Development of high throughput screening technologies in breast cancer

 描述：肿瘤组织中只有极少数的肿瘤细胞是能动的，能够逃逸并引发转移。这些运动的肿瘤起始细胞的基因特征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异质性反应，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）从患者活检中分离高度运动的细胞亚群，2）研究它们对细胞因子靶向药物的反应。用于确定最佳个体化疗法的临床相关方法是筛选药物对移植到小鼠中的患者来源的肿瘤标本（tumorgrafts）的作用。然而，该方法具有四个主要缺点：1）仅28-37%的患者来源的肿瘤移植物在小鼠中生长，2）该方法如果成功，则需要4-12个月，3）其需要大的组织标本，以及4）其检查药物对大量肿瘤细胞群的作用，而不是特异性地对运动的肿瘤起始细胞的作用。因此，迫切需要1）开发一种临床相关技术，使医生能够在初步诊断时准确和快速地评估原发性肿瘤中运动细胞亚群的相对丰度，以及2）了解细胞毒性靶向药物如何影响异质性肿瘤中这些运动细胞的性质和功能，以便开发个性化治疗。这样的技术应该是高通量的、快速的、具有上级检测灵敏度，并且能够研究从患者活组织检查收获的少量肿瘤细胞中分离的能动单细胞的反应。微流控技术满足了所有这些要求。在目标1中，我们将开发、测试和验证一种微流控检测方法，该方法可预测人乳腺癌细胞系和患者来源的乳腺癌细胞的转移倾向。由于癌症患者对细胞骨架靶向药物的异质性反应，我们将使用我们的微流控测定快速评估8种人乳腺癌肿瘤移植物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）。大多数待测试的药物都是FDA批准的或在临床试验中，确保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与非迁移性乳腺癌细胞的形态学和分子特征（Aim 3）。鉴于有令人信服的数据显示巨暗蛋白参与癌症转移，我们将评估巨暗蛋白作为乳腺癌的新生物标志物（目的4）。我们还将研究巨暗蛋白的表达谱如何：1）在迁移细胞与非迁移细胞中改变; 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学和分子特征; 4）暗蛋白信号盒的异位表达如何抑制致瘤性和转移。

 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.