Breast cancer remains a major cause of cancer-related deaths in women worldwide. Chemotherapy-promoted stemness and enhanced stem cell plasticity in breast cancer is a cause for great concern. The discovery of drugs targeting BCSCs was suggested to be an important advancement in the establishment of therapy that improves the efficacy of chemotherapy. In this work, by using single-cell mass cytometry, we observed that stemness in spheroid-forming cells derived from MDA-MB-231 cells was significantly increased after doxorubicin administration and up-regulated integrin αvβ3 expression was also observed. An RGD-included nanoparticle (CS-V) was designed, and it was found that it could promote doxorubicin’s efficacy against MDA-MB-231 spheroid cells. The above observations suggested that the combination of RGD-included nanoparticles (CS-V) with the chemo-drug doxorubicin could be developed as a potential therapy for breast cancer.