Skip to main content
SpringerLink
Log in

In vitro quantification of melanoma tumor cell invasion

  • Published:
Clinical & Experimental Metastasis Aims and scope Submit manuscript

Abstract

In order to quantify the invasiveness of melanoma tumor cellsin vitro, a modification of the amniotic basement membrane (BM) model, described by Liottaet al. (Cancer Letters,11, 141, 1980), was used in combination with radiolabeled tumor cells. B16-F10 metastatic murine melanoma cells and a derived clone (B16-F10L) were prelabeled with 0·1 μCi/ml of [14C]thymidine for 20–24 h in serum-free medium at 37°C. Following incubation, fetal bovine serum was added to a concentration of 5 per cent, and the cells were allowed to grow to confluency for the next 24–28 h. The labeled cells were seeded onto amniotic membranes situated in Membrane Invasion Culture System (MICS) chambers at a density of 2·5×104 per well. At various times points, radioactivity of tumor cells that completely traversed the membrane was determined using an under-the-membrane sampling method. The average percent invasion demonstrated by the B16-F10 line was 2·75 per cent, and 3·65 per cent exhibited by the B16-F10L cell line after 48–53 hin vitro. Since it was apparent that some variability in thickness existed among membrane samples, a morphological analysis was performed on five sectors of a three-inch-diameter sample from four different placentae. Differences and similarities in BM thickness within the same sector were noted by this technique and could possibly contribute to some variability observed in tumor cell invasion in this model. Another parameter examined was the proliferation of tumor cells in the upper and lower wells of the MICS chambers. By 48 h, approximately 32·1 per cent of the B16-F10 cell line as well as the clone had replicated in the upper wells associated with the BMs compared with a 32·9 per cent replication in the lower wells, which reaffirmed the viability of the tumor cells under experimental conditions and insured similarly replicating populations of cells. In order to quantify the invasiveness of radiolabeled tumor cells accurately through a biological membranous barrier, the proper concentration of cells must be used, tumor cell heterogeneity should be taken into consideration, the technique of sampling radiolabeled invasive cells should be critically analysed, and thickness of the membranous barrier should all be considered as possible important factors in the quantitative analyses.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Barsky, S. H., Siegal, G., Jannotta, F., andLiotta, L. A., 1983, Loss of basement membrane components by invasive tumors but not by their benign counterparts.Laboratory Investigation,49, 140–147.

    Google Scholar 

  2. Barsky, S. H., Togo, S., Garbisa, S., andLiotta, L. A., 1983, Type IV collagenase immunoreactivity in invasive breast carcinoma.Lancet,i, 296–297.

    Google Scholar 

  3. Blois, M. S., Sagebiel, R. W., Abarbanel, R. M., Caldwell, T. M., andTuttle, M. S., 1983, Malignant melanoma of the skin.Cancer,52, 1330–1341.

    Google Scholar 

  4. Burger, M. M., 1984, Experimental models for metastasis.Accomplishments in Cancer Research, edited by J. G. Fortner and J. E. Rhoads, (Philadelphia: Lippincott), pp. 174–182.

    Google Scholar 

  5. Fidler, I. J., 1973, Selection of successive tumor lines for metastasis.Nature New Biology,242, 148–149.

    Google Scholar 

  6. Fidler, I. J., Gersten, D. M., andHart, I. R., 1978, The biology of cancer invasion and metastasis.Advances in Cancer Research,28, 149–250.

    Google Scholar 

  7. Gehlsen, K. R., Wagner, H. N., Jr., andHendrix, M. J. C., 1984, Membrane invasion culture system (MICS).Medical Instrumentation,18, 268–271.

    Google Scholar 

  8. Gehlsen, K. R., Kischer, C. W., andHendrix, M. J. C., 1984, The utilization of neural crest cells as controls for melanoma tumor cell invasion through a basement membrane.Journal of Cell Biology,99, 335a.

    Google Scholar 

  9. Glassy, M. C., Handley, H. H., andRoyston, I., 1983, Human monoclonal antibodies to human cancers.Monoclonal Antibodies and Cancer, (New York: Academic Press), pp. 163–170.

    Google Scholar 

  10. Hamburger, A., andSalmon, S. E., 1977, Primary bioassay of human tumor stem cells.Science,197, 461–463.

    Google Scholar 

  11. Hart, I. R., andFidler, I. J., 1978, Anin vitro quantitative assay for tumor cell invasion.Cancer Research,38, 3218–3224.

