Modeling Metastasis and Acquired Drug Resistance Using Circulating Tumor Cells

项目总结 晚期激素受体阳性(HR+)乳腺癌是最常见的亚型，包括 最初对多种内分泌干预有反应，但最终产生抗药性。 循环中的肿瘤细胞(CTCs)是癌症通过血液传播转移的基础，它们 还提供了一种非侵入性来源，在治疗过程中和获得性 抵抗。使用保持CTC活性的微流控浓缩技术，我们成功地 从患有难治性HR+乳房的妇女中建立了15个来自CTC的长期培养的队列 癌症。其中一个子集显示了预期获得的ESR1和PIK3CA突变，已在 与转移性肿瘤活检结果相匹配。然而，最常见、也最不为人所知的是 内分泌耐药是指癌细胞失去雌激素受体α(ER)的表达。在这些情况下， 培养的CTCs概括为ER沉默，有些是双表型，ER+和ER-共存 来自同一患者的亚群。单细胞来源的克隆表明ER+CTCs可以 产生内质网后代，指向可能的表观遗传机制和潜在的细胞可塑性。我们 建议研究ER表达缺失的表观遗传调控及其潜在的恢复， 使用基因组分析和CRISPR功能筛查相结合。在目标1中，我们将比较 等位基因ER+和ER-CTC亚群的染色质景观，定义了它们不同的功能 性质并确定在体外调节它们相互转化的因素。在目标2中，我们将使用Engage CRISPR筛选能够恢复HR+乳腺CTC细胞ER表达的基因 内分泌治疗后失去内源性ER表达的癌症。初步数据 指出这种方法的可行性，我们将探索潜在的修复机制 ER的表达，以及这是否伴随着ER依赖的增殖的回归。一起， 这些目的是解决患者来源的CTCs中ER表达的可塑性 概述临床治疗暴露和肿瘤适应机制。高血压大鼠雌激素受体表达缺失 +乳腺癌还可能导致对替代致癌驱动因素的依赖，这可能构成 难治性HR+乳腺癌的药物靶点。在目标3中，我们在CTC中应用了化学蛋白质组学策略 品系，结合半胱氨酸交联和质谱学来鉴定所有可连接的半胱氨酸残基 在细胞蛋白质中。初步数据显示CTCs中存在多个半胱氨酸靶向蛋白 存在于未经治疗的乳腺癌株系中，我们将使用CRISPR筛查来确定哪些是 增殖，然后从半胱氨酸反应的专门文库中鉴定工具化合物 用于功能分析的共价抑制剂。这些互补的方法共同解决了这一损失 ER在晚期HR+乳腺癌中的表达，目的是克服获得性耐药 内分泌治疗。

PROJECT SUMMARY Advanced hormone receptor positive (HR+) breast cancers, the most common subtype, are initially responsive to multiple endocrine interventions, but they ultimately develop drug resistance. Circulating tumor cells (CTCs) underlie the blood-borne metastatic spread of cancer, and they also provide a noninvasive source to sample tumor cells during the course of therapy and acquired resistance. Using a microfluidic enrichment technology that preserves CTC viability, we successfully established a cohort of 15 CTC-derived longterm cultures from women with refractory HR+ breast cancer. A subset of these shows the expected acquired ESR1 and PIK3CA mutations, confirmed in matched metastatic tumor biopsies. However, the most common, and least well understood, correlate of endocrine resistance is loss of estrogen receptor-α (ER) expression by cancer cells. In these cases, cultured CTCs recapitulate ER silencing, and some are biphenotypic, with coexisting ER+ and ER- subpopulations derived from the same patient. Single cell-derived colonies show that ER+ CTCs can produce ER- progeny, pointing to likely epigenetic mechanisms and underlying cell plasticity. We propose to investigate the epigenetic regulation of ER expression loss and its potential restoration, using genomic analyses combined with CRISPR functional screens. In Aim 1, we will compare chromatin landscapes of isogenic ER+ and ER- CTC subpopulations, define their distinct functional properties and identify factors that modulate their interconversion in vitro. In Aim 2, we will use undertake CRISPR screens to identify genes capable of restoring ER expression to CTC lines from HR+ breast cancers that have lost endogenous ER expression following endocrine therapy. Preliminary data indicate the feasibility of this approach, and we will explore mechanisms underlying restoration of ER expression, and whether this is accompanied by a return to ER-dependent proliferation. Together, these Aims address the plasticity of ER expression in patient-derived cultured CTCs that recapitulate clinical treatment exposures and tumor adaptation mechanisms. Loss of ER expression in HR + breast cancer may also lead to dependence on alternative oncogenic drivers, that may be constitute drug targets in refractory HR+ breast cancer. In Aim 3, we apply a chemical proteomic strategy in CTC lines, combining cysteine cross-linking with mass spectrometry to identify all ligandable cysteine residues within cellular proteins. Preliminary data show multiple cysteine-targetable proteins in CTCs that are not present in untreated breast cancer lines, and we will use CRISPR screens to identify those required for proliferation, followed by identification of tool compounds from a specialized library of cysteine-reactive covalent inhibitors for functional analyses. Together, these complementary approaches address the loss of ER expression in advanced HR+ breast cancers, with the goal of overcoming acquired resistance to endocrine therapy.