The ability to self-renew is one of the key properties of normal stem cells and cancer stem cells. Suspension sphere cultures have been widely used in stem cell biology to identify and enrich stem cells, as theoretically only stem cells can form spheres with an initial phase of symmetric expansion. In cancer, the ability to form tumorspheres in suspension culture is also used to identify cancer stem cells. Here, we showed that treatment with CDDO-Im significantly reduced the sphere forming efficiency of SUM159 cells. The inhibitory effect of CDDO-Im on the sphere forming efficiency was confirmed in other triplenegative and basal-like breast cancer cell lines, SUM149 and MCF10DCIS.com, respectively. In addition, CDDO-Im markedly decreased the size of tumorspheres, which might reflect the altered proliferation/differentiation status of cancer stem cells or the decreased proliferation of progenitor cells by CDDO-Im. Interestingly, secondary tumorspheres from the vehicle treated primary tumorspheres showed significantly higher sphere-forming efficiency than the secondary tumorspheres from the CDDO-Im treated primary tumorspheres. This result might indicate that CDDO-Im inhibits selfrenewal of cancer stem cells in primary tumorspheres, causing the decreased cancer stem cells in seeding cells for the successive secondary sphere culture. In breast cancer, the Notch signaling pathway has been shown to play a critical role in maintaining cancer stem cells by regulating self-renewal. Although most studies have been utilized Notch1 as the readout of Notch signaling, the four Notch receptors, Notch1, Notch2, Notch3 and Notch4, are thought to have different functions in breast cancer. Knockdown of Notch1 or Notch4 inhibited self-renewal and tumorsphere forming ability of breast cancer cells, supporting their roles for the maintenance of cancer stem cells. High activity of Notch3 signaling was associated with aggressive human inflammatory breast cancer and increased lymphovascular invasion, again suggesting tumorigenic activity of Notch3. On the contrary, high mRNA levels of Notch2 were associated with good clinical outcomes. Moreover, ectopic expression of active Notch2 inhibited cell growth and induced apoptosis in triplenegative breast cancer cells, suggesting a tumor suppressor role for Notch2. Interestingly, a Reversine recent study demonstrated that withaferin A, a natural chemopreventive agent which is structurally similar to CDDO with a Michael acceptor group, activated Notch2 but inhibited Notch1 activation. In our study, CDDO-Im also induced the protein level of Notch2, while selectively inhibiting Notch1 and Notch3, indicating differential regulation of Notch receptors by CDDO-Im. Epithelial-mesenchymal transition is a cellular process in which adherent epithelial-type cells transform into mesenchymaltype cells, and induction of EMT in cancer cells generates cells with stem cell-like properties.