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）转化科学和特定的挑战主题，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。我们已经报道了45%的根治性乳腺癌切除术（RP）后5年内无疾病证据（NED）的患者存在休眠播散性肿瘤细胞（DTC）。这些休眠的DTC代表疾病复发的病灶，并且它们的激活可导致骨髓（BM）替换、脊髓压迫、严重骨痛、恶病质和死亡。 我们推测，通过比较休眠和活跃DTC的分子特征，我们将深入了解休眠和休眠细胞激活。为了检验这一假设，我们将使用来自我们的LuCaP PCa异种移植物的休眠肿瘤细胞和来自患者BM的休眠DTC。我们的目标是（1）通过改变微流体室中的微环境来激活休眠DTC，然后恢复休眠，以及（2）在BM中在单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）在培养物中具有细胞抑制性的LuCaP异种移植细胞和（B）来自RP后NED >5年的患者的休眠DTC。细胞将被放置在包被有来自反应性BM基质细胞的细胞外基质的微流体室中，并且使用条件培养基，我们将刺激增殖，然后逆转该过程。由于NED患者的BM抽吸物仅产生约10-20个DTC，因此这些研究仅可能使用微流体。异种移植物细胞和DTC的转录组将使用QuantientTM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个C4-2B细胞的群体扩增和阵列RNA，重复10次，以确定使用一个细胞进行DRIMENTTM阵列分析的代表性、保真度和再现性。然后，我们将应用该技术来扩增和分析来自5名晚期转移性疾病患者中的每一名的BM抽吸物和来自RP后>5年的5名NED患者中的每一名的10个单个DTC的转录组。来自这100个单细胞阵列的数据将确定DTC异质性并鉴定休眠相关基因。 如果单细胞方法没有显示出高保真度，则将使用单细胞或10个或更少DTC的池，通过MixentTM阵列对单个DTC进行分析。这些数据将与来自上述微流体测定和患者DTC的阵列数据组合分析。我们希望这些数据将促进我们对PCa患者BM中肿瘤细胞休眠的理解。 公共卫生相关性：大约90%的晚期前列腺癌患者会发生骨转移，肿瘤细胞扩散至骨可导致骨髓替代、脊髓压迫、骨折、严重骨痛、恶病质和死亡。一些患者在初始治疗和转移证据之间发生的长潜伏期归因于肿瘤细胞休眠。这些休眠的播散性肿瘤细胞代表了疾病复发的病灶，研究肿瘤细胞休眠将显著影响我们在没有疾病证据的个体中理解、识别和靶向肿瘤细胞的能力，这些疾病可能在以后的时间点复发。

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.