Wnt Signaling pp 243-253 | Cite as

Assaying Wnt5A-Mediated Invasion in Melanoma Cells

  • Michael P. O'Connell
  • Amanda D. French
  • Poloko D. Leotlela
  • Ashani T. Weeraratna
Part of the Methods in Molecular Biology™ book series (MIMB, volume 468)


Wnt5A has been implicated in melanoma metastasis, and the progression of other cancers including pancreatic, gastric, prostate, and lung cancers. Assays to test motility and invasion include both in vivo assays and in vitro assays. The in vivo assays include the use of tail vein or footpad injections of metastatic cells, and are often laborious and expensive. In vitro invasion assays provide quick readouts that can help to establish conditions that either activate or inhibit melanoma cell motility, and to assess whether the conditions in question are worth translating into an in vivo model. Here we describe two standard methods for assaying motility and invasion in vitro including wound healing assays and Matrigel invasion assays (Boyden chamber assays). In addition, we and several other laboratories have previously shown that melanoma cells require matrix metalloproteinase (MMP)-2 for their invasion, and have recently shown that Wnt5A treatment can increase the levels of this enzyme in melanoma cells, as demonstrated by gelatin zymography. The use of these techniques can help to assess the migratory capacity of melanoma cells in response to Wnt treatment.

Key words

Melanoma Wnt5A Matrigel Boyden chamber Invasion Wound-healing assays 



We thank Dr. Michel Bernier and Dr. Paritosh Ghosh for helpful comments on this manuscript. Any data represented in this chapter was generated with the support of funds from the Intramural Research Program of the National Institute on Aging, Baltimore, MD.


  1. 1.
    Fidler, I. J. (2002) Critical determinants of metastasis. Semin Cancer Biol 12, 89–96.CrossRefPubMedGoogle Scholar
  2. 2.
    Bourguignon, L. Y. (2001) CD44-medi-ated oncogenic signaling and cytoskeleton activation during mammary tumor progression. J Mammary Gland Biol Neoplasia 6, 287–297.CrossRefPubMedGoogle Scholar
  3. 3.
    Agnantis, N. J., Goussia, A. C., Batistatou, A., and Stefanou, D. (2004) Tumor markers in cancer patients. An update of their prognostic significance. Part II. In Vivo 18, 481–48.PubMedGoogle Scholar
  4. 4.
    Steeg, P. S., Ouatas, T., Halverson, D., Palmieri, D., and Salerno, M. (2003) Metastasis suppressor genes: basic biology and potential clinical use. Clin Breast Cancer 4, 51–62.CrossRefPubMedGoogle Scholar
  5. 5.
    Hofmann, U. B., Westphal, J. R., Van Muijen, G. N., and Ruiter, D. J. (2000) Matrix metalloproteinases in human melanoma. J Invest Dermatol 115, 337–344.CrossRefPubMedGoogle Scholar
  6. 6.
    Hofmann, U. B., Houben, R., Brocker, E. B., and Becker, J. C. (2005) Role of matrix metalloproteinases in melanoma cell invasion. Biochimie 87, 307–314.CrossRefPubMedGoogle Scholar
  7. 7.
    Dissanayake, S. K., Wade, M., Johnson, C. E., O'Connell, M. P., Leotlela, P. D., French A. D., et al. (2007) The Wnt5a/Pkc pathway mediates motility in melanoma cells via the inhibition of metastasis suppressors, and initiation of an epithelial to mesenchymal transition. J Biol Chem 282, 17259–17271.CrossRefPubMedGoogle Scholar
  8. 8.
    Lewis, T. B., Robison, J. E., Bastien, R., Milash, B., Boucher, K., Samlowski, W. E., et al. (2005) Molecular classification of melanoma using real-time quantitative reverse transcriptase-polymerase chain reaction. Cancer 104, 1678–1686.CrossRefPubMedGoogle Scholar
  9. 9.
    Bachmann, I. M., Straume, O., Puntervoll, H. E., Kalvenes, M. B., and Akslen, L. A. (2005) Importance of P-cadherin, beta-catenin, and Wnt5a/frizzled for progression of melanocytic tumors and prognosis in cutaneous melanoma. Clin Cancer Res 11, 8606–8614.CrossRefPubMedGoogle Scholar
  10. 10.
    Rodolfo, M., Daniotti, M., and Vallacchi, V. (2004) Genetic progression of metastatic melanoma. Cancer Lett 214, 133–147.CrossRefPubMedGoogle Scholar
  11. 11.
    Ripka, S., Konig, A., Buchholz, M., Wagner, M., Sipos, B., Kloppel, G., et al. (2007) WNT5A—target of CUTL1 and potent modulator of tumor cell migration and invasion in pancreatic cancer. Carcinogenesis 28, 1178–1187.CrossRefPubMedGoogle Scholar
  12. 12.
    Kurayoshi, M., Oue, N., Yamamoto, H., Kishida, M., Inoue, A., Asahara, T., et al. (2006) Expression of Wnt-5a is correlated with aggressiveness of gastric cancer by stimulating cell migration and invasion. Cancer Res 66, 10439–10448.CrossRefPubMedGoogle Scholar
  13. 13.
    Stanbrough, M., Bubley, G. J., Ross, K., Golub, T. R., Rubin, M. A., Penning, T. M., et al. (2006) Increased expression of genes converting adrenal androgens to testosterone in androgen-independent prostate cancer. Cancer Res 66, 2815–2825.CrossRefPubMedGoogle Scholar
  14. 14.
    Huang, C. L., Liu, D., Nakano, J., Ishikawa, S., Kontani, K., Yokomise, H., et al. (2005) Wnt5a expression is associated with the tumor proliferation and the stro-mal vascular endothelial growth factor—an expression in non-small-cell lung cancer. J Clin Oncol 23, 8765–8773.CrossRefPubMedGoogle Scholar
  15. 15.
    Dejmek, J., Dejmek, A., Safholm, A., Sjolander, A., and Andersson, T. (2005) Wnt-5a protein expression in primary dukes B colon cancers identifies a subgroup of patients with good prognosis. Cancer Res 65, 9142–9146.CrossRefPubMedGoogle Scholar
  16. 16.
    Jonsson, M., Dejmek, J., Bendahl, P. O., and Andersson, T. (2002) Loss of Wnt-5a protein is associated with early relapse in invasive ductal breast carcinomas. Cancer Res 62, 409–416.PubMedGoogle Scholar
  17. 17.
    Bartolome, R. A., Molina-Ortiz, I., Samaniego, R., Sanchez-Mateos, P., Bustelo, X. R., and Teixido, J. (2006) Activation of Vav/Rho GTPase signaling by CXCL12 controls membrane-type matrix metalloproteinase-dependent melanoma cell invasion.Cancer Res 66, 248–258.CrossRefPubMedGoogle Scholar
  18. 18.
    Leotlela, P. D., Wade, M. S., Duray, P. H., Rhode, M. J., Brown, H. F., Rosenthal, D. T., et al. (2006) Claudin-1 overexpression in melanoma is regulated by PKC and contributes to melanoma cell motility. Oncogene 26, 3846–3856.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Michael P. O'Connell
    • 1
  • Amanda D. French
    • 1
  • Poloko D. Leotlela
    • 1
  • Ashani T. Weeraratna
    • 1
  1. 1.Laboratory of Immunology, National Institutes of Health, National Institute on AgingGerontology Research CenterBaltimoreUSA

Personalised recommendations