Molecular Pathology Research for Cancer Diagnostics and Biomarkers

在这个项目中进行研究，以确定和开发新的人类疾病动物模型，并开发更好地确定与人类状况相关的模型的手段。该项目的目标包括对研究技术和标记进行评估。在开发癌症诊断方法和开发和表征人类癌症新模型的研究资源方面取得了进展。开发了用于验证人类癌症动物模型的方法和试剂和技术。该研究项目包括开发癌症模型的分子诊断能力，开发用于定量病理的癌症标本的自动形态图像分析方法，研究S100在卵巢癌中的作用以及Ras癌基因激活的非分裂细胞的细胞周期调节。卵巢癌生物标志物开发的研究进展正在产生有可能作为肿瘤生物标志物的血清蛋白的发现。应用基于质谱的蛋白质组学，我们试图阐明一个悬而未决的生物标志物研究问题，即通过直接从肿瘤细胞发出的卵巢癌生物标志物蛋白来确定肿瘤负荷的能力。由于侵袭性浆液上皮性卵巢癌是导致死亡的主要原因，因此我们建立了一个使用人类SKOV-3浆液性卵巢癌细胞的异种移植模型来模拟扩散性癌的进展。采用低分子量蛋白富集、液相色谱和质谱分析的方法，在晚期实验性卵巢癌小鼠血清中鉴定出了S100的人类特异性肽序列。这种S100的表达在癌症异种移植物和卵巢癌患者组织中都有记录。纵向研究显示，血清S100浓度与肿瘤负荷预测直接相关，这是对从洗涤剂补充抗原捕获免疫测定和全动物生物发光光学成像中获得的数据进行逆回归校准分析得出的结果。动物模型的结果在人类临床材料中得到了证实，因为在晚期卵巢癌妇女的血清中发现，与早期卵巢癌妇女相比，S100家族成员的水平显著升高。S100在卵巢癌和其他癌症组织中表达，但在卵巢癌疾病血清中未见记录。该S100家族成员在血清中的浓度与实验肿瘤负荷和临床疾病分期相关。这些数据表明，S100在与其他生物标志物联合使用时，可能被证明可用于检测和/或监测卵巢癌。在进一步的研究中，MPU利用心脏组织切片验证了成人心肌细胞中致癌Ras表达的小鼠模型，以制定处理有限组织尺寸的质谱蛋白质组学研究的方案。与Ras在典型的细胞增殖反应中所起的作用相反，Ras信号在心肌中响应各种嗜离子性和嗜时性刺激，可能导致心肌肥大。心脏Ras信号也可以表现为致病性心肌肥大和随后的心力衰竭。采用双转基因四环素（tet-off）系统调节M/R小鼠中Ras （H-Ras-G12V）的表达，观察Ras诱导的致病性心肌肥大在去除Ras信号后能否消退。Ras在断奶时激活2周导致肥厚，而激活4-8周导致心肌病和心力衰竭。与持续激活Ras基因的小鼠相比，在早期心肌病发病后停止Ras基因表达可提高生存率和心肌病病变评分，降低心脏/体重比，证明了早期致病性肥厚的可逆性。该系统有望获得Ras癌基因继发于恶性前损伤和肥大心肌细胞愈合过程中重要蛋白信号传导的新信息。本项目使用的重要材料、设备或方法包括重组DNA技术、体外细胞培养、DNA序列分析、免疫诊断、分子成像、形态测量学、计算机辅助图像分析、光学成像、分子病理学和兽医学诊断。

In this project research is conducted to characterize and develop new animal models of human disease and to develop the means to better characterize a models relevance for a human condition. The project aims include the evaluation of research technologies and markers. Progress was made in developing cancer diagnostics and in research resources useful in developing and characterizing new models of human cancer. Methods and reagents were developed and technologies were applied for use in validating animal models of human cancer. This research project included developing capabilities in molecular diagnostics for cancer models, developing methods for automated morphmetric image analysis of cancer specimens for quantitative pathology, investigating the role of S100 in ovarian cancer and cell cycle regulation in nondividing cells undergoing Ras oncogene activation. Research advances in ovarian cancer biomarker development are yielding discovery of serum proteins that have the potential to serve as tumor biomarkers. Applying mass spectrometry-based proteomics, we sought to elucidate an unanswered biomarker research question regarding ability to determine tumor burden detectable by an ovarian cancer biomarker protein emanating directly from the tumor cells. Since aggressive serous epithelial ovarian cancers account for most mortality, a xenograft model using human SKOV-3 serous ovarian cancer cells was established to model progression to disseminated carcinomatosis. Using a method for low molecular weight protein enrichment, followed by liquid chromatography and mass spectrometry analysis, a human-specific peptide sequence of an S100 was identified in sera from mice with advanced-stage experimental ovarian carcinoma. This S100 expression was documented in cancer xenografts as well as from ovarian cancer patient tissues. Longitudinal study revealed that serum S100 concentration is directly related to tumor burden predictions from an inverse regression calibration analysis of data obtained from a detergent-supplemented antigen capture immunoassay and whole-animal bioluminescent optical imaging. The result from the animal model was confirmed in human clinical material as this S100 family member was found to be significantly elevated in the sera from women with advanced stage ovarian cancer compared to those with early stage disease. This S100 is expressed in ovarian and other cancer tissues, but has not been documented previously in ovarian cancer disease sera. This S100 family member is found in serum in concentrations that correlate with experimental tumor burden and with clinical disease stage. The data signify that S100 may prove useful in detecting and/or monitoring ovarian cancer, when used in concert with other biomarkers. In additional research efforts, MPU validated a mouse model of oncogenic Ras expression in adult cardiac myocytes using heart tissue sections to work out protocols for processing limited tissue sizes for mass spectrometry proteomic studies. In contrast to the role oncogenic Ras plays in typical cytoproliferative responses, Ras signaling in the myocardium in response to a variety of ionotropic and chronotropic stimuli, may lead to myocardial hypertrophy. Cardiac Ras signaling can also be manifested as pathogenic myocardial hypertrophy and subsequent heart failure. A two-transgene tetracycline (tet-off) system was used to regulate expression of Ras (H-Ras-G12V) in M/R mice to examine whether Ras-induced pathogenic myocardial hypertrophy could resolve after removal of Ras signaling. Ras activation at weaning for 2-weeks caused hypertrophy, while activation for 4-8 weeks led to cardiomyopathy and heart failure. Discontinuing Ras transgene expression after early cardiomyopathy onset led to improved survival and cardiomyopathy lesion scores, with reduced heart/body weight ratios, compared to mice with continuous Ras activation, demonstrating the reversibility of early pathogenic hypertrophy. This system is anticipated to yield new information on important protein signaling during healing of the cardiac myocyte with pre-malignant injury and hypertrophy secondary to Ras oncogene. The significant materials, equipment or methods in this project include use of recombinant DNA technology, in vitro cell culture, DNA sequence analysis, immunodiagnostics, molecular imaging, morphometrics, computer assisted image analysis, optical imaging, molecular pathology, and veterinary medical diagnosis.