关键词: 乳腺肿瘤；肿瘤干细胞；乳腺癌干细胞微球体；培养液, 无血清；细胞鉴定

Abstract:

Abstract:Objective To culture and identify chemotherapy microspheres (CMSs) isolated from tumor tissues of breast cancer patients after receiving neoadjuvant chemotherapy and provide the theoretical basis for a new method to obtain breast cancer stem cells. Methods After CMSs were isolated from   the breast cancer tissues,the experiments were conducted as follows:① CMSs were plated in DMEM/F12 media containing growth factors and the growth process of CMSs was observed;② Sphere formation assay was performed to determine the self-renewal ability of chemotherapy microspheres-derived cells (CMSDCs);③ The differentiation ability of CMSDCs was observed by culturing them in DMEM/F12 containing 5% fetal bovine serum media;④ The expressions of CK14,CK18 and CK19 of CMSDCs and their differentiated cells were detected by immunocytochemistry;⑤ The ratios of ALDH1+ cells of CMSDCs and their differentiated cells were evaluated by flow cytometry (FCM);⑥ The CMSDCs were implanted into NOD/SCID mice to assess their tumorigenic potential. Results The CMSs could continue to grow in serum-free media and the necrotic cells were distributed over the surface layer of the CMSs after 3 d;The CMSDCs could form new CMSs with strong refraction by serial sphere formation assay;The CMSDCs could differentiate to monolayer of spindle-shaped cells in the media  supplemented with serum;The CMSDCs expressed the breast stem cell marker  CK19,but not CK14 and 18,which marked the myoepithelial and luminal cells,respectively;The ratio of ALDH1+ cells of the CMSDCs accounted for 17.41%-23.57% of all the cells，but which of their differented cells accounted for only 0.50%-1.28% of the total cells;New tumor identical with the primary tumors formed when 103 cells were injected into the mammary fat pad of NOD/SCID mice. Conclusion The CMSs isolated from tumor tissues are rich in breast cancer stem cells and similar to the mammospheres obtained by the suspension culture. It indicats that the isolation of the microspheres in breast cancer stem cells after neoadjuvant chemotherapy may be a new method to obtain breast cancer stem cells.

Key words: breast neoplasms;tumor stem cells;microspheres in breast cancer stem cells;culture media,serum-free;cell identification