancreas with cachexia-inducing and non-cachexia inducing tumor growth using high-resolution quantitative 1H magnetic resonance spectroscopy (MRS) of pancreas tissue obtained from normal, non-cachectic (Panc1) and cachectic (Pa04C) mice bearing pancreatic ductal adenocarcinoma (PDAC) xenografts. Cancer cells were inoculated in the right flank of six to eight week old male severe combined immunodeficient mice. The pancreas was removed from mice following euthanization once tumors were ~300 mm3, snap frozen and stored at -80°C prior to dual phase extraction. 1H MRS was performed on the water phase. All 1H MR spectra with water suppression were acquired on a 750 MHz MR spectrometer using a single pulse sequence. All data processing analyses and quantification were performed with TOPSPIN 3.5 software. All statistical analysis were performed with MetaboAnalyst software [5]. Multivariate analyses performed to analyze the differences in the metabolic profiles among the groups (Control n = 9, Pa04C = 10 and Panc1 = 8) revealed differences in the overall metabolic pattern in the pancreas from normal, cachectic and non-cachectic groups. A significant decrease of leucine, isoleucine, valine, BCAA, glutamate, choline, pyruvate, glycine, niacinamide, fumarate and increase of alanine, pyruvate, and NAD was detected in cachectic mouse pancreas compared to control pancreas. A significant increase in alanine, phenylalanine, pyruvate, phosphocholine and decrease in glycine, NAD, niacinamide was observed in cachectic mouse pancreas compared to non-cachectic mouse pancreas. Pathway impact analysis using MetaboAnalyst web software indicated alterations in branch chain amino acid pathways and glutamate, glutamine metabolism. These results provide new insights into changes in pancreas metabolism with cachexia, and support investigating metabolic targets and biomarkers to reduce cachexia-associated morbidity. Supported by NIH R01CA193365 and R35CA209960. Ref: 1. Fearon K et al, The Lancet Oncology. 2011; 2. Penet MF, Bhujwalla ZM. Cancer journal. 2015; 3. Winnard PT, Jr. et al, Cancer research. 2016; 4. Argiles JM et al, Nature reviews Cancer. 2014; 5. Xia J, Wishart DS. Nature protocols. 2011. Citation Format: Raj Kumar Sharma, Santosh K. Bharti, Paul T. Winnard Jr, Yelena Mironchik, Marie-France Penet, Zaver M. Bhujwalla. 1 H MRS analysis of pancreas metabolites altered by cachexia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5258.

Assessment of Overall Survival in Glioma Patients as Predicted by Metabolomic Criteria.

Objective: We assess the efficacy of the metabolomic profile from glioma biopsies in providing estimates of postsurgical Overall Survival in glioma patients.Methods: Tumor biopsies from 46 patients bearing gliomas, obtained neurosurgically in the period 1992–1998, were analyzed by high resolution 1H magnetic resonance spectroscopy (HR- 1H MRS), following retrospectively individual postsurgical Overall Survival up to 720 weeks.Results: The Overall Survival profile could be resolved in three groups; Short (shorter than 52 weeks, n = 19), Intermediate (between 53 and 364 weeks, n = 19) or Long (longer than 365 weeks, n = 8), respectively. Classical histopathological analysis assigned WHO grades II–IV to every biopsy but notably, some patients with low grade glioma depicted unexpectedly Short Overall Survival, while some patients with high grade glioma, presented unpredictably Long Overall Survival. To explore the reasons underlying these different responses, we analyzed HR-1H MRS spectra from acid extracts of the same biopsies, to characterize the metabolite patterns associated to OS predictions. Poor prognosis was found in biopsies with higher contents of alanine, acetate, glutamate, total choline, phosphorylcholine, and glycine, while more favorable prognosis was achieved in biopsies with larger contents of total creatine, glycerol-phosphorylcholine, and myo-inositol. We then implemented a multivariate analysis to identify hierarchically the influence of metabolomic biomarkers on OS predictions, using a Classification Regression Tree (CRT) approach. The CRT based in metabolomic biomarkers grew up to three branches and split into eight nodes, predicting correctly the outcome of 94.7% of the patients in the Short Overall Survival group, 78.9% of the patients in the Intermediate Overall Survival group, and 75% of the patients in the Long Overall Survival group, respectively.Conclusion: Present results indicate that metabolic profiling by HR-1H MRS improves the Overall Survival predictions derived exclusively from classical histopathological gradings, thus favoring more precise therapeutic decisions.

