Enrichment Of Triple Negative Breast Cancer Stem Cells By Suspension Culture

Objective:In order to acquire triple-negative breast cancer stem cells by adherentMDA-MB-231cells were cultured in serum-free medium.Methods:Human MDA-MB-231breast cancer cell lines were plated in stem cellconditioned culture system to generate mammospheres. Samples of mammospheres in7-9days were taken to evaluate the effects of different MDA-MB-231cell density onthe mammosphere-formation efficiency; the expression levels of stem cellsbiomarker CD44+CD24－/low and CXCR4protein in adherent culture cells andmammosphere cells,and the stemness characteristics of sphere re-formation,colonyformation were assessed,comparing with the adherent culture MDA-MB-231cells.Results:(1).Triple negative breast cancer mammospheres can be enriched in serum-freemedium containing20ng/ml EGF,10ng/ml bFGF,2%b27. The mammosphereformation efficiency was (5.61±0.02)%、(3.23±3.23)%、(2.28±0.48)%、(1.05±1.05)%、(0.91±0.01)%f when the MDA-MB-231cell culturing density was1*104/ml、2*104/ml、3*104/ml、4*104/ml、5*104/ml, respectively, There was asignificantly difference among these groups (p<0.05).(2).The proportion of CD44+CD24-/low in MDA-MB-231adherent cells andmammosheres were (38.54±2.00)%VS (66.35±2.06)%, P=0.003.(3).CXCR4protein expression in mammosheres cells was higher thanMDA-MB-231adherent cells,P=0.03.(4).Single mammosphere cell have the ability to generate sphere again.(5).The colony formation capability of mammosheres need200singlemammoshere cells,while adherent cells need1000single MDA-MB-231cells. Conclusions:1.Triple negative breast cancer stem cells cultured in serum-free medium can beenriched, low cell density is beneficial to the formation of mammosheres.2. Mammosheres had a higher CD44+CD24-/lowproportion than MDA-MB-231adherent cells.3.CXCR4protein expression in mammosheres was higher than in MDA-MB-231adherent cells.