In Vivo Imaging of Hypoxia-Inducible Physiology

描述（由申请人提供）：本提案的目标是应用分子成像和报告基因技术来创建HIF-1和XBP 1活性的体内传感器，并使用它们来研究癌症治疗动物模型中的缺氧诱导细胞信号传导。缺氧诱导的转录因子HIF-1和XBP 1的鉴定使人们深入了解了氧合变化如何激活基因表达，但也证明这些因子可能导致肿瘤恶性程度增加，而不依赖于氧。这些途径作为低氧肿瘤表型的标志物和效应物的重要性已经得到证实，但是缺乏对它们在体内肿瘤微环境中的行为和动力学的完整理解。为了完成这项任务，我们提出了一个为期两年的项目，在技术开发和应用之间取得平衡。我们将首先开发并严格验证一组HIF-1和XBP 1反应的报告构建体在细胞培养中，重点是用于区分报告基因表达与缺氧介导的报告蛋白活性丧失的方法。在评估它们在氧化应激的细胞培养模型中独立测量HIF-1 a稳定性和HIF-1和XBP 1诱导的转录活性的能力后，我们将产生表达每个报告基因的小鼠肿瘤模型，并证明每个构建体在体内条件下报告其靶点的稳健性。然后将用新型抗HIF-1化疗药物治疗这些模型，在此过程中，将通过对报告基因成像来监测HIF-1和XBP 1反应。因此，我们将产生分子生物学工具来研究活体受试者的缺氧诱导生理学，并将这些工具应用于新型靶向化疗的验证和优化，加速其在临床上有用的癌症治疗方面的进展。这项研究将开发报告基因技术，用于检测和成像完整肿瘤内两种转录因子HIF-1和XBP 1的活性。通过开发新的方法来量化这些因素，并将其应用于癌症治疗过程中这些途径的研究，该项目将进一步加深我们对肿瘤生物学的理解，并将加速癌症靶向治疗的开发和评估。

DESCRIPTION (provided by applicant): The goal of this proposal is to apply molecular imaging and reporter gene techniques to create in vivo sensors of HIF-1 and XBP1 activity, and to use them to study hypoxia-inducible cell signaling in animal models of cancer therapy. Identification of the hypoxia-induced transcription factors HIF-1 and XBP1 has given insight into how changes in oxygenation activate gene expression, but has also demonstrated that these factors may cause increased tumor malignancy independent of oxygen. The importance of these pathways as markers as well as effectors of the hypoxic tumor phenotype has been demonstrated, however a complete understanding of their behavior and dynamics in the in vivo tumor microenvironment has been lacking. To accomplish this task, we propose a two-year project balanced between technique development and application. We will first develop and rigorously validate a set of HIF-1- and XBP1-responsive reporter constructs in cell cultures, focusing on methods for distinguishing reporter gene expression from hypoxia-mediated loss of reporter protein activity. After evaluation of their ability to independently measure HIF-1a stabilization and HIF-1- and XBP1-induced transcriptional activity in cell culture models of oxidative stress, we will generate murine tumor models expressing each reporter gene and demonstrate the robustness of each construct to report on its target under in vivo conditions. These models will then be treated with novel anti-HIF-1 chemotherapeutics, over the course of which HIF-1 and XBP1 response will be monitored by imaging the reporters. We will therefore generate molecular biology tools to study hypoxia-inducible physiology in living subjects, and apply these tools towards the validation and optimization of novel targeted chemotherapies, accelerating their progress towards clinically useful cancer treatments. This research will develop reporter gene techniques for detecting and imaging the activity of two transcription factors, HIF-1 and XBP1, within intact tumors. By developing novel methods to quantify these factors and applying them towards the study of these pathways during cancer therapy, this project will further our understanding of tumor biology and will accelerate the development and evaluation of targeted therapies for cancer.