循环肿瘤细胞（CTCs）被认为是肿瘤转移的重要因素，并且能够克服机械性相互作用以获取远处转移的能力。先前的研究已经表明，悬浮状态能够调控乳腺癌细胞（BCCs）的干性。然而，尚未清楚地探索出具体的分子机制。本研究中，我们以MCF-7和MDA-MBA-231 BCCs为模型，在悬浮和贴壁培养条件下进行培养，通过观察悬浮细胞聚集体、分选CD44+/CD24-细胞亚群和检测自我更新能力，进一步阐明了悬浮状态对BCCs的影响。此外，我们发现糖原合成酶激酶-3β（GSK-3β）在MCF-7悬浮细胞中显著下调，同时伴随着Wnt/β-连环蛋白信号传导的激活，而对于MDA-MB-231细胞的情况则相反。随后，GSK-3β在MCF-7悬浮细胞中表达差异显著。当在悬浮MCF-7细胞中过表达GSK-3时，抑制了Wnt/β-连环蛋白信号传导、上皮-间质转化（EMT）和干性。而当GSK-3β下调时，进一步促进了MCF-7细胞的Wnt/β-连环蛋白信号传导、间质特性和干性。本研究证明，悬浮状态通过抑制GSK-3β来激活Wnt/β-连环蛋白信号传导，从而促进上皮性BCCs的干性，为靶向CTCs提供了治疗策略。版权所有© 2023 Elsevier Ltd. 保留所有权利。

Circulating tumor cells (CTCs) are considered an important factor involved in tumor metastasis and can overcome mechanical interactions to gain the ability to distant metastasis. The previous study had shown that the suspension state could regulate the stemness of breast cancer cells (BCCs). However, the specific molecular mechanisms involved have not yet been explored clearly. In this study, MCF-7 and MDA-MBA-231 BCCs were cultured in suspension and adherent. The effect of suspension state on BCCs was further elucidated by observing suspension cell clusters, sorting CD44+/CD24- cell subpopulation and detecting self-renewal ability. Furthermore, it was found that glycogen synthase kinase-3β (GSK-3β) was significantly down-regulated in MCF-7 suspension cells along with the activation of the Wnt/β-catenin signaling, but the converse was true for MDA-MB-231 cells. Subsequently, GSK-3β was differentially expressed in MCF-7 suspension cells. The activation of the Wnt/β-catenin signaling, epithelial-mesenchymal transition (EMT) and stemness were all inhibited when GSK-3 was overexpressed in suspension MCF-7 cells. While GSK-3β was down-regulated, it further promoted the Wnt/β-catenin signaling, mesenchymal characteristic and stemness of MCF-7 cells. This study demonstrated that suspension state could activate the Wnt/β-catenin signaling by inhibiting GSK-3β to promote the stemness of epithelial BCCs, providing a therapeutic strategy for targeted CTCs.Copyright © 2023 Elsevier Ltd. All rights reserved.