Investigation of sub-lineages in pulmonary neuroendocrine cells and identification of the cells of origin of small cell lung cancer

肺神经内分泌细胞亚谱系研究及小细胞肺癌细胞来源鉴定

• 批准号: 10265579
• 负责人: Joyce Huanhuan Chen
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-16 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10265579
• 关键词: Adenoviruses; Automobile Driving; Biological Markers; Biology; CRISPR library; CRISPR screen; Cancer Model; Cancer Patient; Cancer cell line; Cell Lineage; Cell Size; Cells; DNA Sequence Alteration; Data; Diagnosis; Disease; Early Diagnosis; Early treatment; Enterobacteria phage P1 Cre recombinase; Epithelial Cells; Event; Foundations; Frequencies; Genes; Genetic Transformation; Genetically Engineered Mouse; Goals; Health; Heterogeneity; Human; Immunocompromised Host; Immunodeficient Mouse; In Vitro; Investigation; Knockout Mice; Lead; Location; Lung; Maintenance; Malignant Neoplasms; Maps; Methods; Modeling; Molecular; Mus; Mutation; Neuroendocrine Cell; Neuroepithelial Bodies; Oncogenic; Pathogenesis; Pathway interactions; Pattern; Phase; Physiological; Population; Population Heterogeneity; Prevention; Primary Neoplasm; Public Health; RB1 gene; RNA; RNA analysis; Regulation; Research; Resources; Risk Assessment; Sampling; Signal Transduction; Structure of parenchyma of lung; TP53 gene; Testing; Therapeutic; Time; Tumor Suppressor Genes; Tumor Tissue; United States; Variant; airway epithelium; anticancer research; base; cancer initiation; carcinogenesis; cell fate specification; cell type; differential expression; human embryonic stem cell; improved; lung small cell carcinoma; novel; novel strategies; prevent; progenitor; promoter; pulmonary body; screening; single cell technology; single-cell RNA sequencing; stem cells; transcriptome; transcriptomics; tumor; tumorigenesis

PROJECT SUMMARY Small cell lung cancer (SCLC) remains a major challenge in public health because of its frequency, its lethality, and the paucity of convenient models for exploring its pathogenesis and potential therapeutic strategies. Pulmonary neuroendocrine cells (PNECs) are believed to be the putative precursor of SCLC. However, increasing evidence shows that PNECs contain sub-lineages varying in location, cell size, and physiological functions. In my previous research, I developed a novel experimental approach for studying the biology of PNECs - and the initiation of SCLC - by differentiating human embryonic stem cells (hESCs) into the lung lineage, and subsequently perturbing three tumor suppressor genes that are frequently altered in SCLC. By perturbing NOTCH signaling, the lung progenitor cells can be differentiated into PNECs that further undergo oncogenic transformation and form SCLC-like tumors in mice, when RB and P53 expression are reduced. Single cell RNA (scRNA) profiles demonstrated great similarity between the hESC-derived PNECs and the native PNECs in human and mouse lung. Of particular significance, scRNA analysis further revealed sub-lineages within the hESC-derived PNECs. Among them, one profile demonstrated significant similarity to the RNA profiles of early stage human SCLC tumors and SCLC cell lines. The above findings and recent studies by others, led me to further hypothesize that the PNEC sub-lineages have different oncogenic potential, and among them, one specific population serves as the dominant cell of origin of SCLC. I propose to use this model together with other methods such as scRNA transcriptomics, genetically engineering mouse models, to test this hypothesis and to study the origins of SCLC in several ways. First, I will identify the PNEC sub- lineages in normal human and mouse lung tissues that are similar to the ones in the hESC-derived PNECs. Alternatively, sub-lineages of PNECs in mouse lung will be characterized by scRNA profiling and new cell-fate markers identified from the mouse PNEC sub-lineages will be extrapolated to further delineate the heterogeneous populations in human PNECs. Next, I will purify the sub-populations of hESC-derived PNECs and test their transformative potential in culture and in immunocompromised mice by known oncogenic events in SCLC. Alternatively, the PNEC sub-lineages in mouse lung can be tested for their potential to form tumors by using conditional Rb1/Trp53 knockout mice. Through these studies, I expect to identify a specific sub-lineage of PNECs that are most sensitive to SCLC mutations and capable of transformation, which would implicate them as the cell of origin in SCLC. In the R00 phase, I propose to expand the research to explore mechanisms driving the lineage hierarchies of PNECs and their variant oncogenic potentials. These include studying inter-differentiation among the PNEC sub-lineages, the effects of NOTCH, SOX2 and other single pathways on PNEC fate determination, and using CRISPR screening to explore new molecular events important in maintenance of PNEC hierarchical patterns and the regulation of their specific oncogenic capacity. These studies are expected to not only advance our understanding of carcinogenesis of PNECs, but also provide new opportunities to diagnose, categorize, treat, and possibly even prevent this disease more effectively.

