Characterization of Matrix Metalloproteinase Inhibitors: Angiogenesis and Tumor Models

Daniel H. Albert1, Steven K. Davidsen1

1 Abbott Laboratories, Abott Park, Illinois
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 5.23
DOI:  10.1002/0471141755.ph0523s13
Online Posting Date:  August, 2001
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Abstract

Since the matrix metalloproteinases (MMPs) have an essential role in the process of tumor growth, invasion and metastasis, small molecule MMP inhibitors have the ability to modulate tumor progression in animals and the potential to be of therapeutic benefit to cancer patients. The antiangiogenic properties of MMP inhibitors can be assessed by the measurement of hemoglobin content of Matrigel plugs containing angiogenic growth factors introduced into the flanks of mice. A flank tumor growth model using B16 murine melanoma cells provides a useful means of determining the antitumor effects of MMP inhibitors as well as correlating efficacy with the concentration of drug in blood.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Matrigel Plug In Vivo Angiogenesis Model
  • Basic Protocol 2: Syngeneic Tumor Growth Model
  • Commentary
  • Bibliography
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Matrigel Plug In Vivo Angiogenesis Model

 Materials
  • Matrigel (phenol red-free, Becton Dickinson; store frozen up to 9 months at –20°C)
  • 16,000 U/ml heparin (Life Technologies) prepared in PBS (e.g., Life Technologies) containing 0.1% (w/v) bovine serum albumin (store at 4°C)
  • 0.25 µg/ml acidic fibroblast growth factor (aFGF; R&D Systems) in PBS containing 0.1% bovine serum albumin (store frozen in aliquots and thaw each aliquot only once)
  • C57 BL/6 female mice, 22 to 24 g, 6 to 8 weeks old (Charles River)
  • Test compound
  • MMP reference inhibitor ABT-770 (can be obtained from Abbott Laboratories; contact Dr. Daniel H. Albert; daniel.h.albert@abbott.com)
  • 90% (v/v) acetic acid
  • Saline (0.9% NaCl in water)
  • Hemoglobin standard (30 mg/dl, Sigma)
  • 5 mg/ml 3,3¢,5,5¢-tetramethylbenzidine (TMB) in 90% glacial acetic acid (store up to 2 weeks at 4°C)
  • 0.3% hydrogen peroxide
  • Syringes with 25 -G needles
  • Dissecting equipment including scissors and forceps
  • Polytron homogenizer (Brinkmann)
  • 0.25-ml microcentrifuge tubes
  • 96-well flat-bottom microtiter plates
  • Plate reader capable of reading absorbance at or near 600 nm and preferably equipped with data analysis software suitable for kinetic readings (e.g., SPECTRAmax Plus384 with SOFTmax PRO, Molecular Devices)

NOTE: Glacial acetic acid, hydrogen peroxide and TMB can be obtained together in a kit (Hemoglobin, Plasma Chemistry kit) from Sigma.

Basic Protocol 2: Syngeneic Tumor Growth Model

 Materials
  • B16-F10 cells (ATCC #CRL-6475)
  • Dulbecco's modified Eagle's medium (DMEM; Life Technologies) containing 10% FBS and 1% penicillin (5000 U/ml)-streptomycin (5000 µg/ml) (Life Technologies)
  • Versene (Life Technologies)
  • Matrigel (phenol red-free, Becton Dickinson; store frozen up to 9 months at –20°C)
  • 16,000 U/ml heparin (Life Technologies) prepared in PBS (e.g., Life Technologies) containing 0.1% (w/v) bovine serum albumin (store at 4°C)
  • 0.25 µg/ml acidic fibroblast growth factor (aFGF; R&D Systems) in PBS containing 0.1% bovine serum albumin (store frozen in aliquots and thaw each aliquot only once)
  • C57 BL/6 female mice, 22 to 24 g, 6 to 8 weeks old (Charles River)
  • Test compound
  • MMP reference inhibitor ABT-770 (can be obtained from Abbott Laboratories; contact Dr. Daniel H. Albert; daniel.h.albert@abbott.com)
  • Sources of CO2 and O2 to produce 60% CO2/40% O2 mixture
  • 15% (w/v) tripotassium EDTA in phosphate-buffered saline (PBS; Life Technologies)
  • 30% (v/v) methanol
  • Ethyl acetate
  • Source of N2
  • 162- or 175-cm2 tissue culture flasks
  • 15-cm2 conical centrifuge tubes
  • Syringes with 25 -G needles
  • Caliper micrometer
  • Capillary tubes
  • Polytron homogenizer (Brinkmann)

