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

SLIDE 1

Mapping Metabolic Networks in 3D spheroids using Stable Isotope-Resolved Metabolomics (SIRM)

Teresa W.-M. Fan1,*, 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

SLIDE 2

Introduction

2

 2D cell cultures have unrealistic concentration gradients of O2, 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.

SLIDE 3

Results and Discussion

3

SLIDE 4

A549 spheroids are more resistant to SeO3 than 2D counterparts

A549

A549 Ctl A549 SeO3 (10 µM) A549 SeO3 (6.25 µM) A549 Ctl

A549

SLIDE 5

5 Glycolysis

K K A C B D A C B D

Glycolysis

G H F E H F

0.02 0.00

G E

Glycolysis & the Krebs cycle were less impacted by SeO3 in A549 spheroids than 2D counterparts

13C6-Glc

1 6

F6P P

1 6

P

1 3

Pyruvate Lactate

1 3

13C3-lactate

1 3

Pyruvate

1 6

13C6-Glc

Lactate

1 3

13C3-lactate

F6P P

1 6

P CO2 CO2 Glu

1 5

Glu

1 5

Glu-GSH

1 5

Glu-GSH

1 5

CO2 Acetyl CoA

1

Krebs Cycle

Acetyl CoA

1

Krebs Cycle

Asp

1 4

Asp

1 4

J J

0.02 0.00 0.04

I I

0.02 0.00 0.04

Malate Malate Citrate Fumarate Succinate αKetoglutarate Isocitrate CO2 CO2 Oxaloacetate

4

Succinyl CoA

1 1 4 6 1 4 1 5

Citrate Fumarate Succinate αKetoglutarate Isocitrate CO2 CO2 Oxaloacetate

4

Succinyl CoA

1 1 4 6 1 4 1 5

Pyruvate NADPH

1 3

Pyruvate NADPH

1 3

3D Spheroids 2D Cells

SLIDE 6

6

Pyrimidine & the hexosamine biosyn pathways (HBP) were less impacted by SeO3 in A549 spheroids than 2D counterparts

Pyrimidine Synthesis

Asp 1st turn

13C6-Glc

1 6 1 4

NC-Asp Orotate

4 3 2 5 1 6

1

4 3 2 5 1 6

1 1 1 Asp 1st turn

13C6-Glc

1 6 1 4

NC-Asp Orotate

4 3 2 5 1 6

1

4 3 2 5 1 6

1 1 1

HBP

1 6

P NAcGN6P 1

6

P NAcGN1P UDPGlcNAc HN Ac HN Ac

1 1 6

P NAcGN6P 1

6

P NAcGN1P UDPGlcNAc HN Ac

HBP

HN Ac

1

D E D E F F A B C A C B G H I G H I

PPP

5 6 1’ 1 4 3 2

PPP

P OMP CO2 UTP P

5 6 1’ 1 4

P

3 2

P

PPP PPP PPP

P OMP CO2 UTP P

5 6 1’ 1 4

P

3 2

P

PPP

5 6 1’ 1 4 3 2

PPP PPP

Pyrimidine Synthesis

P

1’ 5’

P

1’ 5’

PP R5P PRPP

PPP

P

1’ 5’

P

1’ 5’

PP R5P PRPP

PPP

3D Spheroids 2D Cells

SLIDE 7

PANC1 spheroids are more resistant to SeO3 than 2D counterparts

PANC1

PANC1 SeO3 (10 µM) PANC1 Ctl PANC1 SeO3 (10 µM) PANC1 Ctl

PANC1

SLIDE 8

8 Glycolysis Glycolysis

Glycolysis & the Krebs cycle were less impacted by SeO3 in PANC1 spheroids than 2D counterparts

CO2 CO2 Glu

1 5

Glu

1 5

Glu-GSH

1 5

Glu-GSH

1 5

CO2 Acetyl CoA

1

Krebs Cycle

Acetyl CoA

1

Krebs Cycle

Asp

1 4

Asp

1 4

Pyruvate NADPH

1 3

Pyruvate NADPH

1 3

3D Spheroids 2D Cells

13C6-Glc

1 6

F6P P

1 6

P

1 3

Pyruvate Lactate

1 3

13C3-lactate

1 3

Pyruvate

1 6

13C6-Glc

Lactate

1 3

13C3-lactate

F6P P

1 6

P E D F H F E G H K K D C B *

0.1 0.0

A C B A D Malate Malate Citrate Fumarate Succinate αKetoglutarate Isocitrate CO2 CO2 Oxaloacetate

4

Succinyl CoA

1 1 4 6 1 4 1 5

Citrate Fumarate Succinate αKetoglutarate Isocitrate CO2 CO2 Oxaloacetate

4

Succinyl CoA

1 1 4 6 1 4 1 5

I I J J

SLIDE 9

9

Pyrimidine & the hexosamine biosyn pathways (HBP) were less impacted by SeO3 in PANC1 spheroids than 2D counterparts

Pyrimidine Synthesis

Asp 1st turn

13C6-Glc

1 6 1 4

NC-Asp Orotate

4 3 2 5 1 6

1

4 3 2 5 1 6

1 1 1 Asp 1st turn

13C6-Glc

1 6 1 4

NC-Asp Orotate

4 3 2 5 1 6

1

4 3 2 5 1 6

1 1 1

HBP

1 6

P NAcGN6P 1

6

P NAcGN1P UDPGlcNAc HN Ac HN Ac

1 1 6

P NAcGN6P 1

6

P NAcGN1P UDPGlcNAc HN Ac

HBP

HN Ac

1

PPP

5 6 1’ 1 4 3 2

PPP

P OMP CO2 UTP P

5 6 1’ 1 4

P

3 2

P

PPP PPP PPP

P OMP CO2 UTP P

5 6 1’ 1 4

P

3 2

P

PPP

5 6 1’ 1 4 3 2

PPP PPP

Pyrimidine Synthesis

P

1’ 5’

P

1’ 5’

PP R5P PRPP

PPP

P

1’ 5’

P

1’ 5’

PP R5P PRPP

PPP

3D Spheroids

D E D E F F A B C A B C G I H G H I

2D Cells

SLIDE 10

Conclusions

10

 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 SeO3 treatment that the 2D counterparts in terms of growth.  This drug resistance may be rooted in reduced sensitivity of M3DB spheroids to SeO3 in glycolysis, the Krebs cycle, nucleotide synthesis, and glutathione metabolism, central to cell growth and survival.

SLIDE 11

Acknowledgments

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 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 )