Dynamic Suspension Culture System Reveals HIF-1α-Driven Stemness Maintenance via Dual Suppression of the p53 Pathway in Cancer Stem Cells

Cancer stem cells (CSCs) drive tumor recurrence and therapeutic resistance; however, their investigation remains limited by inefficient enrichment strategies and an incomplete understanding of stemness regulation. In this study, we developed an optimized dynamic suspension culture system that efficiently enriches CSCs. Compared with conventional static culture, this system generated spheroids with increased expression of stemness markers, enhanced proliferative capacity, and improved stability. Transcriptomic analyses further demonstrated that the system mimics fluid shear stress within the tumor microenvironment (TME), thereby activating the HIF-1 signaling pathway. Mechanistically, HIF-1α sustains stem cell activity through dual suppression of the p53 pathway: it directly represses p21 transcription by binding to its promoter, while simultaneously upregulating MDM2 to drive the subsequent degradation of p53. Based on this mechanism, combining the HIF-1α inhibitor PX-478 with the p53 activator doxorubicin synergistically suppressed CSC properties in vitro and significantly inhibited tumor growth in a subcutaneous xenograft model. Collectively, these findings establish an effective CSC enrichment strategy and identify the HIF-1α/p53 axis as a critical regulator of cancer stem cell properties and a promising therapeutic target.