Skip to main content
SpringerLink
Log in

Autophagy in 3D In Vitro and Ex Vivo Cancer Models

  • Protocol
  • First Online:
Autophagy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1880))

  • 4319 Accesses

Abstract

Three-dimensional (3D) models are acquiring importance in cancer research due to their ability to mimic multiple features of the tumor microenvironment more accurately than standard monolayer two-dimensional (2D) cultures. Several groups, including our laboratory, are now accumulating evidence that autophagy in solid tumors is also better represented in 3D than in 2D. Here we detail how we generate 3D models, both in vitro multicellular spheroids generated from cell lines and ex vivo tumor fragment spheroids generated from tumor samples, and how autophagy can be measured in 3D cultures.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 189.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. 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(10):839–845. https://doi.org/10.1038/nrm2236

    Article  CAS  PubMed  Google Scholar 

  2. Nyga A, Cheema U, Loizidou M (2011) 3D tumour models: novel in vitro approaches to cancer studies. J Cell Commun Signal 5(3):239–248. https://doi.org/10.1007/s12079-011-0132-4

    Article  PubMed  PubMed Central  Google Scholar 

  3. Hoffmann OI, Ilmberger C, Magosch S, Joka M, Jauch KW, Mayer B (2015) Impact of the spheroid model complexity on drug response. J Biotechnol 205:14–23. https://doi.org/10.1016/j.jbiotec.2015.02.029

    Article  CAS  PubMed  Google Scholar 

  4. Imamura Y, Mukohara T, Shimono Y, Funakoshi Y, Chayahara N, Toyoda M, Kiyota N, Takao S, Kono S, Nakatsura T et al (2015) Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer. Oncol Rep 33(4):1837–1843. https://doi.org/10.3892/or.2015.3767

    Article  CAS  PubMed  Google Scholar 

  5. Baker BM, Chen CS (2012) Deconstructing the third dimension: how 3D culture microenvironments alter cellular cues. J Cell Sci 125(Pt 13):3015–3024. https://doi.org/10.1242/jcs.079509

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Antoni D, Burckel H, Josset E, Noel G (2015) Three-dimensional cell culture: a breakthrough in vivo. Int J Mol Sci 16(3):5517–5527. https://doi.org/10.3390/ijms16035517

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Barbone D, Cheung P, Battula S, Busacca S, Gray SG, Longley DB, Bueno R, Sugarbaker DJ, Fennell DA, Broaddus VC (2012) Vorinostat eliminates multicellular resistance of mesothelioma 3D spheroids via restoration of Noxa expression. PLoS One 7(12):e52753. https://doi.org/10.1371/journal.pone.0052753

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Edmondson R, Broglie JJ, Adcock AF, Yang L (2014) Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay Drug Dev Technol 12(4):207–218. https://doi.org/10.1089/adt.2014.573

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kim KU, Wilson SM, Abayasiriwardana KS, Collins R, Fjellbirkeland L, Xu Z, Jablons DM, Nishimura SL, Broaddus VC (2005) A novel in vitro model of human mesothelioma for studying tumor biology and apoptotic resistance. Am J Respir Cell Mol Biol 33(6):541–548. https://doi.org/10.1165/rcmb.2004-0355OC

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Thoma CR, Zimmermann M, Agarkova I, Kelm JM, Krek W (2014) 3D cell culture systems modeling tumor growth determinants in cancer target discovery. Adv Drug Deliv Rev 69–70:29–41. https://doi.org/10.1016/j.addr.2014.03.001

    Article  CAS  PubMed  Google Scholar 

  11. Tanner K, Gottesman MM (2015) Beyond 3D culture models of cancer. Sci Transl Med 7(283):283–289. https://doi.org/10.1126/scitranslmed.3009367

    Article  Google Scholar 

  12. do Amaral JB, Rezende-Teixeira P, Freitas VM, Machado-Santelli GM (2011) MCF-7 cells as a three-dimensional model for the study of human breast cancer. Tissue Eng Part C Methods 17(11):1097–1107. https://doi.org/10.1089/ten.tec.2011.0260

    Article  CAS  PubMed  Google Scholar 

  13. Ma XH, Piao S, Wang D, McAfee QW, Nathanson KL, Lum JJ, Li LZ, Amaravadi RK (2011) Measurements of tumor cell autophagy predict invasiveness, resistance to chemotherapy, and survival in melanoma. Clin Cancer Res 17(10):3478–3489. https://doi.org/10.1158/1078-0432.CCR-10-2372

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Gomes LR, Vessoni AT, Menck CF (2015) Three-dimensional microenvironment confers enhanced sensitivity to doxorubicin by reducing p53-dependent induction of autophagy. Oncogene 34(42):5329–5340. https://doi.org/10.1038/onc.2014.461

    Article  CAS  PubMed  Google Scholar 

  15. Koehler BC, Jassowicz A, Scherr AL, Lorenz S, Radhakrishnan P, Kautz N, Elssner C, Weiss J, Jaeger D, Schneider M et al (2015) Pan-Bcl-2 inhibitor Obatoclax is a potent late stage autophagy inhibitor in colorectal cancer cells independent of canonical autophagy signaling. BMC Cancer 15:919. https://doi.org/10.1186/s12885-015-1929-y

