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Biological characterization of breast cancer spheroid formed by fast fabrication method

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Abstract

Engineered three-dimensional (3D) tissue culture platforms are useful for reproducing and elucidating complex in vivo biological phenomena. Spheroids, 3D aggregates of living cells, are produced based on physicochemical or microfabrication technologies and are commonly used even in cancer pathology research. However, conventional methods have difficulties in constructing 3D structures depending on the cell types, and require specialized techniques/lab know-how to reproducibly control the spheroid size and shape. To overcome these issues, we have developed a fabrication method, which enables anyone to make and mature cancer spheroids using a superhydrophobic microwell made of the monolithic porous materials. Here, we characterize the biological behaviors of the breast cancer spheroids fabricated by our method under normoxic and hypoxic conditions. We found that the fabricated spheroid contracted to a certain size via activation of the actomyosin system. Cell proliferation induced a hypoxic state inside the spheroid (elevated expression of the hypoxia-inducible factor HIF-1α), followed by the formation of a necrotic core and cell escape from the spheroid. In addition, we observed a decrease in cancer spheroid contractility and cell escape from spheroids under hypoxic conditions compared to normoxic conditions, which were related to oxygen concentration-dependent cell motility. The fabricated spheroids perform as 3D tumor tissues in a highly reproducible manner and within a short culture period. Our findings indicate that this fabrication method has a wide range of applications in cancer research, such as elucidating the mechanisms of tumor invasion and metastasis and screening anticancer drugs, as with previous methods.

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The authors declare that all data supporting the findings of this study are available within this article and its supplementary information files or from the corresponding author upon reasonable request.

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Acknowledgements

The human pancreatic cancer cell line, MIA Paca2 (RCB2094) and human breast adenocarcinoma cell line, MCF7 (RCB1902), were provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT/AMED, Japan. We thank Dr. Yoshitsugu Aoki, Director of the Department of Molecular Therapy, National Center of Neurology and Psychiatry, for providing the fluorescence microscope system. This study was partly supported by grants from the Nakatani Foundation for Advancement of Measuring Technologies in Biomedical Engineering and the JSPS KAKENHI (No. 21K19893) to D.Y.

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Authors and Affiliations

  1. Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-Cho, Koganei, Tokyo, 184-8588, Japan

    Yuta Iijima

  2. Faculty of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-Cho, Koganei, Tokyo, 184-8588, Japan

    Yuta Iijima & Daisuke Yoshino

  3. Graduate School of Biomedical Engineering, Tohoku University, 6-6-12 Aramaki-Aza-Aoba, Aoba-Ku, Sendai, Miyagi, 980-8579, Japan

    Yuta Iijima, Satomi Hirose & Kenichi Funamoto

  4. Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-Cho, Koganei, Tokyo, 184-8588, Japan

    Norino Uenaka, Mayu Morimoto, Daiki Sato & Daisuke Yoshino

  5. Institute of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-Cho, Koganei, Tokyo, 184-8588, Japan

    Naoyoshi Sakitani & Daisuke Yoshino

  6. Department of Rehabilitation for Movement Functions, National Rehabilitation Center for Persons With Disabilities, 4-1 Namiki, Tokorozawa, Saitama, 359-8555, Japan

    Naoyoshi Sakitani & Masahiro Shinohara

  7. Department of Life Science and Biotechnology, National Institute of Advanced Industrial Science and Technology, 2217-4 Hayashi-Cho, Takamatsu, Kagawa, 761-0395, Japan

    Naoyoshi Sakitani

  8. Institute of Fluid Science, Tohoku University, 2-1-1 Katahira, Aoba-Ku, Sendai, Miyagi, 980-8577, Japan

    Kenichi Funamoto

  9. International Center for Materials Nanoarchitechtonics, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki, 305-0044, Japan

    Gen Hayase

Authors
  1. Yuta Iijima
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  2. Norino Uenaka
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  3. Mayu Morimoto
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  5. Satomi Hirose
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  6. Naoyoshi Sakitani
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  7. Masahiro Shinohara
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  8. Kenichi Funamoto
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  9. Gen Hayase
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  10. Daisuke Yoshino
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Contributions

D.Y. conceived and designed the research. Y.I. conducted most of the experiments. N.U. was in charge of most of the immunoblotting. M.M. and D.S. conducted mathematical modeling and calculation for oxygen concentration in spheroids. Y.I. and S.H. performed the analysis of the distribution of protein fluorescence intensity. N.S. and M.S. provided technical support in Immunohistochemistry. K.F. supported technical methods for hypoxic exposure experiments on cancer spheroids. G.H. developed, prepared, and provided the superhydrophobic substrates for spheroid fabrication. All authors discussed the data. Y.I. and D.Y. wrote the manuscript. D.Y. directed and supervised the project.

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Correspondence to Gen Hayase or Daisuke Yoshino.

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Highlights

- Cancer spheroids of the desired size can be produced in a highly reproducible manner.

- The fabricated MDA-MB-231 spheroid contracts via actomyosin system activation.

- Cell proliferation induces a highly hypoxic state inside the spheroid.

- Necrotic core formation in the inner layer and cell escape from the spheroid can be reproduced.

- Spheroid behavior is influenced by oxygen concentration-dependent changes in cell motility.

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Iijima, Y., Uenaka, N., Morimoto, M. et al. Biological characterization of breast cancer spheroid formed by fast fabrication method. In vitro models 3, 19–32 (2024). https://doi.org/10.1007/s44164-024-00066-3

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