Coculture of breast stromal fibroblasts with primary mammosphere cells Coculture of primary mammosphere cells (1 × 105 cells/dish) with breast stromal fibroblasts

(1 × 105 cells/dish) were performed by using a transwell (BD) cell culture system, which allows free diffusion Linsitinib mouse of substances without contact Pevonedistat order between cancer cells and stromal fibroblasts. Stromal fibroblasts in the insert layer were subcultured on a transwell cell culture membrane (7.5 cm in diameter; pore size: 0.4 μm), and mammosphere cells in the bottom layer were maintained in a 10-cm Petri dish. Stromal fibroblasts were precultured in DMEM/F12 with 10% FBS for 48 h before the start of coculture. Stromal fibroblasts were maintained in fresh serum-free DMEM/F12 medium, and mammosphere cells were cultured in suspension for six days. Coinoculation of mammosphere cells with different stromal fibroblasts in vivo Mammospheres and fibroblasts were collected, enzymatically dissociated, washed in PBS, and kept at 4°C. Mice were

maintained in laminar flow rooms under constant temperature and humidity and received an estradiol supplementation (0.6 mg/kg, s.i., https://www.selleckchem.com/products/pd-0332991-palbociclib-isethionate.html Sigma) every 7 days for 28 days before cell injection. The mammosphere cells (1 × 105) admixed with either CAFs (1 × 105) or NFs (1 × 105) were suspended in 0.1 ml of DMEM/F12 and then inoculated into the mammary fat pad of 5-week-old female NOD/SCID mice (Shanghai Experimental Animal Center, Chinese Academy of Sciences, Shanghai, China). Mice were examined by palpation for tumor formation for up to 12 weeks, and then were sacrificed Methocarbamol by cervical dislocation. The histologic features of the xenografts were examined by hematoxylin and eosin staining. All experimentation performed with NOD/SCID mice, as well as routine care of the animals, was carried out in accordance with the institutional guide of animal care & use committee. Measurement of SDF-1 The baseline level of SDF-1 production was determined by coculture of mammosphere cells with stromal fibroblasts

for six days at a density of 1 × 105/bottle. The concentration of SDF-1 in the supernatant was measured by using a human SDF-1 antibody and enzyme immunoassay kit (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. Statistical analysis Statistical analysis was performed by using GraphPad Prism 4.0 software© (San Diego, CA). Student’s t-test (for comparison between two groups) or ANOVA with Tukey post test (for comparison between more than two groups) were used to determine whether there exists statistically significance. Fisher exact probability test was used to analyze tumorigenicity in NOD/SCID mice. Data is presented as the mean ± SEM. P values of ≤ 0.05 were regarded as being statistically significant.