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、白细胞、 和血液中的血小板。1，2这种疾病的预后很糟糕，因为据估计，2018年有 在过去的十年里，有30,770例新病例和12,770例死亡。 在开发新药和实施联合疗法方面取得了进展，但在 患者之间的治疗反应保持了大约5年的中位生存期，MM仍然存在 一种不治之症。1，2，4 MM不仅被证明是一种高度异质性的疾病，而且在不同的患者之间 患者，但患者也表现出肿瘤内克隆的异质性。4-8为此原因，个性化 治疗是非常可取的，尤其是那些针对特定信号通路的治疗。 1、2、9、10目前，细胞遗传学和分子标记被用来 个性化治疗；然而，这些检测并不直接调查特定的生化活动，即 大多数药物干预的目标。神经鞘氨酸激酶(SK)的作用被广泛接受 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。11-20这方面的知识与 多发性骨髓瘤中越来越多的单细胞研究强调了恶性肿瘤的异质性来自于 患者间和肿瘤内克隆的异质性影响MM细胞对各种药物的敏感性 鞘磷脂途径代谢活性的测量将改善MM的治疗 应对和减少复发频率。4-7，21，22 鞘磷脂途径的生化研究传统上依赖于散装细胞分析，这只是 反映细胞行为的总体平均水平。已经开发了23-26种单细胞分析方法，但即使是高度 自动化分析一直是低吞吐量和技术复杂的。27-29我的目标是解决以下限制 现有的SK通过创建一种吞吐量增加、高度并行化、易于使用的化学物质来进行分析 分离平台，包括用于酶活性测量的化学传感器或报告器。 这项研究中提出的分析化学技术将有助于在 原代MM细胞样本，提供作为治疗方案一部分的SK抑制剂的疗效 以病人为单位。这项研究的中心假设是多发性骨髓瘤细胞在 提出的新型单细胞分析平台将显示SK活性的异质性和响应的多样性 到SK抑制剂。值得注意的是，虽然这项提议将其范围限制在调查MM的SK活动 细胞，这个平台可以很容易地被改造成基本上研究任何疾病的任何代谢途径， 如果有化学探头的话。这项急需的新技术将为研究人员提供 具有易于使用、吞吐量增加的检测方法，可用于评估单细胞内的生化活性。

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.