In Vitro Invasion Assays: Phagocytosis of the Extracellular Matrix

Emma T. Bowden1, Susette Mueller1, Peter J. Coopman2

1 Georgetown University Medical Center, Washington, D.C., 2 Montpellier University II, Montpellier, France
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.13
DOI:  10.1002/0471142956.cy0913s12
Online Posting Date:  May, 2001
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Abstract

Degradation of the extracellular matrix is an essential component of phagocytosis by tumor cells and can be correlated with their invasive capacity. This unit presents flow cytometry based assays for rapid quantitative assessment of both proteolysis and internalization or internalization alone. These assays provide high‐throughput, low‐cost methods to compare cell lines and test the efficacy of treatments designed to stimulate or inhibit invasion.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of the Phagocytic Activity of Cancer Cells on a Cross‐linked FITC‐Conjugated Gelatin Matrix
  • Alternate Protocol 1: Measurement of Phagocytic Activity of Cancer Cells on Cross‐Linked FITC‐Conjugated Gelatin Beads
  • Support Protocol 1: Preparation of FITC‐Conjugated Gelatin Matrix
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measurement of the Phagocytic Activity of Cancer Cells on a Cross‐linked FITC‐Conjugated Gelatin Matrix

  Materials
  • FITC‐conjugated gelatin (see protocol 3)
  • Unlabeled gelatin/sucrose solution (see recipe)
  • 0.5% (v/v) glutaraldehyde/PBS solution (see recipe)
  • PBS ( appendix 2A): ice cold, nonsterile, as well as room temperature, sterile
  • 70% (v/v) ethanol, ice cold
  • Serum‐free cell growth medium with antibiotics: Improved minimal essential medium (IMEM; Life Technologies) with 1× penicillin/streptomycin (from 100× stock; Life Technologies)
  • Cells of interest
  • Trypsin/EDTA solution (Life Technologies)
  • Complete cell growth medium: IMEM with 10% (v/v) heat‐inactivated FBS ( appendix 2A)
  • 3.0% (v/v) formaldehyde/PBS solution (see recipe)
  • 24‐well tissue culture dishes, prechilled to 4°C
  • Filter‐top cell strainer tubes (Falcon)
  • Flow cytometer equipped with 488‐nm argon‐laser excitation and 545‐nm (530 ± 30–nm) band‐pass emission filter

Alternate Protocol 1: Measurement of Phagocytic Activity of Cancer Cells on Cross‐Linked FITC‐Conjugated Gelatin Beads

  • Gelatin type A from porcine skin, 300 bloom (50 to 100 kDa; Sigma)
  • Water‐saturated 1‐butanol (see recipe)
  • 5% (v/v) glutaraldehyde/PBS solution (see recipe)
  • 50°C water bath
  • Kontes Micro Ultrasonic Cell Disruptor (probe type)
  • 12‐well tissue culture dishes

Support Protocol 1: Preparation of FITC‐Conjugated Gelatin Matrix

  Materials
  • Low‐salt conjugation buffer (see recipe)
  • Fluorescein 5‐isothiocyanate (FITC) isomer I (Sigma)
  • Gelatin type A from porcine skin, 300 bloom (50 to 100 kDa; Sigma)
  • High‐salt conjugation buffer (see recipe)
  • PBS ( appendix 2A), nonsterile, 37°C
  • Sucrose
  • Dialysis tubing (MWCO 6000 to 8000; volume/length = 3.3 ml/cm)
  • Spectrophotometer (e.g., Beckman DU‐65)
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Figures

Videos

Literature Cited

Literature Cited
   Bjerknes, R., Bjerkvig, R., and Laerum, O.D. 1987. Phagocytic capacity of normal and malignant rat glial cells in culture. J. Natl. Cancer Inst. 2:279‐288.
   Brown, E.J. 1995. Phagocytosis. Bioessays 2:109‐117.
   Coopman, P.J., Thomas, D.M., Gehlsen, K.R., and Mueller, S.C. 1996. Integrin α3β1 participates in the phagocytosis of extracellular matrix molecules by human breast cancer cells. Mol. Biol. Cell. 11:1789‐1804.
   Coopman, P.J., Do, M.T.H., Thompson, E.W., and Mueller, S.C. 1998. Phagocytosis of cross‐linked gelatin matrix by human breast carcinoma cells correlates with their invasive capacity. Clin. Cancer Res. 4:507‐515.
   Imamura, F., Horai, T., Mukai, M., Shinkai, K., and Akedo, H. 1991. Serum requirement for in vitro invasion by tumor cells. Jpn. J. Cancer Res. 5:493‐496.
   Itoh, K., Yoshioka, K., Akedo, H., Uehata, M., Ishizaki, T., and Narumiya, S. 1999. An essential part for Rho‐associated kinase in the transcellular invasion of tumor cells. Nature Med. 2:221‐225.
   Lochter, A. and Bissell, M.J. 1995. Involvement of extracellular matrix constituents in breast cancer. Semin. Cancer Biol. 3:165‐173.
   MacDougall, J.R. and Matrisian, L.M. 1995. Contributions of tumor and stromal matrix metalloproteinases to tumor progression, invasion and metastasis. Cancer Metast. Rev. 4:351‐362.
   Montcourrier, P., Mangeat, P.H., Valembois, C., Salazar, G., Sahuquet, A., Duperray, C., and Rochefort, H. 1994. Characterization of very acidic phagosomes in breast cancer cells and their association with invasion. J. Cell Sci. 107:2381‐2391.
   Montgomery, A.M.P., Reisfeld, R.A., and Cheresh, D.A. 1994. Integrin αvβ3 rescues melanoma cells from apoptosis in three‐dimensional dermal collagen. Proc. Natl. Acad. Sci. U.S.A. 91:8856‐8860.
   Ruoslahti, E. 1992. The Walter Herbert Lecture. Control of cell motility and tumour invasion by extracellular matrix interactions. Br. J. Cancer. 2:239‐242.
   Stetler‐Stevenson, W.G., Aznavoorian, S., and Liotta, L.A. 1993. Tumor cell interactions with the extracellular matrix during invasion and metastasis. Annu. Rev. Cell Biol. 9:541‐573.
   Van Peteghem, M.C., Mareel, M.M., and De Bruyne, G.K. 1980. Phagocytic capacity of invasive malignant cells in three‐dimensional culture. Virchows Arch. B Cell. Pathol. Mol. Pathol. 2:193‐204.
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