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Analysis of Invasion Dynamics of Matrix-Embedded Cells in a Multisample Format

  • Marleen Van TroysEmail author
  • Paola Masuzzo
  • Lynn Huyck
  • Karima Bakkali
  • Davy Waterschoot
  • Lennart Martens
  • Christophe Ampe
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1749)

Abstract

In vitro tests of cancer cell invasion are the “first line” tools of preclinical researchers for screening the multitude of chemical compounds or cell perturbations that may aid in halting or treating cancer malignancy. In order to have predictive value or to contribute to designing personalized treatment regimes, these tests need to take into account the cancer cell environment and measure effects on invasion in sufficient detail. The in vitro invasion assays presented here are a trade-off between feasibility in a multisample format and mimicking the complexity of the tumor microenvironment. They allow testing multiple samples and conditions in parallel using 3D-matrix-embedded cells and deal with the heterogeneous behavior of an invading cell population in time. We describe the steps to take, the technical problems to tackle and useful software tools for the entire workflow: from the experimental setup to the quantification of the invasive capacity of the cells. The protocol is intended to guide researchers to standardize experimental set-ups and to annotate their invasion experiments in sufficient detail. In addition, it provides options for image processing and a solution for storage, visualization, quantitative analysis, and multisample comparison of acquired cell invasion data.

Key words

Cancer 3D migration Spheroid Live cell imaging Cell tracking CellMissy 

Notes

Acknowledgments

The authors acknowledge funding from the European Union’s Horizon 2020 Programme under Grant Agreement 634107 (PHC32–2014). L.H. was funded by Bijzonder Onderzoeksfonds UGent 01J04806. M.V.T. acknowledges VIB TechWatch for funding of software acquisition.

