Mapping Metabolic Networks in 3D spheroids using Stable - PowerPoint PPT Presentation

Mapping Metabolic Networks in 3D spheroids using Stable Isotope-Resolved Metabolomics (SIRM) Teresa W.-M. Fan 1, *, Salim S. El-Amouri 1 , Jessica K. A. Macedo 1 , and Andrew N. Lane 1 1 Center for Environ. & Systems Biochemistry; Markey Cancer Center; Dept. of Toxicology & Cancer Biology * Corresponding author: twmfan@gmail.com 1

Introduction  2D cell cultures have unrealistic concentration gradients of O 2 , nutrients, and treatment agents.  2D cell cultures lack cell-cell and cell-extracellular cellular matrix interactions (ECM).  3D cell cultures (spheroids in matrigel, hydrogels, micropattern plates, hanging drops, and M3DB systems) can overcome these drawbacks.  Long speroid formation time, variable efficiency, handling complextiy, matrix contamination, and/or scaling-up are of concern for all but the M3DB systems.  The M3DB (Magnetic 3D Bioprinting) method enables spheroid formation by magentizing cells with magnetic nanoparticles, which is easy to handle and can be scaled up readily for metabolomic studies.  3D spheroids display higher drug resistance than 2D cell cultures but the underlying metabolic mechanism is unclear.  Stable Isotope-Resolved Metabolomics (SIRM) is well-suited for exploring drug-induced metabolic perturbations in M3DB spheroid cultures. 2

Results and Discussion 3

A549 spheroids are more resistant to SeO 3 than 2D counterparts A549 Ctl A549 SeO 3 (6.25 µM) A549 A549 SeO 3 (10 µM) A549 Ctl A549

Glycolysis & the Krebs cycle were less impacted by SeO 3 in A549 spheroids than 2D counterparts 3D Spheroids 2D Cells Glycolysis Glycolysis 13 C 3 -lactate 13 C 3 -lactate C B C B A 1 1 P D A 1 1 P D 1 1 1 1 Pyruvate Pyruvate 3 3 3 3 6 P 6 Lactate 6 P 6 CO 2 Lactate CO 2 CO 2 13 C 6 -Glc F6P 13 C 6 -Glc F6P 1 E 1 Acetyl CoA Acetyl CoA E Citrate Citrate 1 1 1 K K 1 4 4 4 4 0.02 6 Asp Oxaloacetate 6 Isocitrate Asp Oxaloacetate F Isocitrate F NADPH 1 NADPH 0.00 1 1 CO 2 1 3 CO 2 3 Pyruvate 4 Krebs Pyruvate 4 α Ketoglutarate Krebs Glu α Ketoglutarate Malate Glu Malate 1 1 Cycle 1 1 Cycle 1 5 5 H 1 5 H 5 CO 2 CO 2 4 4 Fumarate Succinyl CoA I Fumarate Succinyl CoA 0.04 I 0.02 0.00 Succinate Succinate J 0.04 J 1 1 G G 0.02 Glu-GSH Glu-GSH 0.00 5 5 5

Pyrimidine & the hexosamine biosyn pathways (HBP) were less impacted by SeO 3 in A549 spheroids than 2D counterparts 3D Spheroids 2D Cells Pyrimidine Pyrimidine A B C Synthesis A B C Synthesis 4 3 5 4 4 4 3 5 6 2 3 5 4 3 5 4 1 1 1 1 2 6 P P 3 5 5 1 1 6 1 6 1 3 1 2 2 1 1 P P 1’ 1 1 6 1 6 2 2 1 st turn 1’ 1 st turn 4 OMP 1 6 1 4 OMP 6 1 Asp 1 Asp 13 C 6 -Glc P P NC-Asp Orotate 13 C 6 -Glc P P NC-Asp Orotate R5P PPP R5P PP PPP P 5’ P 5’ PP 5’ P P 5’ 1’ 1’ PRPP 1’ CO 2 1’ PRPP CO 2 UTP UTP 4 3 5 4 3 5 2 6 D E F 1 6 PPP D F 2 PPP PPP PPP E 1 PPP PPP PPP PPP 1’ 1’ HN Ac HN Ac P P HN Ac HN Ac P NAcGN1P 6 1 NAcGN6P 6 1 1 P NAcGN1P 6 1 NAcGN6P 6 1 1 UDPGlcNAc HBP UDPGlcNAc HBP H I I G G H 6

