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Generation of Multicellular Breast Cancer Tumor Spheroids: Comparison of Different Protocols

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Abstract

Multicellular tumor spheroids are widely used models in tumor research. Because of their three dimensional organization they can simulate avascular tumor areas comprising proliferative and necrotic cells. Nonetheless, protocols for spheroid generation are still inconsistent. Therefore, in this study the breast cancer cell lines MCF-7, MDA-MB-231 and SK-BR-3 have been used to compare different spheroid generation models including hanging drop, liquid overlay and suspension culture techniques, each under several conditions. Experimental approaches differed in cell numbers (400–10,000), media and additives (25 % methocel, 25 % methocel plus 1 % Matrigel, 3.5 % Matrigel). In total, 42 different experimental setups have been tested. Generation of spheroids was evaluated by light microscopy and the structural composition was assessed immunohistochemically by means of Ki-67, cleaved poly (ADP-ribose) polymerase (cPARP) and mucin-1 (MUC-1) expression. Although the tested cell lines diverged widely in their capacity of forming spheroids we recommend hanging drops supplemented with 25 % methocel as the most reliable and efficient method with regard to success of generation of uniform spheroids, costs, experimental complexity and time expenditure in the different cell lines. MCF-7 cells formed spheroids under almost all analyzed conditions, and MDA-MB-231 cells under only one protocol (liquid overlay technique, 3.5 % Matrigel), while SK-BR-3 did not under neither condition. Therefore, we outline specific methods and recommend the use of adapted and standardized spheroid generation protocols for each cell line.

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Abbreviations

MCTS:

multicellular tumor spheroids

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Acknowledgments

The project is funded by the Wilhelm Sander Foundation (Germany). Karolin Fröhlich receives a Ph.D. grant from the Evangelic Scholarship Department Villigst (Germany). Stella Mary Photini had a Ph.D. grant from the German Academic Exchange Service (DAAD). The Placenta-Labor had grants from the Thuringian Ministry of Education, Science and Arts.

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Authors and Affiliations

  1. Department of Obstetrics, Placenta-Lab, Bachstraße 18, 07743, Jena, Germany

    Karolin Froehlich, Julia Heger, Jana Pastuschek, Stella Mary Photini, Yan Yan, Ekkehard Schleußner, Udo R. Markert & André Schmidt

  2. Department of Anatomy, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173, Hannover, Germany

    Jan-Dirk Haeger & Christiane Pfarrer

  3. Institute of Pharmacology and Toxicology, University Hospital Jena, Drackendorfer Straße 1, 07747, Jena, Germany

    Amelie Lupp

  4. KIMIII Department of Experimental Nephrology, University Hospital Jena, 07743, Jena, Germany

    Ralf Mrowka

Authors
  1. Karolin Froehlich
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  2. Jan-Dirk Haeger
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  3. Julia Heger
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  4. Jana Pastuschek
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  5. Stella Mary Photini
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  6. Yan Yan
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  7. Amelie Lupp
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  8. Christiane Pfarrer
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  10. Ekkehard Schleußner
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  11. Udo R. Markert
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  12. André Schmidt
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Correspondence to Udo R. Markert.

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Froehlich, K., Haeger, JD., Heger, J. et al. Generation of Multicellular Breast Cancer Tumor Spheroids: Comparison of Different Protocols. J Mammary Gland Biol Neoplasia 21, 89–98 (2016). https://doi.org/10.1007/s10911-016-9359-2

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  • DOI: https://doi.org/10.1007/s10911-016-9359-2

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