NOTE: All reagents and equipment coming into contact with live cells must be sterile, and proper sterile technique must be followed accordingly.

NOTE: All culture incubations are performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.
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Figures

  •  FigureFigure 5.23.1 Injection sites for the Matrigel/aFGF/heparin mixture.
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  •  FigureFigure 5.23.2 Effects of ABT-770, administered orally twice daily starting on day 0 through day 4, on FGF-induced angiogenesis in Matrigel plugs. Results are expressed as mean ± SEM, n = 10 per group; * p < 0.05 compared to control on day 5.
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  •  FigureFigure 5.23.3 Effects of ABT-770 on the growth of B16 melanoma cells implanted subcutaneously in the flank of mice. The compound was administered orally twice daily on days 7 to 21. Results are expressed as mean ± SEM, n = 10 per group.
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  •  FigureFigure 5.23.4 Example of exponential growth phase of vehicle- and drug-treated tumors.
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  •  FigureFigure 5.23.5 Blood levels of inhibitory activity on day 21 (14 days of dosing of ABT-770). Results are expressed as mean ± SD, n = 2. Steady-state concentration (Css) was calculated from the linear regression fit of data over the 7- to 8-hr period after the last dose.
    View Image
  •  FigureFigure 5.23.6 Effect of steady-state blood levels (Css) of ABT-770 on efficacy in the B16 tumor growth model. ABT-770 blood levels and efficacy (T/C) were obtained from the experiments described in Figures 5.23.4 and 5.23.5. The minimal biologically significant effect and the maximal effect of the drug are illustrated by dotted lines.
    View Image