Synthesis, chemical characterization and antimicrobial activity of new acylhydrazones derived from carbohydrates

A new series of glycosylated acylhydrazones was synthesized and all the chemical structures were confirmed by High Resolution Mass Spectrometry (HRMS), 1H and 13C Nuclear Magnetic Resonance (1H-NMR; 13C-NMR) and Fourier Transform Infrared (FTIR) spectroscopy methods. The mass accuracy between the calculated and found values observed in HRMS analyses were near or lower than 5 ppm, which are acceptable for proposing a molecular formula using this technique. All of the synthesized compounds were screened for their antibacterial, antifungal and antiviral activities. Five compounds (12, 13, 14, 16 and 19) exerted a modest antifungal activity against the strains evaluated. Derivative 14 showed fungicidal activity against Candida glabrata at 173.8 μM and saccharide unit contributed to the increase of the antifungal potential against this strain. New chemical manipulation of derivative 14 can make it possible to obtain new potentially antimicrobial agents.

Abstract 1680: The immune checkpoint metabolome and its relationship with choline metabolism

Abstract Introduction: Immune checkpoint inhibition has emerged as an exciting treatment option for several cancers. New insights into the role of immune checkpoints in cellular metabolism can be used to optimize their effectiveness in treatment. Programmed cell death-1 ligand (PD-L1) is an immune checkpoint overexpressed in cancers that has been successfully exploited for immune therapy. Here we investigated the relationship between the aberrant choline metabolism observed in most cancers and PD-L1 expression in triple-negative human MDA-MB-231 breast cancer cells, and characterized the metabolic effects of PD-L1 downregulation. Methods: Experiments were performed with MDA-MB-231 cells transiently transfected with small interfering RNA (siRNA) against either luciferase (control siRNA), choline kinase-α (Chk-α), or PD-L1, following standard protocols. Total RNA was isolated, complementary cDNA synthesized, and quantitative real-time PCR performed using IQ SYBR Green supermix and gene-specific primers. Cell extracts were obtained using a dual-phase extraction method and analyzed by high resolution 1H magnetic resonance spectroscopy. 3 to 6 independent experiments were performed for each group. Results: Silencing of Chk-α resulted in a significant increase of PD-L1 mRNA expression and silencing of PD-L1 resulted in a significant increase of Chk-α mRNA expression. These increases were not observed when both PD-L1 and Chk-α were silenced. Transfection with control siRNA did not alter Chk-α mRNA, but induced a small increase of PD-L1 mRNA compared to untreated cells. Transfection of cells with Chk-α and PD-L1 siRNA resulted in a significant decreases of Chk-α and PD-L1 mRNA. Consistent with the mRNA results, a significant increase of phosphocholine (PC) was observed in spectra obtained from cells transfected with PD-L1 siRNA compared to cells transfected with control siRNA, confirming that Chk-α increased in these cells. A significant increase of glutamate, glutathione, alanine and lactate was also observed in cells transfected with PD-L1 siRNA. As expected, cells transfected with a combination of Chk-α and PD-L1 siRNA showed a significant decrease of PC due to downregulation of Chk-α. Discussion: Our data have identified, for the first time, the inverse association between PD-L1 and Chk-α expression following downregulation. These data suggest that treatments that decrease Chk-α and PC could result in cancer cells escaping immune surveillance through increased expression of PD-L1. On the other hand, these cells may become more susceptible to checkpoint inhibitors such as anti-PD-L1 or anti-PD-1 antibodies. PD-L1 downregulation also significantly increased glutathione that acts as a major antioxidant. Our ongoing studies are characterizing the effects of anti-PD-L1 antibody treatment on Chk-α expression. Supported by NIH R35CA209960 and R01CA82337, and the Martin Escudero Foundation and Emerson Foundation. Citation Format: Jesus Pacheco-Torres, Marie-France Penet, Yelena Mironchik, Sridhar Nimmagadda, Balaji Krishnamachary, Zaver M. Bhujwalla. The immune checkpoint metabolome and its relationship with choline metabolism [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 1680.

