Supporting data for “The role of Wnt-β-catenin in cancer stemness and chemoresistance in three-dimensional cultures“

Three-dimensional (3D) culture is increasingly being recognized due to its superior capacity to simulate tissue-like structures compared with two-dimensional (2D) monolayers. Although much is known about the various characteristics of 2D and 3D cultures in certain cancer types, the knowledge on other tumor types is limited. In nasopharyngeal carcinoma (NPC), although Epstein-Barr virus (EBV) is consistently present in undifferentiated NPCs, most studies have focused on EBV-negative NPC cells. In Chapter 2, 3D spheroid models of EBV-positive NPC cells were developed and characterized, and they were compared with conventional 2D cultures, which showed different features of tumor phenotypes and treatment responses. In addition, RNA sequencing analysis indicated substantial transcriptomic differences between 3D spheroids and 2D monolayers. Specifically, Wnt/β-catenin and Eph/ephrin cell signaling pathways were discovered and recognized as activated signals in NPC spheroids as compared to 2D monolayers. United States Food and Drug Administration (FDA)-approved drugs targeting these signaling pathways involved in 3D spheroids can eliminate NPC cell growth/survival. These findings indicate the differential signaling in 3D NPC spheroids of potential therapeutic implications.

Cisplatin and paclitaxel are the main chemotherapeutic agents used for the treatment of ovarian cancer. However, their effectiveness is limited due to inherent and acquired chemoresistance. Cancer stem cells (CSCs) are shown to be chemoresistant. In Chapter 3, a subpopulation of c-Kit (CD117)-positive CSCs was enriched under 3D stem cell-selective conditions to elucidate the critical transcriptional activators of CSCs. The CBP/β-catenin interaction plays a role in the maintenance of cancer stemness. Using coimmunoprecipitation on nuclear β-catenin followed by mass spectrometry analysis, SP100 and HRP2 were identified as novel transcription co-activators that bound β-catenin. SP100- and HRP2-β-catenin interactions were essential for the expansion and drug resistance of CSCs and correlated with poor clinical outcomes. These findings reveal novel interacting partners of β-catenin in cancer stemness and chemoresistance in ovarian cancer.

Together, these findings provide a better understanding of 3D spheroids in NPC and ovarian cancer and demonstrate an essential role of Wnt/β-catenin signaling in these 3D cultures.

三维（3D）细胞培养因其相较于二维（2D）单层培养在模拟组织样结构方面所展现的卓越能力而日益受到重视。尽管关于某些癌症类型中二维和三维细胞培养的各种特性已有诸多了解，但对于其他肿瘤类型的相关知识则相对有限。在鼻咽癌（NPC）中，尽管EB病毒（EBV）在未分化NPC中持续存在，但大多数研究都集中于EBV阴性的NPC细胞。在第二章中，开发并表征了EBV阳性的NPC细胞的3D球体模型，并将其与传统的二维培养进行比较，发现它们在肿瘤表型和治疗反应方面表现出不同的特征。此外，RNA测序分析表明，3D球体与2D单层之间存在显著的转录组差异。具体而言，Wnt/β-连环蛋白和Eph/ephrin细胞信号通路被发现在NPC球体中被激活，相较于二维单层。针对参与3D球体中这些信号通路的美国食品药品监督管理局（FDA）批准的药物可以消除NPC细胞生长/存活。这些发现表明了3D NPC球体中信号传递的差异，并具有潜在的 therapeutic implications。顺铂和多西他赛是治疗卵巢癌的主要化疗药物。然而，由于固有的和获得的耐药性，它们的疗效有限。研究表明，癌细胞干细胞（CSCs）表现出耐药性。在第三章中，通过3D干细胞选择性条件富集了c-Kit（CD117）阳性的CSCs亚群，以阐明CSCs的关键转录激活因子。CBP/β-连环蛋白相互作用在维持癌症干细胞特性中发挥作用。通过在核β-连环蛋白上进行共免疫沉淀，随后进行质谱分析，鉴定了SP100和HRP2作为结合β-连环蛋白的新型转录共激活因子。SP100和HRP2-β-连环蛋白相互作用对于CSCs的扩增和耐药性至关重要，并与不良的临床结果相关。这些发现揭示了β-连环蛋白在癌症干细胞特性及卵巢癌耐药性中的新型相互作用伙伴。总体而言，这些发现加深了对NPC和卵巢癌中3D球体的理解，并证明了Wnt/β-连环蛋白信号在这些三维培养中的关键作用。