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

项目总结： 肿瘤细胞和巨噬细胞之间的相互作用已被证明是促进肿瘤发生的关键。 侵袭和转移。这两种类型的细胞参与旁分泌相互作用，其中肿瘤- 相关巨噬细胞分泌表皮生长因子(EGF)来激活肿瘤细胞。反过来，肿瘤细胞 分泌巨噬细胞反应的集落刺激因子-1(CSF-1)。而可溶性因子则很好 已知在不同细胞之间传递信号，目前尚不清楚这些可溶介质如何导致联合- 巨噬细胞和肿瘤细胞从高浓度的这些因子中迁移出来 原发肿瘤。我们实验室最近的研究揭示了一种新的细胞间机制 巨噬细胞和肿瘤细胞之间的通讯，可以通过 基于膜肌动蛋白的隧道纳米管(TNTs)。这些含有肌动蛋白的薄结构形成得很快，但 具有很长的寿命，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TNTs的形成依赖于肌动蛋白聚合，通过激活 RhoGTP酶CDC42和rac1及其下游效应子Wasp和WAVE。使用基于FRET的 生物传感器，我们观察到了Cdc42和rac1的时空差异激活，表明它们 在巨噬细胞中TNTs的形成过程中起着不同的作用。有趣的是，我们的初步数据显示 巨噬细胞与肿瘤细胞共培养形成异型TNTs。这种新颖的相互作用 巨噬细胞和肿瘤细胞通过TNTs，并可诱导肿瘤细胞形态的变化，与 更具侵袭性的表型。本奖学金F99阶段将完成的拟议工作将是 侧重于巨噬细胞和肿瘤细胞之间异型TNT形成的调节和动力学， 以及TNTs在介导巨噬细胞依赖的肿瘤细胞迁移和侵袭中的作用。我们会 确定巨噬细胞和肿瘤细胞之间形成TNTs的结构和机制。 我们还将确定RhoGTP酶在异型TNTs形成过程中的时空激活 以及TNT的形成对旁分泌环的依赖，我们知道旁分泌环是相互细胞所必需的 入侵。重要的是，我们将分析巨噬细胞TNTs对肿瘤细胞功能的影响，包括EGFR 激活、RhoA激活、内生生物的形成、定向迁移和入侵。除了2D和3D之外 我们将使用一种新开发的一维分析方法来模拟肿瘤细胞和 巨噬细胞沿纤维在体内观察确定TNTs在促进长距离运动中的作用 旁分泌入侵。总体而言，这项研究的结果将增加我们对TNT形成和 在不同的细胞类型中进行调控，并确定是否需要TNTs来持续长距离 巨噬细胞和肿瘤细胞的迁移允许肿瘤进展到转移。本提案的目标3 我将专注于过渡到K00阶段的奖学金，描述我的博士后研究方向 在F99阶段完成之后。

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.