In conclusion, our findings indicate that the NSDHL gene contributes to spheroid formation of ER + human breast cancer cell lines MCF-7 and ZR75-1 and maintains BCSCs/progenitors with CD44+/CD24- and EpCAM+/CD49f + phenotypes and high ALDH activity by regulating TGF-β signaling and key regulators of cancer stemness, SOX2 and NANOG, in MCF-7 spheroids and xenograft tumors. Here, NANOG is linked to breast cancer.