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

开发 3 维人垂体促肾上腺皮质激素肿瘤培养物作为药物发现的临床前模型

基本信息

批准号：
10448514
负责人：
ANTHONY P HEANEY
金额：
$ 34.97万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-09 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10448514
关键词：
3-Dimensional Adrenal Glands Architecture Automobile Driving Blood Catabolism Cell Adhesion Cell model Cells Collagen Corticotropin Development Disease Drug Interactions Drug Screening Evaluation Extracellular Matrix Genes Genetic Genomics Growth Hormone secretion Human Hydrocortisone Immune response Individual Libraries Life Maps Medical Methods Modeling Molecular Monitor Operative Surgical Procedures Pathway interactions Patients Pattern Recognition Pharmaceutical Preparations Pharmacotherapy Pituitary Gland Pituitary Gland Adenoma Pituitary Neoplasms Pituitary-dependent Cushing&apos s disease Pre-Clinical Model Primary Neoplasm Production Proteins Rare Diseases Resected Resources Screening Result Serial Passage Signal Transduction Structure System Testing Time Tissues Translational Research Tumor Tissue Validation Visualization angiogenesis biobank cheminformatics drug discovery drug sensitivity exome sequencing experience experimental study functional genomics genomic platform high throughput screening high-throughput drug screening immunocytochemistry improved individual patient inhibitor miniaturize multidisciplinary neoplastic cell novel therapeutics personalized medicine screening single-cell RNA sequencing small molecule transcriptome transcriptomics tumor

项目摘要

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.

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）分泌垂体腺瘤驱动肾上腺皮质醇过量生产。有大量未满足的医疗需求需要CD治疗。然而，翻译研究由于无法获得任何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）和微阵列通过对手术切除的人促肾上腺皮质激素细胞瘤的转录组分析，我们观察到，促肾上腺皮质激素肿瘤ACTH分泌与减少的血管生成、存活信号和免疫反应一致。与增加的胶原蛋白catalysts，细胞粘附和细胞外基质组织平行的反应。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，第一次，我们已经能够产生分泌ACTH >4个月的3D人促肾上腺皮质激素细胞肿瘤培养物。我们已经组建了一个经验丰富的多学科团队，以完成3个集中的具体目标，首先使用的同类资源。首先，我们将使用全外显子组测序来表征我们的基因组景观。促肾上腺皮质激素细胞3D培养生物库，并比较原始促肾上腺皮质激素细胞之间的基因组和遗传保真度肿瘤、正常血液和来自同一个体患者的匹配的3D促肾上腺皮质激素类肿瘤培养物。单个患者来源的促肾上腺皮质激素细胞肿瘤培养物的连续传代的ScRNAseq分析将监测以下指标：转录组在培养过程中以时间方式变化。我们的组织病理学结构单细胞水平的3D促肾上腺皮质激素细胞培养物将量化组织结构，肿瘤样细胞的组成和分布。第二个目标将采用小型化自动化系统，在我们的3D促肾上腺皮质激素肿瘤培养物中进行高通量药物筛选。化合物将经受严格评价，以确定主要“命中”，并通过使用20个浓度重复筛选三次进行验证从100µM到20 pM（2倍稀释），以可靠地计算每种化合物的EC50。最后，三互补方法，计算化学信息学分析，scRNAseq来描绘转录组学，药物治疗后单细胞水平的变化和功能基因组学将被用来探索验证命中化合物的MOA。这种对我们药物筛选结果和基因的综合询问我们的患者来源的3D肿瘤培养物以及原始肿瘤组织的特征，将使我们能够解开单个药物的作用模式，并直接记录个体化药物的药物敏感性。亲代促肾上腺皮质激素细胞瘤总之，我们将利用我们独特的生物库，特征性垂体促肾上腺皮质激素细胞肿瘤组织和配对衍生的3D促肾上腺皮质激素细胞肿瘤培养物，临床相关化合物库。这种垂体3D肿瘤培养系统在该领域具有变革性由于缺乏任何人垂体促肾上腺皮质激素肿瘤细胞模型，为患有这种可怕的致残性和致命性疾病的患者改进治疗方法，库欣病。