Modeling Metastasis and Acquired Drug Resistance Using Circulating Tumor Cells

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， 这些患者通常不接受干预，但最终会产生耐药性并扩散到多个转移部位。 循环肿瘤细胞（CTC）是癌症血液传播的基础，它们还提供了一种治疗癌症的方法。 非侵入性来源，用于随着患者的发展，真实的实时采样、监测和分析肿瘤演变 具有新转移病灶的进行性耐药疾病。为了能够对 CTC是循环中极其罕见的细胞，我们利用微流体平台， 有效地从血液样本中消耗正常造血细胞，留下富集的 完整的CTC群体，其中一些仍然存活。在过去的资助期内，我们成立了一个 一组源自患者的乳腺癌CTC细胞系（Yu等，科学2014），它提供了一个窗口， 晚期乳腺癌的关键和知之甚少的性质，具有重要的临床意义。 我们证明，这些异质性ER+耐药乳腺癌细胞含有不同的 表型，表达HER 2的增殖状态自发地与Notch 1- 驱动的耐药状态（Jordan等，Nature 2016）。在目标1中，我们将在此观察的基础上定义 调节这种表型转换的可能表观遗传机制。使用活报告基因构建体， 我们将分离单个细胞，因为它们在表型之间切换，以确定早期转录变化， 在条形码混合敲低筛选中，我们将测试染色质调节剂如何影响这种表型 开关，无论是自发的，并在显着增强活性氧（ROS）- 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 用培养的乳腺CTCs制成，即它们在直接血管内注射后获得静止， 接种并传播到肺部。虽然200 CTC接种物可以在肿瘤细胞中引发肿瘤发生， 在乳腺中，尾静脉接种200，000个CTC导致整个细胞的非增殖性单细胞 肺，这一观察结果可能与这些细胞在血流中经历的ROS应激有关 （Zheng等人，Nature Comm，2017）。我们已经使用了汇集的条形码敲低构建体文库， 染色质调节剂，以揭示候选调节剂，这些候选调节剂随着CTC最终启动 在肺中的增殖，这些将单独和组合进行测试，在多个CTC中验证 系，并与从静止到早期过渡的早期阶段的RNA seq转录组相匹配 增生性转移性病变。在目标III中，我们将研究器官特异性途径， 乳腺CTCs在脑中的增殖与骨或肝相比。通过连续接种，我们产生了 与亲本细胞相比，直接植入脑中后有效生长的CTC衍生系 表现出延长延迟的细胞。使用RNA测序和全蛋白质组质量 光谱，我们将确定器官为主的转移，然后将验证调制器 通过功能测定，并与来自器官占优势的患者的原发性CTC相关， 转移总之，这些实验将使用患者来源的转移前体培养物， 更好地了解并最终靶向乳腺癌进展。

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.