PANC1 spheroids are more resistant to SeO 3 than 2D counterparts PANC1 Ctl PANC1 SeO 3 (10 µM) PANC1 PANC1 SeO 3 (10 µM) PANC1 Ctl PANC1

Glycolysis & the Krebs cycle were less impacted by SeO 3 in PANC1 spheroids than 2D counterparts 3D Spheroids 2D Cells Glycolysis Glycolysis 13 C 3 -lactate 13 C 3 -lactate B C B C A D 0.1 1 1 P 1 1 P D 1 A 1 1 1 Pyruvate Pyruvate 0.0 3 3 3 3 * 6 P 6 Lactate 6 P 6 CO 2 Lactate CO 2 CO 2 13 C 6 -Glc F6P 13 C 6 -Glc F6P 1 D 1 Acetyl CoA Acetyl CoA E Citrate Citrate K 1 1 1 K 1 4 4 4 4 E 6 Asp Oxaloacetate 6 Isocitrate Asp Oxaloacetate Isocitrate F NADPH 1 NADPH 1 1 CO 2 1 3 CO 2 3 Pyruvate 4 Krebs Pyruvate 4 α Ketoglutarate Krebs Glu α Ketoglutarate Malate Glu Malate 1 1 Cycle 1 1 Cycle H 1 5 5 1 5 5 H CO 2 CO 2 4 4 Fumarate Succinyl CoA I Fumarate Succinyl CoA I Succinate J Succinate 1 J F 1 G Glu-GSH Glu-GSH 5 5 8

Pyrimidine & the hexosamine biosyn pathways (HBP) were less impacted by SeO 3 in PANC1 spheroids than 2D counterparts 3D Spheroids 2D Cells Pyrimidine Pyrimidine A B C Synthesis A Synthesis B C 4 3 5 4 4 4 3 5 6 2 3 5 4 3 5 4 1 1 1 1 2 6 P P 3 5 5 1 1 6 1 6 1 3 1 2 2 1 1 P P 1’ 1 1 6 1 6 2 2 1 st turn 1’ 1 st turn 4 OMP 1 6 1 4 OMP 6 1 Asp 1 Asp 13 C 6 -Glc P P NC-Asp Orotate 13 C 6 -Glc P P NC-Asp Orotate R5P PPP R5P PP PPP P 5’ P 5’ PP 5’ P P 5’ 1’ 1’ PRPP 1’ CO 2 1’ PRPP CO 2 UTP UTP 4 F 3 5 4 3 5 2 6 D E F 1 6 PPP D E 2 PPP PPP PPP 1 PPP PPP PPP PPP 1’ 1’ HN Ac HN Ac P P HN Ac HN Ac P NAcGN1P 6 1 NAcGN6P 6 1 1 P NAcGN1P 6 1 NAcGN6P 6 1 1 I UDPGlcNAc HBP UDPGlcNAc G H I HBP G H 9

Conclusions  SIRM-mapped metabolic activity in M3DB spheroids was largely comparable to that in the 2D counterparts for both lung A549 and pancreatic PANC1 adenocarcinoma cell lines.  A549 M3DB spheroids were more active in pyrimidine synthesis than the 2D counterparts.  Gluconeogensis was active in PANC1 M3DB spheroids but not in 2D cell cultures.  For both cell lines, M3DB spheroids were more resistant to anti-cancer SeO 3 treatment that the 2D counterparts in terms of growth.  This drug resistance may be rooted in reduced sensitivity of M3DB spheroids to SeO 3 in glycolysis, the Krebs cycle, nucleotide synthesis, and glutathione metabolism, central to cell growth and survival. 10

Acknowledgments  Yan Zhang, Hui Liu, Abagail L. Cornette, Qiushi Sun, and Richard M. Higashi  Markey Foundation, & Kentucky Lung Cancer Foundation  National Institute of Health (1P01CA163223-01A1, 1U24DK097215-01A1 ) 11