[PQC-3] A Metabolic Pathway Activation Marker for Prostate Cancer Prognosis

描述(由申请人提供)：为了优化治疗，在诊断时预测任何局部肿瘤的转移潜力都是有帮助的。对于前列腺癌(PCa)来说，这一点尤为重要。PSA筛查的广泛应用导致越来越多的低级别和小PCA的检测。如果不进行治疗，其中一些PCAS可能会进展并变得致命。然而，他们中的许多人可能会在患者的余生中保持懒惰。格里森评分(GS)系统，目前用于预测 当GS为d7时，PCA进展的可能性不能预测风险。在目前的临床实践中，尚未满足对预测这些局部肿瘤进展可能性的方法的医学需求。假设：有越来越多的证据表明，前列腺癌细胞内产生的活性氧会导致前列腺癌的进展和转移。我们假设，具有高细胞ROS的癌细胞将修改结构蛋白丝状肌动蛋白(F-肌动蛋白)。这样的修饰将使细胞足够灵活，可以挤过肿瘤周围的缝隙，侵入血管和淋巴管进行循环。其中一些带有激活的MMPs和修饰的F-肌动蛋白的循环细胞将逃脱内皮细胞而转移。我们的目标是开发一种综合的预后测试，该测试基于从常规前列腺活检获得的原发肿瘤细胞在肿瘤微环境的体外模型中生长，然后执行FAD荧光定量成像来确定导致前列腺癌进展的特定代谢活动。我们将通过将患者前列腺癌组织的数据与疾病结果相关联来验证这一途径生物标记物(S)。我们的具体目标是：1)量化不同激活水平的野生型和转基因前列腺癌细胞系的荧光特性之间的差异 在一项回顾性的临床验证研究中，通过对基底膜和内皮的微流控模拟中ROS生成途径的研究，能够确定它们的相对侵袭性；2)通过将从多个前列腺TMA收集的FAD荧光强度和寿命数据与患者的预后相关联，建立FAD荧光强度和寿命数据作为预后标志物。将通过测量来自相应组织切片的激光捕获组织中的代谢物水平来确认所观察到的作为酶激活指示器的FAD荧光的变化，3)从知情的、同意的患者的组织样本中制备PCA细胞的单细胞悬浮液，并分析FAD荧光及其在微流体系统中的侵袭特性，如在目标#1中标准化的，b)从从相同患者获得的PCA组织中创建TMA并执行如在目标#2中标准化的FAD荧光分析，C)从相同的TMA切片中激光捕获癌组织，并根据目标2和d)中的标准化估计代谢物，以通过将在a-c中收集的数据与前瞻性临床研究中的患者结果相关联来在临床上建立标记物。

DESCRIPTION (provided by applicant): For best optimization of therapy, it is helpful to predict the metastatic potential of any localized tumor at the time of diagnosis. This is particularly critcal for prostate cancer (PCa). The widespread application of PSA screening is leading to the detection of an increasing number of low grade and small PCas. Left untreated, some of these PCas may progress and become lethal. Many of them, however, may remain indolent for the rest of the patients' lives. The Gleason scoring (GS) system that is currently practiced to predict the possibility of PCa progression fails to predict the risk, when GS is d7. There is unmet medical need for a method for predicting likelihood of progression of these localized tumors without major changes in the current clinical practice. Hypothesis: There is accumulating evidence that production of reactive oxygen species within PCa cells lead to PCa progression and metastasis. We hypothesize that cancer cells with high cellular ROS will modify the structural protein filamentous actin (F-actin). Such modifications will make the cells flexible enough to squeeze through the gaps surrounding the tumor and invade the blood and lymphatic vessels for circulation. Some of these circulating cells with activated MMPs and modified F-actin will escape the endothelia to metastasize. Our goal is to develop an integrated prognostic test based on growing primary tumor cells obtained from routine prostate biopsies in micro-scale in vitro models of tumor microenvironments and then performing quantitative imaging of FAD fluorescence to determine specific metabolic activities that leads to PCa progression. We will validate this pathway biomarker(s) by correlating the data from patient PCa tissues with disease outcome. Our Specific Aims are: 1) To quantitate the difference between fluorescence properties of wild-type and genetically engineered PCa cell lines with varying levels of activation of the ROS generating pathway in a microfluidic mimetic of the basement membrane and endothelium capable of determining their relative invasive proper- ties, 2) To establish FAD fluorescence intensity and lifetime data collected from multiple prostate TMAs as prognostic markers by correlating the data with patient outcome in a retrospective clinical validation study. The observed changes of FAD fluorescence as an indicator of the enzyme activation will be confirmed by measuring the metabolite levels in the laser captured tissues from the corresponding tissue sections, 3) a) To prepare single cell suspensions of PCa cells from the tissue samples from informed, consented patients and analyze FAD fluorescence and their invasive properties in the microfluidic system as standardized in Aim #1, b) To create a TMA from PCa tissues obtained from the same patients and perform FAD fluorescence analysis as standardized in Aim #2, c) To laser capture cancer tissues from the same TMA sections and estimate the metabolites as standardized in Aim #2 and d) To clinically establish the markers by correlating the data collected in a-c with patient outcome in a prospective clinical study.