P5-05-02: Phenotypic Plasticity in the Normal Breast.

Background: The cell of origin of metaplastic carcinoma of the breast (MCB) is an enigma. MCB comprises less than 4% of all breast cancers, and is a member of the subtype of breast cancer referred to as Triple Negative Breast Cancer (TNBC). It is aggressive with a prognosis worse than that that for invasive ductal carcinoma NOS (not otherwise specified), as well as other TNBCs. Identification of the histogenesis of MCB is likely to provide clues to its oncogenesis. Differentiation along non-epithelial lineages is also observed in benign lesions of the breast. Adenomyoepitheliomas, thought to arise from myoepithelial cells, have been observed to contain areas of cartilaginous, sebaceous, squamous, and osseous differentiation. Methods: Healthy woman volunteers from central Indiana were invited to donate breast tissue to the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center. Starting from the 10 gauge tissue cores, 28 normal mammary epithelial (HME) and 33 normal stromal (HMS) cell lines were established using an organoid isolation method after digestion with enzymes for 24 hours. The HME cell lines were characterized by immunohistochemistry (IHC). Ploidy was determined by karyotype and Interphase FISH mapping with centromere probes for Chromosomes X and 17. Cellular morphology was observed both on two-dimensional and in three-dimensional culture systems. The HME cells were subjected to FACS analysis using multiple antibodies including CD24, CD44, Muc1, CD49f, and EpCAM. Results: 96.9% of early passage cells are diploid. The HME cells express vimentin, CK 5/6, p63, CD 10, CK 18, and HER-1 when grown on two dimensional plastic surfaces. Cells placed in the center of a sandwich of Matrigel® uniformly form spheres 37mm-325mm in diameter. Hematoxylin and eosin staining of the formalin-fixed and paraffin-embedded sections of these spheres reveal keratinized squamous differentiation. When the cells are grown on Laminin, Collagen IV, or Fibronectin surfaces multiple cell types are observed including osteoclasts, distinguished by the presence of Tartrate Resistant Acid Phospatase; and chondrocytes, confirmed by staining with Alcian Blue. Other cells with a spindle-shape and cytoplasmic vacuoles turn a dark reddish-brown color when stained with Oil Red O, characteristics of adipocytes. In other areas of the culture, the cells form a syncitium and they express the protein MyoD, a marker of immature muscle. Finally, there are numerous cells with long, dendritic processes. These cells express Nestin, a marker of neural stem cells; glial fibrillary acidic protein (GFAP), expressed by mature astrocytes; and beta-III tubulin produced by differentiated neurons. Using FACS, the HME cells were found to be CD49f positive and EpCAM negative. Multiple nucleoli were confirmed using anti-Nucleostemin IHC. Conclusions: Phenotypic plasticity is common to all the HME cell lines characterized to date. Differentiation into cells of mesodermal and ectodermal origin, CD49+/EpCAM- by FACS, and the presence of multiple nucleoli suggest that the isolated cells are a multipotent/stem cell residing in the normal adult breast. These cells, through a series of yet to be elucidated events, may be the cells of origin of MCB. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-05-02.