Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases

Abstract

Animal models are powerful tools to analyze the mechanism of the induction of human breast cancer. Here we report a detailed analysis of mammary tumor progression in one mouse model of breast cancer caused by expression of the polyoma middle T oncoprotein (PyMT) in the mammary epithelium, and its comparison to human breast tumors. In PyMT mice, four distinctly identifiable stages of tumor progression from premalignant to malignant stages occur in a single primary tumor focus and this malignant transition is followed by a high frequency of distant metastasis. These stages are comparable to human breast diseases classified as benign or in situ proliferative lesions to invasive carcinomas. In addition to the morphological similarities with human breast cancer, the expression of biomarkers in PyMT-induced tumors is also consistent with those associated with poor outcome in humans. These include a loss of estrogen and progesterone receptors as well as integrin-beta1 expression and the persistent expression of ErbB2/Neu and cyclinD1 in PyMT-induced tumors as they progress to the malignant stage. An increased leukocytic infiltration was also closely associated with the malignant transition. This study demonstrates that the PyMT mouse model is an excellent one to understand the biology of tumor progression in humans.

Figure 1.

Early premalignant tumor in PyMT mice is comparable to human normal breast. A: Carmine red-stained whole-mount preparation of mammary gland from a PyMT mouse at 8 weeks of age. PT, primary tumor; second, foci growing on the distant ducts; LN, lymph note. B: H&E staining of a mammary gland prepared from a PyMT mouse at 4 weeks of age. CD, main milk-collecting ducts; H, the hyperplastic lesion; Adi, adipose cells. The inset in B is shown in C. D: H&E staining of a mammary gland section from a normal mouse at 4 weeks of age. E and F: IHC of SMA in a normal mouse mammary duct (E) and a hyperplastic lesion (F). Arrows point to the positively stained myoepithelial cells. G and H: IHC of laminin α1 in a normal mouse mammary duct (G) and a hyperplastic lesion (H). Arrows point to some of the positively stained areas. I: H&E staining of a normal human breast. TDLU, terminal duct lobular unit; SS, subsegmental duct; Ext, extracellular fibers. Note that SS leads to TDLU. The earliest change in the mouse lesion is an alteration in the mammary architecture that produces a lobular-alveolar structure that looks similar to the normal TDLU of the human. Scale bars: 100 μm (B, D, I); 25 μm (H); 10 μm (C, E, F, G). Original magnifications: ×100 (B, D, I); ×1000 (C, E, F, G); ×400 (H).

Figure 2.

Adenoma/MIN in PyMT mice is comparable to human ductal hyperplasia, florid (DHF). A and B: H&E staining of mammary gland sections from a PyMT mouse at 8 weeks of age. A: A primary mammary lesion at adenoma/MIN stage. Arrow points to an infiltration site of leukocytes showing in F at higher magnification. The inset in A is shown in B. C: IHC of SMA in a primary tumor at adenoma/MIN stage. Arrows point to the positively stained myoepithelial cells surrounding the lesion. D and E: IHC of laminin α1 in a normal mammary duct (D) and an adenoma/MIN lesion (E). Arrows point to positively stained areas. F: An enlarged image of the leukocytic infiltration site from A. t, tumor. G: IHC of laminin α1 in an adenoma/MIN-stage lesion consisting of a leukocytic infiltration site. Note that in contrast to the adjacent acini, which are surrounded by laminin α1-positive basement membrane (arrows), the acina adjacent to the leukocytic infiltration site has no positive staining of laminin α (open arrow). H: H&E staining of a human proliferative breast lesion, DHF. The arrow points to infiltrated leukocytes adjacent to the lesion. The inset in H is shown in I. Scale bars: 100 μm (A); 10 (B–F, I); 40 μm (G); 25 (H). Original magnifications: ×100 (A); ×1000 (B, C, D, E, F, I); ×250 (G); ×400 (H).

Figure 3.

