The Suitability of Glioblastoma Cell Lines as Models for Primary Glioblastoma Cell Metabolism
Abstract
:Simple Summary
Abstract
1. Introduction
2. Results
2.1. Baseline Oxygen Consumption Rate (OCR) Comparison of Primary Healthy and GBM Cells, and GBM Cell Lines
2.2. Comparison of Mitochondrial Function in Response to Metabolism-Altering Compounds
2.3. Metabolic Parameters Pertaining to OCR and the Extracellular Acidification Rate (ECAR)
3. Discussion
4. Materials and Methods
4.1. Ethics
4.2. Cell Line Culture
4.3. Short Tandem Repeat Profiling
4.4. Primary Brain Cell Culture
4.5. Metabolic Flux Analysis
4.6. Metabolic Capacity Calculations
4.7. Statistics
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
- Australian Institute of Health and Welfare. Cancer in Australia 2017; AIHW: Canberra, Australia, 2017; pp. 1–204. ISBN 978-1-76054-075-3. [Google Scholar]
- Stupp, R.; Mason, W.P.; van den Bent, M.J.; Weller, M.; Fisher, B.; Taphoorn, M.J.B.; Belanger, K.; Brandes, A.A.; Marosi, C.; Bogdahn, U.; et al. Radiotherapy plus Concomitant and Adjuvant Temozolomide for Glioblastoma. N. Engl. J. Med. 2005, 352, 987–996. [Google Scholar] [CrossRef] [PubMed]
- Sesen, J.; Dahan, P.; Scotland, S.J.; Saland, E.; Dang, V.-T.; Lemarié, A.; Tyler, B.M.; Brem, H.; Toulas, C.; Moyal, E.C.-J.; et al. Metformin Inhibits Growth of Human Glioblastoma Cells and Enhances Therapeutic Response. PLoS ONE 2015, 10, e0123721. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Warburg, O. On the Origin of Cancer Cells. Science 1956, 123, 309–314. [Google Scholar] [CrossRef]
- Bonnet, S.; Archer, S.L.; Allalunis-Turner, J.; Haromy, A.; Beaulieu, C.; Thompson, R.B.; Lee, C.T.; Lopaschuk, G.D.; Puttagunta, L.; Bonnet, S.; et al. A Mitochondria-K+ Channel Axis Is Suppressed in Cancer and Its Normalization Promotes Apoptosis and Inhibits Cancer Growth. Cancer Cell 2007, 11, 37–51. [Google Scholar] [CrossRef] [Green Version]
- McFate, T.; Mohyeldin, A.; Lu, H.; Thakar, J.; Henriques, J.; Halim, N.D.; Wu, H.; Schell, M.J.; Tsang, T.M.; Teahan, O.; et al. Pyruvate Dehydrogenase Complex Activity Controls Metabolic and Malignant Phenotype in Cancer Cells. J. Biol. Chem. 2008, 283, 22700–22708. [Google Scholar] [CrossRef] [Green Version]
- Sun, R.C.; Fadia, M.; Dahlstrom, J.E.; Parish, C.R.; Board, P.G.; Blackburn, A.C. Reversal of the glycolytic phenotype by dichloroacetate inhibits metastatic breast cancer cell growth in vitro and in vivo. Breast Cancer Res. Treat. 2009, 120, 253–260. [Google Scholar] [CrossRef]
- Margareto, J.; Larrarte, E.; Leis, O.; Carrasco, A.; Lafuente, J.V.; Idoate, M.A. Gene expression profiling of human gliomas reveals differences between GBM and LGA related to energy metabolism and notch signaling pathways. J. Mol. Neurosci. 2007, 32, 53–63. [Google Scholar] [CrossRef]
- Marin-Valencia, I.; Yang, C.; Mashimo, T.; Cho, S.; Baek, H.; Yang, X.-L.; Rajagopalan, K.N.; Maddie, M.; Vemireddy, V.; Zhao, Z.; et al. Analysis of Tumor Metabolism Reveals Mitochondrial Glucose Oxidation in Genetically Diverse Human Glioblastomas in the Mouse Brain In Vivo. Cell Metab. 2012, 15, 827–837. [Google Scholar] [CrossRef] [Green Version]
