Objective  To investigate the influence and significance of tumor microenvironment in breast cancer stem-cell culture and identification.

  Methods  Cells isolated from primary breast cancer tissues were cultured in vitro in a serum-free medium PCM-2 and in the supernatant of cultured fibroblasts. The MCF-7 breast cancer cell line was used as the control group. The status of the microspheres was observed, and the proliferative capacity of the cells was detected by methyl thiazolyl tetrazolium assay. The expression of stem cell and epithelial-mesenchymal markers were detected by real-time reverse transcription polymerase chain reaction.

  Results  The diameter of microspheres in PCM-2 gradually increased with prolonged incubation time (t=4.996, P=0.002). The cells in the supernatant of cultured fibroblasts increased daily and mostly exhibited a spindle cell growth. The growth rate of primary breast cancer cells was faster in the supernatant of cultured fibroblasts than in PCM-2 (P=0.004). Compared with the case of cells in the supernatant of cultured fibroblasts, aldehyde dehydrogenase 1 was upregulated in the primary breast cancer cells cultured in serum-free medium PCM-2, whereas E-cadherin and Vimentin were downregulated.

  Conclusion  Serum-free culture can be one of the best methods for enriching breast cancer stem cell-like mammospheres. The tumor micro-environment serves a vital function in the growth and development of tumor cells and in the evolution of breast cancer.