Figure 1. Expression of Mmp13 mRNA is induced at 7 weeks of age in MMTV-PyMT tumors. Mmp13 mRNA levels determined by qPCR during normal development (N) and MMTV-PyMT induced tumor progression (m) in the #4 mammary gland (n = 661 for each time point). Values represent mean6standard error of the mean (SEM). doi:10.1371/journal.pone.0002959.g001 (Fig. 2j). This focal expression of Mmp13 in the tumor core was very different from the expression of Mmp2 and Mmp14, which were found throughout the tumor stroma, including at the tumor MMP13 in PyMT Tumor Fibroblast periphery (Fig. 2 e–h), and in areas with hyperplasia, grade I and grade II MIN (not shown). The localization of the cells that expressed Mmp13 suggested that they were myofibroblasts. Using combined in situ hybridization and immunohistochemical staining, we verified that most of the Mmp13 mRNA positive cells were positive for the myofibroblast marker a- smooth muscle-actin (Fig. 2k), paralleling the expression we found in human breast cancers [12]. Myofibroblasts are prevalent in the invasive MMTV-PyMT tumors, but only a small subpopulation was positive for Mmp13 mRNA. We noted that some of the Mmp13 mRNA positive myofibroblasts were located in narrow bands between tumor cell clusters (Fig. 2l). CD204 positive macrophages did not express Mmp13 mRNA and the location of the Mmp13 mRNA positive cells was not consistent with myoepithelial cells (data not shown), supporting our conclusion that the stromal Mmp13 mRNA expressing cells were myofibroblasts. Early tumor formation does not require MMP13 in MMTV- PyMT The expression levels and patterns of Mmp13 mRNA suggested that the proteinase would play a role in the transition to the invasive stages. To determine the role of MMP13 in mouse mammary tumor progression, we took a genetic approach using Mmp132/2 mice. We verified that the absence of MMP13 did not affect the development of the mammary epithelium (Fig. 3A) and therefore would not affect tumor progression indirectly by Figure 2. Mmp13 mRNA is expressed by a subset of a-smooth muscle actin positive carcinoma-associated fibroblasts. MMTV-PyMT tumors from 13-week-old mice were processed for in situ hybridization for Mmp13 (a–e, i–l), Mmp2 (g), and Mmp14 (h). For clarity the hybridization signals (silver grains) are shown with dark-field illumination in b, d, f, g, and h. (c) and (d) are higher magnification of the area with grade III invasive carcinoma framed in (a) and (b). Mmp13 mRNA signal is seen in the areas with grade III invasive carcinoma (c–d, arrows), but not in the surrounding areas with grade I and II MIN (indicated by I and II in a and b). Mmp13 mRNA expression is restricted to the tumor core (f, white arrows), whereas Mmp2 (g) and Mmp14 (h) mRNAs are seen also in the tumor periphery (blue arrows, f, g, and h are adjacent sections). Stromal septae with Mmp13 mRNA positive signal (i, arrows) are seen in areas close to necrotic tissue (N). Mmp13 mRNA signal in tumor areas with squamous metaplasia showing keratinized (K) epithelium (j, arrows). Expression of Mmp13 mRNA is seen in a-smooth muscle-actin immunoreactive myofibroblasts (k and l, arrows), in some areas restricted to myofibroblasts in the delicate strands of stromal tissue within the cellular tumor (l, arrows). All sections were counter- stained with haematoxylin and eosin except (k and l) that were stained with haematoxylin only. Bars: a, b: 700 mm; c, d: 100 mm; e–i: 400 mm; j: 200 mm; k, l: 50 mm. doi:10.1371/journal.pone.0002959.g002 PLoS ONE | www.plosone.org 4 August 2008 | Volume 3 | Issue 8 | e2959