Development and application of asymmetric-flow field-flow (AF4) technology in fractionation and characterization of exosome subpopulations and novel nanoveiscles in pancreatic cancer model

项目总结/摘要 由于认识到外泌体在免疫调节中的潜在作用，外泌体研究呈指数增长。 病理生理过程，包括癌症。然而，由于技术上的挑战， 构成了一个异质种群，这阻碍了我们对它们的生物起源，分子生物学， 这些化合物在体内的组成、生物分布和功能，并且限制了它们的翻译应用。国家- 最新技术，非对称流场流分馏（AF 4），表现出独特的分离能力， 纳米颗粒，并已被广泛用于表征纳米颗粒和聚合物在药物 工业以及各种生物大分子、蛋白质复合物和病毒。的目的 本研究旨在开发和验证AF4在外来体分离和鉴定新的 使用胰腺癌作为模型系统的纳米囊泡。我们将评估其在分析中的应用 从一组已建立的人胰腺癌细胞系中分离的外来体（Aim 1）。我们将进一步 开发和优化AF4方法，将其应用于复杂的生物样本，如血浆 人类受试者（目标2）。最后，我们将验证AF 4应用程序的分馏， 不同的外来体亚群的表征和其他新的纳米囊泡的鉴定 从新诊断疾病的胰腺患者中分离的标本（血浆和肿瘤组织） 以及在疾病的不同阶段以及正在接受治疗的患者（目标3）。我们预测AF4在 与敏感的分子测定相结合，可以作为一种改进的分析工具， 特定的纳米囊泡亚群，从而解决囊泡异质性的复杂性。

Project Summary/Abstract Exosome research has grown exponentially due to the recognition of the potential roles of exosomes in pathophysiological processes, including cancer. However, due to technical challenges, isolated nanovesicles constitute a heterogeneous population and this has hindered our understanding of their biogenesis, molecular composition, biodistribution, and functions in vivo, and has limited their translational application. The state-of- the-art technology, asymmetric-flow field-flow fractionation (AF4), exhibits unique capability to separate nanoparticles and has been widely utilized to characterize nanoparticles and polymers in the pharmaceutical industry as well as various biological macromolecules, protein complexes and viruses. The objective of this study is to develop and validate the application of AF4 in exosome isolation and identification of novel nanovesicles using pancreatic cancer as a model system. We will evaluate its application in analyzing exosomes isolated from a panel of established human pancreatic cancer cell lines (Aim 1). We will further develop and optimize the AF4 methodology to apply it to complex biological specimens such as blood plasma from human subjects (Aim 2). Lastly, we will validate the AF4 application for the fractionation and characterization of distinct exosome subpopulations and identification of other novel nanovesicles using specimens (blood plasma and tumor tissues) isolated from pancreatic patients with newly diagnosed disease and at different stages of disease as well as patients undergoing treatment (Aim 3). We predict that AF4 in combination with sensitive molecular assays can serve as an improved analytical tool for the isolation of specific nanovesicle subpopulations, thereby addressing the complexities of vesicle heterogeneity.