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Contribution of host MMP-2 and MMP-9 to promote tumor vascularization and invasion of malignant keratinocytes

Véronique Masson^*,1, Laura Rodriguez de la Ballina^*,1, Carine Munaut^*, Ben Wielockx, Maud Jost^*, Catherine Maillard^*, Silvia Blacher^*, Khalid Bajou^*, Takeshi Itoh, Shige Itohara, Zena Werb^||, Claude Libert, Jean-Michel Foidart^* and Agnès Noël^*,2

^* Laboratory of Tumor and Development Biology, University of Liège, Liège, Belgium;
Department for Molecular Biomedical Research, Flanders Interuniversity Institute for Biotechnology and University of Ghent, Ghent, Belgium;
Discovery Research Laboratories, Shionogi & Co., Ltd., Osaka, Japan;
Riken Brain Science Institute, Wako, Japan; and
^|| Department of Anatomy, University of California at San Francisco, California, USA

²Correspondence: Laboratory of Tumor and Development Biology, University of Liège, Tower of Pathology (B23), Sart-Tilman, Liège 4000, Belgium. E-mail: agnes.noel{at}ulg.ac.be

SPECIFIC AIMS

Although matrix metalloproteinases (MMPs) have been shown to play a key role in normal and pathological angiogenesis, specific functions of individual MMPs as antiangiogenic or proangiogenic mediators remain to be elucidated. To more precisely delineate the respective roles of MMP-3, MMP-2, and MMP-9 in tumoral angiogenesis, we assessed the impact of single or combined MMP deficiencies in in vivo and in vitro models of angiogenesis. The malignant keratinocyte transplantation system and the aortic ring assay were applied to single MMP-deficient mice (MMP-2–/–, MMP-3–/–, MMP-9–/–) or double-deficient mice (MMP-3–/–, MMP-9–/–, and MMP-2, MMP-9–/–). MMP expression and production were analyzed by immunohistochemical analysis, in situ zymography, and MMP-9 was mapped using transgenic mice harboring the LacZ reporter gene driven by MMP-9 promoter.

PRINCIPAL FINDINGS

1. MMP-9 promoter of host cells is activated in vivo in tumor transplants
Malignant murine keratinocytes (PDVA cells) cultured on collagen gels were implanted into transgenic MMP-9/LacZ mice. In response to angiogenic stimuli produced by tumor cells, new blood vessels invaded the collagen gel and reached the malignant epithelial layer. Host-derived cells that infiltrated the collagen gel within the first 10 days expressed MMP-9 at the migration front. Three weeks after transplantation, when invasive malignant keratinocytes had invaded the host tissue, host cells displayed MMP-9 promoter activity as assessed by ß-galactosidase staining and expressed endogenous MMP-9 protein. In our model, immunohistochemical analysis revealed that MMP-9 was produced by neutrophils.

2. Deficiency of MMP-9 and/or MMP-3 does not impair in vivo tumor invasion and vascularization
Wild-type and MMP-9-deficient mice were transplanted for 3 wk with malignant PDVA keratinocytes. Neither tumor invasion (Fig. 1 b) nor vascularization (Fig. 2 c) was affected by the single MMP-9 deficiency. Invading tumor sprouts surrounded by a rich capillary network were observed in MMP-9-deficient mice and corresponding wild-type mice (Fig. 1a, b ). Tumor vascularization developed to a similar degree in both genotypes (Fig. 2a, c ).

View larger version (189K): [in this window] [in a new window]   Figure 1. Invasive behavior of malignant mouse keratinocytes (PDVA cells) in vivo 3 wk after transplantation. Tumor cells invade the host tissue of wild-type (a), MMP-9–/– (b), MMP-3–/– (c), MMP-2–/– (d), and MMP-3–/–; MMP-9–/– (e), but not MMP-2–/–; MMP-9–/– mice (f). Sections were stained with hematoxylin eosin. C: Carcinoma cells; G: collagen gel; H: host tissue. Original magnification: x200.

View larger version (40K): [in this window] [in a new window]   Figure 2. Invasive and angiogenic properties of malignant mouse PDVA keratinocytes in vivo. Cells were transplanted for 3 wk into wild-type (a, e), MMP-2–/– (b, f), MMP-9–/– (c, g), or MMP-2–/–; MMP-9–/– mice (d, h). a–d) Malignant cells were stained with anti-keratin Ab (green) and vessels with anti-type IV collagen (red). Collagen type IV collagen labeling was codistributed with immunostaining of endothelial cells recognized by anti-mouse platelet endothelial cell adhesion molecule (PECAM) (data not shown). e–h) In situ zymography detecting gelatinolytic activity (green), cells were counterstained with bisbenzimide (blue). C: carcinoma cells; G: collagen gel; H: host tissue. Original magnification: a–d) x200; e–h) x100.

The impact of the lack of an MMP acting upstream in the MMP activation cascade was evaluated by transplanting malignant cells into MMP-3–/–, MMP-3+/+, double MMP-3–/–; MMP-9–/– mice and corresponding wild-type mice (Fig. 1c, e ). Invasive and angiogenic phenotypes of malignant keratinocytes were preserved in these single or double MMP-deficient mice.

