Profiling and Characterizing Prostate Cancer Tumor Dormancy in the Bone Marrow

描述(由申请人提供)：本申请涉及广泛的挑战领域(15)翻译科学和具体的挑战主题，15-CA-117：肿瘤休眠。前列腺癌(PCa)在90%的晚期疾病患者中转移到骨组织。我们已经报道，在根治性前列腺切除术(RP)后5年无疾病证据(NED)和GT；的患者中，45%存在潜伏的播散性肿瘤细胞(DTC)。这些休眠的DTC是疾病复发的病灶，它们的激活可导致骨髓替代、脊髓受压、严重骨痛、恶病质和死亡。 我们假设，通过比较休眠和活性DTC的分子图谱，我们将深入了解休眠和休眠细胞的激活。为了验证这一假设，我们将使用来自我们的LuCaP PCA异种移植的休眠肿瘤细胞和来自患者BM的休眠DTC。我们的目标是(1)通过改变微流控室内的微环境来激活休眠的DTC，然后恢复休眠，以及(2)在BM的单个细胞水平上表征休眠细胞的转录组。 为了激活休眠的肿瘤细胞，我们将使用(A)在培养中处于细胞抑制状态的LuCaP异种移植细胞，以及(B)来自RP后5年NED&GT；患者的休眠DTC。细胞将被放置在涂有来自反应性BM基质细胞的细胞外基质的微流体室中，使用条件培养液，我们将刺激增殖，然后逆转这一过程。由于NED患者的骨髓抽吸物只产生~10-20个DTC，这些研究只能使用微流体。将使用AgilentTM阵列、实时聚合酶链式反应和统计分析来分析异种移植细胞和DTC的转录组。 接下来，我们将使用10个重复的1、5、10和25个C4-2B细胞群体来扩增和排列RNA，以确定使用一个细胞进行AgilentTM阵列分析的代表性、保真度和重复性。然后，我们将应用该技术从5名晚期转移性疾病患者和5名NED患者的骨髓抽吸物中扩增和分析10个单一DTC的转录组。来自这100个单细胞阵列的数据将确定DTC的异质性并识别休眠相关基因。 单个DTC将由AgilentTM阵列使用单个单元进行分析，如果单单元方法不显示高保真，则在10个DTC或更少的池中进行分析。这些数据将结合来自前述微流控分析和患者DTC的阵列数据进行分析。我们期望这些数据将促进我们对前列腺癌患者骨髓中肿瘤细胞休眠的理解。 公共卫生相关性：前列腺癌在大约90%的晚期疾病患者中转移到骨骼，肿瘤细胞扩散到骨骼可导致骨髓置换、脊髓受压、骨折、严重骨痛、恶病质和死亡。在一些患者中，从最初的治疗到转移的证据之间的长潜伏期被归因于肿瘤细胞休眠。这些休眠的播散性肿瘤细胞是疾病复发的核心，研究肿瘤细胞休眠将显著影响我们对没有疾病证据的个体的肿瘤细胞的理解、识别和靶向，这些疾病可能会在以后的某个时间点复发。

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.