There are no compensatory changes in expression of MMPs or TIMPs in MMTV-PyMT;Mmp132/2 tumors Since we found no changes in tumor progression in the absence of MMP13, we explored the possibility that the lack of MMP13 activity was compensated for by changes in expression of other MMPs or in their inhibitors, the TIMPs. However, there were no significant differences in the mRNA levels for MMP2, -3, -8, -9, - 10, -11, -12, -13, -14 or TIMP1, -2, -3, or -4 in tumors with and without MMP13 from 11-week-old mice by qPCR (Fig. 6). Increased amounts of thin collagen fibers is found in the absence of MMP13 in invasive areas of MMTV-PyMT tumors To determine whether any effects of MMP13 could be seen on its major substrate, fibrillar collagen, we evaluated the fibrillar collagen content in invasive areas of the tumors by picrosirius red staining of tumor sections (Fig. 7A). Although total collagen was not affected by the absence of MMP13, the fraction of thin collagen fibers relative to total collagen was increased significantly by ,30% in the invasive areas of tumors from MMTV-PyMT;Mmp132/2 mice (Fig. 7B, t- test, p = 0.007). Thus, MMP13 played a role in collagen metabolism in invasive areas of MMTV-PyMT tumors, but this did not affect tumor progression. Discussion In this study, we analyzed the effects of absence of MMP13 activity on the progression of MMTV-PyMT mammary carcino- ma. Our previous studies of human DCIS with early invasion led us to hypothesize that MMP13 would be a fibroblast-derived, potential rate-limiting proteinase in the transition from non- invasive to invasive breast carcinoma [12]. Interestingly, a recent MMP13 in PyMT Tumor Fibroblast study reported upregulation of several MMPs, including MMP13, at the transition from non-invasive to invasive breast cancer cells in an in vitro model [33]. However, although Mmp13 mRNA also was strongly induced in a subpopulation of myofibroblasts in MMTV-PyMT mammary carcinomas, concurrent with early transition to invasive carcinoma, there were no effects on tumor cell proliferation, tumor growth, lung metastasis, vascularization or differentiation of the primary tumors by the absence of MMP13. We excluded that the lack of effects was due to compensatory transcriptional changes of other MMPs or their inhibitors expressed in the MMTV-PyMT tumors. MMP13 has a broad substrate profile that includes several fibrillar collagens, tenascin, fibronectin and proteoglycans [11,34,35]. Mmp13 mRNA was found in carcinoma-associated fibroblasts located in the central areas of the MMTV-PyMT tumors, where tumor dedifferentiation and invasion is prominent. In these areas, we found increased thin collagen fibers relative to total collagen in MMTV-PyMT;Mmp132/2 compared to MMTV- PyMT;Mmp13+/+ tumors, indicating that MMP13 influences collagen fiber formation and/or metabolism of fibrillar collagen in the MMTV-PyMT stroma. Mice lacking Mmp13 display no major phenotypic abnormalities [21,36], but a significant contribution of MMP13 in collagen metabolism has been reported during bone development [21,36], skeletal regeneration [37] and in atherosclerotic plaques [38]. We found that most of the Mmp13 mRNA expressing cells in the MMTV-PyMT tumors are carcinoma-associated myofibroblasts. The myofibroblast is an abundant cell type in the MMTV-PyMT tumors (this study) as it is in invasive human breast cancers [39]. There are several reports suggesting that carcinoma-associated myofibroblasts promote cancer progression [40–42]. In addition, proteins expressed by myofibroblasts, such as PAI-1 and uPA, are associated with poor prognosis for breast cancer patients [43,44]. Figure 6. Absence of MMP13 is not compensated for by changes in mRNA expression of other MMPs or TIMPs. Expression of Mmp and Timp mRNAs, relative to 18S ribosomal RNA, in MMTV-PyMT;Mmp13+/+ (white boxes, n = 6) and MMTV-PyMT;Mmp132/2 (gray boxes, n = 6) tumors. In the box and whiskers plot, all values are normalized to the median value of the MMTV-PyMT;Mmp13+/+ group. Horizontal lines indicate the median and the boxes include the 25th and 75th percentiles. The lines outside the boxes correspond to highest or lowest values observed when these did not fall within the quartiles, and circles (white or gray) correspond to outlier values. doi:10.1371/journal.pone.0002959.g006 PLoS ONE | www.plosone.org 8 August 2008 | Volume 3 | Issue 8 | e2959