Re-engineering Tumor Cell-Derived Extracellular Vesicles with Exogenous Tags for Downstream Analysis

胞外囊泡是由细胞和载体持续分泌的磷脂和蛋白质结构。 亲本细胞特有的遗传物质、蛋白质和脂类，可被邻近或有选择地摄取 遥远的目标细胞远离它们的释放。肠道病毒来源于病毒感染等异常情况下的细胞， 神经退行性疾病和癌症具有特定的物质，可以作为潜在的生物标志物。 针对不同的疾病状况。此外，报告还表明，肿瘤EVS作为转移部位发挥了作用。 并对为转移细胞创造有利的微环境或利基环境至关重要。因此，在 为了阐明EVS及其成分的作用方式，在体外和体内追踪EVS是必不可少的。 目前，EVS的检测主要是通过分析跨膜簇等内源性标志蛋白来实现的 分化(CD)蛋白(CD81、CD63和CD9)和肿瘤易感基因101蛋白(TSG101)。 然而，这些标记几乎存在于几乎所有细胞的不同水平上，这导致了分析 异质性。因此，外源标签对于研究现实的细胞相互作用和 跟踪电动汽车。在这一背景下，本提案的主要目标是界定 以外源荧光和金属标记为分析手柄重建EVS以阐明其 重要器官的定量摄取。 最近，PI展示了水溶性阿霉素(DOX)对乳腺肿瘤移植瘤的递送 利用自然杀伤(NK)细胞来源的EVS重建脂质体。结果显示，在治疗方面， 重组EVS的肿瘤抑制率为80%(游离DOX=65%)。同样，当巨噬细胞来源时 用脂质体重建的EVS具有更高的胶体稳定性和载药量。 经过改造的EVS对正常细胞和癌细胞显示出不同的靶向和摄取，从而使 它本身就是潜在的肿瘤靶向药物递送候选者之一。这些突出的发现来自Re- 设计的电动汽车验证了我们的假设，即从亲本细胞获得的特性导航到目标接受者 手机。从重新设计的系统中吸取的教训，我们的总体假设是将外源 近红外(NIR)染料、大环金属和/或放射性药物等成分作为电动汽车的标签 用于下游的定量分析。相关但独立的目标是目标1。重新设计电动汽车 外源标签，并研究其理化性质。目的2.阐明细胞特异性和 重新设计的电动汽车的生物兼容性。目的3.利用乳腺癌模型评价EVS的生物分布。 我们将在人乳腺癌细胞(MCF-7和MDA-MB-)来源的EVS中整合外源标签。 231)研究其在人源化小鼠人乳腺癌异种移植瘤中的相互作用和分布。 这些标签将使用PI实验室建立的最先进的分析技术进行检测。这项研究 该团队预计，这一结果将有助于EVS系统的设计，以加快癌症的诊断和治疗。

Extracellular vesicles (EVs) are phospholipid and protein constructs continuously secreted by cells and carry parent cell-specific genetic materials, proteins, and lipids, which can be selectively taken up by neighboring or distant target cells far from their release. EVs derived from cells with abnormal conditions such as viral infections, neurodegenerative diseases, and cancer have specific cargos which can be exploited as a potential biomarker for the respective disease condition. Moreover, the reports suggest that tumor EVs play a role as metastatic site surveyors and are crucial to create a favorable microenvironment or niche for metastatic cells. Therefore, in order to elucidate the mode of action of EVs and their constituents, tracking EVs in vitro and in vivo is essential. Currently, EVs are detected by analyzing the endogenous marker proteins such as trans-membrane cluster of differentiation (CD) proteins (CD81, CD63, and CD9), and tumor susceptibility gene 101 protein (TSG101). However, these markers are present in nearly almost all cells at different level which results in analysis heterogeneity. Therefore, the exogenous tag is very important to study realistic cellular interaction and tracking of EVs. Within this background, the major goal of this proposal is to define the importance of reconstruction of EVs with exogenous fluorescent and metal tags as an analytical handle to elucidate its quantitative uptake in vital organs. Recently, the PI demonstrated delivery of water-soluble doxorubicin (DOX) to breast tumor xenografts using natural killer (NK) cell-derived EVs reconstructed liposome. Results showed the therapeutic advantage of reconstructed EVs with a tumor inhibition rate of 80% (free DOX = 65%). Similarly when macrophage-derived EVs reconstructed with liposome, a higher order of colloidal stability and drug loading were observed. Engineered EVs showed the differential targeting and uptake against normal and cancerous cells thereby putting itself in the group of potential tumor-targeted drug delivery candidates. These outstanding findings from re- engineered EVs validated our hypothesis that acquired properties from parent cells navigate to target recipient cell. Lesson learned from re-engineered system, our overarching hypothesis is to incorporate exogenous components such as near-infrared (NIR) dye, macrocyclic metal, and/or radiopharmaceuticals as a tag in EVs for downstream quantitative analysis. The related but independent aims are Aim 1. To re-engineer EVs with exogenous tags and study its physicochemical properties. Aim 2. To elucidate the cellular specificity and biocompatibility of re-engineered EVs. Aim 3. To evaluate the biodistribution of EVs using breast cancer models. We will incorporate exogenous tags into the EVs derived from human breast cancer cells (MCF-7 and MDA-MB- 231) to study its interaction and distribution in human breast cancer xenograft developed in humanized mice. These tags will be detected using state-of-art analytical techniques established in the PI’s lab. The research team anticipates the results will assist EVs system designs in accelerating the diagnosis and therapy of cancer.