Figure 1

Phenotypic characterization of single-cell clones obtained from the parental cell line, HMT3909S13, demonstrates G4 as CD44^hi/CD24⁻ with a mesenchymal-like morphology and A4 as CD44^hi/CD24^lo with an epithelial-like morphology capable of forming mammospheres. A4 cells can recapitulate cells similar to the parental cell line in vivo. (A) CD44 and CD24 expression of HMT3909S13 as determined by flow cytometry. The CD44^hi/CD24⁻ G4 and CD44^hi/CD24^lo A4 single-cell clones were obtained from marked gates. G4 exhibits a mesenchymal-like morphology, whereas A4 exhibits an epithelial-like morphology, as determined by phase contrast imaging. (B) Relative CD24 expression of HMT3909S13, A4 and G4 compared with the MDA-MB-231, T47D and MCF7 cell lines, as determined by flow cytometry. HMT3909S13 without primary antibody is shown as a representative negative control. (C) Mammospheres were formed from A4, but not G4, after growth of cells in nonadherent culture flasks at d 7, as depicted by phase contrast imaging. Only loosely adhered clumps of cells were observed in the G4 cell culture flask. Scale bar: 30 µm. (D) ICC analysis of CD24, vimentin, ESA and E-cadherin demonstrating altered expression levels between the three cell lines. Scale bar: 20 µm. (E) IHC staining of primary tumors from immunodeficient mice generated by inoculation of A4 and HMT3909S13 cells, respectively, into the mammary fat pad showing the ability to express the proteins heterogeneously and more similar to the A4 than HMT3909S13 cells in vitro, emphasizing the CSC-like characteristics of A4 clone cells. See also Supplementary Figures S2 and S3 and Supplementary Table S1.