Project 2

项目2

• 批准号: 6748000
• 负责人: BARBARA W. HENDERSON
• 金额: $ 20.08万
• 依托单位: ROSWELL PARK CANCER INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-15 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6748000
• 关键词: cell death; cytokine; cytotoxicity; environmental stressor; enzyme linked immunosorbent assay; flow cytometry; host neoplasm interaction; immunocytochemistry; immunogenetics; immunoprecipitation; inflammation; laboratory mouse; light intensity; microarray technology; neoplasm /cancer blood supply; neoplasm /cancer immunology; neoplasm /cancer photoradiation therapy; neoplastic growth; nonvisual photosensitivity; northern blottings; oxidative stress; photosensitizing agents; photostimulus; respiratory oxygenation; western blottings

Since the inception of this program proiect grant it has been the long-term goal of Project II to bring about a fuller understanding of the complex mechanisms that govern the tissue responses to Photodynamic Therapy (PDT). The Project Leader's laboratory has developed extensive expertise in the study of two major elements of the PDT tissue response: the effects of PDT on tumor oxygenation, and the closely connected effects on the host inflammatory/immune responses directed against the tumor. This renewal application draws on novel discoveries that have emerged from both of these areas of interest. These are (i) that exposure of tumors to PDT greatly affects the expression of a wide spectrum of molecules important for tumor growth; (ii) that PDT regimens can be devised that greatly differ in the type of tumor microenvironment they create, and thus can exert markedly different influences on the regulation of such molecules. Based on these discoveries we have formulated the following hypotheses: (1) The rate at which photodynamic damage occurs will influence the expression of stress-induced genes. (2) The status of tumor oxygenation during and after PDT, largely determined by the parameters of a given PDT regimen, will affect the redoxdependent regulation of molecules important for tumor growth and/or anti-tumor host responses; (3) The mechanism of cell death within the tumor, influenced by a given photosensitizer as well as the microenvironmental conditions created by the PDT regimen will determine the "danger signals" generated and thus will influence tumor cell and anti-tumor host responses. To test these hypotheses we propose the following specific aims: Aim 1 To rationally devise PDT regimens in preclinical tumor models, based on our current knowledge of the different factors important for the PDT response (type of cell death, oxygenation) that are expected to create markedly different tumor microenvironments. Aim 2 To characterize the tumor milieus created by these PDT regimes, with special emphasis on signaling molecules that modulate the inflammatory/immune/angiogenic host response. Aim 3 To determine whether how host cells respond to these signals. Aim 4 To establish the molecular mechanisms by which PDT determines cytokine responsiveness of target cells. Our experilnental approach can be summarized as follows: (1) We will create different tumor microenvironments by, devising PDT regimes utilizing photosensitizers that induce different cell death pathways and by varying light fhience rates that affect tumor oxygenation, (2) We will use molecular techniques (microarray, RT-PCIL. Western blotting. ELISA) to characterize changes in protein expression induced by these PDT regimes; (3) We will assess the response of tumor and host to these changes by employing flow cytometry, immunohistochemistry, adhesion assays to assess host cell recruitment and apoptosis; (4) We will employ immunoblotting for detection of proteins, protein modifications and status of phosphorylation, immunoprecipitation, RT-PCR and Northern blot analyses for mRNA and flow cytometie assessment of cell cycle stage, apoptosis and marker protein expression to determine PDT effects on cytokine signaling. Our long-standing collaboration and multi-disciplinary approach aims to gain a comprehensive view of the mechanisms determining PDT tumor response, thereby possibly developing new treatment paradigms and optimizing treatment design. The subprojects are tightly, linked as they are complementary and necessary extensions of each other. Projects II will provide important input into clinical study design proposed in Project IV. The project is supported by three scientific Cores.

自该项目开始以来，它一直是项目II的长期目标，以更全面地了解控制组织对光动力学疗法（PDT）反应的复杂机制。项目负责人的实验室在PDT组织反应的两个主要因素的研究方面积累了丰富的专业知识：PDT对肿瘤氧合的影响，以及对宿主炎症/免疫反应的密切相关影响。 针对肿瘤的反应。这一更新申请借鉴了从这两个感兴趣的领域出现的新发现。这些是（i）肿瘤暴露于PDT极大地影响了对肿瘤生长重要的广谱分子的表达;（ii）PDT方案可以设计成在它们创建的肿瘤微环境的类型上有很大不同，因此可以对这些分子的调节施加明显不同的影响。基于这些发现，我们提出了以下假设：（1）光动力损伤发生的速率会影响胁迫诱导基因的表达。(2)PDT期间和之后的肿瘤氧合状态，主要由给定PDT方案的参数决定，将影响对肿瘤生长和/或抗肿瘤宿主反应重要的分子的氧化还原依赖性调节;（3）肿瘤内细胞死亡的机制，受给定光敏剂以及所产生的微环境条件的影响 PDT疗法的治疗将决定产生的“危险信号”，从而影响肿瘤细胞和抗肿瘤宿主的反应。为了验证这些假设，我们提出了以下具体目标：目的1合理设计PDT方案在临床前肿瘤模型，基于我们目前的知识的不同因素的重要PDT反应（细胞死亡的类型，氧合），预计将创建显着不同的肿瘤微环境。目的2描述这些PDT方案所产生的肿瘤环境，特别强调 调节炎症/免疫/血管生成宿主反应的信号分子。目的3确定宿主细胞是否对这些信号作出反应。目的4探讨光动力疗法（PDT）影响靶细胞对细胞因子反应性的分子机制。我们的实验方法可以总结如下：（1）我们将通过设计PDT方案来创建不同的肿瘤微环境，该方案利用诱导不同细胞死亡途径的光敏剂，并通过改变影响肿瘤氧合的光通量率，（2）我们将使用分子生物学方法来创建不同的肿瘤微环境。 技术（微阵列，RT-PCIL.蛋白质印迹法。（3）采用流式细胞术、免疫组化、粘附试验等方法检测肿瘤细胞的募集和凋亡，评价肿瘤细胞和宿主细胞对这些变化的反应;（4）我们将采用免疫印迹法检测蛋白质、蛋白质修饰和磷酸化状态，免疫沉淀，RT-PCR和北方印迹分析mRNA和流式细胞术评估细胞周期阶段、凋亡和 标志物蛋白表达以确定PDT对细胞因子信号传导的作用。我们的长期合作和多学科方法旨在全面了解 因此，研究人员可以发现决定PDT肿瘤反应的机制，从而可能开发新的治疗模式并优化治疗设计。这些次级项目相互补充，互为必要的延伸，因此彼此紧密相连。项目II将为项目IV中拟定的临床研究设计提供重要输入。该项目得到三个科学核心的支持。