乳腺癌干细胞（BCSC）被认为在化疗耐药和乳腺癌复发方面有贡献。因此，在开发新的乳腺癌治疗策略中，BCSC代表了一个有前途的目标。近期，BCSC的线粒体动力学被强调为一种可用于针对BCSC的方法。在本研究中，构建了一个三维（3D）培养的乳腺癌干细胞球体模型。流式细胞术和共聚焦显微镜分析了线粒体动力学和功能。我们已经证明，FIS1和Mitofusin 1蛋白水平在BCSC中显著增加。此外，卡匹维斯替（AZD5363）的投与可以抑制BCSC中Mitofusin1的表达。我们使用MitoTracker柑橘红和附加素V双染色实验表明，AZD5363可以诱导BCSC凋亡。 CCK8实验调查了干细胞球对多柿欧文的敏感性，结果表明，AZD5363可以重新使BCSC对Doxo敏感。流式细胞术分析确定，Doxo诱导的BCSC中CD44和CD133的表达可以被AZD5363抑制。结合AZD536，Doxo诱导的BCSC凋亡显著增加。总之，我们的研究首次探索了AZD5363通过调节Mitofusin来靶向3D培养的干细胞球体（BCSC）中的线粒体动力学。© 2023. 作者（S）。

Breast cancer stem cells (BCSCs) have been suggested to contribute to chemotherapeutic resistance and disease relapse in breast cancer. Thus, BCSCs represent a promising target in developing novel breast cancer treatment strategies. Mitochondrial dynamics in BCSCs were recently highlighted as an available approach for targeting BCSCs. In this study, a three-dimensional (3D) cultured breast cancer stem cell spheres model was constructed. Mitochondrial dynamics and functions were analyzed by flow cytometry and confocal microscopy. We have demonstrated that the protein levels of FIS 1 and Mitofusin 1 were significantly increased in BCSCs. Moreover, Capivasertib (AZD5363) administration could suppress Mitofusin1 expression in BCSCs. Our use of MitoTracker Orange and annexin V double-staining assay suggested that AZD5363 could induce apoptosis in BCSCs. The sensitivity of stem cell spheres to doxorubicin was investigated by CCK8 assay, and our results indicated that AZD5363 could re-sensitize BCSCs to Doxo. Flow cytometry analysis identified doxo-induced CD44 and CD133 expression in BCSCs could be suppressed by AZD5363. In combination with AZD536, doxo-induced apoptosis in the BCSCs was significantly increased. In conclusion, our study explored, for the first time, that AZD5363 could target mitochondrial dynamics in 3D cultured stem cell spheres (BCSCs) by regulating Mitofusin.© 2023. The Author(s).