Development of 3-dimensional human pituitary corticotroph tumor cultures as a preclinical model for drug discovery

摘要 库欣病(CD)是一种由促肾上腺皮质激素引起的危及生命的“孤儿病” 促肾上腺皮质激素(ACTH)--分泌性垂体腺瘤导致肾上腺皮质醇分泌过多。有一个大型的未满足的医疗队 需要CD治疗。然而，由于无法获得任何翻译研究，翻译研究受到很大阻碍 人垂体促肾上腺皮质激素肿瘤细胞模型。使用单细胞RNA测序(ScRNAseq)和微阵列 手术切除的人促肾上腺皮质细胞肿瘤的转录组分析，我们观察到垂体腺的缺失 促肾上腺皮质激素肿瘤ACTH分泌与血管生成、生存信号和免疫功能降低相一致 与胶原分解代谢、细胞黏附和细胞外基质组织增加的反应是平行的。 在这些发现的指导下，我们开发了一种独特的三维(3D)垂体瘤培养系统，并针对 第一次，我们已经能够产生3D人类促肾上腺皮质激素肿瘤培养物，分泌ACTH和GT；4个月。 我们组建了一支经验丰富的多学科团队，利用这个第一个完成了3个专注的具体目标 其同类资源。首先，我们将使用整个外显子组测序来描述我们的基因组图景 促肾上腺皮质细胞3D培养生物库和比较原始促肾上腺皮质细胞的基因组和遗传保真度 来自同一患者的肿瘤、正常血液和匹配的3D促肾上腺皮质细胞肿瘤样培养。 对单个患者来源的促肾上腺皮质细胞肿瘤培养的连续传代的ScRNAseq分析将监测 转录组在文化过程中以暂时的方式变化。我们的组织病理学结构 单细胞水平的3D促肾上腺皮质细胞培养将量化组织结构，因此我们可以绘制促肾上腺皮质细胞图谱 瘤样细胞的组成和分布。第二个目标是使用一个小型化的自动化系统来 在我们的3D促肾上腺皮质细胞肿瘤培养中进行高通量药物筛选。化合物将受到 严格的评估，以确定主要的“命中”，并通过使用20种浓度的三份重新筛选进行验证 从100微米到20微米(2倍稀释)，以可靠地计算每种化合物的EC50。最后，三个 互补方法，计算化学信息学图谱，scRNAseq来描绘转录 将利用药物治疗和功能基因组学在单细胞水平上的变化来探索 已确认的HIT化合物的MOA。这是对我们药物筛查结果和基因的综合询问 我们患者衍生的3D肿瘤培养以及原始肿瘤组织的特征，将使我们能够 理清个别药物的作用模式(S)，直接记录个性化的药物敏感性 亲代促皮质细胞肿瘤。总而言之，我们将利用我们独特的生物库全面地从分子上 特征化的垂体促肾上腺皮质细胞肿瘤组织和配对衍生的3D促肾上腺皮质细胞肿瘤培养以测试 临床相关化合物的资料库。这种脑下垂体三维肿瘤培养系统在该领域具有变革性 由于缺乏任何人类垂体促肾上腺皮质激素的肿瘤细胞模型，将为急需 改善了对库欣病这种严重致残、往往是致命疾病的患者的治疗。

Abstract Cushing Disease (CD) is a life-threatening “orphan disease” caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary adenoma driving excess adrenal cortisol production. There is a large unmet medical need for CD treatment. However, translational research has been greatly hampered due to unavailability of any human pituitary corticotroph tumor cell models. Using single cell RNA-sequencing (scRNAseq) and microarray transcriptome analysis of surgically resected human corticotroph tumors, we observed that loss of pituitary corticotroph tumor ACTH secretion coincided with reduced angiogenesis, survival signals and immune responses in parallel with increased collagen catabolism, cell adhesion and extracellular matrix organization. Guided by these findings, we developed a unique 3-dimensional (3D) pituitary tumor culture system and for the first time, we have been able to generate 3D human corticotroph tumor cultures that secrete ACTH >4 months. We have assembled an experienced multidisciplinary team to complete 3 focused specific aims using this first of its kind resource. Firstly, we will use whole exome sequencing to characterize the genomic landscape of our corticotroph 3D culture biobank and compare genomic and genetic fidelity between the original corticotroph tumor, normal blood and matched 3D corticotroph tumoroid cultures from the same individual patient. ScRNAseq analysis of serial passages of individual patient-derived corticotroph tumor cultures will monitor for transcriptome changes in a temporal fashion over the course of culture. The histopathological structure of our 3D corticotroph cultures at the single cell level will quantify tissue architecture so we can map corticotroph tumoroid cellular composition and distribution. A second aim will employ a miniaturized automated system to conduct a high throughput drug screen in our 3D corticotroph tumor cultures. Compounds will be subjected to rigorous evaluation to define primary “hits” and validated by re-screening in triplicate using 20 concentrations from 100µM to 20pM (2-fold dilution) to reliably calculate an EC50 for each compound. Finally, three complementary approaches, computational cheminformatic profiling, scRNAseq to delineate transcriptomic changes at the single cell level following drug treatment and functional genomics will be employed to explore the MOA of validated hit compounds. This integrated interrogation of our drug screen results and the genetic features of our patient-derived 3D tumor cultures as well as that of the original tumor tissue, will allow us to disentangle an individual drug's mode(s) of action, and directly document drug sensitivity of individualized parental corticotroph tumors. In summary, we will use our unique biobank of comprehensively molecularly characterized pituitary corticotroph tumor tissues and paired derived 3D corticotroph tumor cultures to test libraries of clincially relevant compounds. This pituitary 3D tumor culture system is transformative in the field due to the lack of any human pituitary corticotroph tumor cell models and will pave the path for much needed improved therapy for patients with this dreadfully disabling and often fatal disorder, Cushing disease.