THE ROLE OF CSF-1 MEDIATED MACROPHAGE CHEMOTAXIS IN CARCINOMA CELL INVASION

在许多肿瘤中大量存在的肿瘤相关巨噬细胞()似乎也在发挥作用 在促进实体瘤向侵袭性、转移性表型发展过程中起重要作用。它有 最近发现，参与了与癌细胞的旁分泌环路，从而使脑脊液-1- 产生癌细胞和分泌EGF的巨噬细胞(MD)相互作用促进相互趋化 导致癌细胞的侵袭和渗出。在MDS中，PI 3-激酶、CDC42和WASP(Wiskott- Aldrich综合征蛋白)是迁移细胞检测化学诱导剂来源所必需的。它有 已经推测，细胞外梯度的放大是通过细胞内的正离子进行的 包括PI 3-激酶、Rho GTP酶和肌动蛋白组装的反馈环。WASP在WASP中的精确作用 梯度检测尚不清楚。根据初步数据，WASP活性是诱导CSF-1所必需的 肌动蛋白在MD中的聚合可能有助于加强脑脊液中的正反馈环。 1梯度检测(趋化感应)导致有效的趋化作用。在第一个具体目标中，角色 将使用PI3K抑制剂、CSF-1R突变来确定PI3K和CDC42在WASP激活中的作用 缺乏PI3K激活，并通过使用siRNA技术降低内源CDC42水平。这个 WASP的激活机制将通过荧光共振的突变分析来研究 基于能量转移(FRET)的WASP生物传感器。在特定目标2中，WASP介导的肌动蛋白的作用 化学趋化传感中的聚合作用将被确定。WASP在PI3K和PI3K本地化中的作用 Cdc42在化学趋化传感中的作用将通过活细胞成像来确定。此外，我们还将表演 野生型和WASP缺陷型巨噬细胞中CSF-1诱导伪足的生化分离 以确定潜在的WASP相互作用蛋白。在特定目标3中WASP对偏振的影响 MD和癌细胞的运动将使用一种体外实验来检测，该实验重建了 MD与肿瘤细胞的旁分泌相互作用。 相关性：了解MD如何迁移到肿瘤部位以及它们与癌细胞的相互作用 癌症生物学的一个重要研究领域。由于WASP是MD趋化性所特需的 它可能代表一个新的靶点，并可能导致防止转移的新疗法

Tumor-associated macrophages (TAM), which are present in large numbers in many tumors, appear to play an important role in promoting the progression of solid tumors to an invasive, metastatic phenotype. It has been shown recently that TAM participate in a paracrine loop with carcinoma cells such that the CSF-1- producing carcinoma cells and the EGF-secreting macrophages (MD) interact to promote mutual chemotaxis leading to invasion and extravasation of carcinoma cells. In MDs, PI 3-kinase, Cdc42 and WASP (Wiskott- Aldrich syndrome protein) are required for migrating cells to detect the source of a chemoattractant. It has been speculated that amplification of the extracellular gradient occurs through an intracellular positive feedback loop involving PI 3-kinase, Rho GTPases and actin assembly. The precise function of WASP in gradient detection is not known. Based on preliminary data, WASP activity is required for CSF-1 induced actin polymerization in MDs which may contribute to the reinforcement of the positive feedback loop in CSF- 1 gradient detection (chemotactic sensing) leading to efficient chemotaxis. In the first Specific Aim, the role of PI3K and Cdc42 in the activation of WASP will be determined using PI3K inhibitors, CSF-1R mutations that lack PI3K activation, and by reducing endogenous levels of Cdc42 using siRNA technology. The mechanisms of WASP activation will be examined through mutational analysis of a fluorescence resonance energy transfer (FRET) based WASP biosensor. In Specific Aim 2, the role of WASP mediated actin polymerization in chemotactic sensing will be determined. The role of WASP in the localization of PI3K and Cdc42 in chemotactic sensing will be determined by live cell imaging. In addition, we will perform biochemical isolation of CSF-1 elicited pseudopods from wild-type and WASP-deficient macrophages in order to identify potential WASP interacting proteins. In Specific Aim 3 The effect of WASP on polarization and movement of MD and carcinoma cells will be examined using an in vitro assay that reconstitutes the paracrine interaction between MD and tumor cells. Relevance: Understanding how MDs migrate into a tumor site and their interaction with carcinoma cells, is an important area of investigation in cancer biology. Since WASP is specifically required for MD chemotaxis it may represent a novel target and may lead to new therapies to prevent metastasis