sia (MIN), progress into invasive carcinomas at 7–9 weeks after birth [15,16]. Transition to invasive carcinoma takes place in the central core of the tumors, where epithelial atypia and nuclear pleomorphism first appear [15]. Tumors isolated from 13-week-old mice, late stage tumors, show evidence of dedifferentiation and squamous metaplasia and have spread to the lungs and lymph nodes [14,15]. The tumors share both morphological and molecular characteristics with human invasive ductal carcinomas [14,15,17– 19]. Furthermore, we have reported that Mmp2, 3, 11, 13 and 14 mRNAs are expressed in the stromal compartment in a series of late stage MMTV-PyMT tumors, similar to the expression in human ductal breast cancers [18]. In late stage tumors, Mmp13 mRNA shows high, focal expression in stromal cell populations [20]. Since this expression pattern is similar to what we have observed in human breast cancers [12], we chose the MMTV-PyMT mouse breast tumor model to test the functional role of MMP13 in breast cancer progression and metastasis. Materials and Methods Mice MMTV-PyMT and Mmp132/2 mice have previously been described [14,21]. Both strains were backcrossed more than 8 generations to FVB/n. The two strains were intercrossed and the resulting MMTV-PyMT;Mmp13+/2 male offspring were mated with female Mmp13+/2 mice to establish sibling cohorts of MMTV- PyMT;Mmp13+/+ and MMTV-PyMT;Mmp132/2 females. All animal experiments were conducted according to institutional guidelines and approved by the Danish Animal Experiments Inspectorate. A concurrent health report compliant with the guidelines of the Federation of European Laboratory Animal Science Associations revealed no infections. Genotyping Genotyping was performed on chromosomal DNA purified from tail tips [22] in a single PCR reaction using MMTV-PyMT primers, control wt plasminogen primers [23] and Mmp13 primers (MMP13_in5 anti: GGT GGT ATG AAC AAG TTT TCT GAG C, MMP13_in2: CAG ACC CTA CAG TGC CAG ATT TTA G, MMP13_ex5: TGA TGA CGT TCA AGG AAT TCA GTT T). Bands representing MMTV-PyMT transgene (159 bp), plasminogen (268 bp), wt Mmp13 (572 bp) and Mmp13 knock-out allele (672 bp) were identified by agarose gel electrophoresis. Quantitative real time PCR Mice were anesthetized by intraperitoneal administration of a 1:1 mixture of Dormicum (Roche A/S, Basel, Switzerland) and Hypnorm (Janssen-Cilag Ltd, High Wycombe, UK) and sacrificed by intracardial perfusion with 10 ml ice-cold phosphate-buffered saline (PBS). For analysis of Mmp13 mRNA expression during normal mammary gland development and MMTV-PyMT tumorigenesis, the #4 mammary glands were isolated from MMTV-PyMT mice and wild type FVB/n mice at 3, 5, 7, 9, 11 and 13 weeks (n = 5 for each age and genotype). For analyses of the expression of other MMPs and TIMPs, tumors from 11-week old MMTV-PyMT;Mmp13+/+ (n = 6) and MMTV-PyMT;Mmp132/ 2 mice were used (n = 6). Total mRNA was isolated from 100 mg tissue using the Qiagen RNeasy midi Kit (Qiagen, West Sussex, UK). The purified RNA samples were treated with DNase I for 15 minutes at 37uC to remove residual DNA. Reverse transcrip- tion was performed essentially as described [24] followed by TaqMan qPCR performed as described [25] using an ABI7900 (Applied Biosystems, Foster City, CA) and primers specified in [26] except that Mmp13 primers were chosen from the universal MMP13 in PyMT Tumor Fibroblast probe library (number 105, Roche Applied Science, Burgess Hill, UK), left: CTT TTC CTC CTG GAC CAA ACT, right: TCA TGG GCA GCA ACA ATA AA. Gene expression levels relative to 18S were calculated. In situ hybridization 13-week-old mice were anesthetized as above and perfused intracardially with 10 mL PBS followed by 10 mL of 4% paraformaldehyde (PFA). Mammary tumors were dissected from glands #1–3 and #4, fixed in 4% formalin for 5–7 days at 4uC and paraffin embedded. In situ hybridization was performed on samples from MMTV-PyMT;Mmp13+/+ mice (n = 10) with 35S labeled RNA probes for MMP13 as described previously [27]. No Mmp13 mRNA signal was detected in tumors from MMTV- PyMT;Mmp132/2 mice (n = 4). Immunohistochemistry For immunohistochemical staining of a-smooth muscle-actin (mouse mAb, Dako, Glostrup, Denmark) and CD204 (rat mAb clone 2F8, Serotec, Oxford, UK), sections were heat-treated at 98uC in TEG buffer (10 mmol/L Tris, 0.5 mmol/L EGTA, pH 9.0) for 5 minutes in a T/T Micromed microwave processor (Milestone, Sorisol, Italy). The a-smooth muscle-actin antibody was mixed with biotinylated anti-mouse Fab fragments using the Animal Research Kit (Dako) and used for staining following the recommendations of the manufacturer. The rat mAb against CD204 was detected with rabbit anti-rat antibodies followed by Envision-Rabbit reagent (Dako). All antibody incubation steps were performed in an OptiMax automated immunostainer (BioGenex, San Ramon, CA). Immunohistochemical staining combined with in situ hybridization was performed on mouse glands #1–3 tumor samples from MMTV- PyMT;Mmp13+/+ mice (n = 6) as described previously [12]. The sections were developed with DAB chromogene, fixed for 40 min- utes in 4% PFA, and washed three times with sterile water. After dehydration, the sections were incubated with 35S-labeled Mmp13 antisense probes and further treated according to the in situ hybridization as described previously [27]. Morphometric analysis of mammary gland whole mounts The #4 mammary glands were isolated from 4- and 6-week-old MMTV-PyMT;Mmp13+/+ mice (n = 4 and n = 4, respectively) and MMTV-PyMT;Mmp132/2 mice (n = 3 and n = 7, respectively) and prepared for whole mount as described [28]. The whole mounts were photographed using a DFC320 CCD camera (Leica Microsystems, Wetzlar, Germany) and the ductal epithelial invasion determined by measuring the distance from the branch point closest to the nipple to the three most far-reaching ducts and the edge of the fat pad. The penetration is presented as the mean of the three measures of ductal penetration related to the length to the edge of the fat pad. All measurements were done using the Leica IM500 software. Tumor growth and lung metastasis Tumor growth in MMTV-PyMT;Mmp13+/+ mice (n = 38) and MMTV-PyMT;Mmp132/2 mice (n = 25) was followed by weekly palpations of all 10 mammary glands. The length and width of all palpable tumors were measured by caliper. The tumor volume (assuming that tumors took the shape of an ellipsoid) was calculated using the formula: V = (p/6)6W26L, where L = length and W = width. At 13 weeks, MMTV-PyMT;Mmp13+/+ mice (n = 35) and MMTV-PyMT;Mmp132/2 mice (n = 25) were anes- thetized and perfusion fixed (three mice had been terminated before due to extensive tumor burden). The lungs were removed PLoS ONE | www.plosone.org 2 August 2008 | Volume 3 | Issue 8 | e2959