Deletion of Cd151 reduces mammary tumorigenesis in the MMTV/PyMT mouse model

Animal maintenance was in accordance with the Animal Care and Ethics Committee at the Australian BioResources specific pathogen free (SPF) animal breeding facility (Moss Vale, New South Wales). All animal monitoring and experiments were approved by the Animal Care and Ethics Committee at the University of Newcastle. In the tumorigenesis experiments, we used the well-characterized FVB/N (FVB) MMTV/PyMT mouse line (MT#634) carrying a mouse mammary tumor virus promoter-driven polyoma middle T transgene [16]. A pure FVB genetic background is most commonly used for mouse tumorigenesis experiments because of its permissiveness to spontaneous tumor development. Cd151 ^−/− mice are grossly healthy on the C57Bl/6 (B6) background [20] but they develop a severe kidney disease on a pure FVB background [21]. Hence FVB Cd151 ^−/− mice could not be used for the tumorigenesis experiments. Since F1 hybrid FVB x B6 Cd151 ^−/− mice were healthy and did not present any sign of kidney disease onset (monitored for the appearance of proteinuria over a 12 months period, our unpublished data), we conducted the experiments on this hybrid background. This allowed us to keep all experimental animals on a mixed but yet homogenous (50% B6 and 50% FVB) genetic background. FVB Cd151 ^+/− mice were produced by backcross for 10 generations from the original B6 Cd151 ^−/−[20] and maintained as heterozygotes. Heterozygous B6 Cd151 ^+/− females were crossed with FVB Cd151 ^+/− males carrying the MMTV/PyMT transgene (PyMT Cd151 ^+/− males, see Additional file 1: Figure S1 for breeding details) in order to generate the experimental F1 animals. Genotyping was performed as previously described [20]. Experimental and control littermates were co-housed throughout the experiments, in a temperature controlled facility with a 12-h light: dark cycle.

Beginning at weaning (3 weeks of age), female mice were palpated twice weekly for the onset of mammary tumors. For each mouse, tumor palpation was performed in each of the ten mammary glands, in a genotype-blinded fashion. At 15–16 weeks of age, female mice were euthanased by CO₂ inhalation, and all the tumors were dissected and weighed. For each experimental mouse, half of the biggest tumor was fixed in 10% neutral buffered formalin (NBF) for paraffin embedding, one quarter was snap frozen in liquid nitrogen, and the last quarter was snap frozen in OCT compound. At the time of dissection and after excision of the tumors, the lungs were exposed and inflated via tracheal injection of 1 ml of 10% NBF in order to inflate and fix the lung lobes. Lungs were then excised and further fixed in 10% NBF for at least 24 hours before paraffin embedding.

Whole mount analysis was performed by spreading inguinal #4 mammary glands onto poly-lysine slides followed by overnight fixation in 10% NBF, defatting in acetone and overnight staining in carmine alum (0.2% carmine and 0.5% aluminium sulphate) as previously described [22]. The stained glands were then dehydrated in a graded ethanol series, incubated in xylene for 1 hour and stored in methyl salicylate.

Tumor histology/stage was assessed on the largest tumor for each mouse using 5 μm paraffin sections stained with hematoxylin and eosin. Immunohistochemistry (IHC) was performed on 5 μm paraffin sections using a peroxidase VECTASTAIN ABC elite kit and DAB peroxidase substrate kit as per the manufacturer’s recommendations (Vector Laboratories, Burlingame, CA). The antibodies and dilutions used for IHC were rabbit monoclonal anti-Ki67 at 1:200 (Neomarkers, Kalamazoo, MI, USA) and polyclonal rabbit anti-cleaved caspase 3 at 1:800 (Cell Signaling Technology, Danvers, MA, USA).

Immunofluorescence labelings were performed on 5 μm frozen sections as previously described [21]. Primary antibodies and dilutions used for immunofluorescence labeling were rabbit anti-CD151 (LAI-2) at 1:500 [21]; rat anti-CD31 (BD Biosciences, Franklin Lakes, NJ, USA) at 1:200; rabbit anti-α3 integrin (a kind gift from Dr. Fiona Watt, Wellcome Trust Centre for Stem Cell Research, Cambridge, UK) at 1:1000; rat anti-β1 integrin (BD Biosciences) at 1:200; rat anti-β4 integrin (BD Biosciences) at 1:200; rat anti- α6 integrin (Chemicon, Temecula, CA, USA) at 1:200. The double labeling where two rabbit primary antibodies were used (CD151 and α3 integrin) was performed sequentially following established methods as described previously [21].

The largest tumor for each mouse was used to quantitate proliferation, apoptosis and vascularization in a genotype-blinded manner. Tumor cell proliferation was assessed by immunohistochemistry (as described above) for the commonly used Ki67 nuclear marker. Ki67 stained slides were scanned in a digital format using the Aperio™ digital pathology system (Aperio) and 200× magnification snapshots of digital images were generated using Imagescope. Quantitation of proliferation (expressed as percent Ki67 positive nuclear area per total nuclear area) was then performed on the 200× digital images using ImmunoRatio, a publicly available web-based application [23]. The extent of apoptosis in the tumors was quantitated on Aperio images of cleaved-caspase 3 IHC stained slides, using the positive pixel count algorithm at 200× magnification (5 fixed size (300 μm × 300 μm) images were used for each tumor section).

To estimate blood vessel density, CD31 immunofluorescence labeling was performed and Image J was used to quantitate the proportion of CD31 positive area inside the tumors. At least five fluorescent microscope pictures (100× magnification) per tumor were used in the CD31 analysis.