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Bingel C, Koeneke E, Ridinger J, Bittmann A, Sill M, Peterziel H, Wrobel JK, Rettig I, Milde T, Fernekorn U et al (2017) Three-dimensional tumor cell growth stimulates autophagic flux and recapitulates chemotherapy resistance. Cell Death Dis 8(8):e3013. https://doi.org/10.1038/cddis.2017.398

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Russell RC, Yuan HX, Guan KL (2014) Autophagy regulation by nutrient signaling. Cell Res 24(1):42–57. https://doi.org/10.1038/cr.2013.166

    Article  CAS  PubMed  Google Scholar 

  18. Barbone D, Yang TM, Morgan JR, Gaudino G, Broaddus VC (2008) Mammalian target of rapamycin contributes to the acquired apoptotic resistance of human mesothelioma multicellular spheroids. J Biol Chem 283(19):13021–13030. https://doi.org/10.1074/jbc.M709698200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Follo C, Barbone D, Richards WG, Bueno R, Broaddus VC (2016) Autophagy initiation correlates with the autophagic flux in 3D models of mesothelioma and with patient outcome. Autophagy 12(7):1180–1194. https://doi.org/10.1080/15548627.2016.1173799

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, Acevedo Arozena A, Adachi H, Adams CM, Adams PD, Adeli K et al (2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12(1):1–222. https://doi.org/10.1080/15548627.2015.1100356

    Article  PubMed  PubMed Central  Google Scholar 

  21. Karanasios E, Stapleton E, Manifava M, Kaizuka T, Mizushima N, Walker SA, Ktistakis NT (2013) Dynamic association of the ULK1 complex with omegasomes during autophagy induction. J Cell Sci 126(Pt 22):5224–5238. https://doi.org/10.1242/jcs.132415

    Article  CAS  PubMed  Google Scholar 

  22. Barbone D, Ryan JA, Kolhatkar N, Chacko AD, Jablons DM, Sugarbaker DJ, Bueno R, Letai AG, Coussens LM, Fennell DA et al (2011) The Bcl-2 repertoire of mesothelioma spheroids underlies acquired apoptotic multicellular resistance. Cell Death Dis 2:e174. https://doi.org/10.1038/cddis.2011.58

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Xiang X, Phung Y, Feng M, Nagashima K, Zhang J, Broaddus VC, Hassan R, Fitzgerald D, Ho M (2011) The development and characterization of a human mesothelioma in vitro 3D model to investigate immunotoxin therapy. PLoS One 6(1):e14640. https://doi.org/10.1371/journal.pone.0014640

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Barbone D, Follo C, Echeverry N, Gerbaudo VH, Klabatsa A, Bueno R, Felley-Bosco E, Broaddus VC (2015) Autophagy correlates with the therapeutic responsiveness of malignant pleural mesothelioma in 3D models. PLoS One 10(8):e0134825. https://doi.org/10.1371/journal.pone.0134825

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Broaddus VC, Follo C, Barbone D. 3D models of mesothelioma in the study of mechanisms of cell survival. In Asbestos and mesothelioma. Testa JR,. Springer International Publishing, New York 2017:237–257

    Google Scholar 

  26. Folkman J, Moscona A (1978) Role of cell shape in growth control. Nature 273(5661):345–349

    Article  CAS  Google Scholar 

  27. Barth S, Glick D, Macleod KF (2010) Autophagy: assays and artifacts. J Pathol 221(2):117–124. https://doi.org/10.1002/path.2694

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. 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):1–23

    Article  CAS  Google Scholar 

  29. Kunz-Schughart LA, Freyer JP, Hofstaedter F, Ebner R (2004) The use of 3-D cultures for high-throughput screening: the multicellular spheroid model. J Biomol Screen 9(4):273–285. https://doi.org/10.1177/1087057104265040

    Article  CAS  PubMed  Google Scholar 

  30. Hirschhaeuser F, Menne H, Dittfeld C, West J, Mueller-Klieser W, Kunz-Schughart LA (2010) Multicellular tumor spheroids: an underestimated tool is catching up again. J Biotechnol 148(1):3–15. https://doi.org/10.1016/j.jbiotec.2010.01.012

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the Simmons Mesothelioma Foundation; CF was supported also by the Meso Foundation under grant 383573.

Author information

Authors and Affiliations

  1. Zuckerberg San Francisco General Hospital and Trauma Center, University of California San Francisco, San Francisco, CA, USA

    Carlo Follo, Dario Barbone & V. Courtney Broaddus

  2. Division of Thoracic Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    William G. Richards & Raphael Bueno

Authors
  1. Carlo Follo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dario Barbone
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. William G. Richards
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Raphael Bueno
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. V. Courtney Broaddus
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Carlo Follo .

Editor information

Editors and Affiliations

  1. Signalling Programme, Babraham Institute, Cambridge, UK

    Nicholas Ktistakis

  2. Signalling Programme, Babraham Institute, Cambridge, UK

    Oliver Florey

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Follo, C., Barbone, D., Richards, W.G., Bueno, R., Courtney Broaddus, V. (2019). Autophagy in 3D In Vitro and Ex Vivo Cancer Models. In: Ktistakis, N., Florey, O. (eds) Autophagy. Methods in Molecular Biology, vol 1880. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8873-0_31

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8873-0_31

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8872-3

  • Online ISBN: 978-1-4939-8873-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation