ALCAM-mediated cell adhesion and extracellular vesicle biogenesis in bladder cancer

膀胱癌中 ALCAM 介导的细胞粘附和细胞外囊泡生物发生

• 批准号: 10204708
• 负责人: Ariana Kathryn von Lersner
• 金额: $ 3.08万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10204708
• 关键词: 3-Dimensional; ALCAM gene; Activated-Leukocyte Cell Adhesion Molecule; Adaptor Signaling Protein; Adhesions; Adhesives; Affect; Alternative Splicing; Binding; Biogenesis; Biological; Biology; Birds; Bladder; Bladder papilloma; Cancer Patient; Cancer Relapse; Carcinoma; Cell Adhesion; Cell Communication; Cell Line; Cell membrane; Cells; Chemotaxis; Cytometry; Cytoplasmic Tail; DLG4 gene; Data; Detection; Development; Disease; Disease Progression; Electron Microscopy; Embryo; Environment; Event; Goals; Immunoblotting; Immunoglobulin Domain; Immunoglobulins; In Vitro; Intercellular Junctions; Intervention; Laboratories; Length; Leukocyte Adhesion Molecules; Link; Lipid Bilayers; Liquid substance; Literature; Malignant Neoplasms; Malignant neoplasm of urinary bladder; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Membrane; Modeling; Molecular; Monitor; Multivesicular Body; Neoplasm Metastasis; Pathologic; Pathway interactions; Physiology; Plasma; Production; Property; Protein Analysis; Protein Isoforms; Proteins; Publishing; Regulation; Research; Risk; Scaffolding Protein; Site; Techniques; Testing; Tight Junctions; Tumor Cell Migration; Tumor-Derived; Urine; Vesicle; Western Blotting; Work; base; cancer cell; cell motility; clinically relevant; exosome; experimental study; extracellular; extracellular vesicles; in vivo Model; insight; liquid biopsy; member; microvesicles; migration; molecular marker; mortality; nanoparticle; neoplastic cell; novel; particle; prevent; response; synergism; targeted treatment; treatment response; treatment strategy; tumor progression

PROJECT SUMMARY Extracellular vesicles (EVs) are a heterogenous group of lipid bilayer enclosed particles secreted from cells and are found in most, if not all, biological fluids. The term EV encompasses a wide range of vesicle classes, including exosomes (50-150 nm) and microvesicles (150 nm - 1000+ µm), derived from the endosomal pathway after fusion of multivesicular bodies with the plasma membrane or from direct budding from the plasma membrane, respectively. EVs are further characterized by membrane and luminal cargoes that give rise to distinct functional properties. An emerging body of literature suggests that EVs are major contributors in cell-cell communication in normal physiology and pathological events, such as cancer. EVs have been shown to affect cancer cell chemotaxis and metastatic organotropism. Notably, a specific class of EV is dependent upon syntenin-1, an adaptor protein that contains two PSD95/Dlg/zonula occludens 1 (PDZ) domains, for biogenesis and cargo loading through the endosomal pathway. This suggests that upstream effectors which bind to syntenin-1 could modulate its availability, and in turn, regulate exosome biogenesis and cargo loading. Activated Leukocyte Cell Adhesion Molecule (ALCAM) is an adhesion protein in the immunoglobulin superfamily found at sites of cell-cell junctions. ALCAM is a dynamic regulator of cell adhesion through differential shedding of its ectodomain. Our laboratory demonstrated that the full-length ALCAM Isoform (Iso1) resists shedding, promotes adhesion and limits metastasis. Conversely, the alternatively spliced isoform 2 (Iso2) is susceptible to shedding, facilitates motility and metastasis. ALCAM shedding and elevated expression of ALCAM Iso2 correlates with bladder cancer (BCa) disease progression. ALCAM can be linked to EV biogenesis through its association with the cytoplasmic scaffolding protein syntenin-1. Sequestering syntenin-1 via the cytoplasmic tail of ALCAM abrogates motility and metastasis, further underscoring the functional relevance of the regulatory mechanism. Additional preliminary studies revealed that the expression of Iso1 suppresses large EV biogenesis while Iso2-facilitates the biogenesis of large EVs. Based on these findings we hypothesize that ALCAM shedding controls syntenin-dependent EV biogenesis in tumor cells. In addition, we hypothesize that ALCAM-mediated adhesion can control the pro-migratory function of EVs by regulating their cargo. To test this hypothesis, I will utilize cell lines expressing different isoforms of ALCAM and BCa patient-derived plasma to determine the mechanism in which ALCAM-mediated cell adhesion affects EV biology. Experiments proposed in Aim 1 will investigate how modulation of a cells' adhesive state through ALCAM expression alters EV biogenesis and function. EV biogenesis will be assessed through complementary techniques of nanoparticle tracking analysis, western blotting, and microflow cytometry. EV function will be determined with the avian embryo model of metastasis and a 3D organotypic bladder model. Aim 2 will characterize the changes in EV cargos upon ALCAM shedding and disease progression in BCa through palmitoylated-mass spectrometry. Collectively, these data will define a relationship between cell adhesion EV biology, both mechanistically and functionally. Additionally, these findings will identify informative molecular markers to aid in the monitoring of disease state through a non-invasive means.