Abstract 3480: COX-2 alters the metabolic secretome in triple negative human breast cancer xenografts

Abstract Cyclooxygenase-2 (COX-2) is an active mediator of the inflammatory response of cells and plays an important role in the development, progression, invasion, and metastasis of cancers including breast cancer [1]. Tumor interstitial fluid (TIF), the milieu that contains the tumor secretome, is one of the least examined aspects of the TME because of the difficulty in sampling this fluid from tumors. Here, for the first time, we have sampled TIF from COX-2 overexpressing triple negative SUM-149 human breast cancer xenografts and empty vector SUM-149 xenografts. The cloning, construction of a lentivirus vector expressing COX-2 gene, and the establishment of SUM-149 cells stably overexpressing COX-2 (SUM-COX-2) were reported by us previously[2]. A home-built TIF collection chamber was inserted subcutaneously in female SCID mice with 4-6 1-2 mm tumor pieces packed around the chamber. Once tumors were ~ 400 mm3, TIF was collected from chambers. Each chamber yielded ~50μL of TIF that was analyzed with high-resolution 1H magnetic resonance spectroscopy (MRS) at 750 MHz. SUM-COX-2 tumors showed consistently higher COX-2 expression compared to SUM-EV tumors. COX-2 overexpression resulted in a significant increase of lactate, glutamate, acetate, and succinate, and a significant decrease of glucose, glutamine, citrate, formate, and lipids; pyruvate tended to decrease. The changes in lactate and lipids are consistent with our earlier observations where COX-2 downregulation in triple negative MDMB-231 human breast cancer cells resulted in a significant decrease of lactate and an increase of lipids and lipid droplets in intact perfused cells[3]. Here, COX-2 overexpression increased glycolysis. Depletion of pyruvate observed here in COX-2 overexpressing cells would limit production of acetyl-CoA and consequently citrate to diminish fatty acid synthesis/lipids. Increased succinate parallels the increase in glutamate/α-ketoglutarate, the upstream intermediate to succinate in the tricarboxylic acid cycle [1], and indicates increased utilization/depletion of glutamine to supplement the TCA cycle. Importantly, accumulation of succinate inhibits HIF-1α prolyl hydroxylase that stabilizes HIF-1α driven cancer promoting metabolic pathways such as enhanced glycolysis and increased ROS[4]. Moreover, increased succinate also through the succinate/succinate dehydrogenase reaction, provides necessary electrons to the electron transport chain upstream of the COX-2 reaction[5]. These data provide new insights into the role of COX-2 in the metabolic secretome and tumor metabolism, and identify metabolic pathways as potential targets for reducing the effects of COX-2 expression in cancer. Ref. 1. Wang et. al. Nat. Rev. Can. 2010 2. Krishnamachary et al. Oncot. 2017 3. Shah et al. NMR Biomed. 2012 4. Selak et al. Can Cell. 2005 5. Mills et al. Trd in Cell Bio. 2014 Citation Format: Santosh Kumar Bharti, Paul T. Winnard, Yelena Mironchik, Louis Dore-Savard, Balaji Krishnamachary, Zaver M. Bhujwalla. COX-2 alters the metabolic secretome in triple negative human breast cancer xenografts [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 3480.

Low-temperature magnetic resonance imaging with 2.8 μm isotropic resolution

We demonstrate the feasibility of high-resolution 1H magnetic resonance imaging (MRI) at low temperatures by obtaining an MRI image of 20 μm diameter glass beads in glycerol/water at 28 K with 2.8 μm isotropic resolution. The experiments use a recently-described MRI apparatus (Moore and Tycko, 2015) with minor modifications. The sample is contained within a radio-frequency microcoil with 150 μm inner diameter. Sensitivity is additionally enhanced by paramagnetic doping, optimization of the sample temperature, three-dimensional phase-encoding of k-space data, pulsed spin-lock detection of 1H nuclear magnetic resonance signals, and spherical sampling of k-space. We verify that the actual image resolution is 2.7 ± 0.3 μm by quantitative comparisons of experimental and calculated images. Our imaging approach is compatible with dynamic nuclear polarization, providing a path to significantly higher resolution in future experiments.

