Cancer stem cells (CSC) are resistant to chemo and radiotherapies. To validate CSC as therapeutic targets in breast cancer, we analyzed the effect of Lapatinib (Lp, HER2 inhibitor), ATRA or the combined treatments, on growth, cell cycle distribution and metastatic capacity of primary mammospheres (CSC enriched cultures) from HER2 negative cell lines (4T1, MCF-7 and T47D). We determined by WB that HER2 is overexpressed only in CSC subpopulation of all cell lines analyzed. Primary mammospheres were treated for 96 h with Lp1µM for 4T1; 5µM for MCF-7, 2µM for T47D cells combined or not with ATRA 1µM. ATRA treatment alone or combined with Lp only significantly reduced 4T1 mammospheres diameters (p˂0.05 Anova test) and signs of cell death were also observed. The combined treatment induced cell cycle arrest at G0/G1 phase after 48h of treatment in 4T1 mammospheres, analyzed by flow cytometry. However, this combination not significantly induces cell cycle arrest in MCF- 7 mammospheres. Finally, we performed an experimental lung metastasis assay in mice pretreated with ATRA and Lp and observed that combination reduced metastatic potential of 4T1 cells derived from mammospheres (p˂0.05 Kruskal Wallis). In the present work we have demonstrated that the CSC component of HER2 negative cell lines overexpress such receptor. Moreover, Lp and ATRA combined treatment can successfully reduce mammospheres growth and metastatic potential in 4T1 experimental model.