项目概要 细胞外囊泡（EV）是细胞分泌的一组异质脂质双层封闭颗粒， 存在于大多数（如果不是全部）生物体液中 EV 一词涵盖了广泛的囊泡类别，包括外泌体。 (50-150 nm) 和微泡 (150 nm - 1000+ µm)，源自多泡融合后的内体途径 具有质膜的体或从质膜直接出芽的体分别更进一步。 以膜和管腔货物为特征，产生独特的功能特性。 文献表明，EVs 是正常生理和病理事件中细胞间通讯的主要贡献者， 值得注意的是，EV 会影响癌细胞的趋化性和转移性器官倾向。 EV 的类别取决于 Syntenin-1，这是一种接头蛋白，包含两个 PSD95/Dlg/闭合小带 1 (PDZ) 域，通过内体途径进行生物发生和货物装载，这表明上游效应器。 与 syntenin-1 结合可以调节其可用性，进而调节外泌体的生物发生和负载激活。 白细胞细胞粘附分子 (ALCAM) 是免疫球蛋白超家族中的一种粘附蛋白，存在于以下部位： ALCAM 是通过其胞外域的差异脱落来动态调节细胞粘附的。 实验室证明全长 ALCAM Isoform (Iso1) 能够抵抗脱落、促进粘附并限制 离线时，选择性剪接异构体 2 (Iso2) 易于脱落，有利于迁移和转移。 ALCAM 脱落和 ALCAM Iso2 表达升高与膀胱癌 (BCa) 疾病相关。 ALCAM 通过与细胞质支架蛋白的结合与 EV 生物发生相关 syntenin-1 通过 ALCAM 的细胞质尾部隔离，进一步消除了运动和转移。 强调监管机制的功能相关性。 基于这些发现，Iso1 的表达抑制大型 EV 的生物发生，而 Iso2 则促进大型 EV 的生物发生。 我们研究发现 ALCAM 脱落控制肿瘤细胞中依赖于 Syntenin 的 EV 生物发生。 继续认为 ALCAM 介导的粘附可以通过调节 EV 的货物来控制 EV 的促迁移功能。 为了检验这个假设，我将利用表达不同亚型 ALCAM 和 BCa 患者来源血浆的细胞系 确定 ALCAM 介导的细胞粘附影响 EV 生物学的机制。 将研究通过 ALCAM 表达调节细胞粘附状态如何改变 EV 生物发生和功能。 EV生物发生将通过纳米颗粒跟踪分析、蛋白质印迹和免疫印迹等互补技术进行评估 微流式细胞仪将通过禽胚胎转移模型和 3D 器官型来确定 EV 功能。 目标 2 将表征 ALCAM 脱落和 BCa 疾病进展时 EV 货物的变化。 总的来说，这些数据将定义细胞粘附 EV 之间的关系。 此外，这些发现将在机械和功能方面鉴定出信息丰富的分子标记。 通过非侵入性手段帮助监测疾病状态。