Loss of FGFR2 Impaired Self-Renewal of Breast TICs, Resulting in a Decreased TIC Pool.

<p>(A) Flow cytometry analysis of CD24 and CD29 expression for TIC and non-TIC subpopulation frequencies in shRNA-transduced primary MMTV-PyMT breast tumor cells. (B) Quantification of TIC and non-TIC subpopulation frequencies in shRNA transduced breast tumor cells determined by FACS analysis (A). Data (n = 2) represent mean ± s.d. Statistical comparison with shNT (*<i>P</i><0.027; **<i>P</i><0.015). (C) Reduced TIC sphere formation (mean ± SEM., n = 2) of breast tumor cells upon FGFR2 knockdown. Ten thousand FACS-sorted TICs were transduced with lentiviral shRNAs and then subjected to a mammosphere (>50 µm in diameter) forming assay. (D) Differentiation potential of shRNA-transduced breast tumor cells as assessed by immunofluorescence. The shRNA-transduced tumor cells were cultured with a selective drug for 7 days after infection and then were plated them at the same cell density under the differentiation condition for 6 days. The cells were stained for the luminal epithelial marker (K18, green), myoepithelial markers (K14 and SMA, red), and DAPI (nuclei, blue). The scale bars for K18/K14 and SMA represent 40 µm and 140 µm, respectively. (E) Quantification of the immunofluorescence images (D) for the frequency of K18⁺K14⁻ (luminal), K18⁻K14⁺ (myoepithelial), K18⁺K14⁺ (bipotent precursor-like), and SMA⁺ (myoepithelial) cells in shRNA transduced tumor cells. Image quantification was achieved using the multi-wavelength cell scoring analysis module on the ImageXpress Ultra confocal microscope. Statistical comparison with mock infection (*<i>P</i><0.05; **<i>P≤</i>0.01; ***<i>P</i><0.0003). Data (n = 3) represent mean ± SEM.