Department of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota, Mayo Mail Code 480, 420 Delaware Street SE, Minneapolis, MN, 55455, USA.
Present Address: Molecular Diagnostics Laboratory, University of Minnesota Medical Center, Fairview, 420 Delaware St SE, MMC 198, Minneapolis, MN, 55455, USA.
Cell Commun Signal. 2017 Nov 13;15(1):46. doi: 10.1186/s12964-017-0201-2.
Tunneling nanotubes (TNTs) are naturally-occurring filamentous actin-based membranous extensions that form across a wide spectrum of mammalian cell types to facilitate long-range intercellular communication. Valid assays are needed to accurately assess the downstream effects of TNT-mediated transfer of cellular signals in vitro. We recently reported a modified transwell assay system designed to test the effects of intercellular transfer of a therapeutic oncolytic virus, and viral-activated drugs, between cells via TNTs. The objective of the current study was to demonstrate validation of this in vitro approach as a new method for effectively excluding diffusible forms of long- and close-range intercellular transfer of intracytoplasmic cargo, including exosomes/microvesicles and gap junctions in order to isolate TNT-selective cell communication.
We designed several steps to effectively reduce or eliminate diffusion and long-range transfer via these extracellular vesicles, and used Nanoparticle Tracking Analysis to quantify exosomes following implementation of these steps.
The experimental approach outlined here effectively reduced exosome trafficking by >95%; further use of heparin to block exosome uptake by putative recipient cells further impeded transfer of these extracellular vesicles.
This validated assay incorporates several steps that can be taken to quantifiably control for extracellular vesicles in order to perform studies focused on TNT-selective communication.
隧道纳米管（TNTs）是一种自然发生的丝状肌动蛋白为基础的膜延伸物，它在广泛的哺乳动物细胞类型之间形成，以促进长距离细胞间通讯。需要有效的测定法来准确评估 TNT 介导的细胞信号转导在体外的下游效应。我们最近报道了一种改良的 Transwell 测定系统，旨在测试通过 TNTs 进行细胞间传递治疗性溶瘤病毒和病毒激活药物对细胞间转移的影响。本研究的目的是证明这种体外方法的有效性，作为一种新的方法，可有效地排除细胞质内货物的长距离和近距离细胞间扩散形式的转移，包括外泌体/微泡和间隙连接，以分离 TNT 选择性细胞通讯。
我们设计了几个步骤，以有效减少或消除这些细胞外囊泡的扩散和长距离转移，并在实施这些步骤后使用纳米颗粒跟踪分析来定量外泌体。
这里概述的实验方法有效地减少了 >95%的外泌体转运；进一步使用肝素阻止外泌体被假定的受体细胞摄取，进一步阻碍了这些细胞外囊泡的转移。
该验证性测定法结合了几个步骤，可以定量控制细胞外囊泡，以进行专注于 TNT 选择性通讯的研究。
