Imaging Cellular Stress and Treatment Response Using Positron Emission Tomography

描述（由申请人提供）：正电子发射断层扫描（PET）允许人们在体内和无创地测量肿瘤和正常组织的代谢。我们一直在研究胸腺嘧啶及其类似物，最初的目标是成像细胞增殖。正在研究的类似物之一是FMAU{1-（2'-脱氧-2'-氟- β -d -阿拉伯糖脲基）胸腺嘧啶}，我们已经用18F标记并在细胞培养、动物和人类中进行了研究。我们的研究发现，在人类中，FMAU很容易保留在肿瘤中，但不能保留在正常增殖的骨髓中。我们和其他人的研究表明，FMAU是由胸苷激酶2 （TK2）的作用保留的，这是一种线粒体酶。我们发现保留率的增加反映了细胞压力。这与3'-脱氧-3'-氟胸腺嘧啶（FLT）相反，后者由胸腺嘧啶激酶1 （TK1）保留，TK1是与正常DNA合成相关的细胞质酶。在这里，我们将验证这样一个假设，即用18F-FMAU成像细胞应激可能提供癌症治疗反应的早期测量。此外，FMAU可能比正在开发的成像细胞凋亡的药物更容易使用，因为应激的时间过程可能比细胞凋亡的变化更可预测，而细胞凋亡的变化通常是短期的。我们已经开发了18F-FMAU成像的简化方法。为了进一步了解和测试18F-FMAU用于PET成像，我们提出以下目标。目的1将使用细胞培养来确定如何以及何时控制TK2活性和FMAU摄取。我们将扩展我们的初步研究，这些研究表明，在细胞应激过程中会产生一种改变的、更活跃的TK2形式。在顺铂治疗肺癌细胞系的过程中，将使用qRT-PCR测量mRNA水平，Western blots测量蛋白质及其同种异构体，以及酶活性来分析这一点。TK1、细胞增殖、凋亡和线粒体质量的测量也将与FMAU摄取进行比较。目的2将研究将人肺癌细胞系植入免疫缺陷小鼠治疗后发生的变化。将用微pet监测18F-FMAU的摄取，以确定治疗后成像的最佳时间。FMAU摄取将与尸检切除肿瘤的细胞增殖、凋亡和TK2表达进行比较。最后，Aim 3将进一步研究肺癌患者的FMAU摄取。我们将研究FMAU成像的可重复性。我们将在切除早期疾病之前测量FMAU的摄取，并使用切除的标本来检验FMAU摄取与肿瘤TK2活性相关的假设。我们还将测量增殖、TK1和细胞凋亡。本研究将研究接受标准化疗的肺癌患者治疗后FMAU潴留的时间过程。这将验证FMAU摄取早期增加反映细胞应激和对治疗的最终反应的假设。总之，我们的研究试图验证和测试18F-FMAU在细胞培养、小鼠和人类中的应用，作为反映TK2活性和预测治疗反应的细胞应激标志物。

DESCRIPTION (provided by applicant): Positron emission tomography (PET) allows one to measure tumor and normal tissue metabolism in vivo and non-invasively. We have been studying thymidine and its analogs, with the initial goal of imaging cellular proliferation. One of the analogs under study is FMAU {1-(2'-deoxy-2'-fluoro-beta-D- arabinofuranosyl)-thymine}, which we have labeled with 18F and studied in cell culture, animals and humans. Our studies found that in humans FMAU was readily retained in tumors, but not in normally proliferating bone marrow. Our work, and that of others, has demonstrated that FMAU is retained by the action of thymidine kinase 2 (TK2), a mitochondrial enzyme. We have found that increased retention reflects cellular stress. This is in contrast to 3'-deoxy-3'-fluorothymidine (FLT), which is retained by thymidine kinase 1 (TK1) the cytosolic enzyme associated with normal DNA synthesis. Here we will test the hypothesis that imaging cellular stress with 18F-FMAU may provide an early measure of cancer treatment response. Furthermore, FMAU may be simpler to use than agents being developed to image apoptosis, since the time course of stress may be more predictable than changes in apoptosis, which are often short term. We have already developed simplified approaches to 18F-FMAU imaging. To further understand and test 18F-FMAU for PET imaging we propose the following Aims. Aim 1 will use cell cultures to determine how and when TK2 activity and FMAU uptake are controlled. We will extend our preliminary studies, which indicate that an altered, more active form of TK2 is generated during cellular stress. This will be analyzed using qRT-PCR to measure mRNA levels, Western blots to measure the protein and its isoforms, and enzymatic activity during the course of cisplatin treatment of lung cancer cell lines. Measurements of TK1, cell proliferation, apoptosis and mitochondrial mass will also be obtained for comparison with FMAU uptake. Aim 2 will study the changes that occur after therapy using human lung cancer cell lines implanted in immunodeficient mice. Uptake of 18F-FMAU will be monitored with microPET to determine the best time for imaging after therapy. FMAU uptake will be compared to measures of cell proliferation, apoptosis, and TK2 expression from tumors removed at necropsy. Finally, Aim 3 will further study FMAU uptake in humans with lung cancer. We will study the reproducibility of FMAU imaging. We will measure FMAU uptake prior to resection of early stage disease and use the resected specimens to test the hypothesis that FMAU uptake correlates with tumor TK2 activity. We will also measure proliferation, TK1, and apoptosis. This Aim will then study the time course of FMAU retention after treatment in patients receiving standard chemotherapy for lung cancer. This will test the hypothesis that early increases in FMAU uptake reflect cellular stress and ultimate response to therapy. In summary, our studies seek to validate and test the use of 18F-FMAU in cell cultures, mice and humans, as a marker of cellular stress which reflects TK2 activity and predicts treatment response. PUBLIC HEALTH RELEVANCE: The studies being proposed will result in methods to measure tumor activity and treatment-induced changes within hours to days after the start of therapy. Currently there is no routine proven way of doing this early in the course of treatment. If this approach is validated it will be very useful in tailoring therapy that is more successful in killing tumors. While we have chosen to study lung cancer here, FMAU may well find use in the evaluation of other tumors as well. We conjecture that validation of its use in lung tumors will expedite its use with other cancers.