• Application Note

13C Qualitative Flux Analysis with Symphony Software and Polly Software of Non-Small Cell Lung Carcinoma Cells Grown in vitro in Two and Three Dimensions

13C Qualitative Flux Analysis with Symphony Software and Polly Software of Non-Small Cell Lung Carcinoma Cells Grown in vitro in Two and Three Dimensions

  • David Heywood
  • Hans Vissers
  • Abhishek Jha
  • Raghav Sehgal
  • Amrita Cheema
  • Maria Laura Avantaggiati
  • Waters Corporation
  • Elucidata
  • Georgetown University Medical Center, Lombardi Comprehensive Cancer Center

For research use only. Not for use in diagnostic procedures.

Abstract

The direction of glucose in the same cell line can change dramatically depending on whether they are grown as 2D monolayers or 3D spheroids. In this application note, we show the results of a small case study where Symphony was interfaced to PollyPhi for the processing and analysis of isotope labeling metabolic relative flux data of 2D and 3D non-small cell lung carcinoma cells lines treated with 13C-glucose.

Benefits

We illustrate the use of Symphony Software and Polly Software for contrasting the flow of glucose within mammalian cells grown in vitro in two and three dimensions, monolayers and spheroids, respectively. The more commonly used two-dimensional cell cultures are believed to be inadequate to recreate the in vivo physiologically relevant microenvironment. 

Introduction

Mammalian cell lines are commonly used to study human disease and treatment using (2D)/monolayer cell cultures. However, this geometry of multicellular cell cultures is believed to be inadequate to recreate the biological microenvironment of naturally occurring cells, tumor cells in particular.1,2 Three-dimensional (3D)/spheroid cell cultures are considered to be a more viable in vitro alternative since they better mimic the in vivo cellular growth environment. The potentially different outcomes from experiments using 2D and 3D culture systems may have a significant impact on the relevance of experimental findings. For example, the effectiveness of a drug treatment studied in (2D)/monolayers does not necessarily predict equivalent effectiveness in vivo. In this application note, we show the results of a small case study where Symphony was interfaced to PollyPhi for the processing and analysis of isotope labeling metabolic relative flux data of 2D and 3D non-small cell lung carcinoma cells lines treated with 13C-glucose.

Experimental

Growth of monolayers and sphere cultures/cell extracts

H1299 cell lines were grown and extracted as previously described.3 Briefly, cell lines were grown as monolayer in complete Dulbecco’s Modified Eagle’s Medium to generate spheroids, cells were grown in Falcon Bacteriological Petri Dishes coated with 2% pHEMA dissolved in 100% ethanol. Monolayer cells were dissociated using 0.25% Trypsin-EDTA, whereas spheroid cultures were dissociated using StemPro Accutase. Cell extracts from unlabeled cells or from cells labeled with [U-13C] glucose were pelleted and re-suspended in water and lysed by heat shock treatment. Subsequently, chilled MeOH was added followed by the addition of CHCl3. The samples were centrifuged and the resulting phases separated, followed by the addition of chilled acetonitrile and overnight incubation. The samples were centrifuged again and the supernatant collected and dried. Next, the samples were re-suspended and injected onto the LC-MS system. A graphical overview of the experimental design is shown in Figure 1.

Figure 1. Graphical representation of the experimental design for the isotope labeling metabolic relative flux data analysis of 2D and 3D lung cells lines treated with 13C-glucose using Symphony and Polly informatics.

LC conditions

LC system:

ACQUITY UPLC I-Class

Column:

BEH C18, 1.7 μm, 2.1 mm × 50 mm (p/n 186003685)

Column temp.:

40 °C

Sample temp.:

4 °C

Injection volume:

2.0 μL

Flow rate:

0.4 mL/min

Mobile phase A:

0.1% Formic acid in water

Mobile phase B:

0.1% Formic acid in acetonitrile

Mobile phase D:

0.1% Formic acid in IPA/acetonitrile (90/10, v/v)

Gradient:

Time

%B

%D

0.0

5.0

0.0

8.0

98.0

2.0

11.0

98.0

2.0

12.0

5.0

0.0

MS conditions

MS system:

