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3D Spheroid Invasion Assay for High-Throughput Screening of Small-Molecule Libraries

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Chemogenomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2706))

Abstract

Cancer metastasis is a complex cascade that involves the activation of cancer cell migration and invasion of the extracellular space. Cancer-associated fibroblasts (CAFs) are known inducers of cancer cell invasion. However, current in vitro invasion assays such as the Boyden chamber assay are cumbersome and low throughput. Therefore, there is an urgent need for new ex vivo, surrogate invasion assays that can faithfully recapitulate the cancer cell invasion process in vitro and are amenable to large-scale screening of small-molecule libraries in a high-throughput fashion. Here, we describe a well-established high-throughput three-dimensional (3D) spheroid invasion assay as a powerful tool to identify novel molecular targets that can potentially mediate CAF-dependent cancer cell invasion.

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References

  1. Berg S, Kutra D, Kroeger T et al (2019) Ilastik: interactive machine learning for (bio)image analysis. Nat Methods 16:1226–1232

    Article  CAS  PubMed  Google Scholar 

  2. Cacciamali A, Villa R, Dotti S (2022) 3D cell cultures: evolution of an ancient tool for new applications. Front Physiol 13:836480

    Article  PubMed  PubMed Central  Google Scholar 

  3. Chaffer CL, Weinberg RA (2011) A perspective on cancer cell metastasis. Science 331:1559–1564

    Article  CAS  PubMed  Google Scholar 

  4. Elmusrati A, Wang J, Wang CY (2021) Tumor microenvironment and immune evasion in head and neck squamous cell carcinoma. Int J Oral Sci 13:24

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Fennema E, Rivron N, Rouwkema J et al (2013) Spheroid culture as a tool for creating 3D complex tissues. Trends Biotechnol 31:108–115

    Article  CAS  PubMed  Google Scholar 

  6. Fontoura JC, Viezzer C, Dos Santos FG et al (2020) Comparison of 2D and 3D cell culture models for cell growth, gene expression and drug resistance. Mater Sci Eng C Mater Biol Appl 107:110264

    Article  CAS  PubMed  Google Scholar 

  7. Friedl P, Alexander S (2011) Cancer invasion and the microenvironment: plasticity and reciprocity. Cell 147:992–1009

    Article  CAS  PubMed  Google Scholar 

  8. Hanahan D (2022) Hallmarks of cancer: new dimensions. Cancer Discov 12:31–46

    Article  CAS  PubMed  Google Scholar 

  9. Hughes CS, Postovit LM, Lajoie GA (2010) Matrigel: a complex protein mixture required for optimal growth of cell culture. Proteomics 10:1886–1890

    Article  CAS  PubMed  Google Scholar 

  10. Hussain A, Voisin V, Poon S et al (2020) Distinct fibroblast functional states drive clinical outcomes in ovarian cancer and are regulated by TCF21. J Exp Med 217

    Google Scholar 

  11. Johnson DE, Burtness B, Leemans CR et al (2020) Head and neck squamous cell carcinoma. Nat Rev Dis Primers 6:92

    Article  PubMed  PubMed Central  Google Scholar 

  12. Kapalczynska M, Kolenda T, Przybyla W et al (2018) 2D and 3D cell cultures – a comparison of different types of cancer cell cultures. Arch Med Sci 14:910–919

    PubMed  Google Scholar 

  13. Laschke MW, Menger MD (2017) Life is 3D: boosting spheroid function for tissue engineering. Trends Biotechnol 35:133–144

    Article  CAS  PubMed  Google Scholar 

  14. Li S, Zhang Z, Han L (2020) 3D spheroids propel tumor characterization. Trends Cancer 6:622–624

    Article  CAS  PubMed  Google Scholar 

  15. Martinotti S, Ranzato E (2020) Scratch wound healing assay. Methods Mol Biol 2109:225–229

    Article  CAS  PubMed  Google Scholar 

  16. Muller S, Ackloo S, Arrowsmith CH et al (2018) Donated chemical probes for open science. elife 7

    Google Scholar 

  17. Pampaloni F, Reynaud EG, Stelzer EH (2007) The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol 8:839–845

    Article  CAS  PubMed  Google Scholar 

  18. Poon S, Ailles LE (2022) Modeling the Role of cancer-associated fibroblasts in tumor cell invasion. Cancers (Basel) 14

