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Did you struggle to get access to this article? This product could help you. Accessing resources off campus can be a challenge. Lean Library can solve it. If you have the appropriate software installed, you can download article citation data to the citation manager of your choice.

Simply select your manager software from the list below and click on download. The e-mail addresses that you supply to use this service will not be used for any other purpose without your consent. Create a link to share a read only version of this article with your colleagues and friends. Please read and accept the terms and conditions and check the box to generate a sharing link. Osteopontin OPN Spp1 is involved in differentiation of the mammary gland. We engineered mice to overexpress OPN in mammary epithelium and describe an altered mammary phenotype.

Mammary glands from Tg-OPN mice compared to littermate controls showed, at 4 weeks of age, exaggerated terminal end buds; at 8 and 12 weeks, more numerous and complex ducts with increased luminal protein; and at 16 weeks, increased lobulogenesis. Lactational Tg-OPN mammary glands showed more rapid lobulogenesis and lactational changes with slower gland involution and regression following weaning. Ex vivo lobulogenesis was noticeably increased from organoids of Tg-OPN mice.

OPN appears to convey a proliferative stimulus for mammary epithelial cells and alters development and differentiation. The mechanisms involved in dysregulation of mammary gland growth, differentiation, and regression leading to mammary epithelial cell MEC hyperplasia and tumorigenesis are unknown but probably involve the aberrant expression of key genes.

Elucidating why normal MEC proliferation becomes aberrant remains a challenge in the study of breast cancer, but experiments using human derived cell lines have yielded some important findings. Using model systems such as genetically engineered mice provides an in vivo setting to help interrogate the involvement of specific genes in the context and complexity of the mammary gland and its development. Pregnancy induces hormonal changes that cause alveolar cell proliferation and differentiation, resulting in milk production and lactation. While there are many genes and mechanisms involved in MEC proliferation and differentiation, choosing those that have been shown to also play a major role in breast tumorigenesis and overexpressing them in normal tissue could be a good strategy for correlating changes during mammary gland development and differentiation with a preneoplastic phenotype.

OPN has also been shown to alter many aspects of tumorigenesis, especially metastasis. In a recent study, mutant BRCA1 elicited changes involved in metastatic progression in human breast cancer cells via the overexpression of OPN. Also with respect to tumorigenesis, elevated levels of OPN could be observed in tumor cells and surrounding stroma of numerous human cancers and has been correlated with malignant invasion. Here, we describe phenotypic changes in mouse mammary gland development and lactational differentiation as a result of continuous expression of OPN in MEC.

Continuous OPN expression led to increased in vivo and ex vivo mammary gland and organoid lobulogenesis, respectively, with persistent alveolar development and incomplete regression with persistent proliferation. Though continuous OPN expression alone in the context of the developing and differentiating mammary gland may not necessarily confer a frank malignant phenotype, future studies with bitransgenic or polytransgenic mice involving OPN overexpression may be an important strategy for understanding the complexity of advancement to preneoplasia and malignancy.

OPN may be involved in acquisition of invasiveness, a critical step in early stage breast carcinomas. Of 20 live births that carried the transgene, 3 were successfully bred and passed the transgene germ line. Copy analysis showed tandem repeat s of 45, , and of the Opn gene. Eight-week-old nulliparous mammary phenotypes varied between founder lines, but the 2 founder lines with the higher copy s and had similar phenotypes. The low copy founder line had a more subtle phenotype but wild type WT , low copy , and high copy phenotypes could be distinguished in blinded histologic and microscopic review of hematoxylin-eosin-stained sections.

To examine the effect of continuous OPN expression on mammary gland development, nulliparous mice were sacrificed at various time points throughout development. Proliferation and side budding can be seen during the estrus phase, but in the Tg-OPN mice, these changes were seen regardless of stage, suggesting activation of developmental proliferation pathways similar to the estrogen stimulation normally seen in the estrus cycle. The persistence through the estrus cycle also raised the question of a block in regression als. In the OPN transgenic gland there was luminal secretion as well as cytoplasmic excess accumulation.

Proliferation as assessed by immunohistochemistry for Ki67 reveals areas with proliferation exceeding any seen in WT mice Fig. Staining heterogeneity is also consistent with the patchy proliferative phenotype observed in the whole mounts. Figure 1. Tg-OPN transgenic osteopontin alteration of mammary gland structure. Mammary gland 4 whole mounts A from 8-week-old age-matched nulliparous female wild type WT and Tg-OPN littermates were photographed for quantitative image analysis and compared. Figure 2. Quantitation of transgenic osteopontin Tg-OPN duct branching and thickness.