Xevo G2-XS QTof

Ionization mode:

ESI

Acquisition range:

50–1200 m/z

Capillary voltage:

3.0 kV

Acquisition mode:

MSE

Resolution:

30,000 FWHM

Bioinformatics

Data management

MS software:

MassLynx

Informatics:

Symphony, MSConvert, ElMaven, Polly

Figure 2 shows the basic components of a Symphony pipeline, a client/server application that is triggered by the MassLynx data acquisition system. Typically, a server request is executed that consists of a series of tasks that are executed based on input. Here, the pipeline transfers data and converts the native MassLynx format into mzXML using MSconvert (http://proteowizard.sourceforge.net/index.html). Next, it imports the converted data into ElMaven (elucidatainc.github.io/ElMaven) and Polly (elucidata.io) for peak detection, curation, natural abundance correction, and results visualization. In other words, mapping of the relative abundances and distribution of the detected metabolites on metabolic pathways as illustrated by the example shown in Figure 3. The details and benefits of the informatics pipeline have been described in detail elsewhere.4

Figure 2. Symphony pipeline chains data transfer followed by MSconvert, raw to mzXML data conversion, and ElMaven peak detection, integration, annotation, as well as upload.
Figure 3. Pathway mapping and natural abundance isotopic corrected intensity differences (not fractional enriched) between experimental groups for a selected metabolite in PollyPhi Relative LC-MS.

Results and Discussion

Upregulation in glycolysis

The lactate fractional enrichment (13C3 isotopologue) is significantly higher in spheroid cell cultures compared to monolayer cell cultures. Since 13C3-lactate is the major isotopologue produced from 13C6-glucose, the primary energy source, the observations indicate that glycolysis is significantly upregulated in spheroid cell cultures as opposed to monolayers.

Increased glucose contribution to TCA via acetyl-CoA and PDH

In addition, key intermediates in the tricarboxylic acid (TCA) cycle have higher 13C2 isotopologues. 13C2 isotopologues in the TCA cycle are generally formed from pyruvate 13C3 via acetyl-CoA through PDH enzyme. Synthesis of pyruvate 13C3 isotopologue can in turn be attributed to 13C6-glucose. This observation suggests a higher contribution of glucose to the TCA cycle via acetyl CoA in spheroid cell culture as opposed to monolayer cell culture. This is consistent with the finding of upregulated glycolysis in spheroid cells as shown and summarized in Figure 4.

Figure 4. Relatively high lactate 13C3 isotopologue and 13C2 isotopologues in TCA and adjoining metabolites in 3D spheroid cell culture compared to 2D monolayer cell culture indicates an upregulated glycolysis and TCA cycle.

Conclusion

The direction of glucose in the same cell line can change dramatically depending on whether they are grown as 2D monolayers or 3D spheroids. Specifically, increased glucose flow through the glycolysis and TCA pathways were observed when they were grown as spheroids in contrast to monolayers. Such differences in nutrient utilization has significant implications for the translation of findings based on in vitro models to in vivo models. A prominent example has been shown in Figure 4 where the monolayer cell culture of H1299 human non-small cell lung carcinoma cells shows very limited glycolysis. In contrast, the observations and interpretations are very different for lactate. In the spheroid cells, glycolysis rates are increased significantly. Hence, analysis of the monolayer cell culture in isolation would have made translating the findings from the in vitro model to the in vivo model very challenging.

References

  1. Li et al. Survival advantages of multicellular spheroids vs. monolayers of HepG2 cells in vitro. Oncol Rep. 2008 Dec;20(6):1465–71.
  2. Wallace et al. A Model for Spheroid versus Monolayer Response of SK-N-SH Neuroblastoma Cells to Treatment with 15-Deoxy-PGJ2. Comput Math Methods Med. 2016;2016:3628124.
  3. Fernandez et al. The mitochondrial citrate carrier, SLC25A1, drives stemness and therapy resistance in non-small cell lung cancer. Cell Death Differ. 2018 Jul;25(7):1239–1258.
  4. Automating Metabolic Flux Analysis with Symphony and Polly. Waters Corporation. Technical Brief. 720006380EN. 2018.

720006423, November 2018

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