Modeling Metastasis and Acquired Drug Resistance Using Circulating Tumor Cells

使用循环肿瘤细胞模拟转移和获得性耐药性

• 批准号: 10374103
• 负责人: Daniel A. Haber
• 金额: $ 36.67万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10374103
• 关键词: Affect; Bar Codes; Biological; Biological Assay; Blood; Blood Circulation; Blood specimen; Brain; Breast; Breast Cancer Cell; Breast cancer metastasis; Candidate Disease Gene; Cell Line; Cells; Chromatin; Clinical; Deposition; Disease; Disease Resistance; Disseminated Malignant Neoplasm; Distant; Drug or chemical Tissue Distribution; Drug resistance; ERBB2 gene; Engineering; Epigenetic Process; Estrogen Receptors; Estrogen receptor positive; Evolution; Exhibits; Funding; Gene Expression; Genes; Genetic Transcription; Goals; Hematopoietic; Human; Hypoxia; In Vitro; Individual; Injections; Intravenous; Jordan; Libraries; Link; Liver; Lung; Malignant Neoplasms; Mammary gland; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Metastatic breast cancer; Metastatic malignant neoplasm to brain; Metastatic to; Microfluidics; Modeling; Molecular; Molecular Analysis; Monitor; Nature; Neoplasm Circulating Cells; Neoplasm Metastasis; Organ; Pathway interactions; Patients; Phenotype; Population; Primary Neoplasm; Process; Property; Proteome; Proteomics; RNA; Reactive Oxygen Species; Reporter; Resistance; Resistance development; SWI1; Sampling; Science; Site; Source; Specimen; Stains; Stress; Tail; Technology; Testing; Therapeutic Intervention; Time; Tissues; Tropism; Tumor Cell Line; Tumor-Derived; Validation; Veins; Woman; acquired drug resistance; advanced breast cancer; bone; breast cancer progression; cancer cell; candidate validation; drug sensitivity; experience; experimental study; implantation; in vitro testing; in vivo; knock-down; malignant breast neoplasm; mammary; methylation pattern; mouse model; neoplastic cell; notch protein; real time monitoring; reconstitution; response; single-cell RNA sequencing; small hairpin RNA; transcriptome; transcriptome sequencing; tumor; tumor growth; tumor initiation; tumorigenesis; tumorigenic

Project Summary Advanced estrogen receptor (ER)-positive breast cancers are initially responsive to multiple therapeutic interventions, but they ultimately develop drug resistance and disseminate to multiple metastatic sites. Circulating tumor cells (CTCs) underlie the blood-borne spread of cancer and they also provide a noninvasive source to sample, monitor and analyze tumor evolution in real time, as patients develop progressively resistant disease with new metastatic lesions. To enable the detailed molecular study of CTCs, which are extremely rare cells in the circulation, we have made use of microfluidic platforms that efficiently deplete normal hematopoietic cells from blood specimens, leaving behind an enriched population of intact CTCs, some of which remain viable. During the past funding period, we established a panel of patient-derived breast cancer CTC cell lines (Yu et al., Science 2014), which provide a window into critical and poorly understood properties of advanced breast cancer, with significant clinical implications. We demonstrated that these heterogeneous ER+ drug-resistant breast cancer cells contain distinct phenotypes, with a HER2-expressing proliferative state interconverting spontaneously with a Notch1- driven drug resistant state (Jordan et al., Nature 2016). In Aim 1, we will build on this observation to define the likely epigenetic mechanisms that modulate this phenotype conversion. Using live-reporter constructs, we will isolate single cells as they switch between phenotypes to define early transcriptional changes, and in bar-coded pooled knockdown screens, we will test how chromatin modulators affect this phenotype switch, both spontaneously and following the dramatically enhanced reactive oxygen species (ROS)- mediated conversion that we have observed. In Aim 2, we will study another unexpected observation made with cultured breast CTCs, namely their acquired quiescence following direct intravascular inoculation and dissemination to the lung. While a 200 CTC inoculum can initiate tumorigenesis in the mammary gland, tail vein inoculation of 200,000 CTCs leads to non-proliferative single cells throughout the lung, an observation that may be linked to ROS stress experienced by these cells in the bloodstream (Zheng et al., Nature Comm, 2017). We have used pooled bar-coded knockdown construct libraries of chromatin modulators to uncover candidate regulators that are enriched as CTCs eventually initiate proliferation in the lung, and these will be tested individually and in combination, validated in multiple CTC lines, and matched with RNA seq transcriptomes of early stages in the transition from quiescence to early proliferative metastatic lesions. In Aim III, we will examine organ-specific pathways that enable proliferation of breast CTCs in the brain versus bone or liver. By serial inoculation, we have generated derivative lines of CTCs that grow efficiently following direct implantation in brain, compared with parental cells which exhibit a prolonged delay. Using both RNA sequencing and whole proteome mass spectrometry, we will identify modulators of organ-predominant metastasis, which will then be validated through functional assays and correlated with primary CTCs from patients with organ-predominant metastases. All together, these experiments will use patient-derived cultures of metastatic precursors to better understand and ultimately target breast cancer progression.

