Stem-like/CD44^high/CD24^low cells isolated from HMLE cells exhibit attributes of cells that have undergone an EMT (A) Phase-contrast images (left) and immunofluorescence images of CD44^high/CD24^low and CD44^low/CD24^high cells stained using antibodies against E-cadherin, fibronectin or vimentin (right panels). (B) The expression levels of the mRNAs encoding E-cadherin, N-cadherin, vimentin, fibronectin, FOXC2, Slug, SIP1, Twist and Snail in CD44^high/CD24^low cells relative to CD44^low/CD24^high as determined by Real-time RT-PCR. GAPDH mRNA was used to normalize the variability in template loading. The data are reported as mean +/− SEM.

CD44^high/CD24^low cells exhibit many properties of stem cells. (A) CD44^high/CD24^low cells and CD44^low/CD24^high cells were separated by FACS. (B) Phase-contrast images of mammospheres seeded by CD44^high/CD24^low (top) and CD44^low/CD24^high (bottom) cells (C) Quantification of mammospheres formed by cells from the sorted CD44^high/CD24^low and CD44^low/CD24^high populations. The data are reported as mean +/− SEM (P<0.001). (D) Images of mammospheres immunostained with antibodies against basal cytokeratin 14 (K14, red) and luminal cytokeratin 8 (K8, green, top panel) or cytokeratin 18 (K18, green, bottom panel). In the merged images (right), the arrows indicate K8/K14 or K18/K14 double-positive cells (yellow). (E) Images of single-cell clones derived from CD44^high/CD24^low cells (left) or CD44^low/CD24^high cells (right) stained using antibodies against K14 (red) and K18 (green). The arrows indicate K14/K18 double-positive cells (yellow).

EMT induces phenotypes associated with cancer stem cells. (A) Phase-contrast images of NeuNT-Snail-ER, NeuNT-Twist-ER and NeuNT-control vector cells treated with tamoxifen for a period of 10 days as well as images of untreated cells. (B) Western blot analysis of expression of HER2/neu, E-cadherin, fibronectin, and vimentin proteins in the cells shown in panel A. β-actin was used as a loading control. (C) Quantification of the mammospheres seeded by NeuNT-Snail-ER, NeuNT-Twist-ER or NeuNT-control vector cells treated or not treated with tamoxifen for 10 days. The data are reported as mean +/− SD. (D) Images of the colonies formed during soft agar culture of NeuNT-Snail-ER, NeuNT-Twist-ER and NeuNT-control vector cells after being treated with tamoxifen for 10 days. The soft agar assays were performed in the absence of tamoxifen. (E) Quantification of the soft agar colonies shown in panel D. The data are reported as mean +/− SD (** - P<0.01; *** - P<0.001 compared to the control).

The epithelial-mesenchymal transition (EMT) generates cells with properties of stem cells. (A) Phase-contrast images of HMLE cells expressing Snail, Twist or the control vector, as well as HMLE cells treated with recombinant TGF-β1 (2.5 ng/ml) for 12 days (bottom right). (B) Relative expression of the mRNAs encoding E-cadherin, N-cadherin, vimentin, and fibronectin in HMLE cells induced to undergo EMT by the methods outlined in panel A, as determined by Real-time RT-PCR. GAPDH mRNA was used to normalize the variability in template loading. The data are reported as mean +/− SEM). (C) FACS analysis of cell-surface markers, CD44 and CD24, in the cells described in panel A. (D) In vitro quantification of mammospheres formed by cells described in panel A. The data are reported as the number of mammospheres formed/1,000 seeded cells +/− SEM, (* - P < 0.05; *** - P<0.001 comparing to the control).

Primary mouse mammary stem cells, normal human breast stem-like cells, and neoplastic human breast stem-like cells express markers associated with EMT. (A) Primary mouse mammary epithelial cells were separated into CD49f^high/CD24^med and CD49f^high/CD24^med-depleted populations using FACS. (B) A representative image of a cleared mouse mammary fat pad reconstituted using 1000 of CD49f^high/CD24^med cells (top); the same number of CD49f^high/CD24^med-depleted cells failed to reconstitute the fat pad (bottom). (C) Phase-contrast images of CD49f^high/CD24^med and CD49f^high/CD24^med-depleted cells. (D) The expression levels of the mRNAs encoding E-cadherin, N-cadherin, vimentin, Slug and Twist in the CD49f^high/CD24^med relative to CD49f^high/CD24^med-depleted cells, as determined by Real-time RT-PCR. GAPDH mRNA was used to normalize variability in template loading. The data are reported as mean +/− SEM. (E) CD44^high/CD24^low cells (R4) and CD44^low/CD24^high cells (R3) were isolated from human reduction mammoplasty tissues using FACS. (F) The expression levels of the mRNAs encoding E-cadherin, N-cadherin, SIP-1 and FOXC2 in CD44^high/CD24^low cells (R4) relative to CD44^low/CD24^high cells (R3), as determined by Real-time RT-PCR. GAPDH mRNA was used to normalize the variability in template loading. The data are reported as mean +/− SEM. (G) Heat map depicting the expression levels of mRNAs encoding EMT markers in CD44^high/CD24^low cells compared to CD44^low/CD24^high cells, as determined by SAGE analysis. Red and green squares correspond to high and low mRNA levels, respectively.