Brain Tumor Pathology

, Volume 19, Issue 2, pp 69–76 | Cite as

Tracking cell invasion of human glioma cells and suppression by anti-matrix metalloproteinase agent in rodent brain-slice model

  • Daizo Yoshida
  • Kunihiro Watanabe
  • Masahiro Noha
  • Hiroshi Takahashi
  • Akira Teramoto
  • Yuichi Sugisaki
Original Article


Persistent expression of green fluorescent protein (GFP) in human malignant glioma cell clones (U87MG, U251MG, and U373MG) was established using the pEGFP-C1 vector. Tumor spheroid was implanted into the caudate nucleus-putamen of a severely compromised immunodeficient (SCID) mouse brain slice. To allow quantitative assessment of tumor cell invasion, the invasion area index was measured on days 1, 3, 5, and 7 by a fluorescence stereomicroscope and an image analyzer in the presence of varying concentrations of SI-27. In the control group (0μg/ml), all glioma cell lines invaded in a fingerlike fashion, reaching the contralateral hemisphere via the corpus callosum. SI-27 at concentrations of 10, 50, or 100 μg/ml significantly suppressed the index on days 5 and 7 in a dose-dependent manner, whereas 1 μg/ml had no effect. Laser confocal microscopy indicated that the tumor cells penetrated through the brain slice. This model enabled unequivocal periodic tracking of individual invading tumor cells in the normal brain. The significant suppression of glioma cell invasion by SI-27 indicates that anti-matrix metalloproteinase (MMP) treatment may represent an important future therapeutic strategy for malignant cerebral neoplasms.

