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

肿瘤相关巨噬细胞（TAM）在许多肿瘤中大量存在， 在促进实体瘤进展为侵袭性、转移性表型中起重要作用。它有 最近显示TAM参与癌细胞的旁分泌环，使得CSF-1- 产生癌细胞和分泌EGF的巨噬细胞（MD）相互作用以促进相互趋化性 导致癌细胞的侵袭和外渗。在MD中，PI 3-激酶、Cdc42和WASP（Wiskott- 奥德里奇综合征蛋白）是迁移细胞检测化学引诱物来源所必需的。它有 据推测，细胞外梯度的扩增是通过细胞内阳性信号发生的。 反馈环涉及PI 3-激酶、Rho GTPases和肌动蛋白组装。WASP的精确功能 梯度检测是未知的。基于初步数据，WASP活性是CSF-I诱导的细胞凋亡所必需的。 肌动蛋白聚合在MD，这可能有助于加强的正反馈环在CSF- 1梯度检测（趋化传感）导致有效的趋化性。在第一个具体目标中， 将使用PI3K抑制剂、CSF-1R突变和CD42在WASP活化中的作用来确定PI3K和Cdc42在WASP活化中的作用。 缺乏PI3K激活的细胞，并通过使用siRNA技术降低Cdc42的内源性水平。的 WASP激活的机制将通过荧光共振的突变分析来检查 能量转移（FRET）的WASP生物传感器。在特异性目的2中，WASP介导的肌动蛋白的作用 将确定趋化性传感中的聚合。WASP在PI3K定位中的作用， cdc42在趋化传感中的作用将通过活细胞成像来确定。此外，我们将执行 CSF-1的生化分离从野生型和WASP缺陷型巨噬细胞中诱导伪足， 以鉴定潜在的WASP相互作用蛋白。在具体目标3中，WASP对偏振的影响 MD和癌细胞的运动将使用体外测定法进行检查，该测定法重建了 MD和肿瘤细胞之间的旁分泌相互作用。 相关性：了解MD如何迁移到肿瘤部位及其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MD趋化性特别需要WASP 它可能代表一种新的靶点，并可能导致新的治疗方法，以防止转移

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