MMP13 in PyMT Tumor Fibroblast for determination of the metastasis burden by stereological analysis using Cavalieri’s principle as described previously [29]. frames was estimated based on the usage of systematic and randomly positioned reference points [30]. Tumor grading Nuclear grading of the MMTV-PyMT tumors was performed on haematoxylin and eosin (H&E) stained sections from the #1–3 tumors of MMTV-PyMT;Mmp13+/+ mice (n = 34) and MMTV- PyMT;Mmp132/2 mice (n = 24). We defined grade I tumors as tumors with adenoma and atypia in Mammary Intraepithelial Neoplasia (MIN)-like foci and containing up to 5% of MIN with grade II nuclear pleiomorphy. Grade II tumors (‘‘Early carcino- ma’’ [15]) were tumors with widespread invasive carcinoma with grade II nuclear pleiomorphy and contained areas with MIN Grade I/II. Grade III tumors (‘‘Late carcinoma’’) were tumors with focal or widespread invasive carcinoma with grade III nuclear pleiomorphy that also contained areas with Grade I and II carcinoma. Most tumors with grade III carcinoma showed areas of squamous metaplasia. Quantification of angiogenesis Mice were anesthetized as above and 100 mL of 1 mg/mL FITC-conjugated Lycopersicon esculentum (tomato) lectin (Vector labs) injected i.v. in the tail vein. After 5 min, the mice were cardiac perfused with 4% PFA in PBS at 120–140 mm Hg until the flow- through fluid was clear. Tumors were dissected and post-fixed in 4% PFA overnight, incubated in increasing sucrose concentrations (12%, 15%, 18% in PBS), embedded in Tissue-Tek O.C.T. compound and thick sections (30–40 mm) cut. The tissue sections were stained with 1:500 solution of Propidium Iodide in PBS (Molecular probes, P-3566) for 1 hour and the slides were mounted with Gel/Mount (Biomeda, #M01). Confocal image stacks with 1-mm spacing were collected from the most vascularized area in the middle of the tumors using a 20x lens with a Solamere micro-lensed spinning disk confocal microscope (Solamere Technologies, Salt Lake City UT) equipped with an intensified charge-coupled device (ICCD) camera (XRMega- 10EX S-30, Stanford Photonics, Palo Alto, CA). The acquired images were analyzed with Bitplane Imaris version 5.5 for Windows software using ‘filament tracer’ with an approximate filament diameter set at 1.5. Minor manual corrections of the computerized filaments were uniformly done. Vessel (segment) average diameter, length and volume, were exported to a Microsoft Excel file and analyzed using GraphPad 4 statistical software. Eight tumors from MMTV-PyMT;Mmp13+/+ and nine tumor from MMTV-PyMT;Mmp132/2 were analyzed. All image acquisition and analysis was done blindly. Cell proliferation analysis MMTV-PyMT;Mmp13+/+ mice (n = 19) and MMTV- PyMT;Mmp132/2 mice (n = 19) were injected i.p. with 2 mg bromodeoxyuridine (BrdU, Sigma-Aldrich) 2 hours before eutha- nasia. The mice were anesthetized, perfusion fixed, the tumors isolated and fixed in 4% PFA and finally paraffin embedded as described above. For immunohistochemistry, we used a rat mAb against BrdU at 2 mg/mL (clone BU1/75-ICR1, Abcam, Cam- bridge, UK). The BrdU labeled cells were counted as a percentage of the total number of epithelial tumor cells using CastGRID (Visiopharm, Hørsholm, Denmark) essentially as previously described [30]. In brief, frames of 9391 mm2 were systematically but randomly placed over the BrdU stained sections with a final magnification of 658 on the monitor. All BrdU positive neoplastic cells within the frames were counted to give total numbers varying from 250–400. The total number of neoplastic cells within the Collagen evaluation Serial sections (3-mm thick) from mammary tumor glands #1–3 obtained from 13-week-old MMTV-PyMT;Mmp13+/+ mice (n = 26) and MMTV-PyMT;Mmp132/2 mice (n = 21) were stained with haematoxylin and eosin (H&E) or picrosirius red (PSR) [31]. Areas with invasive tumor were identified in H&E stained sections. The same areas were then analyzed in the PSR stained sections by obtaining 2.361.8 mm images with a charge-coupled device (CCD) camera (Leica) under linearly polarized light using a Leica DMRBE microscope. In linearly polarized light, thick collagen bundles appear orange-red and thin collagen fibers green [32]. Images were analyzed with MetaMorph software for collagen content. Total collagen was identified with Hue-Saturation- Intensity (HSI) intervals 0–256, 1–256 and 70–256, respectively. For thin fibrillar collagen, HSI settings of 26–83, 70–135, and 57– 155 were applied to all the images. All image acquisition and analysis was done blindly. The area covered by total collagen, thin fibrillar collagen, and the fraction of thin fibrillar collagen was calculated and used for statistical analysis (t-test). Statistics For statistical analyses of the tumor burdens obtained by caliber measurements, the total tumor volumes were logarithmically transformed and analyzed using a general linear model with repeated measures. Only measurements obtained from week 7 to 13 were included in this analysis. Estimates were obtained using generalized estimating equations (SAS v9.1). Statistical interaction between genotype and time were evaluated for the entire observation period considering repeated measures (SAS). For statistical analyses of the metastasis burdens, the values were logarithmically trans- formed and analyzed using a general linear model. Results Mmp13 is expressed by myofibroblasts in invasive stage MMTV-PyMT tumors Mmp13 mRNA is expressed by a subpopulation of myofibro- blasts in human invasive ductal breast carcinomas, but is rarely expressed in normal breast, benign breast lesions and ductal carcinoma in situ (DCIS) [12]. To determine if the expression level of MMP13 changes during tumor progression in the MMTV- PyMT mouse model, Mmp13 mRNA levels were assessed in tumor tissue from mice at 3-13 weeks of age by qPCR. The level of Mmp13 mRNA was highly increased in MMTV-PyMT tumors after 7-weeks of age, with the highest levels found in older mice, corresponding to late stage tumors (Fig. 1). Mmp13 mRNA levels were low in normal mammary glands at all ages. The increase in Mmp13 mRNA thus took place during the time of transition to invasive stages in this tumor model [15]. To determine where Mmp13 was expressed, we performed in situ hybridization in tumors from 13-week-old mice, containing different stages of nuclear grading. The Mmp13 mRNA signal was seen in stromal fibroblast-like cells located in the tumor core in areas with invasive grade II and III carcinoma (‘‘late carcinoma’’), but was generally absent in the tumor periphery that was dominated by normal-looking mammary tissue, hyperplasia or MIN grade I and II (Fig. 2a–d). In particular, Mmp13 mRNA positive cells were often seen in stromal branches located close to the tumor cores with focal necrosis (Fig. 2i). Sporadic Mmp13 mRNA positive single cells and small cell clusters were also found in areas with strongly keratinized, squamous metaplastic changes PLoS ONE | www.plosone.org 3 August 2008 | Volume 3 | Issue 8 | e2959