Early carcinoma in PyMT mice is comparable to human ductal carcinoma in situ with early invasion (DCIS+EI). A: H&E staining of mammary glands from a PyMT mouse at 9 weeks of age. The inset of A is shown in B. C and D: IHC of laminin α1 in a PyMT mammary tumor at the early carcinoma stage. Note that in C, the positive staining of laminin α1 is located in the periphery (arrows) but not in the center the lesion (open arrows). D: A scattered staining of laminin α1 in the center of a tumor at the early carcinoma stage. E: H&E staining of a early carcinoma showing high density of leukocytic infiltration at the vicinity of the lesion. Open arrows point to the leukocytes. The inset in E is shown in F. Long arrows point to the granulocytes and open green arrows point to blood vessels. G: H&E staining of human proliferative lesion, DCIS+EI. Arrows point to the microinvasions in the tumor stroma. The inset of G is shown in H. Note the nuclear pleomorphism in the area. I: H&E staining of human DCIS+EI showing high density of leukocytic infiltration (arrows). Scale bars: 100 μm (A); 10 μm (B, D, F, H); 25 μm (C, E, G, I). Original magnifications: × 100 (A); ×1000 (B, D, F, H); ×400 (C, E, G, I).

Figure 4.

The late carcinoma in PyMT mice is comparable with human invasive ductal carcinoma. A and B: H&E staining of mammary sections from a PyMT mouse at 15 weeks of age. Note that tumor consists mainly of a solid sheet of cells. Arrows point to the areas adjacent to the primary tumor consisting of early-stage tumor acini. The inset in A is shown in B. C: IHC of laminin α1 in a tumor at late carcinoma stage. Arrows point to positively stained duct in the tumor. D: H&E staining of a human invasive carcinoma. The inset in D is shown in E. Note the pleomorphic nuclei in the section. The arrow in D points to a leukocytic infiltration site, which is shown in F. Scale bars: 100 μm (A, D); 10 μm (B); 25 μm (C, E, F). Original magnifications: ×100 (A, D); ×1000 (B); ×400 (C, E, F).

Figure 5.

Loss of ER and PR during tumor progression to malignancy in the PyMT model. A to L: IHC of ER-α. Positive cells are stained in brown. A: Hyperplastic lesion from a PyMT mouse at 4 weeks of age. The inset in A is shown in B. Arrows point to some of the ER-positive cells. C: TEB adjacent of the hyperplastic lesion shown in A. The neck of the TEB (inset) is shown in D with higher magnification. Arrows point to ER-α-positive stromal cells and open arrows point to ER-α-positive epithelial cells in the TEB. E: ER-α staining of a primary mammary lesion at adenoma/MIN stage from a PyMT mouse at 8 weeks of age. F: A morphologically normal duct adjacent to the primary lesion shown in E. Arrows point to some of the ER-α-positive cells. G to I: Mosaic staining of ER-α in tumor at the early carcinoma stage. G: A tumor section from a PyMT mouse at 14 weeks of age. The inset H is in an area with high density of ER-α-positive cells and shown in H. Inset I is in an area with low-density staining and shown in I. J: A representative staining of ER-α in primary tumor at the late carcinoma stage (18 weeks of age). K: An area from the same tumor consisting of a limited number of ER-α-positive cells (arrow). The inset in K is shown in L with higher magnification. Arrows point to the positively stained cells. M to P: IHC of PR in mammary tumors from PyMT mice. M: A representative staining of PR in the hyperplastic lesion of a PyMT mouse at 6 weeks of age. Arrows point to some of the positively stained cells. N: Staining in an adenoma/MIN from a PyMT mouse at 8 weeks of age. Arrows point to some PR-positive cells. O: A representative PR staining of tumor at the late carcinoma stage. The tumor is from a PyMT mouse at 15 weeks of age and arrows point to PR-positive cells in a residual duct. P: Mammary section from a PyMT mouse at 8 weeks of age showing a representative staining of PR in morphologically normal mammary duct. Scale bars: 25 μm (A, C, K, M, O, P); 10 μm (B, D, E, F, H, I, J, L, N); 100 μm (G). Original magnifications: ×100 (G); ×1000 (B, D, E, F, H, I, J, L, N); ×400 (A, C, K, M, O, P).

Figure 6.

The expression of Neu increases during tumor progression to malignancy. IHC of Neu in mammary gland section of PyMT mice at various ages. A: A representative staining of Neu in a mammary gland carrying the primary lesion at the hyperplasia stage (4 to 5 weeks of age). B: An adjacent morphologically normal duct. C: Neu staining of a mammary gland with a primary lesion at the adenoma/MIN stage (6 weeks of age) and its duct (D). E: A representative staining of Neu in a mammary gland carrying a primary tumor at the late carcinoma stage (13 weeks of age) and its adjacent normal duct, F. Arrows point to some of the positively stained areas. Scale bars, 10 μm.