References

  1. 1.
    Clark AG, Vignjevic DM (2015) Modes of cancer cell invasion and the role of the microenvironment. Curr Opin Cell Biol 36:13–22.  https://doi.org/10.1016/j.ceb.2015.06.004 CrossRefPubMedGoogle Scholar
  2. 2.
    Kramer N, Walzl A, Unger C, Rosner M, Krupitza G, Hengstschläger M, Dolznig H (2013) In vitro cell migration and invasion assays. Mutat Res 752:10–24.  https://doi.org/10.1016/j.mrrev.2012.08.001 CrossRefPubMedGoogle Scholar
  3. 3.
    Masuzzo P, Van Troys M, Ampe C, Martens L (2016) Taking aim at moving targets in computational cell migration. Trends Cell Biol 26:88–110.  https://doi.org/10.1016/j.tcb.2015.09.003 CrossRefPubMedGoogle Scholar
  4. 4.
    Nath S, Devi GR (2016) Three-dimensional culture systems in cancer research: focus on tumor spheroid model. Pharmacol Ther 163:94–108.  https://doi.org/10.1016/j.pharmthera.2016.03.013 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Fayzullin A, Tuvnes FA, Skjellegrind HK, Behnan J, Mughal AA, Langmoen IA, Vik-Mo EO (2016) Time-lapse phenotyping of invasive glioma cells ex vivo reveals subtype-specific movement patterns guided by tumor core signaling. Exp Cell Res 349:199–213.  https://doi.org/10.1016/j.yexcr.2016.08.001 CrossRefPubMedGoogle Scholar
  6. 6.
    Friedl P, Locker J, Sahai E, Segall JE (2012) Classifying collective cancer cell invasion. Nat Cell Biol 14:777–783.  https://doi.org/10.1038/ncb2548 CrossRefPubMedGoogle Scholar
  7. 7.
    Stewart MD, Jang CW, Hong NW, Austin AP, Behringer RR (2009) Dual fluorescent protein reporters for studying cell behaviors in vivo. Genesis 47:708–717.  https://doi.org/10.1002/dvg.20565 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Masuzzo P, Hulstaert N, Huyck L, Ampe C, Van Troys M, Martens L (2013) CellMissy: a tool for management, storage and analysis of cell migration data produced in wound healing-like assays. Bioinformatics 29:2661–2663CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Masuzzo P, Lynn H, Simiczyjew A, Ampe C, Martens L, Van Troys M (2017) An end-to-end software solution for the analysis of high-throughput single-cell migration data. Sci Rep 13:42383.  https://doi.org/10.1038/srep42383 CrossRefGoogle Scholar
  10. 10.
    Masuzzo P, Martens L (2015) An open data ecosystem for cell migration research. Trends Cell Biol 25:55–58.  https://doi.org/10.1016/j.tcb.2014.11.005 CrossRefPubMedGoogle Scholar
  11. 11.
    De Wever O, Hendrix A, De Boeck A, Eertmans F, Westbroek W, Braems G, Bracke ME (2014) Single cell and spheroid collagen type i invasion assay. Methods Mol Biol 1070:13–35.  https://doi.org/10.1007/978-1-4614-8244-4_2 CrossRefPubMedGoogle Scholar
  12. 12.
    Hulkower KI, Herber RL (2011) Cell migration and invasion assays as tools for drug discovery. Pharmaceutics 3:107–124.  https://doi.org/10.3390/pharmaceutics3010107 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Simpson MJ, Treloar KK, Binder BJ, Haridas P, Manton KJ, Leavesley DI, McElwain DLS, Baker RE (2013) Quantifying the roles of cell motility and cell proliferation in a circular barrier assay. J R Soc Interface 10:20130007–20130007.  https://doi.org/10.1098/rsif.2013.0007 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Johnston ST, Simpson MJ, McElwain DLS, (2014) J R Soc Interface 11:20140325.  https://doi.org/10.1098/rsif.2014.0325
  15. 15.
    Glenn HL, Messner J, Meldrum DR (2016) A simple non-perturbing cell migration assay insensitive to proliferation effects. Sci Rep 6:31694.  https://doi.org/10.1038/srep31694 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Raub CB, Unruh J, Suresh V, Krasieva T, Lindmo T, Gratton E, Tromberg BJ, George SC (2008) Image correlation spectroscopy of multiphoton images correlates with collagen mechanical properties. Biophys J 94:2361–2373.  https://doi.org/10.1529/biophysj.107.120006 CrossRefPubMedGoogle Scholar
  17. 17.
    Sabeh F, Shimizu-Hirota R, Weiss SJ (2009) Protease-dependent versus-independent cancer cell invasion programs: three-dimensional amoeboid movement revisited. J Cell Biol 185:11–19.  https://doi.org/10.1083/jcb.200807195 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Wolf K, Alexander S, Schacht V, Coussens LM, von Andrian UH, van Rheenen J, Deryugina E, Friedl P (2009) Collagen-based cell migration models in vitro and in vivo. Semin Cell Dev Biol 20:931–941.  https://doi.org/10.1016/j.semcdb.2009.08.005 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Kunz-Schughart LA, Kreutz M, Knuechel R (1998) Multicellular spheroids: a three-dimensional in vitro culture system to study tumour biology. Int J Exp Pathol 79:1–23.  https://doi.org/10.1046/j.1365-2613.1998.00051.x CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Nagelkerke A, Bussink J, Sweep FCGJ, Span PN (2013) Generation of multicellular tumor spheroids of breast cancer cells: how to go three-dimensional. Anal Biochem 437:17–19.  https://doi.org/10.1016/j.ab.2013.02.004 CrossRefPubMedGoogle Scholar
