外泌体多组学05-EV介导的细胞间通讯研究综述

细胞向胞外环境中释放多种来源于细胞内体或细胞膜的囊泡结构，包括外泌体、微泡等，统称为细胞外囊泡。细胞外囊泡是细胞间信号沟通的一种重要模式，介导了蛋白质，脂质，RNA等活性大分子在细胞间的传递。

文章信息

• 文献标题：Challenges and directions in studying cell–cell communication by extracellular vesicles
• Doi：https://doi.org/10.1038/s41580-022-00460-3
• 发表时间：Published: 08 March 2022
• 作者：Guillaume van Niel 和Pieter Vader

我的问题

• 细胞分泌外泌体是否随机？
• 分泌出去的外泌体的靶向受体细胞是否随机？
• 外泌体介导的细胞通讯与其他方式的细胞通讯有何不同？
• 外泌体运输途径有哪些？

Extracellular vesicle biogenesis

EV由通过多种机制产生的异质膜泡组成。EV的两个主要类型为：ectosomes和exosomes。

• ectosomes：包括oncosomes，microvesicles（在质膜上向外出芽产生的微囊泡）
• exosomes：在细胞内产生，在内吞途径中，通过内吞体膜向内出芽，导致形成内吞体腔内包含的非常小的囊泡状结构

关于EVs的生成涉及到的方面有以下几点值得关注：

• EV subtypes的区分
• 生物发生机制、EV亚型及其内含物的多样性
• EV分泌调节
• 生理背景下的EV生物发生

EV的多种产生机制：

Fig. 1 | State of the art of eV biogenesis and dissemination in the extracellular space

不同的细胞外囊泡亚型以及各自的特征：可以看到这里主要的区别手段是size与marker不同

image-20220422191015381

Box 1 | EV 生成: 关键问题与挑战

• How do we consider the whole complexity of the cell, including regulation of intracellular trafficking and cell metabolism in the study of extracellular vesicle (ev) biogenesis?
• since ev biogenesis is also dependent on interaction with neighbouring cells and matrix in vivo,to which extent doe sthe ‘EV secretome’ change depending on environmental cues?
• are distinct subsets of exosomes with different functions associated with multivesicular endosome (Mve) subpopulations?
• what are the processes that turn subpopulations of Mves into secretory organelles?
• How do chemical parameters, such as pericellular pH, concentration of reactive oxygen species and osmotic pressure, as well as physical constrains of the tissue, such as mechanical pressure due to cell density or stiffening extracellular matrix, affect intraluminal vesicle generation and secretion and plasma membrane budding?
• Are processes described in vitro relevantin vivo?
• From a more practical perspective, when aiming to interfere with ev biogenesis, how can we consider the complexity and diversity of the biogenesis of subpopulations of evs as well as the cell types, culture conditions and level of expression of ev cargoes? For instance, a given pathway, such as ceramide production, may inhibit exosome secretion of one subtype but not of others, while simultaneously affecting other regulatory processes in the cell. Moreover, if the inhibition or impairment of a specific process affects Mve biogenesis, given that not all Mves are secretory, new approaches are needed to clearly distinguish correlation and causality of the importance of the given process during exosome secretion.

The dynamics in extracellular space

EV可以在细胞之间以自分泌、旁分泌或内分泌分泌的方式进行交换。ev介导局部和远处细胞间通信能力的关键是它们在大多数细胞嵌入的细胞胞外和细胞外基质中导航的能力

这个交流方式有以下几点可以关注：

• EVs释放到胞外的过程
• EVs携带的cargos
• EV 在体内的分布情况

这里补充一个知识点：（《细胞生物学》第三版 翟中和）

细胞通讯：

细胞通讯是指一个细胞发出的信息通过介质(又称为配体)传递到另一个细胞并与靶细胞相应的受体相互作用，然后通过细胞信号转导产生细胞内一系列生理生化变化，最终表现为细胞整体的生物学效应的过程。细胞信号转导是细胞间实现通讯的关键过程，对于多细胞生物细胞间功能的协调、控制细胞的生长和分裂、组织发生与形态建成是必需的。

细胞通讯的方式，可以分为三种：

1. 细胞通过分泌化学信号进行细胞间通讯，这是多细胞生物普遍采用的通讯方式(图A-D)
2. 细胞间接触依赖性的通讯，细胞间直接接触，通过与质膜结合的信号分子影响其他细胞(图E)
3. 动物相邻细胞间形成间隙链接，植物细胞间通过胞间连丝是细胞间相互沟通

其中，自分泌方式一般是病理性质的，多见于肿瘤细胞自己分泌刺激因子促进自身分裂增殖

外泌体的交流方式：

Fig. 2 | The dynamics of eVs in extracellular space leading to their uptake and functions in recipient cells

