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：
10653709
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/10653709
关键词：
3-Dimensional Adrenal Glands Architecture Automobile Driving Blood Catabolism Cell Adhesion Cell model Cells Clinical 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.

抽象的库欣病（CD）是一种由促肾上腺皮质激素引起的危及生命的“孤儿病” (ACTH) 分泌性垂体腺瘤导致肾上腺皮质醇产生过多，目前尚有大量未得到满足的医疗问题。然而，由于缺乏任何治疗方法，转化研究受到了极大的阻碍。使用单细胞 RNA 测序 (scRNAseq) 和微阵列建立人垂体促肾上腺皮质激素肿瘤细胞模型。对手术切除的人类促肾上腺皮质激素肿瘤的转录组分析，我们观察到垂体的丧失促肾上腺皮质激素肿瘤 ACTH 分泌与血管生成、生存信号和免疫减少同时发生反应与胶原分解代谢、细胞粘附和细胞外基质组织的增加同时发生。在这些发现的指导下，我们开发了一种独特的 3 维 (3D) 垂体肿瘤培养系统，并用于我们首次能够生成可分泌 ACTH >4 个月的 3D 人类促肾上腺皮质激素肿瘤培养物。我们组建了一支经验丰富的多学科团队，利用这一先决条件完成 3 个重点具体目标首先，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来自同一个体患者的肿瘤、正常血液和匹配的 3D 促肾上腺皮质激素肿瘤样培养物。对个体患者来源的促肾上腺皮质激素肿瘤培养物的连续传代进行 ScRNAseq 分析将监测转录组在文化过程中以时间方式发生变化。单细胞水平的 3D 促肾上腺皮质激素培养物将量化组织结构，以便我们可以绘制促肾上腺皮质激素图谱第二个目标是采用小型化自动化系统来研究肿瘤细胞的组成和分布。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。严格评估以确定主要“命中”，并通过使用 20 个浓度的一式三份重新筛选进行验证从 100μM 到 20pM（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、计算化学信息学分析、scRNAseq 来描绘转录组将利用药物治疗和功能基因组学来探索单细胞水平的变化对我们的药物筛选结果和遗传的综合询问。我们的患者来源 3D 肿瘤培养物以及原始肿瘤组织的特征将使我们能够理清单个药物的作用模式，并直接记录个体化药物的敏感性总之，我们将使用我们亲本独特的全面分子生物库。表征垂体促肾上腺皮质激素肿瘤组织和配对衍生的 3D 促肾上腺皮质激素肿瘤培养物以进行测试该垂体 3D 肿瘤培养系统在该领域具有变革性。由于缺乏任何人类垂体促肾上腺皮质激素肿瘤细胞模型，将为急需的细胞铺平道路改进了对患有库欣病这种可怕的致残且往往致命的疾病的患者的治疗。

项目成果

期刊论文数量（2）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Refractory corticotroph adenomas.

难治性促肾上腺皮质激素腺瘤。

DOI：
发表时间：
2023-06
期刊：
影响因子：
3.8
作者：
Sumal, Amit K S;Zhang, Dongyun;Heaney, Anthony P
通讯作者：
Heaney, Anthony P

Single-cell RNA sequencing in silent corticotroph tumors confirms impaired POMC processing and provides new insights into their invasive behavior.

沉默促肾上腺皮质激素肿瘤中的单细胞 RNA 测序证实了 POMC 处理受损，并为其侵袭行为提供了新的见解。

DOI：
发表时间：
2022-05-12
期刊：
影响因子：
5.8
作者：
Zhang, Dongyun;Hugo, Willy;Bergsneider, Marvin;Wang, Marilene B;Kim, Won;Vinters, Harry V;Heaney, Anthony P
通讯作者：
Heaney, Anthony P