Comprehensive analysis of signal transduction in three-dimensional ECM-based tumor cell cultures
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Abstract
Analysis of signal transduction and protein phosphorylation is fundamental to understand physiological and pathological cell behavior as well as identification of novel therapeutic targets. Despite the fact that more physiological three-dimensional cell culture assays are increasingly used, particularly proteomics and phosphoproteomics remain challenging due to easy, robust and reproducible sample preparation. Here, we present an easy-to-perform, reliable and time-efficient method for the production of 3D cell lysates without compromising cell adhesion before cell lysis. The samples can be used for Western blotting as well as phosphoproteome array technology. This technique would be of interest for researchers working in all fields of biology and drug development.
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References
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2. Nelson CM, Inman JL, Bissell MJ. Three-dimensional lithographically defined organotypic tissue arrays for quantitative analysis of morphogenesis and neoplastic progression. Nat Protoc. 2008;3: 674–8.
3. Fournier M V, Martin KJ. Transcriptome profiling in clinical breast cancer: from 3D culture models to prognostic signatures. J Cell Physiol. 2006;209: 625–30.
4. Storch K, Eke I, Borgmann K, Krause M, Richter C, Becker K, et al. Three-dimensional cell growth confers radioresistance by chromatin density modification. Cancer Res. 2010;70: 3925–34.
5. Eke I, Schneider L, Förster C, Zips D, Kunz-Schughart LA, Cordes N. EGFR/JIP-4/JNK2 signaling attenuates cetuximab-mediated radiosensitization of squamous cell carcinoma cells. Cancer Res. 2013;73: 297–306.
6. Luca AC, Mersch S, Deenen R, Schmidt S, Messner I, Schäfer K-L, et al. Impact of the 3D microenvironment on phenotype, gene expression, and EGFR inhibition of colorectal cancer cell lines. PLoS One. 2013;8: e59689.
7. Eke I, Zscheppang K, Dickreuter E, Hickmann L, Mazzeo E, Unger K, et al. Simultaneous β1 integrin-EGFR Targeting and Radiosensitization of Human Head and Neck Cancer. J Natl Cancer Inst. 2015;107: dju419–.
8. Levin VA, Panchabhai S, Shen L, Baggerly KA. Protein and phosphoprotein levels in glioma and adenocarcinoma cell lines grown in normoxia and hypoxia in monolayer and three-dimensional cultures. Proteome Sci. 2012;10: 5.
9. Lo AT, Mori H, Mott J, Bissell MJ. Constructing three-dimensional models to study mammary gland branching morphogenesis and functional differentiation. J Mammary Gland Biol Neoplasia. 2012;17: 103–10.
10. Matsuda Y, Kawamoto Y, Teduka K, Peng W-X, Yamamoto T, Ishiwata T, et al. Morphological and cytoskeletal alterations of nervous system tumor cells with different culturing methods. Int J Oncol. 2011;38: 1253–8.
11. Eke I, Cordes N. Radiobiology goes 3D: how ECM and cell morphology impact on cell survival after irradiation. Radiother Oncol. 2011;99: 271–8.
12. Martin KJ, Patrick DR, Bissell MJ, Fournier M V. Prognostic breast cancer signature identified from 3D culture model accurately predicts clinical outcome across independent datasets. PLoS One. 2008;3: e2994.
13. Manning G, Plowman GD, Hunter T, Sudarsanam S. Evolution of protein kinase signaling from yeast to man. Trends Biochem Sci. 2002;27: 514–20.
14. Grimsrud PA, Swaney DL, Wenger CD, Beauchene NA, Coon JJ. Phosphoproteomics for the masses. ACS Chem Biol. 2010;5: 105–19.
15. Eke I, Deuse Y, Hehlgans S, Gurtner K, Krause M, Baumann M, et al. β1 Integrin/FAK/cortactin signaling is essential for human head and neck cancer resistance to radiotherapy. J Clin Invest. 2012;122: 1529–40.
16. Eke I, Leonhardt F, Storch K, Hehlgans S, Cordes N. The small molecule inhibitor QLT0267 Radiosensitizes squamous cell carcinoma cells of the head and neck. PLoS One. 2009;4: e6434.
17. Fernandez-Fuente G, Mollinedo P, Grande L, Vazquez-Barquero A, Fernandez-Luna JL. Culture dimensionality influences the resistance of glioblastoma stem-like cells to multikinase inhibitors. Mol Cancer Ther. 2014;13: 1664–72.
18. Schlaepfer DD, Hunter T. Integrin signalling and tyrosine phosphorylation: just the FAKs? Trends Cell Biol. 1998;8: 151–157.
19. Burridge K. Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly. J Cell Biol. 1992;119: 893–903.
20. Parsons JT, Horwitz AR, Schwartz MA. Cell adhesion: integrating cytoskeletal dynamics and cellular tension. Nat Rev Mol Cell Biol. 2010;11: 633–43.
21. Janmey PA. The cytoskeleton and cell signaling: component localization and mechanical coupling. Physiol Rev. 1998;78: 763–81.
22. Fletcher DA, Mullins RD. Cell mechanics and the cytoskeleton. Nature. Nature Publishing Group; 2010;463: 485–492.
23. Eke I, Storch K, Krause M, Cordes N. Cetuximab attenuates its cytotoxic and radiosensitizing potential by inducing fibronectin biosynthesis. Cancer Res. 2013;73: 5869–79.
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Published
Nov 17, 2015
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Eke I, Hehlgans S, Zong Y, Cordes N. Comprehensive analysis of signal transduction in three-dimensional ECM-based tumor cell cultures. J Biol Methods [Internet]. 2015 Nov. 17 [cited 2024 Apr. 25];2(4):e31. Available from: https://jbmethods.org/index.php/jbm/article/view/96
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