- Hambardzumyan, D.; Bergers, G. Glioblastoma: Defining tumour niches. Trends Cancer 2015, 1, 252–265. [Google Scholar] [CrossRef] [Green Version]
- Brunner, G.; Lang, K.; Wolfe, R.A.; McClure, D.B.; Sato, G.H. Selective cell culture of brain cells by serum-free, hormone-supplemented media: A comparative morphological study. Dev. Brain Res. 1981, 2, 563–575. [Google Scholar] [CrossRef]
- Lee, J.; Kotliarova, S.; Kotliarov, Y.; Li, A.; Su, Q.; Donin, N.M.; Pastorino, S.; Purow, B.W.; Christopher, N.; Zhang, W.; et al. Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell 2006, 9, 391–403. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Pollard, S.M.; Yoshikawa, K.; Clarke, I.D.; Danovi, D.; Stricker, S.; Russell, R.; Bayani, J.; Head, R.; Lee, M.; Bernstein, M.; et al. Glioma Stem Cell Lines Expanded in Adherent Culture Have Tumor-Specific Phenotypes and Are Suitable for Chemical and Genetic Screens. Cell Stem Cell 2009, 4, 568–580. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Divakaruni, A.S.; Paradyse, A.; Ferrick, D.A.; Murphy, A.N.; Jastroch, M. Analysis and Interpretation of Microplate-Based Oxygen Consumption and pH Data. Methods Enzymol. 2014, 547, 309–354. [Google Scholar] [CrossRef] [PubMed]
- Herst, P.M.; Berridge, M.V. Cell surface oxygen consumption: A major contributor to cellular oxygen consumption in glycolytic cancer cell lines. Biochim. Biophys. Acta (BBA) Gen. Subj. 2007, 1767, 170–177. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Cataldo, A.M.; Broadwell, R.D. Cytochemical identification of cerebral glycogen and glucose-6-phosphatase activity under normal and experimental conditions. II. Choroid plexus and ependymal epithelia, endothelia and pericytes. J. Neurocytol. 1986, 15, 511–524. [Google Scholar] [CrossRef] [PubMed]
- Pfeiffer-Guglielmi, B.; Fleckenstein, B.; Jung, G.; Hamprecht, B. Immunocytochemical localization of glycogen phosphorylase isozymes in rat nervous tissues by using isozyme-specific antibodies. J. Neurochem. 2003, 85, 73–81. [Google Scholar] [CrossRef] [PubMed]
- Falkowska, A.; Gutowska, I.; Goschorska, M.; Nowacki, P.; Chlubek, D.; Baranowska-Bosiacka, I. Energy Metabolism of the Brain, Including the Cooperation between Astrocytes and Neurons, Especially in the Context of Glycogen Metabolism. Int. J. Mol. Sci. 2015, 16, 25959–25981. [Google Scholar] [CrossRef] [Green Version]
- Nehlig, A.; De Vasconcelos, A.P. Glucose and ketone body utilization by the brain of neonatal rats. Prog. Neurobiol. 1993, 40, 163–220. [Google Scholar] [CrossRef]
- Hao, W.; Chang, C.-P.B.; Tsao, C.-C.; Xu, J. Oligomycin-induced Bioenergetic Adaptation in Cancer Cells with Heterogeneous Bioenergetic Organization. J. Biol. Chem. 2010, 285, 12647–12654. [Google Scholar] [CrossRef] [Green Version]
- Comelli, M.; Pretis, I.; Buso, A.; Mavelli, I. Mitochondrial energy metabolism and signalling in human glioblastoma cell lines with different PTEN gene status. J. Bioenerg. Biomembr. 2017, 50, 33–52. [Google Scholar] [CrossRef]