Box 2 | 胞外空间的动态性: 关键问题与挑战

• what is the interrelationship between extracellular vesicles (evs) and the extracellular matrix?
• are all evs capable of crossing all basement membranes and, if not, is there tissue subpopulation specificity?
• Given the importance of the extracellular matrix in the maintenance of normal tissue function, how do mechanical forces and the local matrix composition influence ev–extracellular matrix interaction?
• to what extent does the composition of the extracellular matrix modulate the composition, and hence function, of evs?
• what types of evs interact with the extracellular matrix, and is release of specific subpopulations into the extracellular matrix directly dependent on plasma membrane–matrix interactions and the associated mechanical forces?
• Do evs carry components/properties that allow them to resist degradation extracellularly and to avoid undesirable interactions with non-target cells, matrix constituents and other interstitial factors?
• How important are the biophysical properties of evs (for example, their size and compressibility) in traversing through the complex extracellular microenvironment?
• after traversing the extracellular matrix, evs, or at least a subpopulation thereof, appear to have the capacity to cross several biological barriers. is this capability influenced by specific pathological situations (for example, the increase in vascular permeability)?
• Do specific cell types interact with evs more efficiently, and does this interaction differ between different ev subpopulations?
• Most of our understanding ofthe fate of EVsin vivo comesfrom studies using exogenously administered evs. However, ev isolation, storage, method of formulation and labelling may affect their targeting specificity and ultimate fate. thus, are the results obtained using exogenously administered evs representative of the physiological fate of endogenously generated evs?
• Multiple approaches have been used to assessthe biodistribution of EVs in vivo, including fluorescent labelling of lipids and proteins, immunofluorescence, bioluminescence, Pet, sPeCt, Mri and Ct imaging59. all of these approaches have limitations in tracking the fate of evs, and novel approaches with large dynamic ranges of both temporal and spatial resolution are required to overcome these.

Functional delivery of extracellular vesicle cargo

很多研究都观察到不同细胞类型对EV的摄取现象，EV摄取可以被视为一系列的步骤（图2，5-9），但引导EV进入受体细胞的过程目前尚不清楚，以下几点值得关注：

• EV 与细胞表面的作用
• 进入细胞的途径
• EV胞内的运输与传递
• 细胞与细胞间通过EVs通讯

Fig. 3 | Key questions and challenges in studying eV biology

假设这下面两个人是两个细胞，中间的是细胞外囊泡，那两个细胞的EV交流方式是不是就像下面这样口若悬河滔滔不绝？

Box 3 | EV cargo的摄取与功能传递: 关键问题与挑战

• which extracellular vesicle (ev)-associated factors are critical for dictating function (defined by signal-induction and/or cell entry)?
• Can we define the rules that govern ev targeting, and can we manipulate this knowledge in future translational studies using ev-based medicines?
• to what extent is the nature of the protein corona controlled by the native ev composition and how important is the corona in determining interactions with recipient cells or whole systems?
• How do we overcome the technical challenge of small ev size and scarcity of cargo, posing issues on directimaging of functional delivery of cargo by native EVs in vivo?
• are different ev subpopulations processed differently in recipient cells and do they exert different cellular functions? Can this heterogeneity be addressed by studying ev subpopulations individually, or do they cooperate in a synergistic manner?
• How do we provide direct evidence that observed physiological effects are truly ev mediated and, if so, what ev cargoes are responsible? in this regard, an overlooked aspect is the relative contribution of evs compared to other communication means such as cell–cell contact, nanotubes orsoluble factorslike cytokines or extracellular rNa.
• when attempting to dissect the relative potency of evs in directing cell communication from other secretome constituents, how important are the different non-vesicular secreted factors, such as extracellular matrix proteins and cytokines, in co-operation with evs in dictating the final and complex cellular response?

未来的研究方向

• Technical challenges
• Standardization of research
• Applications and translational opportunities

厉害的文章必有大牛：

• 自 1998 年以来，Guillaume van Niel 一直在研究外泌体及其起源区室、多泡体。
• 在 Necker 研究所（法国巴黎）攻读博士学位期间，他报告并研究了肠上皮细胞分泌的外泌体的分泌和功能。
• 2003 年，他在乌得勒支内侧中心（荷兰乌得勒支）展示了 MHC II 分子的泛素化，这是一种用于分选树突细胞中多泡体的关键翻译后修饰。
• 2005 年，他加入居里研究所（法国巴黎），并于 2008 年在该团队获得 CNRS 永久职位，以研究色素细胞中多泡体的生物发生。他特别报道了腔内囊泡在生理淀粉样蛋白原纤维生成中的作用。
• 自 2017 年以来，他担任巴黎精神病学和神经科学研究所（法国巴黎）的团队负责人，开发新工具来可视化外泌体，尤其是在体内，并了解内体动力学在淀粉样蛋白相关病理（阿尔茨海默病和胶质母细胞瘤发展等神经病）中降解和分泌之间的作用。
• 他在外泌体的细胞生物学方面拥有丰富的专业知识，并使用广泛不同的成像技术，其中包括电子显微镜。