Generation of Breast Cancer Stem Cells through Epithelial-Mesenchymal Transition

Recently, two novel concepts have emerged in cancer biology: the role of so-called “cancer stem cells” in tumor initiation, and the involvement of an epithelial-mesenchymal transition (EMT) in the metastatic dissemination of epithelial cancer cells. Using a mammary tumor progression model, we show that cells possessing both stem and tumorigenic characteristics of “cancer stem cells” can be derived from human mammary epithelial cells following the activation of the Ras-MAPK pathway. The acquisition of these stem and tumorigenic characters is driven by EMT induction

CD24⁻ cells display tumorigenic and stem-like properties.

<p>(A) Colony assay. Growth in soft agar. Number of colonies are indicated for 5×10³ plated cells (+/− standard deviation, n = 3, 40x magnification). (B) Tumorigenicity assay. Athymic <i>nude</i> mice received a single injection of 10⁶ CD24⁺ or CD24⁻ cells to a mammary fat pad. Tumor growth was monitored every 3 days and volume measured every 15 days. Blue lines indicate CD24⁻ cells and red lines indicate CD24⁺ cells, marker shapes represent duplicate injections in separate animals from three independent clones for both cell populations. (C) In contrast to CD24⁺ cells, CD24⁻ cells formed mammospheres in low-adherent conditions. Scale bars = 100 µm.</p

Characterization of the different steps of the <i>in vitro</i> model of HMEC transformation.

<p>(A) Schematic representation of the successive steps of the transformation. (B,C,D,E) Evaluation of the ability of HMEC-derived cell lines to grow as non-adherent mammospheres. Scale bars = 100 µm. FACS analysis of CD24 and CD44 markers in HMEC-derived cell lines (F,G,H,I). B and F: Primary Human Mammary Epithelial Cell, (HMECs); C and G: hTERT- immortalized HMECs (HME); D and H: HME expressing SV40 small t and large T antigens (HMLE); E and I: H-Ras^V12-infected HMLE cells (HMLER).</p

Oncogenic versions of Ras promote a CD24⁺ to CD24⁻ transition and EMT in MCF10A and HMLE cell lines.

<p>(A) FACS analysis of CD24 and CD44 markers in MCF10A cells infected either with a K-Ras^V12 retroviral construct or the corresponding empty vector as a control. (B) Cell morphology of MCF10A cells infected either with a K-Ras^V12 retroviral construct or the corresponding empty vector as a control (upper panel) and HMLE infected either with a H-Ras^V12 retroviral construct or the corresponding empty vector as a control (lower panel). Images shown at 40x magnification. (C) CD24⁺ and CD24⁻ display epithelial and mesenchymal features, respectively. Top: Cell morphology. Bottom: Expression analysis as assessed by western-blotting of epithelial (E-cadherin, β-catenin) and mesenchymal (vimentin, fibronectin) markers in two independent clones for both cell populations. Images shown at 40x magnification.</p

Oncogenic Ras and TGFβ1 cooperate to promote the CD24⁺ to CD24⁻ cells.

<p>HMLE cells were infected with an H-Ras^V12 retroviral expression construct or the empty vector (pBabe) as a control. Two days post-infection, experimental cells were treated with TGFβ1. Percentage of CD24⁻ cells was assessed at different times following infection. Error bars indicate +/− standard deviation of triplicates.</p