    Google Scholar 

  12. Hendrix, M. J. C., 1984, A morphological characterization of melanoma tumor cell interaction with a basement membrane model in the absence and presence of an anticancer agent.Scanning Electron Microscopy,4, 1973–1982.

    Google Scholar 

  13. Hill, R. P., Chambers, A. F., andLing, V., 1984, Dynamic heterogeneity: Rapid generation of metastatic variants in mouse B16 melanoma cells.Science,224, 998–1001.

    Google Scholar 

  14. Ito, S., andKarnovsky, M. J., 1968, Formaldehyde-glutaraldehyde fixatives containing trinitro compounds.Journal of Cell Biology,39, 168a.

    Google Scholar 

  15. Lacovara, J., Cramer, E. B., andQuigley, J. P., 1984, Fibronectin enhancement of directed migration of B16 melanoma cells.Cancer Research,44, 1657–1663.

    Google Scholar 

  16. Liotta, L. A., Lee, C. W., andMorakis, D. J., 1980, New method for preparing large sufaces of intact human basement membrane for tumor invasion studies.Cancer Letters,11, 141–152.

    Google Scholar 

  17. Liotta, L. A., Thorgeirsson, U. P., andGarbisa, S., 1982, Role of collagenases in tumor cell invasion.Cancer Metastasis Reviews,1, 277–288.

    Google Scholar 

  18. Liotta, L. A., andHart, I. R., 1982,Invasion and Metastasis, (The Hague: Martinus Nijhoff).

    Google Scholar 

  19. Liotta, L. A. Rao, C. N., andBarsky, S. H., 1983, Tumor invasion and the extracellular matrix.Laboratory Investigation,49, 636–649.

    Google Scholar 

  20. Ludwig, R., Alberts, D. S., Miller, T. P., andSalmon, S. E., 1984, Evaluation of anti-cancer drug schedule dependency using anin vitro human tumor clonogenic assay.Cancer Chemotherapy and Pharmacology,12, 135–141.

    Google Scholar 

  21. Markus, G., Kohga, S., Camiolo, S. M., Madeja, J. M., Ambrus, J. L., andKrakousis, C., 1984, Plasminogen activators in human malignant melanoma.Journal of the National Cancer Institute,72, 1213–1222.

    Google Scholar 

  22. McCarthy, J. B., andFurcht, L. T., 1984, Laminin and fibronectin promote the haptotactic migration of B16 mouse melanoma cellsin vitro.Journal of Cell Biology,98, 1474–1480.

    Google Scholar 

  23. Meyn, R. E., Hewitt, R. R., andHumphrey, R. M., 1973, Evaluation of S phase synchronization by analysis of DNA replication in 5-bromodeoxyuridine.Experimental Cell Research,82, 137–142.

    Google Scholar 

  24. Meyskens, F. L., Jr., Loescher, L., Moon, T. E., Takasugi, B., andSalmon, S. E., 1984, Relation ofin vitro colony survival to clinical response in a prospective trial of single-agent chemotherapy for metastatic melanoma.Journal of Clinical Oncology,2, 1223–1228.

    Google Scholar 

  25. Nicolson, G. L., 1984, Tumor progression, oncogenes and the evolution of metastatic phenotypic diversity.Clinical and Experimental Metastasis,2, 85–105.

    Google Scholar 

  26. Nicolson, G. L., Brunson, K. W., andFidler, I. J., 1978, Specifically of arrest, survival, and growth of selected metastatic variant cell lines.Cancer Research,38, 4105–4111.

    Google Scholar 

  27. Parton, J. W.,Merriman, R. L., andTanzer, L. R., 1984, Anin vitro assay to quantitatively measure tumor cell invasion. FASCEB, 601a.

  28. Pauli, B. U., Schwartz, D. E., Thonar, E. J. M., andKuettner, K. E., 1983, Tumor invasion and host extracellular matrix.Cancer Metastasis Reviews,2, 129–152.

    Google Scholar 

  29. Persky, V., Guzzino, K., Ostrowski, L., andSchultz, R. M., 1984, Inter-placental invasion rates for B16 F10 murine melanoma cells in the human amnion basement membrane model.Journal of Cell Biology,99, 335a.

    Google Scholar 

  30. Poste, G., 1982, Variation in the metastatic properties of cells from malignant tumors and the control of the metastatic phenotype.Tumour Progression and Markers, Proceedings of the Sixth Meeting of the European Association for Cancer Research (Amsterdam: Kugler Publications), pp. 197–210.

