Development of a patient-derived tumoroid culture system to explore novel medical treatments for refractory prolactinomas

开发患者源性肿瘤培养系统，探索难治性泌乳素瘤的新疗法

• 批准号: 10643450
• 负责人: ANTHONY P HEANEY
• 金额: $ 21.88万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10643450
• 关键词: 3-Dimensional; Address; Affect; Agonist; Biological Assay; Biomedical Engineering; CRISPR library; Catabolism; Cell Adhesion; Cell model; Cells; Collagen; Custom; Data; Development; Digit structure; Disease remission; Dopamine Agonists; Dose; Drug Targeting; Event; Excision; Exclusion; Exhibits; Extracellular Matrix; Hormone secretion; Human; Hyperprolactinemia; Hypogonadism; Immune response; In Vitro; Lead; Libraries; Literature; Medical; Methodology; Modeling; Molecular; Morphology; Mus; Neuroendocrine Tumors; Operative Surgical Procedures; Pathway interactions; Patients; Peripheral; Pharmaceutical Preparations; Phenotype; Pituitary Gland; Pituitary Neoplasms; Poison; Pre-Clinical Model; Primary Neoplasm; Prolactin; Prolactin Secreting Pituitary Gland Neoplasm; Prolactinoma; Proliferating; RNA library; Rattus; Refractory; Reproductive system; Research Personnel; Rotation; STAT3 gene; Signal Transduction; Silicon Dioxide; Specificity; System; Testing; Time; Toxic effect; Transcript; Translational Research; Tumor Subtype; Validation; Work; angiogenesis; biobank; drug discovery; drug use screening; experience; follow-up; high throughput screening; high-throughput drug screening; human disease; individual patient; inhibitor; innovation; interest; matrigel; multidisciplinary; neoplastic cell; novel; novel therapeutics; pharmacologic; pre-clinical; research and development; response; segregation; side effect; small hairpin RNA; small molecule inhibitor; statistics; subfertility; success; therapy development; transcriptome; transcriptome sequencing; transcriptomic profiling; tumor

ABSTRACT Prolactinomas (PRL-omas), the most common secreting pituitary tumors, frequently cause hypogonadism and subfertility. Although dopamine agonist therapy often works well initially and 1/3 of patients experience long-term remission after 2 years of therapy, 1/3 of patients require long-term D2 agonist therapy to maintain normal PRL levels and a further 1/3 of patients are either refractory to DA therapy or intolerant of their side effects. Although surgical resection can be offered in some, remission rates in large locally invasive tumors is <50%. Translational research has been hampered by the lack of any human PRL-oma cell models. Using current methodologies, human PRL-oma cultures only survive for 7-10 days in vitro. There is an urgent unmet need for establishment of a human PRL-secreting tumor model that retains its highly differentiated phenotype and allows sufficient long-term expandability of cells for use in preclinical translational research and development of novel treatment options. We compared the global transcriptome landscape in consecutive passages from two human prolactin-secreting pituitary tumor primary cultures using bulk RNA-seq to understand the molecular events leading to loss of hormone secretion during in vitro human prolactinoma culture. As pituitary tumor cells lost hormone secretion, we observed a reduction in 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) PRL-oma culture system and for the first time, we have generated long-term (> 6 months) expandible PRL-secreting 3D human pituitary tumor cultures (>6 months). In the first of two aims, we will use our unique patient-derived 3D PRL-oma culture model in a high throughput screen to identify small molecule inhibitors of PRL secretion and proliferation. We have already demonstrated the feasability of this approach and identified a compound of interest in a pilot screen and now wish to conduct an expanded HTS to identify novel therapies for patients with refractory PRL-omas. Our primary screen will include pharmacological validation and repurposing-, targeted-, lead-like- and diverse- libraries. Initial hits will be selected using robust z-score statistics and transitioned to aim 2 for further development. Aim 2 will employ a cascade of follow-up assays to validate potential hit compounds. These will include dose-response curves in both rat and human PRL-oma cells, assessment and exclusion of non-specific overly toxic compounds. And tests of specificity on hormone secretion in various murine and human neuroendocrine tumors. Additionally, in-silica target prediction will be combined with RNA-seq transcriptome profiling to segregate drug target pathways and deconvolute the MOA of resultant hits. Finally, potential actions of hits on identified pathway targets will be corroborated by pathway disruption using short hairpin RNA (shRNA) and CRISPR libraries in both rat and human PRL-omas to define a group of first-in-class hits.

摘要 催乳素瘤(PRL-OMA)是最常见的分泌性垂体瘤，常引起 性腺功能减退和不育症。尽管多巴胺激动剂治疗最初通常效果良好，三分之一的患者 经过2年的治疗后长期缓解，三分之一的患者需要长期的D2激动剂治疗 维持正常的PRL水平，另外三分之一的患者要么对DA治疗无效，要么对他们的 副作用。虽然在某些情况下可以进行手术切除，但在局部侵袭性较大的患者中，缓解率 肿瘤的发病率为50%。由于缺乏任何人类PRL-OMA细胞模型，翻译研究一直受到阻碍。 使用目前的方法，人类PRL-OMA培养在体外只能存活7-10天。有一件急事 尚未满足的建立保持高度分化的人催乳素分泌肿瘤模型的需求 表型，并允许细胞有足够的长期可扩充性，用于临床前的转化研究和 开发新的治疗方案。我们连续比较了全球转录组的情况 用Bulk RNA-Seq从两种分泌催乳素的人垂体瘤原代培养细胞传代 了解导致体外培养的人催乳素瘤激素分泌丧失的分子事件 文化。随着垂体瘤细胞失去激素分泌，我们观察到血管生成减少，存活率下降。 与胶原分解代谢、细胞黏附和细胞外增加并行的信号和免疫反应 矩阵组织。在这些发现的指导下，我们开发了一种独特的三维(3D)PRL-OMA培养 系统，第一次，我们已经产生了长期(6个月)可扩展的分泌PRL的3D人类 垂体瘤培养(&gt；6个月)。 在两个目标中的第一个目标中，我们将使用我们独特的患者衍生的3D PRL-OMA培养模型 吞吐量筛选以确定PRL分泌和增殖的小分子抑制物。我们已经这么做了 演示了此方法的可行性，并在试点屏幕中确定了感兴趣的化合物，现在 希望进行一项扩大的HTS，以确定难治性PRL-OMA患者的新疗法。我们的 主要筛选将包括药理验证和再利用-、靶向、类铅和多样化- 图书馆。最初的命中将使用稳健的z-Score统计数据进行选择，并过渡到目标2以进一步 发展。AIM 2将采用一系列后续测试来验证潜在的命中化合物。这些遗嘱 包括大鼠和人PRL-OMA细胞的剂量-反应曲线、非特异性的评估和排除 剧毒化合物。以及不同小鼠和人激素分泌的特异性试验 神经内分泌肿瘤。此外，硅胶靶标预测将与rna-seq转录组相结合。 分析以分离药物靶向通路，并解开所产生的命中的MOA。最后，潜在的行动 使用短发夹状RNA的通路中断将证实在已识别的通路目标上的命中 (ShRNA)和CRISPR文库，以定义一组一流的命中。