Intraperitoneal dissemination of ovarian cancers is preceded by the development of chemoresistant tumors with malignant ascites. Despite the high levels of chemoresistance and relapse observed in ovarian cancers, there are no in vitro models to understand the development of chemoresistance in situ. METHOD We describe a highly integrated approach to establish an in vitro model of chemoresistance and stemness in ovarian cancer, using the 3D hanging drop spheroid platform. The model was established by serially passaging non-adherent spheroids. At each passage, the effectiveness of the model was evaluated via measures of proliferation, response to treatment with cisplatin and a novel ALDH1A inhibitor. Concomitantly, the expression and tumor initiating capacity of cancer stem-like cells (CSCs) was analyzed. RNA-seq was used to establish gene signatures associated with the evolution of tumorigenicity, and chemoresistance. Lastly, a mathematical model was developed to predict the emergence of CSCs during serial passaging of ovarian cancer spheroids. RESULTS Our serial passage model demonstrated increased cellular proliferation, enriched CSCs, and emergence of a platinum resistant phenotype. In vivo tumor xenograft assays indicated that later passage spheroids were significantly more tumorigenic with higher CSCs, compared to early passage spheroids. RNA-seq revealed several gene signatures supporting the emergence of CSCs, chemoresistance, and malignant phenotypes, with links to poor clinical prognosis. Our mathematical model predicted the emergence of CSC populations within serially passaged spheroids, concurring with experimentally observed data. CONCLUSION Our integrated approach illustrates the utility of the serial passage spheroid model for examining the emergence and development of chemoresistance in ovarian cancer in a controllable and reproducible format.

中文翻译：

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癌症干细胞富集和化学抗性的工程3D模型。

卵巢癌的腹膜内扩散是伴随着具有恶性腹水的化学耐药性肿瘤的发展。尽管在卵巢癌中观察到高水平的化学抗药性和复发，但尚无体外模型来了解化学抗药性的原位发展。方法我们描述了一种高度集成的方法，该方法使用3D悬垂式球体平台建立卵巢癌化学耐药性和干性的体外模型。该模型是通过连续传代非粘附球体而建立的。在每次传代中，均通过增殖，对顺铂和新型ALDH1A抑制剂治疗的反应来评估模型的有效性。同时，分析了癌干样细胞（CSCs）的表达和肿瘤启动能力。RNA-seq用于建立与致瘤性和化学抗性进化相关的基因标记。最后，开发了数学模型来预测卵巢癌球体连续传代过程中CSC的出现。结果我们的连续传代模型显示出细胞增殖增加，CSC富集以及铂抗性表型的出现。体内肿瘤异种移植测定法表明，与早期传代球体相比，更高传代球体的晚期传球体具有更高的致瘤性。RNA-seq显示了支持CSC，化学抗药性和恶性表型出现的几个基因特征，并与不良的临床预后相关。我们的数学模型与实验观察到的数据一致，预测了CSC族群在连续传代的球体中的出现。