Stigmasterol Attenuates Triple-negative Breast Cancer Stem Cell Properties by Inhibiting JAK3

<b>Background:</b> Breast cancer stem-like cells (BCSCs) are considered a source of tumor origins, metastasis and drug resistance, thereby limiting current treatment regimens. Stigmasterol has been reported to inhibit various cancer processes, but its effects and mechanisms in BCSCs have not been investigated. <b>Methods:</b> To generate spheroids, we enriched parental and SUM159 cells with BCSCs in a serum-free medium. The effects on the stemness, metastasis and drug resistance of CSC-enriched SUM159 cells were detected for the first time by <i>in vivo</i> and <i>in vitro</i> experiments. <b>Results:</b> CSC-enriched SUM159 and 4T1 cells demonstrated higher potential for tumorigenesis and metastasis. Stigmasterol suppresses BCSCs' spheroid formation, cell viability, and migration ability and promotes cell apoptosis. Stigmasterol also inhibited BCSCs-originated cancer formation in rat models. Stigmasterol also attenuated the growth of TNBC organoids from human breast cancer tissues. These data revealed the inhibitory effects of stigmasterol on BCSC traits. In the meantime, we found that JAK3 was upregulated in BCSCs, and Stigmasterol could effectively inhibit its expression. In addition, JAK3 was evidenced to negatively regulate BCSC activity and stemness both <i>in vitro</i> and <i>in vivo</i>. More importantly, the results indicated that Stigmasterol suppresses BCSC activity by inhibiting JAK3 expression. <b>Conclusion:</b> This study is the first to demonstrate that Stigmasterol inhibited metastasis and stemness of BCSCs by downregulating JAK3, which might provide a new method for the clinical application of Stigmasterol in breast cancer.