Videos

Literature Cited

 Literature Cited
    Bergers, G., Brekken, R., McMahon, G., Vu, T.H., Itoh, T., Tamaki, K., Tanzawa, K., Trope, P., Itohara, S., Werb, Z., and Hanahan, D. 2000. Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nature Cell. Biol. 2:737-744.
    Bottomley, K.M., Johnson, W.H. and Walter, D.S. 1998. Matrix metalloproteinase inhibitors in arthritis. J. Enzyme Inhib. 13:79-101.
    Brown, P.D. 1997. Matrix metalloproteinase inhibitors in the treatment of cancer. Med. Oncol. 14:1-10.
    Donovan, J. and Brown, P. 1995. Euthanasia. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 1.8.1-1.8.4. John Wiley & Sons, New York.
    Duivenvoorden, W.C., Hirte, H.W., and Singh, G. 1997. Use of tetracycline as an inhibitor of matrix metalloproteinase activity secreted by human bone-metastasizing cancer cells. Invasion Metastasis 17:312-322.
    Gross, J. and Lapiere, C.M. 1962. Collagenolytic activity in amphibian tissues: A tissue culture assay. Proc. Natl. Acad. Sci. U.S.A. 48:1014-1022.
    Houchens, D.P., Ovejera, A.A., and Barker, A.D. 1978. Use of athymic nude mice in cancer research (D.P. Houchens and A.A. Ovejera, eds.) pp. 267-295. Gustav Fisher, New York.
    Johnson, M.D., Kim, H.R., Chesler, L., Tsao-Wu, G., Bouck, N. and Polverini, P.J. 1994. Inhibition of angiogenesis by tissue inhibitor of metalloproteinase. J. Cell Physiol. 160:194.
    Kähäri, V.-M. and Saarialho-Kere, U. 1999. Matrix metalloproteinases and their inhibitors in tumour growth and invasion. Ann. Med. 31:34-45.
    Lynch, K. 1999. Theoretical and practical difficulties of developing a tumouristatic drug in the treatment of cancer. Int. J. Pharm. Med. 13:127-136.
    Nagase, H. and Woessner, J.F. 1999. Matrix metalloproteinases. J. Biol. Chem. 274:21491-21494.
    Nelson, A.R., Fingleton, B., Rothenberg, M.L., and Matrisian, L.M. 2000. Matrix metalloproteinases: Biologic activity and clinical implications. J. Clin. Oncol. 18:1135-1149.
    Ovejera, A.A. and Houchens, D.P. 1981. Human tumor xenografts in athymic nude mice as a preclinical screen for anticancer agents. Semin. Oncol. 8:386-393.
    Passaniti, A., Taylor, R.M., Pili, R., Guo, Y., Long, P.V., Haney, J.A., Pauly, R.R., Grant, D.S., and Martin, G.R. 1992. A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin, and fibroblast growth factor. Lab. Invest. 67:519-528.
    Plantner, J.J., Smine, A., and Quinn, T.A. 1998. Matrix metalloproteinases and metalloproteinases in human interphotoreceptor matrix and vitreous. Curr. Eye Res. 17:132-140.
    Schnaper, H.W., Grant, D.S., Stetler-Stevenson, W.G. 1993. Type IV collagenase(s) and TIMPs modulate endothelial cell morphogenesis in vitro. J. Cell Physiol. 156:235-246.
    Shalinsky, D.R., Brekken, J., Zou, H., Bender, S., Zook, S., Appelt, K., Webber, S., and Varki, N.V. 1998. Increased apoptosis in human androgen-independent prostatic PC-3 tumors following oral administration of a novel matrix metalloproteinase (MMP) inhibitor AG3340, in male nude mice. Proc. AACR 39:646.
    Snyder, L.R., Glajch, J.L., and Kirkland, J.J. 1998. Practical HPLC Method Development. John Wiley & Sons, New York.
    Steel, G.G., Courtaney, V.D., and Peckman, M.J. 1983. The response to chemotherapy of a variety of human tumor xenografts. Br. J. Cancer 47:1-13.
    Stetler-Stevenson, W.G. 1999. Matrix metalloproteinases in angiogenesis. A moving target for therapeutic intervention. J.Clin. Invest. 103:1237-1241.
    Tallarida, R.J., Raffa, R.B., and McGonigle, P. 1998. Principles in General Pharmacology. Springer-Verlag, New York.
    Timm, K. 1980. Peri-orbital, bleeding technique for the mouse, hamster, and rat: Anatomical consideration. Synapse 13:15-16.
    Tomayko, D.P. and Reynolds, C.P. 1989. Determination of subcutaneous tumor size in athymic nude mice. Cancer Chemother. Pharmacol. 24:148-154.
    Vu, T.H. and Werb, Z. 2000. Matrix metalloproteinases: Effectors of development and normal physiology. Genes Dev. 14:2123-2133.
    Whittaker, M., Floyd, C.D., Brown, P., and Gearing, A.J.H. 1999. Design and therapeutic application of matrix metalloproteinases inhibitors. Chem. Rev. 99:2735-2776.
    Yu, A.E., Hewitt, R.E., Connor, E.W., and Stetler-Stevenson, W.G. 1997. Matrix metalloproteinases. Novel targets for directed cancer therapy. Drugs Aging 11:229-244.
 Key Reference
    Passaniti et al., 1992. See above.

Original description of the Matrigel angiogenesis model.

 Internet Resource
    http://www.keystonescientific.com

Provides access to useful technical bulletins on HPLC techniques.

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