Invasion Assays

Hynda K. Kleinman1, Karin Jacob1

1 National Institute of Dental Research/NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 12.2
DOI:  10.1002/0471143030.cb1202s00
Online Posting Date:  May, 2001
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Abstract

Basement membrane is a thin extracellular matrix that underlies epithelia and endothelia and separates them from the stroma. Tumor cells must cross this membrane to invade stroma and establish distant metastases. They do this by producing proteases that degrade the matrix. Of several in vitro models, those using Matrigel are the most reliable, reproducible, and representative of in vivo invasion. In the assay presented here, tumor cells are placed in the upper chamber of Boyden migration chamber. The upper and lower chambers are separated by a porous membrane coated with Matrigel. A chemoattractant in the lower well stimulates migration. After an interval, tumor cells are recovered from or counted on the lower surface of the membrane.

     
 
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Table of Contents

  • Basic Protocol 1: Measuring Invasion Through a Matrix
  • Support Protocol 1: Preparation of Matrigel‐Coated Filters
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measuring Invasion Through a Matrix

  Materials
  • 3T3‐conditioned medium (see recipe) or selected chemoattractant or inhibitor
  • Tumor cells in culture
  • 0.5 M EDTA, pH 8.0 ( appendix 2A)
  • Culture medium containing 0.1% (w/v) recipeBSA (varies by cell type)
  • Diff‐Quik fixative and stains (Baxter)
  • Boyden chambers (Neuro Probe; available as single or 48‐well chambers)
  • Polyvinylpyrrolidone (PVP)‐free polycarbonate membranes, 8‐ or 12‐µm pore size (e.g., Nucleopore filters, Neuro Probe), sized to fit Boyden chambers (13‐mm for single chambers and 25 × 80–mm for 48‐well chambers) coated with Matrigel (see protocol 2Support Protocol)

Support Protocol 1: Preparation of Matrigel‐Coated Filters

  Materials
  • PVP‐free polycarbonate membranes, 8‐ or 12‐µm pore size (e.g., Nucleopore filters, Neuro Probe), sized to fit Boyden chambers (13‐mm for single chambers; 25 × 80–mm for 48‐well chambers)
  • Matrigel (unit 10.2; Matrigel can also be obtained commercially from Becton Dickinson Labware or Sigma)
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Figures

Videos

Literature Cited

Literature Cited
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   Chambers, S.K., Wang, Y., Gertz, R.E., and Kacinski, B.M. 1995. Macrophage colony stimulating factor mediates invasion of ovarian cancer cells through urokinase. Cancer Res. 55:1578‐1585.
   Hasegawa, Y., Takada, M., Yamamoto, M., and Saitoh, Y. 1994. The gradient of basic fibroblast growth factor concentration in human pancreatic cancer cell invasion. Biochem. Biophys. Res. Commun. 200:1435‐1439.
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   Koochepour, S., Merzak, A., and Pilkington, G.J. 1995. Extracellular matrix proteins inhibit proliferation, upregulate migration and induce morphological changes in human glioma cell lines. Eur. J. Cancer 31A:375‐380.
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   Nicolson, G.L., Nakajima, M., Herrmann, J.L., Menter, D.G., Cavanaugh, P.G., Park, J.S., and Marchetti, D. 1993. Malignant melanoma metastasizes to brain: Role of degradative enzymes and responses to paracrine growth factors. J. Neurooncol. 18:139‐149.
   Reich, R., Thompson, E.W., Iwamoto, Y., Martin, G.R., Deason, J.R., Fuller, G.C., and Miskin, R. 1988. Effects of inhibitor of plasminogen activator, serine proteases and collagenase IV on invasion of basement membrane by metastatic cells. Cancer Res. 48:3307‐3312.
   Rong, S., Segal, S., Avner, M., Ressau, J.H., and Van der Woude, G.F. 1994. Invasiveness and metastasis of NIH 3T3 cells induced by Met‐hepatocyte growth factor/scatter factor autocrine stimulation. Proc. Natl. Acad. Sci. U.S.A. 91:4731‐4735.
   Stahl, A. and Meuller, B.M. 1994. Binding of urokinase to its receptor promotes migration and invasion of human melanoma cells in vitro. Cancer Res. 54:3066‐3071.
   Sunitha, I., Meighen, D.L., Hartman, D.P., Thompson, E.W., Byers, S.W., and Avigan, M.I. 1994. Hepatocyte growth factor stimulates invasion across reconstituted basement membrane by a new human small intestinal cell line. Clin. Exp. Metastasis 12:1143‐1154.
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