项目摘要 小细胞肺癌（SCLC）仍然是公共卫生的主要挑战，因为它的频率，它的致命性， 缺乏方便的模型来探讨其发病机制和潜在的治疗策略。肺 神经内分泌细胞（PNEC）被认为是SCLC的推定前体。然而，越来越多的证据表明， PNEC含有在位置、细胞大小和生理功能上不同的亚系。 在我以前的研究中，我开发了一种新的实验方法来研究PNEC的生物学， 启动SCLC -通过将人胚胎干细胞（hESC）分化为肺谱系，随后 干扰三种在SCLC中经常改变的肿瘤抑制基因。通过干扰NOTCH信号， 祖细胞可以分化成PNEC，其进一步经历致癌转化并形成SCLC样肿瘤 在小鼠中，当RB和P53表达降低时。单细胞RNA（scRNA）谱显示出在 人和小鼠肺中hESC衍生的PNEC和天然PNEC。特别重要的是，scRNA分析 进一步揭示了hESC衍生的PNEC中的亚系。其中，一个剖面显示出显著的相似性 早期人类SCLC肿瘤和SCLC细胞系的RNA谱。上述发现和最近的研究， 其他人，使我进一步假设PNEC亚系具有不同的致癌潜力，其中，一个 特定的细胞群是SCLC的优势起源细胞。 我建议将这个模型与其他方法一起使用，如scRNA转录组学，基因工程， 小鼠模型，以验证这一假设，并从几个方面研究SCLC的起源。首先，我将确定PNEC子- 正常人和小鼠肺组织中的谱系与hESC衍生的PNEC中的谱系相似。可选择地， 小鼠肺中PNEC的亚系将通过scRNA谱和从PNEC中鉴定的新细胞命运标志物来表征。 小鼠PNEC亚系将被外推以进一步描述人PNEC中的异质群体。 接下来，我将纯化hESC衍生的PNEC的亚群，并在培养物和细胞中测试它们的转化潜力。 免疫功能低下的小鼠通过已知的致癌事件在SCLC。或者，小鼠肺中的PNEC亚系可以 通过使用条件性Rb 1/Trp 53敲除小鼠测试它们形成肿瘤的潜力。通过这些研究，我希望 鉴定对SCLC突变最敏感并能够转化的PNEC的特定亚系， 表明它们是小细胞肺癌的起源细胞 在R 00阶段，我建议扩展研究，探索PNEC谱系层次的驱动机制 及其变异的致癌潜能。其中包括研究PNEC亚系之间的相互分化， NOTCH，SOX 2和其他单一途径对PNEC命运确定的影响，并使用CRISPR筛选来探索 在维持PNEC分级模式和调节其特异性致癌基因中重要的新分子事件 容量这些研究不仅有助于我们进一步了解PNEC的致癌作用， 诊断、分类、治疗甚至可能更有效地预防这种疾病的新机会。