The cocultured medium of primary mammosphere cells with CAFs had higher SDF-1 expression The marked effects of cancer stromal niche promote us to investigate the molecular mechanisms by which CAFs increased the tumorigenicity of mammosphere cells. Recent reports have indicated that SDF-1 boosts the proliferation of several cancer cell lines in culture, including breast carcinoma cells . In order to
determine HSP990 whether SDF-1 involved in the proliferation of CD44+CD24- cells, the production of SDF-1 in mammosphere cultures subject to various treatments were measured by ELISA. The result indicated elevated levels of SDF-1 protein in the medium conditioned by the CAFs as compared with that by mammosphere cells alone (426.4 ± 30.6 pg/ml vs. 283.6 ± 35.1 pg/ml, P < 0.05). In addition, the cocultured medium of mammosphere cells with NFs significantly decreased the production of SDF-1 (52.9. ± 13.1 pg/ml vs. 283.6 ± 35.1 pg/ml, P <0.01) (Fig. 4). These results exhibited the similar trend as MFE, NU7026 solubility dmso generation of CD44+CD24- cells and tumorigenicity of mammosphere cells by CAFs, implying that the elevated production of SDF-1 by CAFs may be the reason for the promoted MFE, generation of CD44+CD24- cells and tumorigenicity of mammosphere cells. Figure 4 The SDF-1 protein expression in cocultured medium of mammosphere cells with CAFs and NFs. The SDF-1 protein level in the medium conditioned
by the CAFs was (426.4 ± 30.6) (pg/ml) (middle), compared to the levels
produced by mammosphere cells alone (283.6 ± 35.1) (pg/ml) (left), P <0.05. The cocultured Tenoxicam medium of mammosphere cells with NFs (right) showed a far lower level of SDF-1(52.9. ± 13.1) (pg/ml) secretion when compared with mammosphere cells alone, P <0.01. The SDF-1 level was measured three times in each experiment. CXCR4 antagonist reduced the generation of CD44+CD24- cells In order to further prove whether enhanced generation of CD44+CD24- cells by CAFs is mediated by SDF-1 and its receptor CXCR4, we detected CXCR4 expression in mammosphere cells and monolayer cells by qRT-PCR. The results showed that CXCR4 mRNA expression was higher in mammosphere cells than that in monolayer cells, (P < 0.01, Fig. 5), and CXCR4 antagonist AMD3100 could decrease CXCR4 gene expression in both cells. Moreover, AMD3100 significantly reduced MFE and mammosphere cell number in monoculture mammospheres and cocultured mammospheres with CAFs and NFs (Table 3), and decreased the proportion of CD44+CD24- cells (Fig. 6, and see Additional file 2). These results collectively demonstrated that CAFs enhanced generation of CD44+CD24- cells in mammospheres may be caused by SDF-1/CXCR4 signaling. Figure 5 Mammosphere cells and monolayer cells were cultured in the presence of 1 μg/ml AMD3100 for 48 h. qRT-PCR showed that CXCR4 mRNA expression in mammosphere cells was 3.9 fold higher than that in monolayer cells, (P <0.