    Google Scholar 

  31. Poste, G., Doll, J., Brown, A. E., Tzeng, J., andZeidman, I., 1982, Comparison of the metastatic properties of B16 melanoma clones isolated from cultured cell lines, subcutaneous tumors, and individual lung metastases.Cancer Research,42, 2770–2778.

    Google Scholar 

  32. Poste, G., Doll, J., Hart, I. R., andFidler, I. J., 1980,In vitro selection of murine B16 melanoma variants with enhanced tissue-invasive properties.Cancer Research,40, 1636–1644.

    Google Scholar 

  33. Poste, G., andFidler, I. J., 1980, The pathogenesis of cancer metastasis.Nature,283, 139–146.

    Google Scholar 

  34. Russo, R. G., Liotta, L. A., Thorgeirsson, U., Brundage, R., andSchiffmann, E., 1981, Polymorphonuclear leukocyte migration through human amnion membrane.Journal of Cell Biology,91, 459–467.

    Google Scholar 

  35. Salmon, S. E., Hamburger, A. W., Soehnlen, B. J., Durie, B. G. M., Alberts, D. V., andMoon, T. E., 1978, Quantitation of differential sensitivity of human tumor stem cells to anticancer drugs.New England Journal of Medicine,298, 1321–1327.

    Google Scholar 

  36. Salo, T., Liotta, L. A., Keski-Oja, J., Turpeenniemi-Hujanen, T., andTryggvason, K., 1982, Secretion of basement membrane collagen degrading enzyme and plasminogen activator by transformed cells—role in metastasis.International Journal of Cancer,30, 669–673.

    Google Scholar 

  37. Schleich, A., Tchao, R., Frick, M., andMayer, A., 1981, Interaction of human carcinoma cells with an epithelial layer and the underlying basement membrane. A new model.Archiv fur Geschwulstforschung,51, 40–44.

    Google Scholar 

  38. Sheridan, J. W., andSimmons, R. J., 1981, Studies on a human melanoma cell line: Effect of cell crowding and nutrient depletion on the biophysical and kinetic characteristic of the cells.Journal of Cell Physiology,107, 85–100.

    Google Scholar 

  39. Snedecor, G. W., andCochran, W. G., 1967,Statistical Methods (Ames, Iowa: Iowa State University Press), pp. 135–198.

    Google Scholar 

  40. Tchao, R., Schleich, A. B., Frick, M., andMayer, A., 1980, A newin vitro model to study tumour cell invasion.Metastasis, Clinical and Experimental Aspects, edited by K. Hellmann, P. Hilgard and S. Eccles (The Hague: Martinus Nijhoff), pp. 28–32.

    Google Scholar 

  41. Thorgeirsson, U. P., Turpeenniemi-Hujanen, T., Neckers, L. M., Johnson, D. W., andLiotta, L. A., 1984, Protein synthesis but not DNA synthesis is required for tumor cells invasionin vitro.Invasion and Metastasis,4, 73–83.

    Google Scholar 

  42. Tveit, K. M., Fodstead, O., andPihl, A., 1981, The usefulness of human tumor cell lines in the study of chemosensitivity. A study of malignant melanomas.International Journal of Cancer,28, 403–408.

    Google Scholar 

  43. Weiss, L., andWard, P. M., 1983, Cell detachment and metastasis.Cancer Metastasis Reviews,2, 111–127.

    Google Scholar 

  44. Woolley, D. E., andGrafton, C. A., 1980, Collagenase immunolocalization studies of cutaneous secondary melanomas.British Journal of Cancer,42, 260–265.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Anatomy, College of Medicine, University of Arizona, 85724, Tucson, AZ, USA

    Mary J. C. Hendrix, Kurt R. Gehlsen & Herbert N. Wagner Jr

  2. Department of Internal Medicine, University of Arizona, 85724, Tucson, AZ, USA

    Frank L. Meyskens Jr

  3. Arizona Cancer Center, 85724, Tucson, AZ, USA

    Steven R. Rodney & Frank L. Meyskens Jr

  4. Department of Surgery, College of Medicine, 85724, Tucson, AZ, USA

    Ronald L. Misiorowski

Authors
  1. Mary J. C. Hendrix
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kurt R. Gehlsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Herbert N. Wagner Jr
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Steven R. Rodney
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ronald L. Misiorowski
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Frank L. Meyskens Jr
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hendrix, M.J.C., Gehlsen, K.R., Wagner, H.N. et al. In vitro quantification of melanoma tumor cell invasion. Clin Exp Metast 3, 221–233 (1985). https://doi.org/10.1007/BF01585078

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01585078

Keywords

Search

Navigation