All the lung lobes were dissected and processed for paraffin embedding. Five microns paraffin sections were stained with hematoxylin and eosin and slides were scanned in a digital format using the Aperio™ digital pathology system (Aperio). The lung area per section was measured using Scanscope and the metastatic burden (mm² of metatastases/cm² lung) was calculated for each animal, using the data from 3 sections at least 100 μm apart, as previously described [24].

Before investigating the effect of Cd151 deletion on mammary tumorigenesis it was necessary to determine whether mammary gland development and differentiation was normal in Cd151 knock-out mice. For this purpose, we performed whole mount analysis of #4 inguinal mammary glands in virgin, pregnant and lactating FVB Cd151 ^+/+ and Cd151 ^−/− mice (Figure 1). Ductal outgrowth and mammary gland differentiation appeared grossly normal in the Cd151 ^−/− females at the different stages of development investigated.

Immunofluorescence labeling of 6-week old virgin mammary glands and confocal microscopy revealed strong expression of CD151 in the mammary ducts in a basolateral pattern (Figure 2), reminiscent of the surrounding basal layer of myoepithelial cells and in accordance with what has been previously reported in humans [11]. Fainter CD151 labeling was also noticeable in the basolateral membrane of ductal luminal cells (Figure 2A). Moreover, CD151 labeling in the mammary ducts colocalized to some extent with the α3-, α6-, β1- and β4- chains of integrins, suggesting association of CD151 with the α3β1, α6β1 and α6β4 integrin heterodimers in the mammary gland. Similarly to the situation in the normal glands, CD151 appeared to be expressed strongly by the myoepithelial cell layer surrounding the tumor nodules of the early stage PyMT tumors, while the cancer cells in the nodules were only faintly labeled (Figure 3). The CD151 labeling overlapped to some extent with the β1- and β4- integrin immunolabelings in the tumors suggesting as expected some degree of colocalization.

To avoid the kidney phenotype that has been described in FVB Cd151 ^−/− mice [21], we chose to investigate the effects of Cd151 deletion on mammary tumorigenesis on a F1 (FVB×B6) background (see explanations in Methods). Consistent with previous reports showing increased PyMT tumor latency on the B6 background [24, 25], the onset of mammary tumors was significantly delayed by about 20 days on the (FVBxB6) F1 background in comparison to the pure FVB background (Additional file 2: Figure S2).

Investigation of mammary tumor onset by palpation showed no statistically significant difference in tumor latency between Cd151 ^+/+ and Cd151 ^−/− PyMT mice (Figure 4A). There was however a trend towards increased tumor latency in Cd151 ^−/− (T₅₀ = 67 days; n = 25) as compared to Cd151 ^+/+ (T₅₀ = 61.5 days; n = 26) that was approaching significance (Log-rank test P-value =0.0536). Interestingly, the median age to tumor onset in the heterozygous Cd151 ^+/− mice was in between the values reported for Cd151 ^+/+ and Cd151 ^−/− PyMT mice (Cd151 ^+/− T₅₀ = 63, n = 21). However, the difference between the median tumor latency of Cd151 ^+/− versus Cd151 ^+/+ mice was not significant (Log-rank test P = 0.7533).

At 15 to 16 weeks of age (i.e. 6 to 7 weeks after initial tumor palpation), the mice were euthanased and all the tumors were collected and weighed for each mouse. Interestingly both the total tumor weight (P = 0.0003) and tumor multiplicity (P = 0.0008) per mouse were significantly decreased in the Cd151 ^−/− group as compared to Cd151 ^+/+ group (Figure 4B and 4C). Individual tumor weight (as shown with weight of the largest tumor per mouse Figure 4D) was also clearly decreased in Cd151 ^−/− mice (P = 0.0023), suggesting an effect of Cd151 gene deletion on both mammary tumor induction and growth/proliferation in vivo. Regardless of the Cd151 genotype, at least part of the largest tumor of each mouse had progressed to the carcinoma stage as determined by H&E staining (illustrated in Figure 5). Immunolabelings for Ki67, cleaved caspase-3 and CD31 were performed on representative primary tumor samples in order to compare the rates of cell proliferation, apoptosis and tumor angiogenesis between both groups of mice, respectively (Figure 6). These experiments did not reveal any significant differences between Cd151 ^+/+ and Cd151 ^−/− tumors.

Since the mammary tumors of MMTV/PyMT mice primarily metastasize to the lungs, we examined the lungs of Cd151 ^+/+ and Cd151 ^−/− PyMT mice at the time of dissection, which corresponds to 6 to 7 weeks after tumor onset. The incidence of lung metastasis was similar in both groups with 22 out of 26 Cd151 ^+/+ (85%) and 20 out of 24 Cd151 ^−/− (83%) mice developing lung metastases (Figure 7A). In addition, the median number of lung metastases (Mann Whitney test P-value = 0.3554) and the average size of individual metastasis per mouse (Mann Whitney test P-value = 0.6757) were similar between the two groups (Figure 7B and 7D, respectively). The distribution of the number of metastases in the Cd151 ^+/+ group, was much more heterogeneous (Figure 7B), with a large subset of the Cd151 ^+/+ but not Cd151 ^−/− group of mice containing high numbers of metastases (Chi-square P-value = 0.0329, Figure 7C). On closer observation, it could be noted that the mice with high number of metastases in the Cd151 ^+/+ group were also the mice with higher tumor burden (Figure 7E) suggesting that tumour burden rather than Cd151 status is the likely explanation. In conclusion, ablation of Cd151 per se did not appear to affect the number or size of metastases in the MMTV/PyMT mice.