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

描述（由申请人提供）：肿瘤相关巨噬细胞（TAM）在许多肿瘤中大量存在，似乎在促进实体瘤进展为侵袭性、转移性表型方面发挥重要作用。最近已经表明，TAM参与癌细胞的旁分泌环，使得产生CSF-1的癌细胞和分泌EGF的巨噬细胞（MF）相互作用以促进相互趋化性，从而导致癌细胞的侵袭和外渗。在MF中，PI 3-激酶、Cdc 42和WASP（Wiskott-Aldrich综合征蛋白）是迁移细胞检测化学引诱物来源所必需的。据推测，细胞外梯度的扩增通过涉及PI 3-激酶、Rho GTP酶和肌动蛋白组装的细胞内正反馈回路发生。WASP在梯度检测中的精确功能尚不清楚。基于初步数据，在MF中CSF-1诱导的肌动蛋白聚合需要WASP活性，这可能有助于CSF- 1梯度检测（趋化性传感）中正反馈环的加强，从而导致有效的趋化性。在第一个特定目的中，将使用PI 3 K抑制剂、缺乏PI 3 K活化的CSF-1 R突变以及通过使用siRNA技术降低Cdc 42的内源性水平来确定PI 3 K和Cdc 42在WASP活化中的作用。WASP激活的机制将通过基于WASP生物传感器的荧光共振能量转移（FRET）的突变分析来检查。在具体目标2中，将确定WASP介导的肌动蛋白聚合在趋化传感中的作用。WASP在趋化传感中PI 3 K和Cdc 42的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缺陷型巨噬细胞中进行CSF-1诱导的伪足的生化分离，以鉴定潜在的WASP相互作用蛋白。具体目标3。WASP对MF和癌细胞的极化和运动的影响将使用重建MF和肿瘤细胞之间的旁分泌相互作用的体外测定来检查。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重要领域。由于WASP是MF趋化性的特异性要求，它可能代表一个新的靶点，并可能导致新的治疗方法，以防止转移

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