Functional and molecular characterisation of mammary side population cells

Abstract

Background: Breast cancer is thought to arise in mammary epithelial stem cells. However, the identity of these stem cells is unknown.

Methods: Studies in the haematopoetic and muscle systems show that stem cells have the ability to efflux the dye Hoechst 33342. Cells with this phenotype are referred to as the side population (SP). We have adapted the techniques from the haematopoetic and muscle systems to look for a mammary epithelial SP.

Results: Of mammary epithelial cells isolated from both the human and mouse mammary epithelia, 0.2-0.45% formed a distinct SP. The SP was relatively undifferentiated but grew as typical differentiated epithelial clones when cultured. Transplantation of murine SP cells at limiting dilution into cleared mammary fat pads generated epithelial ductal and lobuloalveolar structures.

Conclusion: These data demonstrate the existence of an undifferentiated SP in human and murine mammary epithelium. Purified SP cells are a live single-cell population that retain the ability to differentiate in vitro and in vivo. Studies of haematopoetic cells have suggested that the SP phenotype constitutes a universal stem cell marker. This work therefore has implications for mammary stem cell biology.

Figure 1

Flow-cytometric analysis of human breast epithelial cells stained with 5 μM Hoechst 33342. (a) Trace demonstrating the presence of a side population (box). (b) Addition of 20 μM verapamil (+v), resulting in a 12-fold reduction in the side population. X axis, Hoechst red fluorescence intensity (FL5); Y axis, Hoechst blue fluorescence intensity (FL4).

Figure 2

Analysis of mouse side population (SP) cells. Flow cytometric analysis of mouse cells stained with Hoechst 33342: (a) SP in mouse mammary epithelial cells (box), (b) SP in mouse bone marrow cells. X axis, Hoechst red fluorescence intensity (FL5); Y axis, Hoechst blue fluorescence intensity (FL4). (c) RT-PCR analysis of ABC transporter cassette proteins in SP and non-SP cells from mouse mammary epithelial cells. M, molecular weight marker; Brcp1, breast cancer resistance protein; Mrp, multidrug resistance-associated protein. Sizes of bands: Brcp1, 327 bp; Mrp1, 701 bp; Mrp3, 301 bp; Mrp4, 222 bp.

Figure 3

Histology and immunocytochemistry of two representative side population (SP) outgrowths. (a), (b) Wholemount morphology of carmine-stained outgrowths from transplanted mouse mammary side population cells. Lobuloalveolar structures indicated by dashed outlines. Bar = 750 μm. (c), (d) H & E-stained section of SP outgrowth. (e), (f) Immunocytochemical staining of the section of SP outgrowth for the myoepithelial cell marker α-smooth muscle actin. (g), (h) Immunohistochemical staining of the section of SP outgrowth for the luminal epithelial cell marker cytokeratin 19. (c)–(h) Bar = 350 μm.