Topic 359: A Device Prototype for Isolation of Melanoma Exosomes for Diagnostics and Treatment Monitoring

拟议的工作旨在开发用于临床诊断实验室的外泌体分离装置的预商业原型。我们将推进该设备，以促进下游液体活检测试的外泌体分离，这些测试需要分析蛋白质、DNA或microRNA，这些都依赖于获得完整和纯的外泌体。该器械策略将设计为分离总外泌体，然后分离肿瘤源性外泌体（TEX）和非恶性外泌体（non-TEX）的离散群体。该设备策略将在拟议的实验中用于黑色素瘤外泌体分离，但预计也适用于其他癌症。为了实现本研究的目标，我们将开发和优化一种结合GNA血液净化器的过滤系统，用于从生物流体中分离总外泌体（具体目标1）。GNA血液净化器从不同生物液体中分离黑色素瘤TEX和非TEX的能力将通过评价外泌体去除性能来表征（具体目标2）。然后，我们将构建一种新的设备，CSPG 4盒，用于从总外泌体中分离TEX，然后测试从起始生物流体样品到最终TEX分离的最终工作流程（具体目标3）。该项目的成功完成将产生一个外泌体分离系统，该系统将适用于高通量外泌体分离，并适用于在肿瘤学临床护理环境中从不同复杂性的各种生物流体中分离TEX和非TEX。

The proposed work aims to develop a pre-commercial prototype of an exosome isolation device to be used in clinical diagnostic laboratories. We will advance the device to facilitate exosome isolation for downstream liquid biopsy tests requiring analysis of protein, DNA, or microRNA, which rely upon acquisition of intact and pure exosomes. The device strategy will be designed to isolate total exosomes and then to separate discrete populations of tumor-derived exosomes (TEX) and non-malignant exosomes (non-TEX). The device strategy will be advanced for melanoma exosome isolation in the proposed experiments but is expected to be applicable for other cancers. To achieve this study's objectives, we will develop and optimize a filtration system incorporating the GNA Hemopurifier for isolation of total exosomes from biofluids (Specific Aim 1). The capabilities of the GNA Hemopurifier for isolating melanoma TEX and non-TEX from diverse biofluids will be characterized by evaluating exosome depletion performance (Specific Aim 2). We will then construct a novel device, the CSPG4 cartridge, for the isolation of TEX from total exosomes, followed by testing the final workflow from the starting biofluid sample to the final TEX isolation (Specific Aim 3). Successful completion of this project will produce an exosome isolation system that will be applicable for high throughput exosome isolation and for TEX and non-TEX separation from diverse biofluids of varying complexity in a clinical care setting in oncology.