Quantitation was performed using an original unprocessed image of duct growth A. In B , blue dots represent branch points, red line represents duct skeleton, and green lines represents duct outline. The white outline is the area used by AngioTool to find the duct structure. Mammary gland 4 whole mounts from 6-week-old age-matched nulliparious female wild type WT and Tg-OPN littermates were photographed for qualitative image analysis and compared quantitatively for junctions per millimeter C , D and thickness E , F.

Figure 3. Immunohistochemistry against murine OPN top panels shows the abundant expression of OPN in normal mammary glands with secretion into the ductal lumen arrows. In the OPN transgenic gland, there is luminal secretion arrows as well as cytoplasmic excess accumulation arrowhe. Proliferation as assessed by immunohistochemistry for Ki67 middle panels reveals areas with proliferation exceeding any seen in wild type mice. In the representative areas there is one positive nucleus in the wild type arrowhead , whereas in the Tg-OPN there are many positive nuclei arrowhe mark only some of the positive cell nuclei.

In relatively young 4 weeks of age mammary glands, Tg-OPN mice appeared to have exaggerated TEBs when compared to littermate controls not expressing the transgene Fig. In some TEBs, excessive luminal degraded cells can be seen inset , possibly representing secondary remodeling via anoikis. Figure 4. Osteopontin OPN -induced epithelial hyperplasia in young nulliparous mammary gland. More rapid lobulogenesis was apparent in pregnant Tg-OPN mice compared to littermate controls to the extent that there appeared to be little ductal extension mostly alveolar in Tg-OPN mice Fig. In addition, lactational changes were noted in the transgenic mammary gland.

With respect to regression, Tg-OPN mice demonstrated slower gland involution and regression of lactational changes following pregnancy when compared to littermate controls Fig. Figure 5. Osteopontin OPN -induced proliferation in prelactating mammary gland. Figure 6. Osteopontin OPN mammary epithelial cell overexpression induces mammary gland persistence long after pregnancy.

Five months postpartum PP , wild type WT gland top 2 panels has completely regressed compared to transgenic Tg -OPN gland 4 months after a second pregnancy. Areas in the Tg-OPN mammary glands appeared prelactating middle 2 panels persistently lactating lower left panel and persistently acinar lower right. Middle right panel shows persistent milk production M in the transgenic mouse.

Parous OPN overexpressing animals given a normative time to regress at least 3 months had areas in the mammary glands that appeared prelactating, persistently lactating, and persistently acinar Fig. Lipid production was still apparent. Since we have observed increased mammary gland lobulogenesis with persistent alveolar development and incomplete regression with persistent proliferation, we hypothesized that the effect of continuous OPN expression in the mouse mammary epithelium may be related to changes in MEC populations, including stem and progenitor cells.

Figure 7. The precocious lobulogenesis observed in vivo was also observed repetitively in an ex vivo lobulogenesis assay. Figure 8. Transgenic osteopontin Tg-OPN induces ex vivo lobulogenesis. Organoids were measured and placed into 1 of 6 Suppl. The percentage of total organoids for each category and the mean size are depicted in A , B , C , and D.

Osteopontin is required for normal physiologic function but has also been shown to play a role in cancer progression and other diseases. Its contribution to tumor growth, angiogenesis, and metastasis has been vigorously studied, but a role for OPN in induction of neoplasia needs further attention.

These would form a basis for future studies by defining the phenotypic changes in mammary gland and comparing those changes with what may occur in gland predisposed to neoplasia. Regulated expression of OPN is essential in normal mammary gland development, but when dysregulated, OPN can play a role in mammary tumorigenesis.

The proliferation stimulus provided by OPN is unknown but led to larger TEBs in early development compared to wild type, more numerous TEBs, and larger ducts with more numerous side branching as development progressed and increased lobulogenesis at later time points. Continuous OPN expression also led to observable mammary gland changes in pregnancy and lactation and appeared to drive persistence with slower and especially incomplete involution. In a study where OPN expression was suppressed in the mammary epithelium, development of normal lobuloalveolar structures was ificantly decreased with only few, sparsely distributed individual patches throughout the fat pad leading to a virtual lack of milk synthesis in lactating animals.

Indeed, OPN expression has been shown to vary with low to moderate levels of expression in the nulliparous and pregnant glands but extremely high levels in the lactating gland, 7 so timing of expression is critical as well. It is not surprising that OPN expression is crucial for mammary gland development and can cause major changes if dysregulated based on its role in cell migration, invasion, and survival, critical steps for developing epithelial tissues embedded in a stroma.

This phenotype is also associated with a shift toward EMT and greater tissue plasticity. EMT is also important in mouse and human mammary tumors. After reimplantation into pseudopregnant female mice, there were 20 live births that carried the transgene, 3 of which were successfully bred and passed the transgene germ line.

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Emergent Complications of Assisted Reproduction: Expecting the Unexpected