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Assessment of Stiffness-Dependent Autophagosome Formation and Apoptosis in Embryonal Rhabdomyosarcoma Tumor Cells

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Methods in Molecular Biology

Part of the book series: Methods in Molecular Biology

Abstract

Remodeling of the extracellular matrix (ECM) eventually causes the stiffening of tumors and changes to the microenvironment. The stiffening alters the biological processes in cancer cells due to altered signaling through cell surface receptors. Autophagy, a key catabolic process in normal and cancer cells, is thought to be involved in mechano-transduction and the level of autophagy is probably stiffness-dependent. Here, we provide a methodology to study the effect of matrix stiffness on autophagy in embryonal rhabdomyosarcoma cells. To mimic stiffness, we seeded cells on GelMA hydrogel matrices with defined stiffness and evaluated autophagy-related endpoints. We also evaluated autophagy-dependent pathways, apoptosis, and cell viability. Specifically, we utilized immunocytochemistry and confocal microscopy to track autophagosome formation through LC3 lipidation. This approach suggests that the use of GelMA hydrogels with defined stiffness represents a novel method to evaluate the role of autophagy in embryonal rhabdomyosarcoma and other cancer cells.

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Acknowledgments

SG (Saeid Ghavami) and AZ (Ali Zarrabi) conceptualized and designed the study. SS (Serap Sezen) and SA (Sevin Adiguzel) performed the experiments. SG and SS analyzed the data. SS and A Kh (Arezoo Khosravi) wrote the manuscript. SS, SG, AZ, SA, Joseph W. Gordon (JWG), Atefeh Zarepour (AtZ), and AKh reviewed and edited the manuscript. AZ funded and SG supervised the experiments.

Funding

This study was funded in part by URGP (University of Manitoba) (SG) and 1002-short term R&D funding program of The Scientific and Technological Research Council of Türkiye (TÜBİTAK, Project No: 221Z107) (AZ).

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Author notes

  1. Authors Saeid Ghavami and Ali Zarrabi have senior authorship.

Authors and Affiliations

  1. Department of Molecular Biology, Genetics and Bioengineering, Faculty of Engineering and Natural Sciences, Sabanci University, Tuzla, Istanbul, Türkiye

    Serap Sezen

  2. Nanotechnology Research and Application Center, Sabanci University, Tuzla, Istanbul, Türkiye

    Serap Sezen & Sevin Adiguzel

  3. Department of Research Analytics, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, Tamil Nadu, India

    Atefeh Zarepour

  4. Department of Genetics and Bioengineering, Faculty of Engineering and Natural Sciences, Istanbul Okan University, Istanbul, Türkiye

    Arezoo Khosravi

  5. Department of Human Anatomy and Cell Science, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, Canada

    Joseph W. Gordon & Saeid Ghavami

  6. Faculty of Medicine in Zabrze, University of Technology in Katowice, Zabrze, Poland

    Saeid Ghavami

  7. Research Institute of Oncology and Hematology, Cancer Care Manitoba, University of Manitoba, Winnipeg, MB, Canada

    Saeid Ghavami

  8. Department of Biomedical Engineering, Faculty of Engineering and Natural Sciences, Istinye University, Istanbul, Türkiye

    Ali Zarrabi

  9. Graduate School of Biotechnology and Bioengineering, Yuan Ze University, Taoyuan, Taiwan

    Ali Zarrabi

Authors
  1. Serap Sezen
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  2. Sevin Adiguzel
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  3. Atefeh Zarepour
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  4. Arezoo Khosravi
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  5. Joseph W. Gordon
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  6. Saeid Ghavami
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  7. Ali Zarrabi
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Corresponding author

Correspondence to Ali Zarrabi .

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Sezen, S. et al. (2024). Assessment of Stiffness-Dependent Autophagosome Formation and Apoptosis in Embryonal Rhabdomyosarcoma Tumor Cells. In: Methods in Molecular Biology. Springer, New York, NY. https://doi.org/10.1007/7651_2024_538

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  • DOI: https://doi.org/10.1007/7651_2024_538

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  • Publisher Name: Springer, New York, NY

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