Development of high throughput screening technologies in breast cancer

 描述：在肿瘤组织中，只有极小%的肿瘤细胞能够活动，并能够逃逸和启动转移。这些能动的肿瘤启动细胞的基因标志可以预测乳腺癌患者的临床结果。患者对靶向细胞骨架的药物表现出不同的反应，细胞骨架是旨在抑制肿瘤细胞生长、运动和侵袭的治疗的常见靶点。到目前为止，还没有临床试验来1)从患者活检组织中分离出高运动性细胞亚群，2)研究它们对细胞骨架靶向药物的反应。一种确定最佳个体化治疗的临床相关方法是筛选药物对移植于小鼠(肿瘤移植物)的患者来源的肿瘤标本的影响。然而，这种方法有四个主要缺陷：1)只有28-37%的患者来源的肿瘤移植物在小鼠身上生长；2)这种方法如果成功，需要4-12个月；3)它需要大量的组织标本；4)它检查药物对大部分肿瘤细胞的影响，而不是对可移动的肿瘤启动细胞的影响。因此，当务之急是1)开发一种临床上相关的技术，使医生能够在最初诊断时准确和快速地评估原发肿瘤中活动细胞亚群的相对丰度，以及2)了解细胞骨架靶向药物如何影响异质肿瘤中这些活动细胞的特性和功能，以便开发个性化的治疗方法。这种技术应该是高通量、快速和高检测灵敏度的，并能够研究从患者活检组织中提取的少量肿瘤细胞中分离出的可移动的单个细胞的反应。微流控技术满足了所有这些要求。在目标1中，我们将开发、测试和验证一种预测人乳腺癌细胞系和患者来源的乳腺癌细胞转移倾向的微流控分析方法。由于癌症患者对细胞骨架靶向药物的反应不一，我们将使用我们的微流控分析来快速评估8个人乳腺癌移植瘤以及患者来源的乳腺癌细胞对不同细胞骨架调节剂的反应，以确定最佳的个性化治疗(目标2)。大多数被测试的药物都是FDA批准的或正在进行临床试验，这确保了这项研究可以迅速影响乳腺癌的临床治疗。由于细胞特性调节迁移、侵袭和转移，我们将使用一种新的高通量、单细胞成像技术来定义通过流体分析分离的迁移性和非迁移性乳腺癌细胞的形态和分子特征(目标3)。鉴于令人信服的数据显示巨型黑素参与癌症转移，我们将评估巨型黑素作为乳腺癌的一种新的生物标记物(目标4)。我们还将研究巨型黑素的表达谱如何：1)在迁移细胞和非迁移细胞中发生变化；2)调节乳腺癌细胞对细胞骨架药物的反应；3)影响乳腺癌细胞的形态和分子特征；以及4)黑素信号盒的异位表达如何抑制肿瘤的发生和转移。

 DESCRIPTION: Only a tiny % of tumor cells within the tumor tissue is motile and capable of escaping and initiating metastasis. The gene signature of these motile tumor-initiating cells predicts clinical outcome in breast cancer patients. Patients exhibit heterogeneous responses to drugs targeting the cytoskeleton, which is a common target for therapies aiming to inhibit tumor cell growth, motility and invasion. To date, there is no clinical assay to 1) isolate the highly motile cell subpopulations from patient biopsies, and 2) investigate their responses to cytoskeleton-targeting drugs. A clinically relevant method for identifying optimal individualized therapies is to screen the effects of drugs on patient-derived tumor specimens transplanted in mice (tumorgrafts). However, this method suffers from four major drawbacks: 1) only 28-37% of patient-derived tumorgrafts grow in mice, 2) this method, if successful, requires 4-12 months, 3) it requires large tissue specimens, and 4) it examines the effects of drugs on the bulk tumor cell population, and not specifically on the motile tumor-initiating cells. It is thus urgent 1) to deveop a clinically relevant technology that will enable physicians to accurately and rapidly assess the relative abundance of motile cell subpopulations in primary tumors at the time of initial diagnosis, and 2) to understand how cytoskeletal-targeting drugs affect the properties and function of these motile cells within a heterogeneous tumor in order to develop personalized therapies. Such technology should be high- throughput, rapid with superior detection sensitivity, and capable of studying the responses of motile single cells isolated from a small number of tumor cells harvested from patient biopsies. The microfluidic technology meets all these requirements. In Aim 1, we will develop, test and validate a microfluidic assay that predicts the metastatic propensity of human breast cancer cell lines and patient-derived breast cancer cells. Because of the heterogeneous responses of cancer patients to cytoskeleton-targeting drugs, we will use our microfluidic assay to rapidly assess the responses of 8 human breast cancer tumorgrafts as well as patient-derived breast cancer cells to different cytoskeletal modulators to identify optimal personalized therapies (Aim 2). Most of the drugs to be tested are FDA-approved or in clinical trials, ensuring that this study could rapidly impact the clinical treatmen of breast cancer. Since cell properties regulate migration, invasion and metastasis, we will use a novel high-throughput, single-cell imaging technology to define the morphological, and molecular signatures of migratory vs. non-migratory breast cancer cells isolated by the ¿-fluidic assay (Aim 3). In view of compelling data showing the involvement of giant obscurins in cancer metastasis, we will evaluate giant obscurins as a novel biomarker for breast cancer (Aim 4). We will also examine how the expression profile of giant obscurins: 1) is altered in migratory versus non-migratory cells; 2) regulates the responses of breast cancer cells to cytoskeletal drugs; 3) affects the morphological and molecular signatures of breast cancer cells; and 4) how ectopic expression of an obscurin signaling cassette can suppress tumorigenicity and metastasis.