Preservation of stemness in high-grade serous ovarian cancer organoids requires low Wnt environment

High-grade serous ovarian cancer (HGSOC) creates a unique clinical challenge due to late detection, limited therapeutic options and wide-spread resistance to chemotherapy, all of which lead to very low survival rates. It is now widely recognized that this cancer emerges as metastasis from the fallopian tube and the lack of suitable in vitro disease models poses a major obstacle for efforts to improve our understanding of this deadly disease. We used organoids of healthy human FT epithelium as a model for stepwise genetic modification towards malignancy, using shRNA-mediated knockdown (KD) of three major HGSOC driver genes: P53, PTEN and Retinoblastoma (RB). Moreover, we successfully established patient-derived HGSOC organoids from solid tumor deposits. After combinatorial screening approach of different media compositions we have established 15 organoid lines which depend on the absence of exogenous Wnt3a in the medium. We could show that those cultures closely resemble the parental tumor tissue and express hallmarks of HGSOC phenotype as revealed by histology and global gene expression profiles of the matching organoid and tissue samples. Expression and sequencing analysis revealed that all samples were characterized by p53 mutation and/or aberrant p53 expression. Interestingly, the addition of Wnt agonists to cancer organoids leads to rapid growth arrest and a sharp drop in stemness, as proven by cell viability assay and the number of CD133 positive cells, respectively. Complementary to this finding, we can show that the medium optimized for ovarian cancer organoids also improves the growth of the modified organoid lines, showing that changes within the niche environment are required to promote stemness upon depletion of P53/PTEN/RB. Taken together, our data reveal that critical changes in the paracrine environment are likely to represent early events during HGSOC development and provide a starting point to further analyze how specific mutations and paracrine signals influence pathways of stemness regulation and thus drive malignant transformation in ovarian carcinogenesis. Microarray experiments were performed as single-color hybridizations on Agilent 4x44K Mouse Genome Microarrays.

高级别浆液性卵巢癌（High-grade serous ovarian cancer, HGSOC）因确诊延迟、治疗手段匮乏且普遍存在化疗耐药性，导致患者生存率极低，是一类极具临床挑战性的恶性肿瘤。目前学界已广泛达成共识，该类癌症起源于输卵管上皮的转移灶，而缺乏合适的体外疾病模型，是阻碍我们深入解析这一致命疾病的核心障碍。本研究以健康人输卵管上皮类器官（organoids）为模型，通过短发夹RNA介导的敲低（short hairpin RNA-mediated knockdown, KD）技术，靶向敲除三个关键的HGSOC驱动基因：P53、PTEN与视网膜母细胞瘤基因（Retinoblastoma, RB），逐步诱导其向恶性表型转化。此外，我们还成功从实体肿瘤转移灶中建立了患者来源的HGSOC类器官系。通过对多种培养基组分开展组合筛选，我们最终获得了15株依赖于培养基中外源Wnt3a缺失的类器官系。实验结果证实，这些类器官与亲本肿瘤组织高度同源，且可通过组织病理学（histology）及匹配类器官与组织样本的全局基因表达谱（global gene expression profiles），验证其具备HGSOC的典型表型特征。表达谱与测序分析显示，所有受试样本均存在p53突变及/或p53表达异常。值得关注的是，向癌症类器官中添加Wnt激动剂可诱导其快速生长停滞，并伴随干细胞干性的显著降低，该结论分别通过细胞活力实验与CD133阳性细胞计数得到验证。与此发现互为佐证的是，我们发现针对卵巢癌类器官优化的培养基，同样可促进经基因修饰的类器官系的生长，这表明在P53/PTEN/RB基因敲除后，微环境的改变是维持干细胞干性所必需的。综上，本研究数据表明，旁分泌微环境的关键改变可能是HGSOC发生发展过程中的早期事件，本研究也为进一步解析特定突变与旁分泌信号如何调控干细胞干性通路、进而驱动卵巢癌恶性转化过程，提供了重要的研究基础。本研究采用安捷伦4x44K小鼠基因组基因芯片（Agilent 4x44K Mouse Genome Microarrays）开展单通道杂交的基因芯片实验。