Profiling and Characterizing Prostate Cancer Tumor Dormancy in the Bone Marrow

骨髓中前列腺癌肿瘤休眠的分析和特征

• 批准号: 7809196
• 负责人: ROBERT Louis VESSELLA
• 金额: $ 49.92万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7809196
• 关键词: Address; Area; Aspirate substance; Biological Assay; Bone Marrow; Bone Pain; Cachexia; Cell Count; Cells; Cessation of life; Coculture Techniques; Compression Fracture; Conditioned Culture Media; Cytostatics; Data; Disease; Extracellular Matrix; Gene Expression; Gene Expression Profile; Genes; Genomics; Growth; Heterogeneity; Individual; Lead; Malignant neoplasm of prostate; Methodology; Methods; Microfluidics; Molecular Profiling; Neoplasm Metastasis; No Evidence of Disease; Oligonucleotide Microarrays; PC3 cell line; Patients; Pattern; Physiologic pulse; Population; Process; RNA; Radical Prostatectomy; Reagent; Recurrence; Reporting; Reproducibility; Sampling; Seeds; Series; Stromal Cells; Techniques; Testing; Time; Translational Research; Xenograft procedure; advanced disease; bone; insight; neoplastic cell; public health relevance; restoration; spinal cord compression; tumor

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (15) Translational Science and specific Challenge Topic, 15- CA-117: Tumor dormancy. Prostate cancer (PCa) metastasizes to the bone in ~90% of patients with advanced disease. We have reported dormant disseminated tumor cells (DTC) are present in 45% of patients with no evidence of disease (NED) >5 years after radical prostatectomy (RP). These dormant DTC represent a nidus for disease recurrence and their activation can lead to the replacement of bone marrow (BM), spinal cord compression, severe bone pain, cachexia and death. We hypothesize that by comparing the molecular profile of dormant and active DTC we will gain insight into dormancy and dormant cell activation. To test this hypothesis, we will use dormant tumor cells from our LuCaP PCa xenografts and dormant DTC from patient BM. Our aims are (1) activate dormant DTC by altering the microenvironment in microfluidic chambers and then restore dormancy, and (2) characterize the transcriptome of dormant cells at a single cell level in the BM. To activate dormant tumor cells, we will use (a) LuCaP xenograft cells that are cytostatic in culture and (b) dormant DTC from patients who are NED >5 years post RP. Cells will be placed in microfluidic chambers coated with extracellular matrix from reactive BM stromal cells, and using conditioned media, we will stimulate proliferation and then reverse the process. Since BM aspirates from NED patients only yield ~10-20 DTC, these studies are only possible using microfluidics. The transcriptome of both the xenograft cells and DTC will be analyzed using AgilentTM arrays, real-time PCR, and statistical analyses. Next, we will amplify and array RNA using a population of 1, 5, 10, and 25 C4-2B cells in replicates of 10, to determine the representation, fidelity, and reproducibility of using one cell for AgilentTM array analysis. We will then apply the technique to amplify and profile the transcriptome of ten single DTC from BM aspirates of each of 5 patients with advanced metastatic disease and from each of 5 NED patients >5 years post RP. Data from these 100 single cell arrays will determine DTC heterogeneity and identify dormancy related genes. The individual DTC will be profiled by AgilentTM arrays using single cells or in pools of 10 DTC or less if the single cell methodologies don't show high fidelity. These data will be analyzed in combination with the array data from the aforementioned microfluidic assays and patient DTC. We expect that these data will advance our understanding of tumor cell dormancy in the BM of patients with PCa. PUBLIC HEALTH RELEVANCE: Prostate cancer metastasizes to the bone in approximately 90% of patients with advanced disease and dissemination of tumor cells to the bone can lead to replacement of bone marrow, spinal cord compression, fracture, severe bone pain, cachexia and death. The long latency period that occurs in some patients between initial treatment and evidence of metastases is attributed to tumor cell dormancy. These dormant disseminated tumor cells represent a nidus for disease recurrence and studying tumor cell dormancy will significantly impact our ability to, understand, identify and target tumor cells in individuals with no evidence of disease that may recur at a later point in time.

描述（由申请人提供）：此申请应解决广泛的挑战领域（15）转化科学和特定挑战主题，15- CA-117：肿瘤休眠。前列腺癌（PCA）在约90％的晚期疾病患者中转移到骨骼。我们报道说，没有疾病证据（NED）> 5年后，有45％的患者存在休眠的肿瘤细胞（DTC）。这些休眠的DTC代表了疾病复发的NIDU，它们的激活可能导致骨髓（BM），脊髓压缩，严重的骨痛，病虫气和死亡的替代。 我们假设通过比较休眠和活性DTC的分子谱，我们将深入了解休眠和休眠细胞活化。为了检验该假设，我们将使用来自LUCAP PCA异种移植物的休眠肿瘤细胞和患者BM的休眠DTC。我们的目的是（1）通过改变微流体腔中的微环境，然后恢复休眠，以及（2）表征BM中单个细胞水平的休眠细胞转录组，从而激活休眠DTC。 为了激活休眠的肿瘤细胞，我们将使用（a）在培养物中具有细胞抑制性的Lucap异种移植细胞，以及（b）RP后5年> 5年的患者的休眠DTC。细胞将放置在来自反应性BM基质细胞的细胞外基质的微流体腔中，并使用条件培养基，我们将刺激增殖，然后逆转过程。由于来自NED患者的BM吸入仅产生约10-20 DTC，因此仅使用微富集剂才能进行这些研究。异种移植细胞和DTC的转录组将使用AgilentTM阵列，实时PCR和统计分析进行分析。 接下来，我们将使用1、5、10和25 c4-2b细胞的群体以10的重复分析和阵列RNA，以确定使用一个细胞用于AgilentTM阵列分析的表示，保真度和可重复性。然后，我们将应用该技术来扩增和介绍5例患有晚期转移性疾病患者的BM抽吸物中的十个单个DTC的转录组，以及RP后5年> 5年的5例NED患者中的每名患者。来自这100个单细胞阵列的数据将确定DTC异质性并确定与休眠相关的基因。 如果单细胞方法不显示高忠诚度，则使用单个单元格或更少的池或更少的池中，通过敏捷TM阵列对单个DTC进行介绍。这些数据将与上述微流体分析和患者DTC的阵列数据结合进行分析。我们预计这些数据将提高我们对PCA患者BM肿瘤细胞休眠的理解。 公共卫生相关性：大约90％患有晚期疾病的患者的前列腺癌向骨骼转移，肿瘤细胞向骨骼传播可能会导致骨髓，脊髓压缩，骨折，严重的骨痛，卡氏症和死亡的骨髓替代。在初始治疗和转移证据之间，某些患者发生的长潜伏期归因于肿瘤细胞休眠。这些休眠的散布肿瘤细胞代表了疾病复发的Nidus，研究肿瘤细胞休眠将显着影响我们的能力，理解，识别，识别和靶向没有疾病证据的个体中可能会在以后重复出现的肿瘤细胞。