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

描述（由申请人提供）：为了最佳优化治疗，在诊断时预测任何局部肿瘤的转移潜力是有帮助的。这对于前列腺癌（PCa）尤其重要。PSA筛查的广泛应用导致越来越多的低级别和小前列腺癌的检测。如果不进行治疗，其中一些PCAs可能会进展并致命。然而，他们中的许多人可能会在病人的余生中保持懒惰。格里森评分（GS）系统目前用于预测 当GS为d 7时，PCa进展的可能性不能预测风险。对于预测这些局部肿瘤进展的可能性而不对当前临床实践进行重大改变的方法存在未满足的医学需求。假设：越来越多的证据表明，PCa细胞内活性氧的产生导致PCa进展和转移。我们假设，具有高细胞活性氧的癌细胞会修饰结构蛋白丝状肌动蛋白（F-actin）。这样的修饰将使细胞足够灵活，可以通过肿瘤周围的间隙挤压并侵入血液和淋巴管进行循环。这些具有活化的MMP和修饰的F-肌动蛋白的循环细胞中的一些将逃离内皮而转移。我们的目标是开发一种综合的预后测试，其基于在肿瘤微环境的微尺度体外模型中从常规前列腺活检获得的生长的原发性肿瘤细胞，然后进行FAD荧光的定量成像以确定导致PCa进展的特定代谢活动。我们将通过将来自患者PCa组织的数据与疾病结局相关联来验证该途径生物标志物。我们的具体目标是：1）定量具有不同活化水平的野生型和基因工程化PCa细胞系的荧光性质之间的差异 在基底膜和内皮的微流体模拟物中的ROS产生途径，能够确定它们的相对侵袭特性，2）通过在回顾性临床验证研究中将从多种前列腺TMA收集的FAD荧光强度和寿命数据与患者结果相关联，建立FAD荧光强度和寿命数据作为预后标志物。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，将确认观察到的作为酶活化指示剂的FAD荧光的变化。3）a）为了从来自知情同意的患者的组织样品制备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入性质，如目标#1中标准化的，B）从获自相同患者的PCa组织产生TMA并进行如目标#2中标准化的FAD荧光分析，c）从相同TMA切片激光捕获癌组织并估计如目标#2中标准化的代谢物，以及d）通过将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临床建立标志物。

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.