Abstract 2510: Interrogating liver metabolic stress due to cancer-induced cachexia

Abstract Cancer-induced cachexia accounts for approximately 20% of all cancer deaths 1. In pancreatic cancer, the syndrome affects nearly 80% of patients 2,3. Cachectic patients experience a wide range of symptoms affecting the function of several organs such as muscle, liver, brain, and heart, that decrease quality of life and worsen prognosis. A major characteristic of cachexia is the accelerated skeletal muscle and fat storage wasting causing nutrient mobilization both directly as lipid and amino acids, and indirectly as glucose derived from the exploitation of liver gluconeogenesis that reaches the tumor through the bloodstream4. Patients with cachexia develop a wide range of metabolic stress from increased proteins and fat tissue burning resulting in increased energy expenditure. Here, for the first time, we have performed high-resolution quantitative 1H magnetic resonance spectroscopy (MRS) of liver tissue obtained from normal, noncachectic and cachectic mice bearing PDAC that are cachectic (Pa04C) and noncachectic (Panc1). A significant reduction in liver weight and significant changes in 1H MRS derived metabolite profiles were detected with cachexia. Human pancreatic cancer cell lines, Panc1 and Pa04C, were used for the study. Six to 8 week old male severe combined immunodeficient mice were inoculated in the right flank with cancer cells (5×106) and in the right hind leg muscle with reporter myoblasts (2×106) to monitor the development of cachexia in mice5. Live animal optical imaging was done using a Xenogen IVIS® Spectrum (PerkinElmer) optical scanner. Dual phase solvent extraction was performed on liver tissue to extract water soluble metabolites. All 1H MR spectra were acquired on an Avance 750 MHz Bruker NMR spectrometer. We found, for the first time, that the liver in Pa04C tumor bearing mice underwent a profound weight loss; although Panc1 tumor bearing mice showed some liver weight loss this was not as profound as observed with Pa04C tumors. Significant decreases in lactate, glucose and glutathione were observed in cachectic mouse liver compared to noncachectic mouse liver, and the liver from healthy control mice. The significant decrease of these metabolites in cachectic livers may reflect increased utilization of glucose, lactate and glutathione by the tumor or other organs during the cachexia cascade4. These results provide new insights into changes in liver metabolism during cachexia, and support investigating metabolic strategies such as supplementing glutathione or glucose to reduce cachexia associated morbidity.

18F](2S,4R)4-Fluoroglutamine PET Detects Glutamine Pool Size Changes in Triple-Negative Breast Cancer in Response to Glutaminase Inhibition.

Glutaminolysis is a metabolic pathway adapted by many aggressive cancers, including triple-negative breast cancers (TNBC), to utilize glutamine for survival and growth. In this study, we examined the utility of [18F](2S,4R)4-fluoroglutamine ([18F]4F-Gln) PET to measure tumor cellular glutamine pool size, whose change might reveal the pharmacodynamic (PD) effect of drugs targeting this cancer-specific metabolic pathway. High glutaminase (GLS) activity in TNBC tumors resulted in low cellular glutamine pool size assayed via high-resolution 1H magnetic resonance spectroscopy (MRS). GLS inhibition significantly increased glutamine pool size in TNBC tumors. MCF-7 tumors, with inherently low GLS activity compared with TNBC, displayed a larger baseline glutamine pool size that did not change as much in response to GLS inhibition. The tumor-to-blood-activity ratios (T/B) obtained from [18F]4F-Gln PET images matched the distinct glutamine pool sizes of both tumor models at baseline. After a short course of GLS inhibitor treatment, the T/B values increased significantly in TNBC, but did not change in MCF-7 tumors. Across both tumor types and after GLS inhibitor or vehicle treatment, we observed a strong positive correlation between T/B values and tumor glutamine pool size measured using MRS (r2 = 0.71). In conclusion, [18F]4F-Gln PET tracked cellular glutamine pool size in breast cancers with differential GLS activity and detected increases in cellular glutamine pool size induced by GLS inhibitors. This study accomplished the first necessary step toward validating [18F]4F-Gln PET as a PD marker for GLS-targeting drugs. Cancer Res; 77(6); 1476-84. ©2017 AACR.