项目概要 晚期雌激素受体（ER）阳性乳腺癌最初对多种治疗有反应 干预措施，但它们最终会产生耐药性并传播到多个转移部位。 循环肿瘤细胞 (CTC) 是癌症血源性传播的基础，它们还提供了 随着患者病情的发展，实时采样、监测和分析肿瘤演变的非侵入性来源 疾病逐渐耐药并出现新的转移病灶。为了能够进行详细的分子研究 CTC 是循环系统中极其罕见的细胞，我们利用微流体平台 有效地消耗血液样本中的正常造血细胞，留下富集的 完整的 CTC 群体，其中一些仍然存活。在过去的资助期间，我们建立了 患者来源的乳腺癌 CTC 细胞系组（Yu 等人，Science 2014），这提供了一个窗口 晚期乳腺癌的关键和知之甚少的特性，具有重大的临床意义。 我们证明这些异质 ER+ 耐药乳腺癌细胞含有不同的 表型，表达 HER2 的增殖状态与 Notch1 自发地相互转换 驱动的耐药状态（Jordan 等人，Nature 2016）。在目标 1 中，我们将基于这一观察来定义 调节这种表型转换的可能的表观遗传机制。使用现场记者构造， 我们将分离单细胞，因为它们在表型之间切换，以定义早期转录变化，并且 在条形码合并敲低筛选中，我们将测试染色质调节剂如何影响这种表型 开关，既可以自发地进行，也可以在活性氧 (ROS) 显着增强后进行- 我们观察到的介导转化。在目标 2 中，我们将研究另一个意想不到的观察结果 由培养的乳腺 CTC 制成，即直接血管内注射后获得的静止状态 接种并传播至肺部。虽然 200 个 CTC 接种物可以在 乳腺，尾静脉接种200,000个CTC导致整个过程中单细胞不增殖 肺部，这一观察结果可能与这些细胞在血液中经历的活性氧应激有关 （Zheng 等人，Nature Comm，2017）。我们使用了汇集的条形码敲低构建库 染色质调节剂，以发现随着 CTC 最终启动而丰富的候选调节剂 肺部增殖，这些将进行单独和组合测试，并在多个 CTC 中进行验证 线，并与从静止到早期过渡的早期阶段的 RNA seq 转录组相匹配 proliferative metastatic lesions.在目标 III 中，我们将检查器官特异性途径，使 乳腺 CTC 在大脑中与骨或肝脏中的增殖。通过连续接种，我们产生了 与亲本相比，CTC 的衍生品系在直接植入大脑后可有效生长 表现出长时间延迟的细胞。同时使用 RNA 测序和全蛋白质组质量 光谱测定法，我们将鉴定器官为主转移的调节剂，然后对其进行验证 通过功能测定并与器官为主的患者的原发性 CTC 相关 转移。总之，这些实验将使用源自患者的转移前体培养物来 更好地了解并最终针对乳腺癌的进展。