https://openworks.mdanderson.org/sumexp21/1227/thumbnail.jp

Azordegan, Hawk

Bae, Seong-Woo

Bhattacharya, Pratip K., PhD

Enriquez, José Santiago

Manning, Henry Charles

Wen, Xiaoxia

English

OpenWorks @ MD Anderson

BackgroundGlutamine is the most abundant, free amino acid in plasma, utilized by cancer cells for survival and rapid reproduction. Cancer cells employ glutamine through a transporter (ASCT2)  and metabolizing it through a catalyzing enzyme. During glutaminolysis, the amino acid transporter, SLC1A5 (ASCT2), metabolizes glutamine with a catalyzing enzymeV-9302 treatment in-vivo• Mice with with Cre/+; Kras/+; Smad4 -/- were divided into three groups:Group 1 (low dose) consisted of 2 male mice and 2 female mice. Group 2 (control) consisted of 2 male mice.Group 3 (high dose) consisted of 2 male mice and 2 female mice.• Mice were given intraperitoneal injection of V-9302: Group 1 – V9302, 12.5mg/kg, twice a day for 4 weeksGroup 2 – Vehicle, 2% DMSO, twice a day for 4 weeksGroup 3 – V9302, 75mg/kg, single dose for 4 hoursMaterials and methodsMouse selection by genotypingABFig. 1 V-9302.HypothesisMouse using the developed inhibitor of the ASCT2 amino acid transporter, V-9302, malignant tumors will be unable to metabolize glutamine leading to difficulties in proliferation and an imbalance of metabolites in the microenvironment.Fig. 2 Breeding and genotyping. A. Mice with Cre/+;Kras/+;Smad4f/f are the only ones that will grow tumor with only a 25% chance of occurring. B. Genotyping through gel electrophoresis allows for the identification of the groups in the study. References1) Choi, Y. K., et al. G. Targeting Glutamine Metabolism for Cancer Treatment. Biomolecules & therapeutics, 26(1), 19–28 (2018).2) Jin, L., et al. Glutaminolysis as a target for cancer therapy. Oncogene 35, 3619–3625 (2016). 3) Schulte, M. L., et al. Pharmacological blockade of ASCT2-dependent glutamine transport leads to antitumor efficacy in preclinical models. Nature medicine, 24(2), 194–202 (2018). NMR-based Metabolic Profiling of Pancreatic Cancer and Response to Glutamine Transport InhibitionHawk Azordegan, Xiaoxia Wen, Seong-Woo Bae, Jose S. Enriquez, Pratip K. Bhattacharya, Henry C. ManningThe University of Texas, MD Anderson Cancer Center, Department of Cancer Systems Imaging, Houston, TX, USAMaterials and methods (cont.)Tumor tissue collection • Mice were euthanized after treatment. Tumors were extracted, half of the tissue was preserved in 10% formalin for histological analysis, the other half was flash frozen in liquid nitrogen for 1HNMR assay. Mice were 11 weeks old at dissection in group 1 (A) and 2 (B), and 9 weeks old in group 3 (C).Nuclear Magnetic Resonance (NMR)• Generally utilized to determine the content, molecular structure, and purity of a sample. Allowed us to gather information on the metabolites in the tumor microenvironment. • Prepared the sample through derivatization and buffering before following usual NMR protocol. Hematoxylin and eosin staining• Slides were prepared by preserving, staining, dehydration, infiltration, embedding, mirotoming, and mounting. Conclusions• This investigation attempted to treat malignant cancer cells with V-9302 in different ways and found some changes in metabolic profile. • It is possible that interference with inhibiting glutamine transporter changes cancer characteristics. • However, it is essential to gain a better understanding of glutaminolysis as well as other cancer metabolisms before determining the most effective usage of V-9302 and what manipulations can improve its benefits in treating cancer.  Fig. 6 Representation of general results and takeaways of V-9302 treatment. V-9302 was able to inhibit glutaminolysis and thus decreases were noticed in lactate, glutamine, glutamate, choline, and acetate. Mouse #335 Mouse #360AMouse #372 Mouse #375 Mouse #376 Mouse #379 Mouse #336 Mouse #339 Mouse #342 Mouse #350 BCFig. 3 Photographs of extracted tumor tissues. A. Group 1. B. Group 2 C. Group 3ResultsNuclear Magnetic Resonance (NMR)Group 1 Group 2 Group 3Fig. 5 Histology of cancer cells (2.5x magnification). Darker purple represents healthy pancreatic cells, lighter purple is fibrosis, and white areas inside the area of the tumor are necrosis. Fig. 4 Measurement of Metabolites by Nuclear Magnetic Resonance (NMR). A. Glutamine, B. Glutamate, C. Total Choline, D. Lactate, E. Acetate from all three groups in the study. Group 1Group 2Group 30.00000.00050.00100.00150.00200.0025GlutamineNormalized Glutamine (a.u)Group 1Group 2Group 3Group 1Group 2Group 30.0000.0020.0040.006GlutamateNormalized Glutamate (a.u)Group 1Group 2Group 3✱Group 1Group 2Group 30.0000.0050.0100.0150.0200.025Total CholineNormalized Total Choline (a.u)Group 1Group 2Group 3Group 1Group 2Group 30.0000.0050.0100.0150.020LactateNormalized Lactate (a.u) Group 1Group 2Group 3✱Group 1Group 2Group 30.00000.00020.00040.00060.00080.0010AcetateNormalized Acetate (a.u)Group 1Group 2Group 3Results (cont.)• After treatment, the metabolism profile changed.• NMR showed lower levels of glutamine, glutamate, and total choline in treated mice compared to the control group. • Lactate was lower at elevated doses of V9302.• Acetate displayed lower levels in group 3 while group 1 saw no change.Histology• Histologically, chronic exposure of V9302 led to tumors that were less advanced than tumors from the acute dose control groups.

NMR-based Metabolic Profiling of Pancreatic Cancer and Response to Glutamine Transport Inhibition

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1227&context=sumexp21

NMR-based Metabolic Profiling of Pancreatic Cancer and Response to Glutamine Transport Inhibition

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