Figure 7.

Increased expression of cyclin D1 during tumor progression to malignancy. A and B: Representative IHC of cyclin D1 in a PyMT-induced mouse mammary tumor at the adenoma/MIN stage (6 weeks of age). A: Positively stained cells are mainly found as a rim in the vicinity of the lesion. The inset in A is shown in B. Arrows point to cyclin D1-positive cells and open arrows point to adjacent acini that consist of mainly negatively stained cells. C and D: Cyclin D1 staining of an adjacent TEB and duct. Arrows point to positively stained cells. E: Cyclin D1 staining of a mammary tumor at the early carcinoma stage (9 weeks of age). The inset in E is shown in F. Arrows point to some of the positively stained cells. G and H: Staining of a primary tumor at the late carcinoma stage. The inset in G is shown in H. I: A mammary gland section stained with secondary antibody only as a negative control for the cyclin D1 IHC. Scale bars: 25 μm (A, C, E, G, I); 10 μm (B, D, F, H). Original magnifications: ×400 (A, C, E, G, I); ×1000 (B, D, F, H).

Figure 8.

Lost expression of integrin-β1 in PyMT-induced lesions. A to C: IHC of integrin-β1 in mammary gland of a PyMT mouse at 4 weeks of age. A: Staining of primary lesion. Note that the positive staining is in the baseline of a morphologically normal duct (arrows) but no such staining is in the adjacent hyperplastic lesion (open arrows). The inset in A is shown in B. Arrows point to the positively stained areas and open arrows point to negatively stained adjacent hyperplastic lesions. C: The staining of a morphologically normal duct in the same mammary gland shown in A. Arrows point to positively stained areas. D and E: Staining for integrin-β1 of a PyMT-induced mammary tumor at late carcinoma stage. D: The primary tumor. Arrows point to the center of the tumor with negative staining of integrin-β1. E: Staining of secondary lesions in the same mammary gland. Arrows point to the positive stained area. Scale bars: 25 μm (A, C); 10 μm (B, D, E). Original magnifications: ×400 (A, C); ×1000 (B, D, E).

Figure 9.

Western blot analysis of biomarker expression in mammary gland and tumor. A: Western blot analysis of Neu and cyclin D1 expression. Lanes 1 to 4, normal mammary glands from mice at 5, 9, 12, and 18 weeks of age, respectively. Lanes 5 to 7, mammary tumors at adenoma/MIN stage from three individual PyMT mice at 6, 6, and 10 weeks of age, respectively. Lanes 8 to 10, mammary tumors at early carcinoma stage isolated from three individual PyMT mice at 14 weeks of age. Lanes 11 to 13, mammary tumors at late carcinoma stage from PyMT mice at 18, 18, and 15 weeks of age, respectively. Ninety-six-kd Neu and 36-kd cyclin D1 bands are indicated. In this experiment, β-tubulin expression is used as the protein loading control. B: Western blot analysis of ER-α and PR in PyMT tumor and normal mammary glands. Uterus (lane 1) and mammary gland (lane 2) from an ovariectomized mouse treated with estrogen and progesterone for 2 days (16 weeks of age). Lanes 3 to 5, mammary glands from normal mice at 9, 12, and 18 weeks of age, respectively. Lanes 6 to 8, mammary tumors from PyMT mice at 10 (adenoma/MIN, lane 6), 14 (early carcinoma, lane 7), and 15 weeks of age (late carcinoma, lane 8).

Figure 10.

Summary of tumor progression and biomarker expression in PyMT mouse model of breast cancer. Top: Gross, displays the overall development of lesions in mammary glands of PyMT mice. Tumor lesions are indicated by blue dots. The H&E panel displays the corresponding histology of primary lesions at different stages of tumor progression. The cellular morphology panel schematically illustrates changes in the cytology of the cells as well as the integrity of the basement membrane and the presence or absence of myoepithelial and focal inflammation. Moreover, the changes in biomarkers during tumor progression is summarized in the panel of biomarkers. T/D, the ratio of Neu expression between lesions and normal ducts in age-matched mammary glands.