(PQD5) Mass Profiling Melanoma Responses to Improve Therapy Choices and Prognosis

描述（由申请人提供）：本提案解决了D组挑衅性问题（PQD 5）：由于目前预测候选新药在人体中的疗效或毒性的方法通常不准确，我们能否开发新方法来测试潜在的治疗药物，从而更好地预测反应？我们将解决预测治疗反应的关键缺陷，以预测肿瘤复发和改善患者的预后，这通常是基于肿瘤的异质性。我们将通过开发和应用一种新的单细胞反应测量技术来实现这一目标，该技术被称为高通量筛选活细胞干涉仪（HTS-LCI），以量化单细胞生物量在药物暴露之前和期间的时间变化。利用10，000秒的时间依赖性生物量曲线，我们将快速表征肿瘤对治疗的异质动力学反应，以提供定量统计分类器。我们的建议是变革性的，对所有类型的癌症都有广泛的影响，但在这里，我们专注于转移性黑色素瘤（主要是III-IV期），因为1）它是一种常见的癌症，发病率不断增加，2）通常迅速致命，3）关于靶向治疗和耐药性的了解很多。具体而言，MAPK通路激活BRAF丝氨酸/苏氨酸激酶突变存在于约50%的黑素瘤中。重要的是，良好表征的BRAF抑制剂（BRAFi）敏感和耐药细胞系和新鲜的患者黑色素瘤样品可随时用于原理验证临床前研究。个性化医疗的方法依赖于静态生物标志物、基因组和表观遗传参数来改进治疗选择和预测预后，但它们都未能纳入治疗反应，这是一个关键的遗漏。经验证的个体化肿瘤细胞反应谱可能对治疗效果、快速癌症诊断、预后和肿瘤复发预测产生巨大影响。为了达到这一目标，我们提出了一种具有三个创新组成部分的新方法，包括1）工程化HTS-LCI以真实的时间量化响应治疗剂的肿瘤细胞生物量变化; 2）使用配对的BRAFi敏感和抗性患者来源的转移性黑素瘤细胞系，其已经被我们的合作者广泛表征为基因组、表观基因组和表达谱;和3）通过与Jonsson综合癌症中心临床医生及其正在进行的早期临床试验的合作，利用我们立即获得去识别患者样本。我们的提议的具体目的是：目的1：生成BRAFi敏感性和抗性配对黑素瘤细胞系统计分类器。目的2：设计HTS-LCI，用于36孔板格式中的多药物生长速率分析。目的3：评估用于BRAFi敏感和抗性品系的快速反应检测的HTS-LCI。目的4：将HTS-LCI平台应用于新鲜黑素瘤患者样本的生物量分析。

DESCRIPTION (provided by applicant): This proposal addresses the Group D Provocative Question (PQD5): Since current methods to predict the efficacy or toxicity of new drug candidates in humans are often inaccurate, can we develop new methods to test potential therapeutic agents that yield better predictions of response? We will address critical shortcomings in predicting therapeutic responses to anticipate tumor recurrence and improve patient outcome, which is usually based on tumor heterogeneity. We will accomplish this goal by developing and applying a novel single-cell response measuring technology, termed a High-Throughput Screening Live Cell Interferometer (HTS-LCI), to quantify single-cell biomass changes temporally, before and during drug exposure. With 10,000s of time-dependent biomass profiles, we will rapidly characterize a tumor's heterogeneous kinetic response to therapy in order to provide a quantitative statistical classifier. Our proposal is transformative with broad implications for all types of cancer, but here we focus on metastatic melanoma (mainly stage III-IV) because 1) it is a common cancer with increasing incidence, 2) is often rapidly fatal, and 3) much is known about targeted therapy and resistance. Specifically, MAPK pathway-activating BRAF serine/threonine kinase mutations are present in ~50% of melanomas. Importantly, well-characterized BRAF-inhibitor (BRAFi) sensitive and resistant cell lines and fresh patient melanoma samples are readily available for proof-of-principle preclinical studies. Approaches in personalized medicine rely on static biomarker, genomic, and epigenetic parameters to refine therapy choice and predict prognosis, but they all fail to incorporate therapeutic response, which is a critical omission. Validated, individualized tumor cell response profiling could have enormous impact on therapeutic efficacy, rapid cancer diagnosis, prognosis, and prediction of tumor recurrence. To reach this goal we propose a new approach with three innovative components that include 1) engineering the HTS-LCI to quantify tumor cell biomass changes in response therapeutic agents, in real time; 2) using paired BRAFi sensitive and resistant patient-derived metastatic melanoma cell lines that have been extensively characterized for genomic, epigenomic, and expression profiling by our collaborators; and 3) utilizing our immediate access to de-identified patient samples through collaboration with Jonsson Comprehensive Cancer Center clinicians and their ongoing early phase clinical trials. The Specific Aims of our proposal are: Aim 1: To generate a BRAFi sensitive and resistant paired melanoma cell line statistical classifier. Aim 2: To engineer the HTS-LCI for multi-drug growth rate profiling in a 36-well plate format. Aim 3: To evaluate the HTS-LCI for rapid response detection of BRAFi sensitive and resistant lines. Aim 4: To apply the HTS-LCI platform for biomass profiling of fresh melanoma patient samples.