1. Journal Club Paper
Matrix metalloproteinase 2 (MMP-2) attenuates brain tumour growth, while
promoting macrophage recruitment and vascular repair
Pierrot Tremblay, Marie-Jos´ee Beaudet, Eve Tremblay, Naika Rueda, Tina Thomas and Luc Valli`eres
J Pathol 2011; 224: 222–233
DOI: 10.1002/path.2854
Speaker : Ahmad Usama
艾哈曼
Date: Fri-Feb.01,2013
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2. Introduction
* MMP-2(Gelatinase A):
- An extracellular protein-degrading enzyme widely believed to be involved in the
invasion of brain tumour cells.
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3. Introduction
* MMP-2(Gelatinase A):
- MMP2 is a zinc-dependent endopeptidase expressed in response to injury or disease.
- There is two isoforms of MMP-2 , they are MMP-2α & MMP-2β.
MMP-2 & MMP-9 structures
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http://arthritis-research.com/content/figures/ar2532-1-l.jpg

4. Introduction
* Astrocytoma:
- Astrocytomas are a type of cancer of the brain. They originate in a particular kind of
glial cells, star-shaped brain cells in the cerebrum called astrocytes.
* GFAP:
- GFAP (Glial Fibrillary Acidic Protein) intermediate filament protein that expressed is
astrocytes and ependymal cells in CNS (Astrocytic marker).
* CD 31: Blood vessel endothelium marker .
* Anti- Ki 67: Cell proliferation marker .
* Anti- Cleaved Caspase 3: Apoptotic cells marker .
* Anti-Iba-1: Macrophages marker .
* DAPI: Nuclei marker .
* GFP: Cell morphology marker .
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5. Materials & Methods
1) DNA microarray .
2) Real-time quantitative PCR (qPCR).
3) Western blotting.
4) Flow cytometry.
5) Cell invasion assay.
6) Cell adhesion assay.
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6. Materials & Methods
7) DNA construction.
8) Intracerebral implantation of human astrocytoma cells.
9) Immunohistochemistry (IHC).
10) In situ hybridization.
11) Stereological analyses (for the estimation of tumour volume , tumour vessel
parameters (calibre , density , sprouts ….) , cell counting , cell size ).
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7. Results
1) In vivo characterization of astrocytoma cells.
2) Expression of MMP2 in astrocytoma cell lines and biopsies.
3) Knock down or overexpression of MMP2 in astrocytoma cells.
4) Effects of MMP2 on astrocytoma cells in culture.
5) Effects of MMP2 on intracranial astrocytomas.
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8. 1) In vivo characterization of astrocytoma cells.
Characterization of GFP+ astrocytoma cells∗ 3 weeks after intracerebral implantation in mice (n ≥ 6; 75 103 cells injected per 8
mouse)

9. 1) In vivo characterization of astrocytoma cells.
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10. Mini-Conclusion
Vascular destabilization results from a biochemical property of the cells rather than
a physical disruption of the tissue.
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11. 2) Expression of MMP2 in astrocytoma cell lines and biopsies.
* Which genes that might confer the ability to destabilize the vasculature?
*Are astrocytoma cells produce MMP2 not only in culture, but also after
transplantation in vivo?
* Are normal cells express MMP2 in the tissue surrounding the tumour?
*Which MMP2 isoforms are expressed in astrocytomas?
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12. 2) Expression of MMP2 in astrocytoma cell lines
and biopsies.
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13. Mini-Conclusion
1) At the in vitro scale , lack of MMP2 leads to the inability of the U343 line to
destabilize the vasculature.
2) MMP2 expression is maintained after in vivo transplantation and is restricted to
the tumour tissue.
3) MMP-2 is upregulated in brain tumours and this is due to MMP-2α isoform not
MMP-2β
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14. 3) Knock down or overexpression of MMP2 in
astrocytoma cells
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15. 4) Effects of MMP2 on astrocytoma cells in
culture
Ex vivo properties of SKI 1 cells underexpressing or overexpressing 15

16. Mini-Conclusion
The deficiency in MMP2 slows the growth of astrocytoma cells in culture
by affecting their proliferation, but not their survival.
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17. 5) Effects of MMP2 on intracranial astrocytomas
* What is the effect of MMP-2 on astroytoma gtowth in vivo?
*If the differences in the tumours size could be related to changes in cell proliferation
and/or survival?
*If the increase in tumour volume could be related to changes in
cell size or density (eg due to oedema)?
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18. 5) Effects of MMP2 on intracranial astrocytomas
Influence of MMP-2 on the growth of intracrinal astrocytomas 18

19. 5) Effects of MMP2 on intracranial astrocytomas
Influence of MMP-2 on the growth of intracrinal astrocytomad 19

20. 5) Effects of MMP2 on intracranial astrocytomas
Influence of MMP-2 on the growth of intracrinal astrocytomad 20

21. Mini-Conclusion
The inhibition of MMP2 enhances the growth of intracranial astrocytomas likely by
increasing tumour cell proliferation.
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22. 5) Effects of MMP2 on intracranial astrocytomas
*If MMP2 contributes the vasculature destabilization in vivo ?
*If the absence of MMP2 had affected the number of Tumour Associated
Macrophages (TAMs)?
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23. 5) Effects of MMP2 on
intracranial astrocytomas
Influence of MMP-2 on blood vessels and macrophages in astrocytoma 23
xenografts

24. 5) Effects of MMP2 on intracranial astrocytomas
Influence of MMP-2 on blood vessels and macrophages in astrocytoma
xenografts 24

25. Mini-Conclusion
1) MMP2 limits the extent of vascular destabilization and
regression induced by astrocytomas.
2) MMP2 inhibition promotes brain tumour growth due to
the reduction in the recruitment of TAMs.
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26. Final Conclusion
1) MMP2 slows vessel destabilization and astrocytoma growth. This finding contrasts with
the widespread belief that MMP2 promotes astrocytoma cell invasion, which has been
inferred mainly from in vitro studies and this underscores the necessity of confirming in
vitro results with more relevant, in vivo models.
2) The deficiency in MMP2 promotes astrocytoma growth and this effect might be
attributable to a reduction in macrophage recruitment and/or vascular repair. In addition, it
refutes the hypothesis that MMP2 contributes to the phenomenon of vascular
destabilization that is commonly associated with malignant brain tumours.
3) MMP2 is not the cause of vascular atypia in malignant brain tumours, but is involved in
a tissue repair response that tends to limit the growth of these tumours.
4) According to these points of view , this indicates that MMP2 is not an appropriate target
for the treatment of brain cancer.
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