Functional Analysis of Hypoxia-Induced Genes in Promoting Malignant Growth of Glioblastoma

缺氧诱导基因促进胶质母细胞瘤恶性生长的功能分析

• 批准号: 10611372
• 负责人: Valerie Marallano
• 金额: $ 2.04万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-13 至 2023-09-12
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10611372
• 关键词: Address; Adhesions; Affect; Automobile Driving; Biology; Blood Vessels; CXCR4 gene; Candidate Disease Gene; Cell Communication; Cell Hypoxia; Cell Line; Cells; Complex; Data; Engineering; Environment; Future; Genes; Genetic; Genetic Transcription; Glioblastoma; Glioma; Growth; Heterogeneity; Hypoxia; Hypoxia Inducible Factor; Immune; Immunosuppression; In Vitro; Infiltration; Ionizing radiation; Label; Laboratories; Link; Magnetic Resonance Imaging; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of brain; Mediating; Mesenchymal; Messenger RNA; Metabolic; Molecular; Outcome; Oxygen; Pathway interactions; Patients; Pimonidazole; Population; Positron-Emission Tomography; Prodrugs; Prognosis; Proliferating; Proteins; Reporter; Reporting; Resistance; Resolution; Response Elements; Role; Synapses; Testing; Therapeutic; Transcript; Transplantation; Tumor Biology; Tumor-associated macrophages; Validation; Work; Xenograft Model; angiogenesis; brain tissue; candidate validation; cell motility; chemokine receptor; chemoradiation; cytotoxicity; detection sensitivity; efficacy evaluation; experimental study; fluorophore; genetic signature; improved; in vivo; in vivo Model; insight; irradiation; knock-down; migration; neoplastic cell; novel; oxidation; promoter; response; single-cell RNA sequencing; small hairpin RNA; stem cells; synergism; therapy resistant; transplant model; tumor; tumor growth; tumor hypoxia; tumor microenvironment; vector

Project Summary Tumor hypoxia is linked to worse outcome of glioblastoma (GBM), the most malignant type of brain cancer. Hypoxic GBM cells are thought to resist therapy, and to have high migratory capacity to move away from hypoxic zones, infiltrating normal brain tissue. Aside from these cell-autonomous attributes, hypoxic tumor cells also contribute to GBM progression by exerting non-cell autonomous functions, in particular inducing angiogenesis and reprograming tumor microenvironment (TME) into an immunosuppressive and tumor-supporting state. To better understand the gene signatures of GBM cells in hypoxic niches and their interactions with TME cells, it is critical to conduct studies with in vivo models. However, earlier approaches to track hypoxic cells, such as pimonidazole or HRE-GFP have limited sensitivity and resolution. To facilitate improved in vivo tracking of hypoxic tumor cells during GBM progression, I will apply a genetic reporter with HRE (hypoxia response element promoter) driving expression of UnaG, a fluorescent protein that does not require oxygen for fluorophore maturation, thus offering superior sensitivity over GFP in hypoxic conditions. The UnaG mRNA transcript also allows me to distinguish hypoxic from normoxic cells in single cell (sc) RNA-seq studies. Indeed, my preliminary data with a patient-derived GBM stem cell line engineered with HRE-UnaG showed sensitive reporting of hypoxia in vivo in intracranial transplant experiments. Excitingly, using immunohistological analysis combined with scRNA-seq, I have collected preliminary evidence supporting the central hypothesis that GBM cells in hypoxic niches in vivo are relatively quiescent and display a shift to a mesenchymal state, both features that are linked to therapy resistance and migratory potency. I have also identified top differentially regulated genes in hypoxic GBM cells in vivo, with potential roles in promoting migration and enhancing proliferation of neighboring tumor cells. This proposal will expand upon my initial studies to further define in vivo GBM hypoxia gene signatures. A key focus in my first aim will be the interaction of hypoxic tumor cells with surrounding TME, in particular tumor associated macrophages (TAM) in hypoxic niches. To dissect the molecular mechanisms of hypoxia-driven GBM malignancy, I will conduct for my second aim functional analyses of two candidate genes, CXCR4 and NXPH4, which are highly upregulated in hypoxic GBM cells. I will test the hypothesis that CXCR4 promotes migration of hypoxic tumor cells, while NXPH4 may support the growth of neighboring GBM cells. Lastly, for my third aim, I will explore a therapeutic strategy to target the hypoxic population to evaluate its efficacy to sensitize GBM cells to irradiation therapy. I plan to carry out my studies with two patient-derived GBM stem cell lines of different transcriptional subtypes (mesenchymal and proneural), to assess how different GBM subtypes respond to hypoxia. In sum, my comprehensive in vivo studies, combining HRE-UnaG reporter, in vivo intracranial transplants, scRNA-seq, immunohistology, functional validations, and therapeutic strategies will provide new insights into the biology of hypoxia-induced GBM malignancy and will delineate pathways for future treatments.

