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

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Cellular and Molecular Immunology - B (CMI)
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Marino, Pamela
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Albert Einstein College of Medicine
Anatomy/Cell Biology
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United States
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Miskolci, Veronika; Hodgson, Louis; Cox, Dianne (2017) Using Fluorescence Resonance Energy Transfer-Based Biosensors to Probe Rho GTPase Activation During Phagocytosis. Methods Mol Biol 1519:125-143
Hanna, Samer J; McCoy-Simandle, Kessler; Miskolci, Veronika et al. (2017) The Role of Rho-GTPases and actin polymerization during Macrophage Tunneling Nanotube Biogenesis. Sci Rep 7:8547
McCoy-Simandle, Kessler; Hanna, Samer J; Cox, Dianne (2016) Exosomes and nanotubes: Control of immune cell communication. Int J Biochem Cell Biol 71:44-54
Miskolci, Veronika; Wu, Bin; Moshfegh, Yasmin et al. (2016) Optical Tools To Study the Isoform-Specific Roles of Small GTPases in Immune Cells. J Immunol 196:3479-93
Wu, Bin; Miskolci, Veronika; Sato, Hanae et al. (2015) Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences. Genes Dev 29:876-86
Miskolci, Veronika; Hodgson, Louis; Cox, Dianne et al. (2014) Western analysis of intracellular interleukin-8 in human mononuclear leukocytes. Methods Mol Biol 1172:285-93
Cammer, Michael; Cox, Dianne (2014) Chemotactic responses by macrophages to a directional source of a cytokine delivered by a micropipette. Methods Mol Biol 1172:125-35
Hind, Laurel E; Mackay, Joanna L; Cox, Dianne et al. (2014) Two-dimensional motility of a macrophage cell line on microcontact-printed fibronectin. Cytoskeleton (Hoboken) 71:542-54
Hanna, Samer; Miskolci, Veronika; Cox, Dianne et al. (2014) A new genetically encoded single-chain biosensor for Cdc42 based on FRET, useful for live-cell imaging. PLoS One 9:e96469
Park, Haein; Dovas, Athanassios; Hanna, Samer et al. (2014) Tyrosine phosphorylation of Wiskott-Aldrich syndrome protein (WASP) by Hck regulates macrophage function. J Biol Chem 289:7897-906

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