- Mai, W.X.; Gosa, L.; Daniels, V.W.; Ta, L.; Tsang, J.E.; Higgins, B.; Gilmore, W.B.; Bayley, N.A.; Harati, M.D.; Lee, J.T.; et al. Cytoplasmic p53 couples oncogene-driven glucose metabolism to apoptosis and is a therapeutic target in glioblastoma. Nat. Med. 2017, 23, 1342–1351. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ishii, N.; Tada, M.; Hamou, M.-F.; Janzer, R.C.; Meagher-Villemure, K.; Wiestler, O.D.; De Tribolet, N.; Van Meir, E.G. Cells with TP53 mutations in low grade astrocytic tumors evolve clonally to malignancy and are an unfavorable prognostic factor. Oncogene 1999, 18, 5870–5878. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kennedy, C.R.; Tilkens, S.B.; Guan, H.; Garner, J.A.; Or, P.M.; Chan, A.M. Differential sensitivities of glioblastoma cell lines towards metabolic and signaling pathway inhibitions. Cancer Lett. 2013, 336, 299–306. [Google Scholar] [CrossRef] [PubMed]
- Hardie, D.G.; Ross, F.A.; Hawley, S.A. AMPK: A nutrient and energy sensor that maintains energy homeostasis. Nat. Rev. Mol. Cell Biol. 2012, 13, 251–262. [Google Scholar] [CrossRef] [Green Version]
- Wang, Q.; Hu, B.; Hu, X.; Kim, H.; Squatrito, M.; Scarpace, L.; deCarvalho, A.C.; Lyu, S.; Li, P.; Li, Y.; et al. Tumor evolution of glioma-intrinsic gene expression subtypes associates with immunological changes in the microenvironment. Cancer Cell 2017, 32, 42–56.e46. [Google Scholar] [CrossRef] [Green Version]
- Brennan, C.W.; Verhaak, R.G.W.; McKenna, A.; Campos, B.; Noushmehr, H.; Salama, S.R.; Zheng, S.; Chakravarty, D.; Sanborn, J.Z.; Berman, S.H.; et al. The Somatic Genomic Landscape of Glioblastoma. Cell 2013, 155, 462–477. [Google Scholar] [CrossRef]
- Wise, D.R.; DeBerardinis, R.J.; Mancuso, A.; Sayed, N.; Zhang, X.-Y.; Pfeiffer, H.K.; Nissim, I.; Daikhin, E.; Yudkoff, M.; McMahon, S.B.; et al. Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction. Proc. Natl. Acad. Sci. USA 2008, 105, 18782–18787. [Google Scholar] [CrossRef] [Green Version]
- Orcutt, K.P.; Parsons, A.D.; Sibenaller, Z.A.; Scarbrough, P.M.; Zhu, Y.; Sobhakumari, A.; Wilke, W.W.; Kalen, A.L.; Goswami, P.; Miller, F.J.; et al. Erlotinib-Mediated Inhibition of EGFR Signaling Induces Metabolic Oxidative Stress through NOX4. Cancer Res. 2011, 71, 3932–3940. [Google Scholar] [CrossRef] [Green Version]
- Conn, V.M.; Gabryelska, M.; Marri, S.; Stringer, B.W.; Ormsby, R.J.; Penn, T.; Poonnoose, S.; Kichenadasse, G.; Conn, S.J. SRRM4 Expands the Repertoire of Circular RNAs by Regulating Microexon Inclusion. Cells 2020, 9, 2488. [Google Scholar] [CrossRef]
- McCarthy, K.D.; De Vellis, J. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. Cell Biol. 1980, 85, 890–902. [Google Scholar] [CrossRef] [Green Version]
- Banker, G.A.; Cowan, W.M. Rat hippocampal neurons in dispersed cell culture. Brain Res. 1977, 126, 397–425. [Google Scholar] [CrossRef]
Comparison | Parameter | Healthy | 1′ GBM | U87MG | U251MG | U373MG | D54 | T98G |
---|---|---|---|---|---|---|---|---|
Compared to 1′ Healthy Cells | Non-mito respiration | 6.57 ± 0.21 | 6.11 ± 0.19 | p = 0.007 15.95 ± 0.76 | 2.82 ± 0.21 | 4.79 ± 0.62 | p < 0.001 0.51 ± 0.22 | p = 0.012 15.61 ± 0.89 |
Basal mito rate | 5.30 ± 0.28 | p = 0.043 2.76 ± 0.19 | p < 0.001 0.02 ± 0.01 | p = 0.003 2.00 ± 0.33 | 2.82 ± 0.31 | 3.92 ± 0.24 | p < 0.001 0.05 ± 0.25 | |
