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

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

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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.

抽象的库欣疾病（CD）是一种威胁生命的“孤儿疾病”，由肾上腺皮质激素引起（ACTH） - 分泌垂体腺瘤驱动超过肾上腺皮质醇的产生。有一个很大的未得到医学需要CD处理。但是，由于没有任何内容，翻译的研究受到了极大的阻碍人垂体皮质营养肿瘤细胞模型。使用单细胞RNA-Sequencing（SCRNASEQ）和微阵列对手术切除的人皮质营养肿瘤的转录组分析，我们观察到垂体的丧失皮质营养肿瘤ACTH分泌与血管生成，生存信号和免疫与胶原蛋白分解代谢，细胞粘合剂和细胞外基质组织同时的反应。在这些发现的指导下，我们开发了独特的3维（3D）垂体肿瘤培养系统第一次，我们能够产生3D人类皮质营养肿瘤培养物，使ACTH> 4个月。我们已经组建了一支经验多学科团队，以使用这一目标来完成3个重点的特定目标同类资源。首先，我们将使用整个外显子组测序来表征我们的基因组景观皮质营养3D培养生物库，并比较原始皮质营养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皮质营养性瘤培养物。 scRNASEQ分析单个患者衍生的皮质营养肿瘤培养的串行通道将监测转录组在文化过程中以临时方式变化。我们的组织病理结构单细胞水平的3D皮质营养培养物将量化组织结构，因此我们可以绘制皮质营养肿瘤细胞组成和分布。第二个目标将采用微型自动化系统在我们的3D皮质营养肿瘤培养物中进行高通量药物筛查。化合物将受到严格的评估以定义主要的“命中”，并通过使用20个浓度一式三份重新筛选来验证从100μm到下午20点（2倍稀释），可靠地计算每种化合物的EC50。最后，三个互补方法，计算化学图谱分析，scrnaseq以描绘转录组药物治疗和功能基因组学后，单细胞水平的变化将被雇用以探索经过验证的HIT化合物的MOA。对我们的药物筛查结果和遗传的综合询问我们患者衍生的3D肿瘤培养物以及原始肿瘤组织的特征将使我们得以解散单个药物的作用模式，并直接记录个性化的药物敏感性亲本皮质营养肿瘤。总而言之，我们将全面地使用我们独特的生物库表征垂体皮质营养肿瘤组织和成对的衍生的3D皮质营养肿瘤培养物进行测试临床相关化合物的库。这种垂体3D肿瘤培养系统在现场具有变化由于缺乏任何人垂体皮质营养肿瘤细胞模型，将为急需的路径铺平道路改善了这种可怕的残疾和经常致命疾病的患者的治疗，库什疾病。