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

项目总结 细胞外小泡(EVS)是一组由细胞分泌的异质性脂质双层包裹颗粒， 即使不是全部，也存在于大多数生物体液中。EV一词涵盖了广泛的囊泡类别，包括外体 (50-150 nm)和微泡(150 nm-1000微米)，来自多囊泡融合后的内体途径 有质膜的小体或由质膜直接萌发的小体。电动汽车走得更远 以具有不同功能特性的膜和管腔为特征的。一个新兴的团体 文献表明，在正常生理和病理事件中，EV是细胞间通讯的主要贡献者， 比如癌症。EVS已被证明影响癌细胞的趋化和转移性器官趋化。值得注意的是，一个特定的 EV的类型依赖于Syntenin-1，这是一种含有两个PSD95/DLG/zonula occludens 1(PDZ)的接头蛋白 结构域，用于生物发生和通过内体途径装载货物。这表明上游效应器 与Syntenin-1的结合可以调节其可用性，进而调节外切体的生物发生和货物装载。已激活 白细胞黏附分子(ALCAM)是免疫球蛋白超家族中的一种黏附蛋白，发现于 细胞-细胞连接。AlCaM是一种细胞黏附的动态调节因子，通过其胞外结构域的差异脱落来调节细胞的黏附。我们的 实验室证明，全长alcam异构体(Iso1)防止脱落，促进黏附和限制 转移。相反，选择性剪接的异构体2(Iso2)容易脱落，有助于运动和 转移。Alcam脱落和Alcam Iso2表达升高与膀胱癌的相关性 进步。Alcam可通过与细胞质支架蛋白的结合而与EV的生物发生联系 Syntenin-1。通过alcam的细胞质尾巴隔离syntenin-1可进一步抑制运动和转移 强调监管机制的功能相关性。其他初步研究显示， Iso1的表达抑制了大型EV的生物发生，而Iso2-促进了大型EV的生物发生。基于这些发现 我们假设alcam的脱落控制了肿瘤细胞中依赖于Syntenin的EV的生物生成。此外，我们 假设alcam介导的黏附可以通过调节电动汽车的载重量来控制电动汽车的亲迁移功能。至 验证这一假设，我将利用表达不同亚型的alcam和BCA患者来源的血浆的细胞系来 确定alcam介导的细胞黏附影响EV生物学的机制。目标1中建议的实验 将研究通过alcam表达对细胞黏附状态的调节如何改变EV的生物发生和功能。 EV的生物发生将通过纳米颗粒跟踪分析、Western blotting和 微流细胞术。EV的功能将通过转移的禽类胚胎模型和3D器官型来确定 膀胱模型。目标2将描述肠易激综合征脱落时EV货物的变化和BCA的疾病进展 通过棕榈酰化的质谱学。总的来说，这些数据将定义细胞黏附EV之间的关系 生物学，无论是机械的还是功能的。此外，这些发现将识别信息丰富的分子标记，以 通过非侵入性手段帮助监测疾病状态。

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.