MMP13 in PyMT Tumor Fibroblast influencing the number of mammary epithelial cells. We next crossed the MMTV-PyMT mice with Mmp132/2 mice and measured the total hyperplastic area on whole-mounts of mammary glands from mice at age 4 or 6 weeks, before the transition to invasive carcinoma. No difference was found between MMTV-PyMT;Mmp13+/+ and MMTV-PyMT;Mmp132/2 mice (Fig. 3B), consistent with the lack of Mmp13 mRNA expression at the early cancer stage. MMP13 is not required for tumor growth, tumor vascularization or metastasis in MMTV-PyMT mammary carcinomas Mmp13 mRNA was induced in tumors from mice of 7–9 weeks-of- age, and we therefore expected that MMP13 would be required in the later stages of tumorigenesis, possibly affecting tumor growth and metastasis. We followed tumor growth in a cohort of littermate MMTV-PyMT;Mmp13+/+ (n = 38) and MMTV-PyMT;Mmp132/2 females (n = 25) by weekly palpations and caliper measurements until 13 weeks of age, when tumors were isolated for histological examination and the lungs isolated to determine metastasis burden. A minor reduction in the growth of the tumors in MMTV- PyMT;Mmp132/2 mice were seen at 8, 9, 11, 12 weeks (Fig. 4A), but this was not reproduced in a different cohort (Fig. 4B). Furthermore, there were no differences in cancer cell proliferation between tumors from MMTV-PyMT;Mmp13+/+ and MMTV-PyMT;Mmp132/2 mice at 8 or 11 weeks of age as determined by BrdU-labeling (Fig. 5C). In addition, histological examination and nuclear grading of the tumors revealed no significant differences in the percentages of tumors that had progressed to grade III, late-stage carcinoma (Fig. 4C, 8% of the MMTV-PyMT;Mmp132/2 vs. 18% of MMTV-PyMT;Mmp13+/+ tumors). Furthermore, analyses of H&E stained sections revealed no differences in the presence of necrosis or stroma between tumors from MMTV-PyMT;Mmp132/2 and MMTV-PyMT;Mmp13+/+ mice (not shown). Finally, no effects were seen on tumor vascularization, when analyzing vessel diameter, length, or volume in 3D reconstructions of vessels in tumors of lectin-perfused mice (Fig. 5A, B). Thus, the absence of MMP13 did not influence progression in the primary tumors. Figure 3. Mammary gland development and early tumor development is not influenced by MMP13. (A) Morphometric analysis of ductal epithelial penetration during normal mammary gland development. Representative whole mount-stained #4 mammary glands from Mmp13+/+ (n = 4 and n = 4 at 4 and 6 weeks of age, respectively), and Mmp132/2 mice (n = 3 and n = 7 at 4 and 6 weeks of age, respectively. The distances from the nipple to the three most far-reaching ducts and to the edge of the fat pad were measured. The mean ductal epithelial penetration as percentage of the whole fat pad is shown with the SEM. (B) Morphometric analysis of early MMTV-PyMT tumor growth. Representative whole mount-stained #4 gland tumors from MMTV- PyMT;Mmp13+/+ and MMTV-PyMT;Mmp132/2 mice at 6-weeks-of-age. The tumor area was measured in 4- and 6-week-old MMTV-PyMT;Mmp13+/+ (n = 5 and n = 7, respectively) and MMTV-PyMT;Mmp132/2 mice (n = 3 and n = 10, respectively) and are presented as mean with the SEM. doi:10.1371/journal.pone.0002959.g003 PLoS ONE | www.plosone.org 5 August 2008 | Volume 3 | Issue 8 | e2959