CD29^highCD24⁺ Cells Have Self-Renewal Capacity and Contain Bipotent Precursor-like Cells.

<p>(A) Mammosphere assay for the four FACS-sorted subpopulations from MMTV-PyMT breast tumors. The number of spheres (>50 µm in diameter) formed from 2000 or 4000 seeded cells was measured 3 weeks after seeding. Statistical comparison with CD29^highCD24⁺ subpopulation (*<i>P</i><0.02; **<i>P</i><0.01). Data (n = 3) represent mean ± SEM. Scale bars in the sphere images represent 50 µm. (B) Differentiation potential of breast tumor subpopulations assessed by immunofluorescence. The breast tumor cells from the four FACS-sorted subpopulations were cultured under the differentiation condition. The sorted cells from various populations were plated at the same cell density on collagen-coated plates. The differentiated cells were stained for the luminal epithelial marker (K18, green), the myoepithelial markers (K14 and SMA, red), and DAPI (nuclei, blue). A significant portion of CD29^highCD24⁺ cells contains K18⁺K14⁺ (bipotent precursor-like), whereas the majority of cells from the other subpopulations contain lineage-restricted cells. Magnifications of the boxed regions are shown below the figure. Scale bars represent 32 µm. (C) Myoepithelial lineage-specific differentiation assessed by immunofluorescence. The differentiated tumor cells from FACS-sorted subpopulations were stained for myoepithelial markers (SMA, red), and DAPI (nuclei, blue). Scale bars represent 120 µm. (D) Quantification of the immunofluorescence images (B and C) for the frequency of K18⁺K14⁻ (luminal), K18⁻K14⁺ (myoepithelial), K18⁺K14⁺ (bipotent precursor-like), and SMA⁺ (myoepithelial) cells. Image quantification was achieved using the multi-wavelength cell scoring analysis module on the ImageXpress Ultra confocal microscope. An average of three values from three images (different positions) per group is presented. Statistical comparison with CD29^highCD24⁺ subpopulation (*<i>P≤</i>0.05; **<i>P≤</i>0.01; ***<i>P</i><0.001). Data (n = 3) represent mean ± SEM.