Indeed, Mmp13 mRNA is induced by hypoxia in various cell lines [47–49] including fibroblasts [49], and vasculature of ossification sites and migration of endothelial cells into cartilage is impaired in Mmp132/2 mice [50]. However, our measurements of vascular diameter, length and volumes in MMTV-PyMT tumors did not reveal any effect of MMP13 on tumor vascularization. It has been reported that Mmp8 and Mmp9 mRNAs are upregulated during bone development in the ossification sites in Mmp132/2 mice [36] and that Mmp8 mRNA is upregulated during skin wound healing in Mmp132/2 mice [51]. We tested whether the mRNA expression levels of several MMPs were changed in MMTV-PyMT;Mmp132/2 tumors, but found no significant changes. We found that tumor progression in the MMTV-PyMT model was associated with strong upregulation of the Mmp13 mRNA level as compared to the normal mammary gland. Preliminary results suggest that Mmp10 and Mmp12 mRNA also are upregulated, while Mmp2, -3, -8, -9, -11, and -14 are not (TXP, CP, DE, unpublished data). This suggests that the three MMPs are upregulated by a common mechanism and potentially implicates MMP10 and MMP12 in tumorigenesis in the MMTV- PyMT model even though MMP10 and MMP12 mRNAs were not significantly upregulated in the MMTV-PyMT;Mmp132/2 tumors compared to MMTV-PyMT wild type tumors. However, we cannot exclude that differences exist at the level of enzyme activity or that other classes of proteolytic enzymes compensate for the absence of MMP13. The expression of Mmp13 mRNA is strongly restricted compared to the expression of Mmp2 and Mmp14 mRNAs that are expressed in a broader group of (myo)fibroblasts. We have previously reported that mRNAs for several MMPs, including Mmp2, Mmp3, Mmp11 and Mmp14 are expressed in the MMTV-PyMT tumor stroma [18]. Of these MMPs, MMP2 and MMP14 may have substrates in common with MMP13, for example, both are also collagenases in vivo [3]. Therefore MMP2, MMP14 or the activity of other MMPs may compensate for the lack of MMP13 in the MMTV-PyMT;Mmp132/2 tumors. It is possible that MMP13 plays both promoting and inhibiting roles during cancer progression, resulting in a neutral net effect in its absence in the MMTV-PyMT model. Tumor promoting and inhibiting functions have been found for MMP9 in the human papilloma virus (HPV) 16 skin cancer model where the absence of Mmp9 reduced the number of tumors, but the tumors that formed were more aggressive [4]. A tumor protective role of a number of matrix degrading proteases, including MMP8 and MMP12, has been described [52]. These studies emphasize the need for better understanding the role not only of the individual proteases but also of their separate activities in cancer progression. References 1. Ferguson JE, Schor AM, Howell A, Ferguson MW (1992) Changes in the extracellular matrix of the normal human breast during the menstrual cycle. Cell and Tissue Research 268: 167–177. 2. Lochter A, Bissell MJ (1995) Involvement of extracellular matrix constituents in breast cancer. Semin Cancer Biol 6: 165–173. 3. Egeblad M, Werb Z (2002) New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2: 163–176. 4. Coussens LM, Tinkle CL, Hanahan D, Werb Z (2000) MMP-9 supplied by bone marrow-derived cells contributes to skin carcinogenesis. Cell 103: 481–490. 5. Deryugina EI, Quigley JP (2006) Matrix metalloproteinases and tumor metastasis. Cancer Metastasis Rev 25: 9–34. 6. Almholt K, Green KA, Juncker-Jensen A, Nielsen BS, Lund LR, et al. (2007) Extracellular Proteolysis in Transgenic Mouse Models of Breast Cancer. J Mammary Gland Biol Neoplasia 12: 83–97. 7. Szabova L, Chrysovergis K, Yamada SS, Holmbeck K (2007) MT1-MMP is required for efficient tumor dissemination in experimental metastatic disease. Oncogene. 