    Google Scholar 

  19. Principe S, Mejia-Guerrero S, Ignatchenko V et al (2018) Proteomic analysis of cancer-associated fibroblasts reveals a paracrine role for MFAP5 in human oral tongue squamous cell carcinoma. J Proteome Res 17:2045–2059

    Article  CAS  PubMed  Google Scholar 

  20. Rawal P, Tripathi DM, Nain V et al (2022) VEGF-mediated tumour growth and EMT in 2D and 3D cell culture models of hepatocellular carcinoma. Oncol Lett 24:315

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Sahai E, Astsaturov I, Cukierman E et al (2020) A framework for advancing our understanding of cancer-associated fibroblasts. Nat Rev Cancer 20:174–186

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Vigneswaran N, Williams MD (2014) Epidemiologic trends in head and neck cancer and aids in diagnosis. Oral Maxillofac Surg Clin North Am 26:123–141

    Article  PubMed  PubMed Central  Google Scholar 

  24. Vinci M, Box C, Eccles SA (2015) Three-dimensional (3D) tumor spheroid invasion assay. J Vis Exp:e52686

    Google Scholar 

  25. Wang Y, Jeon H (2022) 3D cell cultures toward quantitative high-throughput drug screening. Trends Pharmacol Sci 43:569–581

    Article  PubMed  Google Scholar 

  26. Yamada KM, Cukierman E (2007) Modeling tissue morphogenesis and cancer in 3D. Cell 130:601–610

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

Thanks to Dr. Christina Karamboulas, Dr. Ngoc Bui and Jalna Meens for their help and training in the Ailles laboratory. Thanks to Dr. Claudia Tredup and Dr. Bennedict Tillman-Berger from SGC-Frankfurt and Dr. Suzanne Ackloo and Albina Bolotokova from SGC-Toronto laboratories for chemical probe management, sourcing, and distribution. Tissues used to generate the CAF lines were collected from HNSCC patients who underwent surgical resection of their tumors at the University Health Network (UHN) and provided written consent under UHN Research Ethics Board approval (REB no. 12-5639). Thanks to Drs. Rodger Tiedemann, Shane Harding and Robert Rottapel for generously allowing us to use their Incucyte imaging system. Thanks to Dr. Carman K M Ip and Dr. Troy Ketela at the Princess Margaret Genomics Centre (PMGC) for helping to make the chemical probe daughter plates using the Echo liquid handler. The authors appreciate the excellent research environment and the research facilities provided by the Princess Margaret Cancer Centre and the SGC. Kunal Karve was supported by a Mitacs Accelerate fellowship. Stephanie Poon was supported by Ontario Student Opportunity Trust Funds endowed by the Princess Margaret Cancer Center, Ontario Institute for Cancer Research, the University of Toronto and the Province of Ontario. This work was partly funded by a research contract awarded to Drs. Ailles and the SGC from Merck KGaA, Darmstadt, Germany, and by an Investigator Award from the Ontario Institute for Cancer Research. The SGC is a charity (register number 1097737) that received funding from AbbVie, Bayer AG, Boehringer Ingelheim, Bristol Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute [OGI-196], Janssen, Merck KGaA (aka EMD in Canada and USA), Pfizer, Takeda, and the Innovative Medicines Initiative 2 Joint Undertaking (IMI2 JU; EUbOPEN grant 875510) from the European Union and European Federation of Pharmaceutical Industries and Associations (EFPIA).

Author information

Authors and Affiliations

  1. Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada

    Kunal Karve, Stephanie Poon & Laurie Ailles

  2. Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada

    Kunal Karve & Panagiotis Prinos

  3. Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada

    Stephanie Poon & Laurie Ailles

Authors
  1. Kunal Karve
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  2. Stephanie Poon
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  3. Panagiotis Prinos
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  4. Laurie Ailles
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Corresponding author

Correspondence to Laurie Ailles .

Editor information

Editors and Affiliations

  1. Department of Pharmacy, Ludwig-Maximilians-Universität München, Munich, Germany

    Daniel Merk

  2. Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Frankfurt am Main, Germany

    Apirat Chaikuad

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Karve, K., Poon, S., Prinos, P., Ailles, L. (2023). 3D Spheroid Invasion Assay for High-Throughput Screening of Small-Molecule Libraries. In: Merk, D., Chaikuad, A. (eds) Chemogenomics. Methods in Molecular Biology, vol 2706. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3397-7_15

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  • DOI: https://doi.org/10.1007/978-1-0716-3397-7_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3396-0

  • Online ISBN: 978-1-0716-3397-7

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