Skip to main content
SpringerLink
Log in

Protocols for Migration and Invasion Studies in Prostate Cancer

  • Protocol
  • First Online:
Prostate Cancer

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1786))

Abstract

Prostate cancer is the most common malignancy diagnosed in men in the western world. The development of distant metastases and therapy resistance are major clinical problems in the management of prostate cancer patients. In order for prostate cancer to metastasize to distant sites in the human body, prostate cancer cells have to migrate and invade neighboring tissue. Cancer cells can acquire a migratory and invasive phenotype in several ways, including single cell and collective migration. As a requisite for migration, epithelial prostate cancer cells often need to acquire a motile, mesenchymal-like phenotype. This way prostate cancer cells often lose polarity and epithelial characteristics (e.g., expression of E-cadherin homotypic adhesion receptor), and acquire mesenchymal phenotype (for example, cytoskeletal rearrangements, enhanced expression of proteolytic enzymes and other repertory of integrins). This process is referred to as epithelial-to-mesenchymal transition (EMT). Cellular invasion, one of the hallmarks of cancer, is characterized by the movement of cells through a three-dimensional matrix, resulting in remodeling of the cellular environment. Cellular invasion requires adhesion, proteolysis of the extracellular matrix, and migration of cells. Studying the migratory and invasive ability of cells in vitro represents a useful tool to assess the aggressiveness of solid cancers, including those of the prostate.

This chapter provides a comprehensive description of the Transwell migration assay, a commonly used technique to investigate the migratory behavior of prostate cancer cells in vitro. Furthermore, we will provide an overview of the adaptations to the Transwell migration protocol to study the invasive capacity of prostate cancer cells, i.e., the Transwell invasion assay. Finally, we will present a detailed description of the procedures required to stain the Transwell filter inserts and quantify the migration and/or invasion.

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 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 179.99
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. Siegel RL, Miller KD, Jemal A (2015) Cancer statistics, 2015. CA Cancer J Clin 65(1):5–29. https://doi.org/10.3322/caac.21254

    Article  PubMed  Google Scholar 

  2. Bastian PJ, Carter BH, Bjartell A, Seitz M, Stanislaus P, Montorsi F, Stief CG, Schroder F (2009) Insignificant prostate cancer and active surveillance: from definition to clinical implications. Eur Urol 55(6):1321–1330. https://doi.org/10.1016/j.eururo.2009.02.028

    Article  PubMed  Google Scholar 

  3. Pienta KJ, Bradley D (2006) Mechanisms underlying the development of androgen-independent prostate cancer. Clin Cancer Res 12(6):1665–1671. https://doi.org/10.1158/1078-0432.ccr-06-0067

    Article  PubMed  CAS  Google Scholar 

  4. Harris WP, Mostaghel EA, Nelson PS, Montgomery B (2009) Androgen deprivation therapy: progress in understanding mechanisms of resistance and optimizing androgen depletion. Nat Clin Pract Urol 6(2):76–85. https://doi.org/10.1038/ncpuro1296

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Friedl P, Gilmour D (2009) Collective cell migration in morphogenesis, regeneration and cancer. Nat Rev Mol Cell Biol 10(7):445–457. https://doi.org/10.1038/nrm2720. nrm2720 [pii]

    Article  PubMed  CAS  Google Scholar 

  6. Larue L, Bellacosa A (2005) Epithelial-mesenchymal transition in development and cancer: role of phosphatidylinositol 3′ kinase/AKT pathways. Oncogene 24(50):7443–7454. https://doi.org/10.1038/sj.onc.1209091

    Article  PubMed  CAS  Google Scholar 

  7. Nakaya Y, Sheng G (2013) EMT in developmental morphogenesis. Cancer Lett 341(1):9–15. https://doi.org/10.1016/j.canlet.2013.02.037

    Article  PubMed  CAS  Google Scholar 

  8. Kalluri R, Neilson EG (2003) Epithelial-mesenchymal transition and its implications for fibrosis. J Clin Invest 112(12):1776–1784. https://doi.org/10.1172/jci20530

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. van der Pluijm G (2011) Epithelial plasticity, cancer stem cells and bone metastasis formation. Bone 48(1):37–43. https://doi.org/10.1016/j.bone.2010.07.023. S8756-3282(10)01367-0 [pii]

    Article  PubMed  CAS  Google Scholar 

  10. Hugo H, Ackland ML, Blick T, Lawrence MG, Clements JA, Williams ED, Thompson EW (2007) Epithelial–mesenchymal and mesenchymal–epithelial transitions in carcinoma progression. J Cell Physiol 213(2):374–383. https://doi.org/10.1002/jcp.21223

    Article  PubMed  CAS  Google Scholar 

  11. Williams LV, Veliceasa D, Vinokour E, Volpert OV (2013) miR-200b inhibits prostate cancer EMT, growth and metastasis. PLoS One 8(12):e83991. https://doi.org/10.1371/journal.pone.0083991