ATP-linked resp rate | 4.95 ± 0.36 | p < 0.001 1.86 ± 0.16 | 1.33 ± 0.51 | 4.36 ± 0.36 | 5.18 ± 0.99 | 4.41 ± 0.62 | 6.11 ± 0.27 | |
Proton leak | 0.07 ± 0.23 | 0.78 ± 0.13 | p = 0.006 0.00 ± 0.00 | 2.34 ± 0.21 | 2.14 ± 0.36 | 0.04 ± 0.22 | p = 0.006 0.01 ± 0.00 | |
Reserve capacity | 4.74 ± 0.17 | p = 0.037 10.17 ± 0.29 | 5.22 ± 0.43 | 3.51 ± 0.49 | 0.04 ± 0.46 | 7.10 ± 0.48 | 0.15 ± 0.43 | |
Maximal mito resp rate | 9.80 ± 0.24 | 12.99 ± 0.38 | 11.99 ± 0.56 | 9.15 ± 0.41 | 9.48 ± 0.38 | 12.59 ± 0.46 | p < 0.001 0.00 ± 0.01 | |
Compared to 1′ GBM | Non-mito respiration | - | - | p = 0.004 | - | - | p = 0.002 | p = 0.007 |
Basal mito rate | p = 0.043 | - | p = 0.002 | - | - | - | p = 0.020 | |
ATP-linked resp rate | p < 0.001 | - | p = 0.004 | p = 0.004 | p < 0.001 | p < 0.001 | p < 0.001 | |
Proton leak | - | - | p = 0.011 | - | - | - | p = 0.011 | |
Reserve capacity | p = 0.037 | - | - | p < 0.001 | p < 0.001 | - | p < 0.001 | |
Maximal resp rate | - | - | - | - | - | - | p < 0.001 |
Comparison | Parameter | Healthy | 1′ GBM | U87MG | U251MG | U373MG | D54 | T98G |
---|---|---|---|---|---|---|---|---|
Compared to 1′ Healthy Cells | Basal glycolysis | 4.29 ± 0.30 | 4.05 ± 0.35 | p = 0.006 0.16 ± 0.36 | p = 0.006 0.31 ± 0.34 | p = 0.009 2.07 ± 0.23 | p = 0.009 0.06 ± 0.26 | 5.19 ± 0.27 |
Glycolytic capacity | 3.30 ± 0.22 | 4.73 ± 0.29 | p = 0.038 1.00 ± 0.32 | 3.02 ± 0.22 | p < 0.001 0.84 ± 0.18 | p < 0.001 0.00 ± 0.28 | 4.02 ± 0.20 | |
Krebs cycle capacity | 1.01 ± 0.22 | p = 0.002 3.63 ± 0.13 | 0.56 ± 0.21 | p = 0.002 0.00 ± 0.03 | 0.92 ± 0.20 | 0.01±0.26 | 2.14 ± 0.17 | |
Compared to 1′ GBM | Basal glycolysis | - | - | p = 0.004 | p < 0.001 | p = 0.025 | P < 0.001 | - |
Glycolytic capacity | - | - | p = 0.035 | - | p < 0.001 | p < 0.001 | - | |
Krebs cycle capacity | p = 0.002 | - | p = 0.005 | p < 0.001 | p = 0.001 | p < 0.001 | - |
Category | Parameter | Healthy | U87MG | U251MG | U373MG | D54 | T98G |
---|---|---|---|---|---|---|---|
OCR related | Non-mito respiration | ✓ | - | ✓ | ✓ | - | - |
Basal mito rate | - | - | ✓ | ✓ | ✓ | - | |
ATP-linked resp rate | - | - | - | - | - | - | |
Proton leak | ✓ | - | ✓ | ✓ | ✓ | - | |
Reserve capacity | - | ✓ | - | - | ✓ | - | |
Maximal mito resp rate | ✓ | ✓ | ✓ | ✓ | ✓ | - | |
ECAR related | Basal glycolysis | ✓ | - | - | - | - | ✓ |
Glycolytic capacity | ✓ | - | ✓ | - | - | ✓ | |
Krebs cycle capacity | - | - | - | - | - | ✓ |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Arthurs, A.L.; Keating, D.J.; Stringer, B.W.; Conn, S.J. The Suitability of Glioblastoma Cell Lines as Models for Primary Glioblastoma Cell Metabolism. Cancers 2020, 12, 3722. https://doi.org/10.3390/cancers12123722
Arthurs AL, Keating DJ, Stringer BW, Conn SJ. The Suitability of Glioblastoma Cell Lines as Models for Primary Glioblastoma Cell Metabolism. Cancers. 2020; 12(12):3722. https://doi.org/10.3390/cancers12123722
Chicago/Turabian StyleArthurs, Anya L., Damien J. Keating, Brett W. Stringer, and Simon J. Conn. 2020. "The Suitability of Glioblastoma Cell Lines as Models for Primary Glioblastoma Cell Metabolism" Cancers 12, no. 12: 3722. https://doi.org/10.3390/cancers12123722