Development of an Ultrasensitive Microdevice for the Investigation of Sphingosine Kinase Activity in Single Cells

项目摘要 多发性骨髓瘤（MM）是一种致命的疾病，其特征是红细胞、白色细胞， 1，2这种疾病的预后很差，因为据估计，2018年 30，770例新发病例和12，770例死亡病例由MM引起。 在开发新药和实施联合疗法方面取得了长足进展，但 患者之间的治疗反应使中位生存期保持在约5年，MM仍然是 一种无法治愈的疾病。1，2，4 MM不仅被证明是一种患者间高度异质性的疾病， 患者，但患者也表现出肿瘤内克隆异质性。4 -8因此，个性化 治疗是非常需要的，特别是那些靶向特定信号传导途径的治疗， 目前，细胞遗传学和分子标记被用于告知 然而，这些测定并不直接研究特定的生物化学活性，即， 大多数药物干预措施的目标。人们普遍认为，鞘氨醇激酶（SK） 在多发性骨髓瘤的发生、发展和耐药性中起着至关重要的作用。 越来越多的MM单细胞研究强调， 患者间和肿瘤内克隆异质性影响MM细胞对各种药物的敏感性， 鞘脂途径中代谢活性的测量将改善MM治疗 缓解并降低复发频率。4 - 7，21，22 鞘脂途径的生物化学研究传统上依赖于大量细胞测定，其仅 反映了细胞行为的群体平均值。23 -26已经开发了单细胞测定法，但即使是高度 自动化检测的通量低，技术复杂。27 -29我的目标是解决 现有的SK检测，通过创建一个增加的吞吐量，高度并行化，简单易用的化学 分离平台，其结合了用于酶活性测量的化学传感器或报告器。 本研究中提出的分析化学技术将有助于测量SK活性， 原代MM细胞的样本，提供了SK抑制剂作为治疗方案的一部分的疗效的见解 一个病人接一个病人这项研究的中心假设是，在细胞上分析的MM细胞， 提出的新型单细胞分析平台将表现出SK活性的异质性和应答的多样性， SK抑制剂。重要的是要注意，虽然这项建议将其范围限制在调查MM的SK活动， 细胞，该平台可以很容易地适用于研究基本上任何疾病的任何代谢途径， 只要有化学探针就行这项急需的新技术将为研究人员提供 具有易于使用的、增加的通量测定，用于评估单细胞内的生物化学活性。

Project Summary Multiple myeloma (MM) is a deadly disease characterized by a shortage of red blood cells, white blood cells, and platelets in the blood.1,2 The disease has a grim prognosis, as it is estimated that in 2018 there were 30,770 new cases and 12,770 deaths resulting from MM.3 In the past decade there have been significant strides made in the development of new drugs and implementation of combination therapies, but variability in treatment response between patients has kept median survival approximately 5 years and MM remains an incurable disease.1,2,4 MM has not only been shown to be a highly heterogenous disease from patient-to- patient, but patients have also exhibited intra-tumor clonal heterogeneity.4–8 For this reason, personalized therapies are highly desirable, particularly those targeted at specific signaling pathways exhibiting aberrant behavior in a given patient.1,2,9,10 Presently, cytogenetic and molecular markers are used to inform personalized therapies; however, these assays do not directly investigate specific biochemical activity i.e. the target for most pharmacologic interventions. It is well-accepted that the enzyme sphingosine kinase (SK) plays a crucial function in MM initiation, progression, and drug resistance.11–20 This knowledge, in combination with the increasing number of single-cell studies in MM highlighting that the heterogeneity of the malignancy from patient-to-patient and intra-tumor clonal heterogeneity affects MM cell sensitivity to various drugs, suggests that measurements of metabolic activity in the sphingolipid pathway will improve MM treatment response and decrease the frequency of relapse.4–7,21,22 Biochemical investigations of the sphingolipid pathway have traditionally relied on bulk cell assays, which only reflect a population average of cell behavior.23–26 Single-cell assays have been developed, but even highly automated assays have been low throughput and technically complex.27–29 I aim to address the limitations of existing SK assays by creating an increased-throughput, highly parallelizable, simple-to-use chemical separations platform that incorporates a chemical sensor or reporter for enzyme activity measurement. The analytical chemistry technology proposed in this research will facilitate measurements of SK activity within samples of primary MM cells, providing insights into the efficacy of SK inhibitors as part of a therapy regimen on a patient-by-patient basis. The central hypothesis of this research is that MM cells analyzed on the proposed novel single-cell analysis platform will exhibit heterogeneity in SK activity and diversity in responding to SK inhibitors. It is important to note that while this proposal limits its scope to investigating SK activity in MM cells, the platform can easily be adapted to investigate essentially any metabolic pathway for any disease, provided that chemical probes are available. This urgently needed novel technology will provide researchers with an easy-to-use, increased throughput assay for evaluating biochemical activity within single cells.