8. Freije JM, Diez Itza I, Balbin M, Sanchez LM, Blasco R, et al. (1994) Molecular cloning and expression of collagenase-3, a novel human matrix metalloprotei- nase produced by breast carcinomas. J Biol Chem 269: 16766–16773. MMP13 in PyMT Tumor Fibroblast Breast cancer progression in humans may have a larger contribution from the stromal environment than murine tumors, since human tissue have a more fibroblast-rich stroma than murine [6]. Nevertheless, the MMTV-PyMT breast cancer model shares many histological and molecular characteristics with human luminal breast cancer [14,15,17], but it differs from human ductal carcinoma in one interesting aspect: the myoepithelial cells, which represent a cellular barrier for tumor cells and are an important source for basement membrane, are partly lost early during MMTV-PyMT oncogene-induced tumor progression [15]. The absence of an intact myoepithelial barrier may lead to pre-invasive precursor lesions different from human DCIS and render MMP13 a dispensable proteinase in the critical transition phase from non- invasive to invasive carcinoma. Thus, despite the presence of a heterogeneous stromal environment and the appearance of a considerable myofibroblast population in the MMTV-PyMT tumors, myofibroblasts may play a minor role for the overall progression of the MMTV-PyMT tumor compared to human breast cancer. Therefore, models in which transition from DCIS to invasive carcinoma occurs similar to in human tumors are needed to definitely rule out a role of MMP13 in breast cancer. In conclusion, we found that the absence of MMP13 in the MMTV-PyMT mice did not result in any differences in tumor progression to invasive and metastatic breast carcinoma. Carci- noma-associated fibroblasts have been shown to facilitate tumor progression through stimulation of angiogenesis, growth factor secretion and invasion [53–55]. However, there are no good markers allowing systematic sub-classification of these cells. Thus, even though MMP13 itself apparently is dispensable for tumor progression in the MMTV-PyMT model, its restricted expression in myofibroblasts in invasive regions suggests that it marks a subpopulation of carcinoma-associated fibroblasts that are in- volved in the transition to invasive carcinoma. Acknowledgments Charlotte Lønborg, Lotte Frederiksen, O¨znur Turan and Birthe Larsen for excellent technical assistance and John Post for photographic assistance. Author Contributions Conceived and designed the experiments: BSN ME ZW LRL. Performed the experiments: BSN ME CJP TXP LRL. Analyzed the data: BSN ME FR HAA CJP TXP IJC LRL. Contributed reagents/materials/analysis tools: DRE ZW. Wrote the paper: BSN ME. Critically revised the manuscript: DRE ZW LL. 9. Knauper V, Will H, Lopez-Otin C, Smith B, Atkinson SJ, et al. (1996) Cellular mechanisms for human procollagenase-3 (MMP-13) activation. Evidence that MT1-MMP (MMP-14) and gelatinase a (MMP-2) are able to generate active enzyme. J Biol Chem 271: 17124–17131. 10. Cowell S, Knauper V, Stewart ML, D’Ortho MP, Stanton H, et al. (1998) Induction of matrix metalloproteinase activation cascades based on membrane- type 1 matrix metalloproteinase. Biochem J 331 (Pt 2): 453–458. 11. Knauper V, Lopez-Otin C, Smith B, Knight G, Murphy G (1996) Biochemical characterization of human collagenase-3. J Biol Chem 271: 1544–1550. 12. Nielsen BS, Rank F, Lopez JM, Balbin M, Vizoso F, et al. (2001) Collagenase-3 (MMP-13) expression in breast myofibroblasts as a molecular marker of transition of ductal caricnoma in situ lesions to invasive ductal carcinomas. Cancer Res 61: 7091–7100. 13. Nielsen BS, Rank F, Illemann M, Lund LR, Danø K (2007) Stromal cells associated with early invasive foci in human mammary ductal carcinoma in situ co-express urokinase and urokinase receptor. Int J Cancer 120: 2086–2095. 14. Guy CT, Cardiff RD, Muller WJ (1992) Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease. Mol Cell Biol 12: 954–961. PLoS ONE | www.plosone.org 10 August 2008 | Volume 3 | Issue 8 | e2959