3A). In addition, we used flow cytometry to assess the proportion of BCSCs that has the phenotypic marker of CD44+CD24-, and found that CAFs significantly increased the proportion of CD44+CD24- cells in mammospheres (21.4 ± 1.8% vs. 17.2 ± 2.3%, P < 0.05); while NFs decreased the proportion of CD44+CD24- cells in mammospheres (8.7 ± 0.9% vs. 17.2 ± 2.3%, P < 0.01) (Fig. 3B, and see Additional file 1), which Cabozantinib exhibited similar trend as MFE. These

results suggest that CAFs have positive effects on the generation of CD44+CD24- cells, while NFs have negative effects on CD44+CD24- cell formation. Table 1 Different MFE and cell number when cocultured with different stromal fibroblasts Culture Condition MFE (%) Cell Number (× 105) Monoculture 8.1 ± 0.7 1.51 ± 0.43 Mammosphere + CAFs 13.5 ± 1.2** 3.82 ± 0.41** Mammosphere + NFs 5.2 ± 0.6* 0.65 ± 0.22* *P < 0.05, **P < 0.01 compared with monoculture Figure 3 Mammosphere cells were cocultured with different stromal fibroblasts and flow cytometry was

used to measure CD44 and CD24 expression. (A) Mammosphere cells (1 × 105 cells/dish) cocultured with different stromal fibroblasts (1 × 105 cells/dish) using transwells for six days, and mammosphere cells cocultured with CAFs (middle) had the highest MFE (13.5 ± 1.2%), compared with monoculture mammosphere cells (left) (8.1 ± 0.7%), P < 0.01. (B) Flow cytometry analysis to measure CD44 and CD24 expression of cells derived from monoculture mammosphere cells and cocultured mammosphere ZD1839 cell line cells. The expression of CD44+CD24- in monoculture mammosphere cells (left) was (17.2 ± 2.3%). Compared to monoculture mammosphere cells, the expression of CD44+CD24- in cocultured mammosphere cells with CAFs (middle) was (21.4 ± 1.8%), P < 0.05, and the expression of CD44+CD24- in cocultured mammosphere cells with NFs (right) was (8.7 ± 0.9%), P < 0.01. The data were provided as the mean ± SD. Each experiment was performed three times. CAFs had a positive role on the tumorigenicity of mammosphere

cells To investigate whether altered stromal niche could influence the tumorigenicity in vivo, we evaluated the tumor formation in NOD/SCID mice by inoculation of mammosphere cells with or without CAFs and NFs. The results revealed that inoculation of 1 × 105 mammosphere cells from alone resulted in tumor formation in 60% of mice (3/5), and coinoculation of 1 × 105 mammosphere cells with 1 × 105 CAFs significantly improved tumor formation (5/5). Interestingly, coinoculation of 1 × 105 mammosphere cells with 1 × 105 NFs sharply decreased tumorigenicity, only 20% mice developed tumors (1/5, Table 2). These data strongly suggested that cancer stromal fibroblast significantly promote the tumorigenicity of mammosphere cells. Table 2 Incidence of tumors by coinoculation of mammosphere cells with CAFs and NFs in NOD/SCID mice Cells Inoculated Mammosphere Mammosphere + CAFs Mammosphere + NFs Tumors 3/5 5/5* 1/5* *P < 0.