The role of tunneling nanotubes as a novel mode of tumor cell-macrophage communication

项目概要： 肿瘤细胞和巨噬细胞之间的相互作用已被证明是至关重要的促进肿瘤 侵袭和转移。这两种细胞类型参与了旁分泌相互作用，其中肿瘤- 相关的巨噬细胞分泌表皮生长因子（EGF）以激活肿瘤细胞。反过来，肿瘤细胞 分泌巨噬细胞应答的集落刺激因子-1（CSF-1）。虽然可溶性因子 已知在不同细胞之间传递信号，目前还不清楚这些可溶性介质如何导致共 巨噬细胞和肿瘤细胞迁移远离高浓度的这些因素存在于 原发性肿瘤我们实验室最近的研究揭示了一种新的细胞间 巨噬细胞和肿瘤细胞之间的通信，可以通过长距离传输信号， 膜肌动蛋白为基础的隧道纳米管（TNTs）。这些薄肌动蛋白含有结构形成迅速， 具有长的寿命，并且长度可以达到几个细胞直径。我们的初步数据表明， 巨噬细胞中TNT的形成依赖于肌动蛋白聚合，通过激活 RhoGTP酶Cdc 42和Rac 1及其下游效应物WASP和WAVE。使用基于FRET的 生物传感器，我们已经观察到Cdc 42和Rac 1的差异时空激活，表明它们 在巨噬细胞中TNTs的形成过程中发挥不同的作用。有趣的是，我们的初步数据显示， 在共培养中，在巨噬细胞和肿瘤细胞之间形成异型TNT。这种新的互动， 巨噬细胞和肿瘤细胞通过TNT，并可以诱导肿瘤细胞形态的变化，符合 更具侵袭性的表型拟在该研究金F99阶段完成的工作将包括： 专注于调节异型TNT形成和巨噬细胞与肿瘤细胞之间的动力学， 以及TNT在介导巨噬细胞依赖性肿瘤细胞迁移和侵袭中的作用。我们将 确定巨噬细胞和肿瘤细胞之间形成TNT的结构和机制。 我们还将确定异型TNT形成过程中RhoGTPases的时空激活 以及TNT形成对旁分泌环的依赖性，我们知道这是相互细胞所必需的。 入侵重要的是，我们将分析巨噬细胞TNT对肿瘤细胞功能的影响，包括EGFR 激活、RhoA激活、侵入伪足形成、定向迁移和侵入。除了2D和3D 分析，我们将采用新开发的一维分析，模拟肿瘤细胞的共迁移， 巨噬细胞沿着纤维，如在体内看到的，以确定TNTs在促进长距离 旁分泌侵袭总的来说，这项研究的结果将增加我们对TNT形成的理解， 调节不同类型的细胞，并确定是否需要TNT的持久的长距离 巨噬细胞和肿瘤细胞的迁移允许肿瘤进展至转移。本提案的目标3 我将着重介绍我的博士后研究方向，并将其过渡到K 00阶段 在F99阶段完成后。

PROJECT SUMMARY: The interaction between tumor cells and macrophages has been shown to be crucial in promoting tumor invasion and metastasis. These two cell types are engaged in a paracrine interaction in which tumor- associated macrophages secrete epidermal growth factor (EGF) to activate tumor cells. In turn, tumor cells secrete colony-stimulating factor-1 (CSF-1) to which macrophages respond. While soluble factors are well known to transmit signals between different cells, it is unclear how these soluble mediators can lead to the co- migration of macrophages and tumor cells away from the high concentration of these factors present in the primary tumor. Recent studies done in our lab have revealed a novel mechanism of intercellular communication between macrophages and tumor cells that can transmit signals over long distances through membranous actin-based tunneling nanotubes (TNTs).These thin actin containing structures form rapidly but have long lifetimes and can be up to several cell diameters in length. Our preliminary data demonstrates that the formation of TNTs in macrophages is dependent on actin polymerization, through activation of the RhoGTPases Cdc42 and Rac1 and their downstream effectors WASP and WAVE. Using FRET-based biosensors, we have observed differential spatiotemporal activation of Cdc42 and Rac1 indicating that they play different roles during the formation of TNTs in macrophages. Interestingly, our preliminary data shows that heterotypic TNTs form between macrophages and tumor cells in co-culture. This novel interaction between macrophages and tumor cells via TNTs and can induce changes in tumor cell morphology consistent with a more invasive phenotype. The proposed work to be completed during the F99 phase of this fellowship will be focused on the regulation of heterotypic TNT formation and dynamics between macrophages and tumor cells, as well as the role of TNTs in mediating macrophage-dependent tumor cell migration and invasion. We will determine the structure and mechanism by which TNTs are formed between macrophages and tumor cells. We will also determine the spatiotemporal activation of RhoGTPases during the formation of heterotypic TNTs as well as the dependence of TNT formation on the paracrine loop which we know is required for mutual cell invasion. Importantly, we will analyze the effects of macrophage TNTs on tumor cell function including EGFR activation, RhoA activation, invadopodia formation, directional migration and invasion. In addition to 2D and 3D assays, we will employ a newly developed 1D assay that mimics the co-migration of tumor cells and macrophages along fibers as seen in vivo to determine the role of TNTs in the promotion of long distance paracrine invasion. Overall, the results of this study will increase our understanding of TNT formation and regulation in different cell types and determine whether TNTs are required to the persistent long range migration of macrophages and tumor cells allowing for tumor progression to metastasis. Aim 3 of this proposal will focus on the transition into the K00 phase of the fellowship describing my postdoctoral research direction following the completion of the F99 phase.