UNS:Direct measurement of DUB activity in intact single cells using a droplet microfluidic array

骨髓瘤患者面临的最大挑战之一是对目前用于治疗该疾病的药物的耐药性。这个跨学科的项目包括开发一种微流体装置和人造的基于肽的生物传感器，以特异性地识别肿瘤中发现的不同的癌细胞亚群，包括那些对特定化疗药物具有耐药性的细胞。最终，这项提议的目标是开发一种新的诊断工具，可以为高风险骨髓瘤患者提供个性化的治疗方案，显著提高与癌症作斗争的个体的预后。分子靶向治疗和个性化医疗极大地提高了癌症患者的预后。在多发性骨髓瘤的情况下，使用特异性靶向与泛素蛋白酶体系统（UPS）相关的酶的药物已经取得了巨大的成功。去泛素化酶（DUBs）就是这样一类酶，因为它们能够促进多发性骨髓瘤患者的耐药性。在本提案中，跨学科方法将应用于开发一种新方法，直接测量异质人群（如肿瘤活检）中完整单细胞中的DUB活性。一个长寿命，细胞渗透性，DUB特异性荧光报告将开发直接测量DUB活性。这种基于肽的报告蛋白的一个特点是包含了一个<s:1>发夹？保护肽？，既赋予dub特异性底物的稳定性，又作为一种有效的细胞穿透肽（CPP）。这种新颖的报告方案将被纳入微流控液滴阵列，将开发以促进骨髓瘤细胞群体的高通量筛选（HTS）。本提案中设计的微流控液滴阵列将允许芯片上封装，然后实时定量完整单细胞中的DUB活性。生化分析将作为开发新技术的第一步，允许1)确定患者是否会从dub靶向治疗中获益，2)确定理想剂量的药物以最大限度地提高疗效，同时最大限度地减少副作用，以及3)分析异质样本以识别不同的耐药细胞亚群，这无法通过批量测量进行。该奖项由CBET部门的生物技术和生化工程项目颁发，由刺激竞争研究的实验项目（EPSCoR）共同资助。

1509713 Melvin, AdamOne of the greatest challenges facing myeloma patients is the resistance to the drugs currently used to treat the disease. This interdisciplinary project involves the development of a microfluidic device and man-made peptide-based biosensors to specifically identify distinct sub-populations of cancer cells found in a tumor, including those that are resistant to particular chemotherapy drugs. Ultimately, the goal of this proposal is to develop a new diagnostic tool that can lead to a personalized treatment protocol for high risk myeloma patients, dramatically increasing the prognosis of individuals battling cancer.Molecularly-targeted therapeutics and personalized medicine have dramatically increased the prognosis of patients suffering from cancer. In the case of multiple myeloma, there has been great success using drugs that specifically target enzymes associated with the ubiquitin proteasome system (UPS). Deubiquitinating enzymes (DUBs) are one such class of enzyme due to their ability to promote drug resistance in multiple myeloma patients. In this proposal, an interdisciplinary approach will be applied to develop a new method to directly measure DUB activity in intact single cells across a heterogeneous population such as tumor biopsy. A long-lived, cell permeable, DUB-specific fluorescent reporter will be developed to directly measure DUB activity. One hallmark of this peptide-based reporter is the inclusion of a â-hairpin ?protectide?, serving to both confer stability onto the DUB-specific substrate, and to act as a potent cell penetrating peptide (CPP). This novel reporting scheme will be incorporated into a microfluidic droplet array that will be developed to facilitate high-throughput screening (HTS) of a population of myeloma cells. The microfluidic droplet array designed in this proposal will allow for on-chip encapsulation followed by real-time quantification of DUB activity in intact single cells. The biochemical assay will serve as a first step in developing a new technique allowing to 1) determine if patients would benefit from a DUB-targeted therapy, 2) identify an ideal dose of drug to maximize efficacy while minimizing side effects, and 3) analyze a heterogeneous sample to identify distinct subpopulations of drug-resistant cells, which cannot be performed using bulk measurement.This award by the Biotechnology and Biochemical Engineering Program of the CBET Division is co-funded by the Experimental Program to Stimulate Competitive Research (EPSCoR).