3. A combined deficiency of MMP-2 and MMP-9 impairs tumor invasion and vascularization
Tumor cells transplanted into MMP-2–/– mice recruited angiogenesis and infiltrated into host tissue to similar extents as observed in wild-type mice (Fig. 1a, d ). In sharp contrast, when malignant cells were transplanted into double MMP-2–/–; MMP-9–/– mice, host-derived blood vessels were unable to migrate toward the tumor cell layer and remained confined beneath the collagen gel. In addition, the tumor cells failed to invade the collagen gel and remained as an irregular stratified epithelium on top of the matrix (Fig. 1f , Fig. 2d ). In situ zymography revealed that the gelatinolytic activity was confined to the host tissue and no activity could be detected in tumor layers (Fig. 2) . This activity was completely absent in double MMP-2;MMP-9-deficient mice (Fig. 2h ), strongly reduced in MMP-2–/– mice (Fig. 2f ), but still evident in MMP-9–/– mice (Fig. 2g ). Gelatinolytic activity was differently distributed in each of the gelatinase-deficient mice: largely distributed in the stroma of MMP-9–/– mice and much more dispersed in MMP-2–/– mice, in keeping with stromal expression of MMP-2 and inflammatory cell (neutrophil) expression of MMP-9.

4. Deficiency of MMPs does not impair the angiogenesis in vitro
To determine whether the impact of MMP deficiency in angiogenesis was a direct effect on vascular cells or was indirect through the stromal and inflammatory cells present in the growing tumor mass, we next evaluated in vitro angiogenesis by using aortic ring assay. Expression of MMP-9 by vascular cells was first assessed by using aortic rings from MMP-9/LacZ mice. Under the culture conditions, we observed the spreading out of ß-galactosidase positive staining in microvessels, indicating that MMP-9 is expressed during the course of endothelial cells sprouting. We then compared aortic explants resected from the MMP-deficient mice and their corresponding wild-type mice that were cultured in collagen gels in the presence of autologous serum (2.5%). Objective quantification of cell spreading was performed by computer-assisted image analysis. At day 6, neoangiogenesis was maximal and similar vascular networks were observed in all MMP-deficient and wild-type mice. The double MMP-2;MMP-9 deficiency did not affect in vitro vessel outgrowth.

CONCLUSIONS AND SIGNIFICANCE

Several lines of evidence support the contribution of MMPs to the course of tumoral angiogenesis. However, their individual contribution and putative synergistic or even opposite effects are not well known. By using a tumor transplantation system allowing the study of host-tumor interactions, we investigated the contribution of host MMP-2, MMP-3, and MMP-9 to invasion and vascularization of skin carcinomas. The angiogenic and invasive phenotypes of malignant keratinocytes were not affected by the single deficiency of host MMP-2, MMP-3, or MMP-9; or the combined deficiency of MMP-3 and MMP-9. Thus, although MMP-3 has a skin wound-healing phenotype and can activate MMP-9, acting upstream in the cascade of MMP activation, it has no significant contribution in this tumor model. Our study demonstrates for the first time, in an experimental cancer model, that MMP-2 and MMP-9 are both required to promote tumor progression. The invasive and angiogenic phenotypes of malignant keratinocytes were impaired by the combined deficiency in both gelatinases, but not by their single deficiencies. Of interest is the identification of neutrophils as the inflammatory cell origin of MMP-9 in our experimental model. In sharp contrast, MMP-2 was produced by stromal cells, highlighting the entirely different cellular distribution of the two gelatinases. In situ gelatin zymography revealed a partial and a strong reduction of gelatinolytic activity in MMP-9- or MMP-2-deficient mice, respectively, suggesting a lack of compensation of one gelatinase by the other. Although MMP-2 and MMP-9 are endowed with similar enzymatic activities in vitro, these MMPs may have distinct activities in vivo against nonmatrix substrates such as chemokines, cytokines, or growth factors. The requirement of both MMP-2 and MMP-9 for successful tumor invasion and vascularization might reflect the necessity of specific interactions between stromal cells producing MMP-2 and inflammatory cells secreting MMP-9. The intact in vitro outgrowth of microvessels from aortic rings of mice with a combined gelatinase deficiency demonstrates that endothelial cell migration in a pure collagen matrix can occur in the absence of gelatinases. In sharp contrast, in vivo, endothelial cell activation and migration might require gelatinases produced by other host cells to generate angiogenic or chemoattractant factors. This emphasizes the importance of different host cells for creating a microenvironment favorable to angiogenesis.

Our work illustrates that different host cell types produced MMP-2 and MMP-9 and highlights the contribution of neutrophils in MMP-9 production and in cancer progression. By demonstrating the requirement of both gelatinases for malignant keratinocyte invasion and vascularization, our data provide a new insight into skin tumor biology and identify MMP-2 and MMP9 as suitable targets for anticancer therapy. They demonstrate the importance to develop double MMP-deficient mice for a better understanding of the individual functions of MMPs, their putative redundancy, and/or their cooperative or opposite effects in cancer progression.

View larger version (34K): [in this window] [in a new window]   Figure 3. Schematic diagram of the hypothetical mechanisms of host gelatinase contribution to the invasive and angiogenic phenotypes of malignant mouse PDVA keratinocytes in vivo. MMP-2 and MMP-9 are produced by different host cells; both gelatinases can degrade common substrates such as type IV collagen of the basement membrane or cleave-specific substrates such as chemokines or cytokines, and contribute to processes associated with tumor progression: 1) extracellular matrix (ECM) degradation; 2) cell surface remodeling; 3) release of growth factor (GF) from the ECM; and 4) activation or inactivation of soluble messagers such as chemokines/cytokines (C). While the invasive and angiogenic phenotypes of malignant keratinocytes were preserved in the absence of either MMP-2 or MMP-9, a lack of both gelatinases profoundly affected tumor invasion and vascularization.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2140fje;

¹ These authors contributed equally to this work.

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