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: 9920676
• 负责人: Daniel A. Haber
• 金额: $ 37.42万
• 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/9920676
• 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 Culture Techniques; 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; 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.

项目摘要 晚期雌激素受体(ER)阳性乳腺癌最初对多种治疗有效 但它们最终会产生抗药性，并传播到多个转移部位。 循环中的肿瘤细胞(CTC)是癌症通过血液传播的基础，它们还提供了一种 非侵入性来源，随着患者的发展，实时采样、监测和分析肿瘤演变 有新转移灶的渐进性耐药疾病。为了使详细的分子研究成为可能 CTCs是循环中极其罕见的细胞，我们利用微流控平台 有效地从血液样本中耗尽正常的造血细胞，留下丰富的 大量完好的四氯化碳，其中一些仍然活着。在过去的资助期间，我们建立了一个 乳腺癌患者来源的CTC细胞系(Yu等人，《科学》2014)，这提供了一个了解 晚期乳腺癌的关键和鲜为人知的特性，具有重要的临床意义。 我们证明了这些异质ER+耐药乳腺癌细胞含有截然不同的 表型，表达HER2的增殖状态与Notch1- 驱使抗药性状态(Jordan等人，《自然》2016)。在目标1中，我们将在此观察的基础上定义 可能的表观遗传机制，调节这种表型转换。使用现场报道构建， 我们将分离单个细胞，因为它们在表型之间转换，以定义早期转录变化，以及 在条码池击倒屏幕中，我们将测试染色质调节剂如何影响这种表型 开关，无论是自发的，还是在活性氧物种(ROS)显著增强后- 我们观察到的中介转化。在目标2，我们将研究另一个意想不到的观察 用培养的乳腺CTCs制作的，即它们直接血管内注射后的获得性静止 接种和传播到肺部。而接种200ct的CTC疫苗可以在 乳腺、尾静脉接种200,000个CTCs可诱导全程非增殖性单个细胞 肺，这一观察可能与血流中的这些细胞经历的ROS应激有关 (郑等人，《自然通讯》，2017)。我们已经使用了池条码击倒构造库 染色质调节剂发现随着CTCs最终启动而丰富的候选调节器 肺中的增殖，这些将单独和组合进行测试，在多个CTC中进行验证 品系，并与从静止到早期过渡的早期阶段的RNA序列转录本匹配 增生性转移灶。在AIM III中，我们将研究器官特异性通路，这些通路能够 乳腺CTCs在大脑中的增殖，而不是骨骼或肝脏。通过连续接种，我们已经产生了 直接植入脑内后高效生长的CTCs衍生系与亲本相比 表现出较长延迟的细胞。同时使用RNA测序和全蛋白质组质量 光谱分析，我们将确定器官为主的转移的调节器，然后进行验证 通过功能分析，并与器官占优势的患者的初级CTC进行相关性分析 转移瘤。总而言之，这些实验将使用患者来源的转移前体培养来 更好地了解并最终针对乳腺癌的进展。