Table 3 AMD3100 significantly inhibited MFE and cell number when

Table 3 AMD3100 significantly inhibited MFE and cell number when cocultured with different stromal fibroblasts Culture Condition MFE (%) Cell Number (× 105) Monoculture 1.6 ± 0.1 0.22 ±

0.07 Mammosphere + CAFs 2.3 ± 0.2 0.43 ± 0.14 Mammosphere + NFs 1.5 ± 0.2 0.28 ± 0.08 *P < 0.01 compared with no treatment of AMD3100. Figure 6 Mammosphere cells were cocultured with different stromal fibroblasts with the administration of AMD3100 selleck inhibitor and flow cytometry was used to measure CD44 and CD24 expression. (A) Mammosphere cells were cocultured with different stromal fibroblasts with the administration of AMD3100 (1 μg/ml) for six days. As a result, MFE in monoculture mammosphere cells (left), cocultured mammosphere cells with CAFs (middle) and NFs (right) was significantly reduced to (1.6 ± 0.1%), (2.3 ± 0.2%) and (1.5 ± 0.2%), respectively. (B) Flow cytometry analysis was used to measure CD44 and CD24 expression of cells derived from mammosphere cells. The expression of CD44+CD24- in monoculture mammosphere cells (left), cocultured mammosphere cells with stromal CAFs (middle) and NFs (right) was (2.2 ± 0.3%), (4.4 ± 0.8%) and (2.7 ± 0.3%), respectively. The data were provided as the mean ± SD. Each experiment was performed three times. Discussion Mammosphere culture system is now widely used for stem cell

culture. Dontu and his colleagues had developed an in vitro cultivation system that allowed for the proliferation of undifferentiated human mammary epithelial cells in suspension. When cultured on nonadherent surfaces in the presence of growth factors, nonadherent mammospheres were enriched in cells with functional S63845 clinical trial characteristics of stem/progenitor cells [18]. Another study also showed that breast tumorigenic cells with self-renewal could be propagated in vitro as nonadherent mammospheres [7]. Consistent with the above reports, our study shows that mammosphere cells could be cultured in suspension and generate BCSCs with the CD44+CD24- phenotype. Thus, long-term cultures of mammosphere in vitro may represent a suitable model to study BCSCs. Stem Montelukast Sodium cell properties in normal and malignant tissues are tightly regulated

by the Wnt, Shh and Notch signaling pathways [19–21]. Notch signaling has been implicated in the regulation of cell-fate decisions such as self-renewal of adult stem cells and selleckchem differentiation of progenitor cells along a particular lineage. Dontu and his colleagues demonstrated that Notch activation promoted mammary stem cell self-renewal, but modulation of this pathway had no significant effect on differentiated mammary epithelial cells [20]. In breast cancers, it was found that BCSCs preferentially expressed some “”stemness”" genes, including Notch1 and β-catenin [18]. Our qRT-PCR analysis obtained the similar result that Notch2 and β-catenin were expressed at higher levels in mammosphere cells than in monolayer cells, suggesting that Notch2 and β-catenin are involved in BCSC regulation.

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