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.

自从该计划授予赠款的成立以来，这是项目II的长期目标，即对控制光动力疗法（PDT）的组织反应的复杂机制有更深入的了解。该项目负责人的实验室在研究PDT组织反应的两个主要元素方面已经建立了广泛的专业知识：PDT对肿瘤氧合的影响以及对宿主炎症/免疫的紧密联系的影响 针对肿瘤的反应。这种更新应用借鉴了从这两个感兴趣的领域中出现的新发现。这些（i）暴露于PDT的暴露会极大地影响对肿瘤生长重要的广泛分子的表达； （ii）可以设计出PDT方案在它们创建的肿瘤微环境的类型上有很大差异，因此对这种分子的调节产生明显不同的影响。基于这些发现，我们提出了以下假设：（1）发生光动力损伤的速率将影响应激诱导的基因的表达。 （2）在PDT期间和之后的肿瘤氧化状态，主要由给定PDT方案的参数确定，将影响对氧化还原依赖的分子调节，对肿瘤生长和/或抗肿瘤宿主反应很重要； （3）肿瘤内细胞死亡的机理，受给定的光敏剂以及产生的微环境条件的影响 PDT方案将确定产生的“危险信号”，从而影响肿瘤细胞和抗肿瘤宿主反应。为了检验这些假设，我们提出了以下特定目的：目标1基于我们当前对PDT反应（细胞死亡类型，氧合）对不同因素重要的知识，在临床前肿瘤模型中制定PDT方案，这些因素有望产生明显不同的肿瘤微环境。 AIM 2来表征这些PDT制度所产生的肿瘤环境，并特别强调 关于调节炎症/免疫/血管生成宿主反应的信号分子。目标3以确定宿主细胞如何响应这些信号。目标4以建立PDT决定靶细胞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）我们将通过设计诱导不同细胞死亡途径的光敏剂来设计不同的肿瘤微环境，并通过影响肿瘤氧气的光率不同，（2）我们将使用分子（2） 技术（微阵列，RT-PCIL。Westernblotting。ELISA）表征了这些PDT状态引起的蛋白质表达的变化； （3）我们将通过使用流式细胞术，免疫组织化学，粘附测定法评估肿瘤和宿主对这些变化的反应，以评估宿主细胞募集和凋亡； （4）我们将采用免疫印迹来检测蛋白质，蛋白质修饰和磷酸化，免疫沉淀，RT-PCR和北印迹分析的mRNA和北印迹分析，用于细胞周期，凋亡和凋亡和流式细胞仪评估 标记蛋白表达确定PDT对细胞因子信号的影响。我们长期的合作和多学科方法旨在全面了解 确定PDT肿瘤反应的机制，从而可能开发新的治疗范式并优化治疗设计。子弹的紧密连接，因为它们是互补的且必要的扩展。项目II将为IV项目提出的临床研究设计提供重要的意见。该项目得到了三个科学核心的支持。