  21. 21.
    Fennema E, Rivron N, Rouwkema J, van Blitterswijk C, De Boer J (2013) Spheroid culture as a tool for creating 3D complex tissues. Trends Biotechnol 31:108–115.  https://doi.org/10.1016/j.tibtech.2012.12.003 CrossRefPubMedGoogle Scholar
  22. 22.
    Napolitano AP, Dean DM, Man AJ, Youssef J, Ho DN, Rago AP, Lech MP, Morgan JR (2007) Scaffold-free three-dimensional cell culture utilizing micromolded nonadhesive hydrogels. BioTechniques 43:494–500.  https://doi.org/10.2144/000112591 CrossRefPubMedGoogle Scholar
  23. 23.
    Leung BM, Lesher-Perez SC, Matsuoka T, Moraes C, Takayama S (2015) Media additives to promote spheroid circularity and compactness in hanging drop platform. Biomater Sci 3:336–344.  https://doi.org/10.1039/C4BM00319E CrossRefPubMedGoogle Scholar
  24. 24.
    Surolia R, Li FJ, Wang Z, Li H, Liu G, Zhou Y, Luckhardt T, Bae S, Liu R, Rangarajan S, de Andrade J, Thannickal VJ, Antony VB (2017) 3D pulmospheres serve as a personalized and predictive multicellular model for assessment of antifibrotic drugs. JCI Insight 2:e91377.  https://doi.org/10.1172/jci.insight.91377 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Raub CB, Suresh V, Krasieva T, Lyubovitsky J, Mih JD, Putnam AJ, Tromberg BJ, George SC (2007) Noninvasive assessment of collagen gel microstructure and mechanics using multiphoton microscopy. Biophys J 92:2212–2222.  https://doi.org/10.1529/biophysj.106.097998 CrossRefPubMedGoogle Scholar
  26. 26.
    Gobeaux F, Mosser G, Anglo A, Panine P, Davidson P, Giraud-Guille MM, Belamie E (2008) Fibrillogenesis in dense collagen solutions: a physicochemical study. J Mol Biol 376:1509–1522.  https://doi.org/10.1016/j.jmb.2007.12.047 CrossRefPubMedGoogle Scholar
  27. 27.
    Yang YL, Motte S, Kaufman LJ (2010) Pore size variable type I collagen gels and their interaction with glioma cells. Biomaterials 31:5678–5688.  https://doi.org/10.1016/j.biomaterials.2010.03.039 CrossRefPubMedGoogle Scholar
  28. 28.
    Fraley SI, Feng Y, Krishnamurthy R, Kim D, Celedon A, Longmore GD, Wirtz D, Louis S (2010) A distinctive role for focal adhesion proteins in three-dimensional cell motility. Nat Cell Biol 12:598–604.  https://doi.org/10.1038/ncb2062.A CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Bertier L, Boucherie C, Zwaenepoel O, Vanloo B, Van Troys M, Van Audenhove I, Gettemans J (2017) Inhibitory cortactin nanobodies delineate the role of NTA- and SH3-domain-specific functions during invadopodium formation and cancer cell invasion. FASEB J 31:2460–2476.  https://doi.org/10.1096/fj.201600810RR CrossRefPubMedGoogle Scholar
  30. 30.
    Mitchell CB, O’Neill G (2016) Cooperative cell invasion: matrix metalloproteinase–mediated incorporation between cells. Mol Biol Cell 27:3284–3292CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Blacher S, Erpicum C, Lenoir B, Paupert J, Moraes G, Ormenese S, Bullinger E, Noel A (2014) Cell invasion in the spheroid sprouting assay: a spatial organisation analysis adaptable to cell behaviour. PLoS One 9:e97019.  https://doi.org/10.1371/journal.pone.0097019 CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Kumar KS, Pillong M, Kunze J, Burghardt I, Weller M, Grotzer MA, Schneider G, Baumgartner M (2015) Computer-assisted quantification of motile and invasive capabilities of cancer cells. Sci Rep 5:15338.  https://doi.org/10.1038/srep15338 CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez J-Y, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682.  https://doi.org/10.1038/nmeth.2019 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Marleen Van Troys
    • 1
    • 2
    Email author
  • Paola Masuzzo
    • 1
    • 2
    • 3
  • Lynn Huyck
    • 1
    • 4
  • Karima Bakkali
    • 1
  • Davy Waterschoot
    • 1
  • Lennart Martens
    • 1
    • 2
    • 3
  • Christophe Ampe
    • 1
    • 2
  1. 1.Department of Biochemistry, Faculty of Medicine and Health CareGhent UniversityGhentBelgium
  2. 2.Cancer Research Institute Ghent (CRIG)GhentBelgium
  3. 3.Medical Biotechnology Center, VIBGhentBelgium
  4. 4.UZ Gent VINRADGhentBelgium

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