Evidence of progenitor cells of glandular and myoepithelial cell lineages in the human adult female breast epithelium: a new progenitor (adult stem) cell concept

Abstract

Although experimental data clearly confirm the existence of self-renewing mammary stem cells, the characteristics of such progenitor cells have never been satisfactorily defined. Using a double immunofluorescence technique for simultaneous detection of the basal cytokeratin 5, the glandular cytokeratins 8/18 and the myoepithelial differentiation marker smooth muscle actin (SMA), we were able to demonstrate the presence of CK5+ cells in human adult breast epithelium. These cells have the potential to differentiate to either glandular (CK8/18+) or myoepithelial cells (SMA+) through intermediary cells (CK5+ and CK8/18+ or SMA+). We therefore proceeded on the assumption that the CK5+ cells are phenotypically and behaviourally progenitor (committed adult stem) cells of human breast epithelium. Furthermore, we furnish evidence that most of these progenitor cells are located in the luminal epithelium of the ductal lobular tree. Based on data obtained in extensive analyses of proliferative breast disease lesions, we have come to regard usual ductal hyperplasia as a progenitor cell-derived lesion, whereas most breast cancers seem to evolve from differentiated glandular cells. Double immunofluorescence experiments provide a new tool to characterize phenotypically progenitor (adult stem) cells and their progenies. This model has been shown to be of great value for a better understanding not only of normal tissue regeneration but also of proliferative breast disease. Furthermore, this model provides a new tool for unravelling further the regulatory mechanisms that govern normal and pathological cell growth.

Figure 1

Normal breast epithelium. (a) Small duct and adjacent lobule. Note that the normal epithelium consists of a bilayer with luminal glandular and basal myoepithelial cells (haematoxylin and eosin). (b) Double‐fluorescence staining of the epithelium of a small duct for CK5 (FITC, green) and CK8/18/19 (Cy3, red) displaying few CK5⁺ progenitor cells (green signal), intermediary cells (hybrid signal) and CK8/18⁺ glandular cells (red signal). (c) Double‐fluorescence staining of the epithelium of two terminal ducts and adjacent lobules of a resting breast for CK5 (green signal for FITC) and CK8/18 (Cy3, red) revealing the same cellular components as seen in the small duct in (b), indicating a relatively immature glandular epithelium. (d) Double‐fluorescence staining of the epithelium of a small duct and adjacent lobule of a resting breast for CK5 (FITC, green) and SMA (Cy3, red). The arrow marks a progenitor cell expressing CK5 alone. Note that most cells in the outer layer represent intermediary myoepithelial cells expressing both CK5 and SMA (hybrid colour). The differentiated myoepithelial express only SMA (green signal). Note also that the inner epithelium of both duct and of two acini for CK5 (FITC, green) and SMA (Cy3, red) contain precursor cells. (e) Double‐fluorescence staining of the epithelium of lobular acini (red signal) Panels for SMA (FITC, green) and CK8/18 (Cy3, red) clearly showing a glandular and myoepithelial cell lineage. Note the absence of any transitional cells, indicating that there is no transdifferentiation between those two lineages.

Figure Figur 2

HRP immunohistochemical labelling of CK5 immunohistochemistry of a duct and several adjacent lobules of normal resting breast. Note that the inner epithelium of the duct and of the lobules on the right‐hand side stain intensively, indicating that this epithelium is immature and contains many putative CK5⁺ (precursor?) cells. In contrast, the lobule on the left is not stained at all. This epithelium has lost CK5.

Figure 3

Lactating mammary‐gland epithelium compared with epithelium of normal resting breast. Double‐labelling for CK5 and CK8/18 of terminal ducts (ID) and adjacent lobules. The lobules contain differentiated CK8/18⁺ cells only, in contrast to the normal breast, as shown in Fig. 1(c).

Figure 4

Usual ductal hyperplasia and ductal carcinoma in situ. Double‐fluorescence staining for CK5 (greenl) and CK8/18 (red). Note that UDH (a) contains the whole spectrum of cells, including CK5⁺ progenitor (green signal), intermediary glandular cell (hybrid signal) and differentiated glandular cells (red signal), whereas DCIS (b) displays a purely glandular phenotype (red). (c) Western blotting of breast tissue preparations and cultured cells. Lane 1, A‐431 cells (total cellular proteins); lane 2, A‐549 cells (total cellular proteins); lane 3, normal breast tissue (microdissected terminal ducts and lobular units); lane 4 and 5, usual ductal hyperplasia and DCIS (microdissected ductal lesions). Western blotting immunoreaction with anti‐CK5 and anti‐CK8. Note the appearance of CK5 and CK8 bands in preparations of normal breast epithelium and of UDH (lanes 3 and 4). The CK8 band occurs in preparations from DCIS only.

Figure 5

A new progenitor cell concept based on our fluorescence immunolabelling and Western blotting findings. CK5⁺ progenitor cells (yellow) give rise to both glandular cells (CK8/18⁺ green) and myoepithelial cells (SMA⁺ red) via intermediary cells that coexpress CK5 with the lineage‐specific marker (either CK8/18⁺ or SMA⁺).

Figure 6

Architectural distribution of the cells of the new progenitor cell model in the normal resting breast epithelium.