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Kong D, Li Y, Wang Z, Banerjee S, Ahmad A, Kim HR, Sarkar FH (2009) miR-200 regulates PDGF-D-mediated epithelial-mesenchymal transition, adhesion, and invasion of prostate cancer cells. Stem Cells (Dayton, Ohio) 27(8):1712–1721. https://doi.org/10.1002/stem.101

    Article  CAS  Google Scholar 

  13. Li P, Yang R, Gao WQ (2014) Contributions of epithelial-mesenchymal transition and cancer stem cells to the development of castration resistance of prostate cancer. Mol Cancer 13:55. https://doi.org/10.1186/1476-4598-13-55

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Nauseef JT, Henry MD (2011) Epithelial-to-mesenchymal transition in prostate cancer: paradigm or puzzle? Nat Rev Urol 8(8):428–439. https://doi.org/10.1038/nrurol.2011.85

    Article  PubMed  PubMed Central  Google Scholar 

  15. Haeger A, Wolf K, Zegers MM, Friedl P (2015) Collective cell migration: guidance principles and hierarchies. Trends Cell Biol 25(9):556–566. https://doi.org/10.1016/j.tcb.2015.06.003

    Article  PubMed  Google Scholar 

  16. Das T, Safferling K, Rausch S, Grabe N, Boehm H, Spatz JP (2015) A molecular mechanotransduction pathway regulates collective migration of epithelial cells. Nat Cell Biol 17(3):276–287. https://doi.org/10.1038/ncb3115

    Article  PubMed  CAS  Google Scholar 

  17. Ilina O, Friedl P (2009) Mechanisms of collective cell migration at a glance. J Cell Sci 122(Pt 18):203–3208. https://doi.org/10.1242/jcs.036525

    Article  CAS  Google Scholar 

  18. Kramer N, Walzl A, Unger C, Rosner M, Krupitza G, Hengstschlager M, Dolznig H (2013) In vitro cell migration and invasion assays. Mutat Res 752(1):10–24. https://doi.org/10.1016/j.mrrev.2012.08.001

    Article  PubMed  CAS  Google Scholar 

  19. Keller R (2005) Cell migration during gastrulation. Curr Opin Cell Biol 17(5):533–541. https://doi.org/10.1016/j.ceb.2005.08.006

    Article  PubMed  CAS  Google Scholar 

  20. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144(5):646–674. https://doi.org/10.1016/j.cell.2011.02.013

    Article  PubMed  CAS  Google Scholar 

  21. Justus CR, Leffler N, Ruiz-Echevarria M, Yang LV (2014) In vitro cell migration and invasion assays. J Vis Exp (88). https://doi.org/10.3791/51046

  22. Yu D, Zhong Y, Li X et al (2015) ILs-3, 6 and 11 increase, but ILs-10 and 24 decrease stemness of human prostate cancer cells in vitro. Oncotarget 6(40):42687–42703. https://doi.org/10.18632/oncotarget.5883

    Article  PubMed  PubMed Central  Google Scholar 

  23. Wang Z, Gao Y, Liu Y, Chen J, Wang J, Gan S, Xu D, Cui X (2015) Tectonic1 contributes to the growth and migration of prostate cancer cells in vitro. Int J Mol Med 36(4):931–938. https://doi.org/10.3892/ijmm.2015.2313

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Banerjee S, Li G, Li Y, Gaughan C, Baskar D, Parker Y, Lindner DJ, Weiss SR, Silverman RH (2015) RNase L is a negative regulator of cell migration. Oncotarget 6(42):44360–44372. https://doi.org/10.18632/oncotarget.6246

    Article  PubMed  PubMed Central  Google Scholar 

  25. Guo W, Keener AL, Jing Y, Cai L, Ai J, Zhang J, Fisher AL, Fu G, Wang Z (2015) FOXA1 modulates EAF2 regulation of AR transcriptional activity, cell proliferation, and migration in prostate cancer cells. Prostate 75(9):976–987. https://doi.org/10.1002/pros.22982

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Zoni E, van der Horst G, van de Merbel AF et al (2015) miR-25 modulates invasiveness and dissemination of human prostate cancer cells via regulation of alphav- and alpha6-integrin expression. Cancer Res 75(11):2326–2336. https://doi.org/10.1158/0008-5472.can-14-2155

    Article  PubMed  CAS  Google Scholar 

  27. Kroon J, in’t Veld LS, Buijs JT, Cheung H, van der Horst G, van der Pluijm G (2014) Glycogen synthase kinase-3beta inhibition depletes the population of prostate cancer stem/progenitor-like cells and attenuates metastatic growth. Oncotarget 5(19):8986–8994. https://doi.org/10.18632/oncotarget.1510

    Article  PubMed  Google Scholar 

  28. van den Hoogen C, van der Horst G, Cheung H et al (2010) High aldehyde dehydrogenase activity identifies tumor-initiating and metastasis-initiating cells in human prostate cancer. Cancer Res 70(12):5163–5173. https://doi.org/10.1158/0008-5472.can-09-3806