项目摘要 肿瘤缺氧与胶质母细胞瘤（GBM）（最恶性的脑癌类型）的预后不良有关。 低氧GBM细胞被认为抵抗治疗，并且具有高迁移能力以远离低氧环境。 区域，渗入正常脑组织除了这些细胞自主属性，缺氧肿瘤细胞也 通过发挥非细胞自主功能，特别是诱导血管生成，促进GBM进展 以及将肿瘤微环境（TME）重编程为免疫抑制和肿瘤支持状态。到 为了更好地了解GBM细胞在缺氧环境中的基因特征及其与TME细胞的相互作用， 这对于用体内模型进行研究至关重要。然而，早期追踪低氧细胞的方法，如 pimonidazole或HRE-GFP具有有限的灵敏度和分辨率。为了促进改善的体内追踪， 在GBM进展过程中缺氧肿瘤细胞，我将应用具有HRE（缺氧反应元件）的遗传报告基因 启动子）驱动UnaG的表达，UnaG是一种荧光蛋白，其荧光团不需要氧 成熟，从而在缺氧条件下提供优于GFP的上级灵敏度。UnaG mRNA转录物还 允许我在单细胞（sc）RNA-seq研究中区分低氧细胞和常氧细胞。事实上，我的初步 用HRE-UnaG工程化的患者来源的GBM干细胞系的数据显示了缺氧的敏感报告 在体内颅内移植实验中。令人兴奋的是，使用免疫组织学分析结合 scRNA-seq，我已经收集了初步证据，支持GBM细胞缺氧的中心假设， 在体内的小生境是相对静止的，并显示出向间充质状态的转变，这两个特征都与 治疗抵抗力和迁移能力。我还发现了缺氧性脑损伤中最重要的差异调节基因， 体内GBM细胞，具有促进邻近肿瘤迁移和增强增殖的潜在作用 细胞这个建议将扩大我的初步研究，以进一步确定在体内GBM缺氧基因签名。一 在我的第一个目标的重点将是缺氧肿瘤细胞与周围的TME，特别是肿瘤细胞的相互作用， 相关巨噬细胞（TAM）在缺氧壁龛。剖析缺氧驱动GBM的分子机制 恶性肿瘤，我将进行我的第二个目标的两个候选基因，CXCR 4和NXPH 4的功能分析， 其在缺氧GBM细胞中高度上调。我将测试CXCR 4促进细胞迁移的假设。 NXPH 4可以促进缺氧肿瘤细胞的生长，而NXPH 4可以支持邻近GBM细胞的生长。最后，我的第三个目标是， 将探索一种针对缺氧人群的治疗策略，以评估其致敏GBM细胞的功效 到放射治疗。我计划用两种不同的患者来源的GBM干细胞系进行研究。 转录亚型（间充质和前神经），以评估不同的GBM亚型如何响应 缺氧综上所述，我的综合体内研究，结合HRE-UnaG报告基因， 移植，scRNA-seq，免疫组织学，功能验证和治疗策略将提供新的 深入了解缺氧诱导的GBM恶性肿瘤的生物学，并将描绘未来治疗的途径。