Key words

Brain slice Cell invasion Green fluorescent protein Malignant glioma Matrix metalloproteinase 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Engebraaten O, Hjortland GO, Hirschberg H, et al (1999) Growth of precultured human glioma specimens in nude rat brain. J Neurosurg 90:125–132PubMedGoogle Scholar
  2. 2.
    Fillmore HL, Shum J, Furqueron P, et al (1999) An in vivo rat model for visualizing glioma tumor cell invasion using stable persistent expression of the green fluorescent protein. Cancer Lett 141:9–19PubMedCrossRefGoogle Scholar
  3. 3.
    Goldbrunner RH, Bendszus M, Sasaki M, et al (2000) Vascular endothelial growth factor-driven glioma growth and vascularization in an orthotopic rat model monitored by magnetic resonance imaging. Neurosurgery 47:921–929PubMedCrossRefGoogle Scholar
  4. 4.
    Kaiser MG, Andrew AT, Fine RL, et al (2000) Tissue distribution and antitumor activity of topotecan delivered by intracerebral clysis in a rat glioma model. Neurosurgery 47:1391–1399PubMedCrossRefGoogle Scholar
  5. 5.
    Gory L, Montel MC, Zagorec M (2001) Use of green fluorescent protein to monitorLactobacillus sakei in fermented meat products. FEMS Microbiology Letters 194:127–133PubMedCrossRefGoogle Scholar
  6. 6.
    Penar PL, Koshomn S, Bushan A, et al (1997) Inhibition of epidermal growth factor-associated tyrosine kinase blocks glioblastoma invasion of the brain. Neurosurgery 40:141–151PubMedCrossRefGoogle Scholar
  7. 7.
    Goldbrunner RH, Bernstein JJ, Plate KH, et al (1999) Vascularization of human glioma spheroids implanted into rat cortex is conferred by two distinct mechanisms. Cancer Lett 55:486–495Google Scholar
  8. 8.
    Grill J, Beusechem VWV, VanDerValk P et al (2001) Combined targeting of adenovirus to integrins and epidermal growth factor receptors increases gene transfer into primary glioma cells and spheroid. Clin Cancer Res 7:641–650PubMedGoogle Scholar
  9. 9.
    Murakami M, Goto S, Yoshikawa M, et al (2001) The invasive features of glial and non-central nervous system tumor cells are different on organotypic brain slices from newborn rats. Int J Oncol 18:721–727PubMedGoogle Scholar
  10. 10.
    Birkedal-Hansen H, Moore WGI, Bodden MK, et al (1993) Matrix metalloproteinases: a review. Crit Rev Oral Biol Med 4:197–250PubMedGoogle Scholar
  11. 11.
    Nakagawa T, Kubota T, Kabuto M, et al (1994) Production of matrix metalloproteinases and tissue inhibitor of metalloproteinases-1 by human brain tumors. J Neurosurg 81:69–77PubMedCrossRefGoogle Scholar
  12. 12.
    Sang QX (1998) Complex role of matrix metalloproteinases in angiogenesis (review). Cell Research 8:171–177PubMedGoogle Scholar
  13. 13.
    Sato H, Takino T, Okada Y, et al (1994) A matrix metalloproteinase expressed on the surface of invasive tumor cells. Nature 370:61–65PubMedCrossRefGoogle Scholar
  14. 14.
    Noha M, Yoshida D, Watanabe K, et al (2000) Suppression of cell invasion on human malignant glioma cell lines by a novel matrix-metalloproteinase inhibitor SI-27: in vitro study. J Neuro-Oncol 48:217–223CrossRefGoogle Scholar
  15. 15.
    Yoshida D, Noha M, Watanabe K, et al (2002) SI-27, a novel inhibitor of matrix metalloproteinases with antiangiogenic activity; detection with a variable-pressure scanning electron microscope. Neurosurgery 50:578–587PubMedCrossRefGoogle Scholar
  16. 16.
    Yoshida D, Watanabe K, Noha M, et al (2002) Suppression of matrix metalloproteinase activity by sl-27: detection by a new activity assay with s-2444, a specific chromogenic peptide. J Neuro-Oncol 58:1–11CrossRefGoogle Scholar
  17. 17.
    Farson D, Witt R, McGuiness R, et al (2001) A new-generation stable inducible packaging cell line for lentiviral vectors. Hum Gene Ther 12:981–997PubMedCrossRefGoogle Scholar
  18. 18.
    Jung S, Ackerly C, Ivanchuk S, et al (2001) Tracking the invasiveness of human astrocytoma cells by using green fluorescent protein in an organotypic brain slice model. J Neurosurg 94:80–89PubMedCrossRefGoogle Scholar
  19. 19.
    Matsumura H, Ohnishi T, Kanemura Y, et al (2000) Quantitative analysis of glioma cell invasion by confocal laser scanning microscopy in a novel brain slice model. Biochem Biophys Res Commum 269:513–520CrossRefGoogle Scholar
  20. 20.
    Ohnishi T, Matsumura H, Izumoto S, et al (1998) A novel model of glioma cell invasion using organotypic brain slice culture. Cancer Res 58:2935–2940PubMedGoogle Scholar
  21. 21.
    Yong VW, Krekoski CA, Forsyth PA, et al (1998) Matrix metalloproteinases and diseases of the central nervous system. Trends Neurosci 21:75–80PubMedCrossRefGoogle Scholar
  22. 22.
    Chitala SK, Tonn JC, Rao JS (1999) Matrix metalloproteinases and their function in human gliomas. Int J Develop Neurosci 17:495–502CrossRefGoogle Scholar
  23. 23.
    Edwards DR, Beaudry PP, Laing TD, et al (1996) The roles of tissue inhibitors of metalloproteinases in tissue remodeling and cell growth. Int J Obesity 20:s9-s15Google Scholar
  24. 24.
    Forsyth PA, Wong H, Laing TD, et al (1999) Gelatinase-A (MMP-2), gelatinase-B (MMP-9) and membrane type matrix metalloproteinase-1 (MT1-MMP) are involved in different aspects of the pathophysiology of malignant gliomas. Br J Cancer 79:1828–1835PubMedCrossRefGoogle Scholar
  25. 25.
    Im SA, Gomez-Manazano C, Fueyo J, et al (1999) Antiangiogenesis treatment for gliomas: transfer of antisense-vascular endothelial growth factor inhibits tumor growth in vivo. Cancer Res 59:895–900PubMedGoogle Scholar
  26. 26.
    Nakano A, Tani E, Miyazaki K, et al (1995) Matrix metalloproteinases and tissue inhibitor of metalloproteinases in human gliomas. J Neurosurg 83:298–307PubMedCrossRefGoogle Scholar
  27. 27.
    Reict A, Rucklidge GJ (1992) Invasion of brain tissue by primary glioma: evidence for the involvement of urokinase-type plasminogen activator as an activator of type IV collagenase. Biochem Biophys Res Commun 186:348–354CrossRefGoogle Scholar
  28. 28.
    Rao JS, Steck PA, Mohanan S, et al (1993) Elevated leved ofMr 92,000 type IV collagenase in human brain tumor. Cancer Res 53:2208–2211PubMedGoogle Scholar
  29. 29.
    Watanabe K, Yoshida D, Noha M, et al (2001) Suppression of matrix metalloproteinase-2 and-9 mediated cell invasiveness by a novel matrix metalloproteinase inhibitor, BE-16627B on human malignant glioma cell lines; in vitro study (in press)Google Scholar
  30. 30.
    Naito K, Kanbayashi N, Nakajima S, et al (1994) Inhibition of growth of human tumor cells in nude mice by a metalloproteinase inhibitor. Int J Cancer 58:730–735PubMedGoogle Scholar
  31. 31.
    MacDonald TJ, Tabrizi P, Shimada H, et al (1998) Detection of brain tumor invasion and micrometastasis in vivo by expression of enhanced green fluorescent protein. Neurosurgery 43:1437–1442PubMedCrossRefGoogle Scholar
  32. 32.
    Hong YK, Chung DS, Joe YA, et al (2000) Efficient inhibition of in vivo human malignant glioma growth and angiogenesis by interferon-beta treatment at early stage of tumor development. Clin Cancer Res 6:3354–3360PubMedGoogle Scholar
  33. 33.
    Lampson LA, Lampson MA, Dunne AD (1993) Exploiting the lacZ reporter gene for quantitative analysis of disseminated tumor growth within the brain: use of a lacZ gene product as a tumor antigen, for evaluation of antigenic modulation, and to facilitate image analysis of tumor growth in situ. Cancer Res 53:176–182PubMedGoogle Scholar
  34. 34.
    Torti SV, Golden-Fleet M, Willingham MC, et al (2000) Use of green fluorescent protein to measure tumor growth in an implanted bladder tumor model. J Urol 167:724–728CrossRefGoogle Scholar
  35. 35.
    Chattejee TK, Fisher RA (2000) Cytoplasmic, nuclear and golgi localization of RGS proteins. Evidence for N-terminal and RGS domain sequence as intracellular targeting motifs. J. Biol Chem 275:24013–24021CrossRefGoogle Scholar

Copyright information

© The Japan Society of Brain Tumor Pathology 2002

Authors and Affiliations

  • Daizo Yoshida
    • 1
  • Kunihiro Watanabe
    • 1
  • Masahiro Noha
    • 1
  • Hiroshi Takahashi
    • 1
  • Akira Teramoto
    • 1
  • Yuichi Sugisaki
    • 2
  1. 1.Department of NeurosurgeryNippon Medical SchoolTokyoJapan
  2. 2.Central Institute for Electron MicroscopyNippon Medical SchoolTokyoJapan

Personalised recommendations