    Article  PubMed  Google Scholar 

  29. Gillies RJ, Didier N, Denton M (1986) Determination of cell number in monolayer cultures. Anal Biochem 159(1):109–113

    Article  CAS  PubMed  Google Scholar 

  30. Chiba K, Kawakami K, Tohyama K (1998) Simultaneous evaluation of cell viability by neutral red, MTT and crystal violet staining assays of the same cells. Toxicol In Vitro 12(3):251–258

    Article  CAS  PubMed  Google Scholar 

  31. Sun C, Zhao X, Xu K, Gong J, Liu W, Ding W, Gou Y, Xia G, Ding Q (2011) Periostin: a promising target of therapeutical intervention for prostate cancer. J Transl Med 9:99. https://doi.org/10.1186/1479-5876-9-99

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Rodriguez LG, Wu X, Guan JL (2005) Wound-healing assay. Methods Mol Biol (Clifton, NJ) 294:23–29

    Google Scholar 

  33. Liang CC, Park AY, Guan JL (2007) In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2(2):329–333. https://doi.org/10.1038/nprot.2007.30

    Article  PubMed  CAS  Google Scholar 

  34. Menon MB, Ronkina N, Schwermann J, Kotlyarov A, Gaestel M (2009) Fluorescence-based quantitative scratch wound healing assay demonstrating the role of MAPKAPK-2/3 in fibroblast migration. Cell Motil Cytoskeleton 66(12):1041–1047. https://doi.org/10.1002/cm.20418

    Article  PubMed  CAS  Google Scholar 

  35. Yarrow JC, Perlman ZE, Westwood NJ, Mitchison TJ (2004) A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods. BMC Biotechnol 4:21. https://doi.org/10.1186/1472-6750-4-21

    Article  PubMed  PubMed Central  Google Scholar 

  36. Marshall J (2011) Transwell((R)) invasion assays. Methods Mol Biol (Clifton, NJ) 769:97–110. https://doi.org/10.1007/978-1-61779-207-6_8

    Article  CAS  Google Scholar 

  37. Pawar SC, Demetriou MC, Nagle RB, Bowden GT, Cress AE (2007) Integrin alpha6 cleavage: a novel modification to modulate cell migration. Exp Cell Res 313(6):1080–1089. https://doi.org/10.1016/j.yexcr.2007.01.006

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Gang X, Wang G, Huang H (2015) Androgens regulate SMAD ubiquitination regulatory factor-1 expression and prostate cancer cell invasion. Prostate 75(6):561–572. https://doi.org/10.1002/pros.22935

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Albini A, Benelli R (2007) The chemoinvasion assay: a method to assess tumor and endothelial cell invasion and its modulation. Nat Protoc 2(3):504–511. https://doi.org/10.1038/nprot.2006.466

    Article  PubMed  CAS  Google Scholar 

  40. Albini A, Benelli R, Noonan DM, Brigati C (2004) The “chemoinvasion assay”: a tool to study tumor and endothelial cell invasion of basement membranes. Int J Dev Biol 48(5-6):563–571. https://doi.org/10.1387/ijdb.041822aa

    Article  PubMed  CAS  Google Scholar 

  41. Wu HC, Hsieh JT, Gleave ME, Brown NM, Pathak S, Chung LW (1994) Derivation of androgen-independent human LNCaP prostatic cancer cell sublines: role of bone stromal cells. Int J Cancer J 57(3):406–412

    Article  CAS  Google Scholar 

  42. Wu TT, Sikes RA, Cui Q et al (1998) Establishing human prostate cancer cell xenografts in bone: induction of osteoblastic reaction by prostate-specific antigen-producing tumors in athymic and SCID/bg mice using LNCaP and lineage-derived metastatic sublines. Int J Cancer 77(6):887–894

    Article  CAS  PubMed  Google Scholar 

  43. Thalmann GN, Anezinis PE, Chang SM, Zhau HE, Kim EE, Hopwood VL, Pathak S, von Eschenbach AC, Chung LW (1994) Androgen-independent cancer progression and bone metastasis in the LNCaP model of human prostate cancer. Cancer Res 54(10):2577–2581

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Urology, Leiden University Medical Center, Leiden, The Netherlands

    Arjanneke F. van de Merbel, Geertje van der Horst, Jeroen T. Buijs & Gabri van der Pluijm

Authors
  1. Arjanneke F. van de Merbel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Geertje van der Horst
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jeroen T. Buijs
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gabri van der Pluijm
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gabri van der Pluijm .

Editor information

Editors and Affiliations

  1. Department of Urology, Innsbruch Medical University, Innsbruck, Austria

    Zoran Culig

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

van de Merbel, A.F., van der Horst, G., Buijs, J.T., van der Pluijm, G. (2018). Protocols for Migration and Invasion Studies in Prostate Cancer. In: Culig, Z. (eds) Prostate Cancer. Methods in Molecular Biology, vol 1786. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7845-8_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7845-8_4

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7843-4

  • Online ISBN: 978-1-4939-7845-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation