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

描述(申请人提供)：肿瘤相关巨噬细胞()，它大量存在于许多肿瘤中，似乎在促进实体瘤向侵袭性、转移性表型发展过程中发挥重要作用。最近的研究表明，参与了与癌细胞的旁分泌环路，使产生脑脊液-1的癌细胞和分泌表皮生长因子的巨噬细胞相互作用，促进相互趋化，导致癌细胞的侵袭和渗出。在MFS中，迁移细胞需要PI3-激酶、CDC42和WASP(Wiskott-Aldrich综合征蛋白)来检测趋化物质的来源。已经推测，细胞外梯度的放大是通过细胞内的正反馈环发生的，该环涉及PI3-激酶、Rho GTP酶和肌动蛋白组装。WASP在梯度检测中的确切作用尚不清楚。根据初步数据，WASP活性是CSF-1诱导的MFS肌动蛋白聚合所必需的，这可能有助于加强CSF-1梯度检测(趋化检测)中的正反馈环，从而导致有效的趋化。在第一个特定目标中，将使用PI3K抑制剂、缺乏PI3K激活的CSF-1 R突变以及通过siRNA技术降低内源性CDc42水平来确定PI3K和CDC42在WASP激活中的作用。将通过对基于荧光共振能量转移(FRET)的WASP生物传感器的突变分析来研究WASP的激活机制。在具体目标2中，将确定WASP介导的肌动蛋白聚合在趋化感觉中的作用。WASP在PI3K和CDC42定位中的作用将通过活细胞成像来确定。此外，我们将从野生型和WASP缺失的巨噬细胞中对CSF-1诱导的伪足进行生化分离，以确定潜在的WASP相互作用蛋白。在特定目的3.WASP对MF和癌细胞的极化和运动的影响将通过重建MF和肿瘤细胞之间旁分泌相互作用的体外实验来检测。相关性：了解MFS如何迁移到肿瘤部位以及它们与癌细胞的相互作用，是癌症生物学研究的一个重要领域。由于WASP是MF趋化所必需的，它可能代表一个新的靶点，并可能导致新的治疗方法来防止转移

DESCRIPTION (provided by applicant): 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 (MF) interact to promote mutual chemotaxis leading to invasion and extravasation of carcinoma cells. In MFs, 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 MFs 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-1 R 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 MF and carcinoma cells will be examined using an in vitro assay that reconstitutes the paracrine interaction between MF and tumor cells. Relevance: Understanding how MFs 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 MF chemotaxis it may represent a novel target and may lead to new therapies to prevent metastasis