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Interactions of Breast Cancer Cells with Macrophages in Controlled 3D in vitro Microenvironments

Final Report Summary - CANCER-3DMATRIX-EGF (Interactions of Breast Cancer Cells with Macrophages in Controlled 3D in vitro Microenvironments)

The leading cause of death for cancer patients is metastasis. During metastasis, cancer cells interact with various molecules and cells. It has been shown that a group of macrophages facilitates metastasis. Our goal is to determine the mechanism of interaction between epidermal growth (EGF) produced by macrophages and epidermal growth factor receptor (EGFR) expressed by breast cancer cells. Soluble EGF or macrophage conditioned medium does not stimulate breast cancer cell invasion into collagen matrix and 6 kDa EGF is not detected in conditioned medium or cell lysates. Various growth factors are known to bind the extracellular matrix (ECM) and cells can move by haptotaxis (chemo-attractants are substrate-bound). What is more, breast cancer cells do not invade into collagen matrix when macrophages are not present. Based on the above, we will test the following hypotheses:
1) Breast cancer cells show chemotaxis to freely diffusing EGF in ECM.
2) Breast cancer cells show haptotaxis to ECM bound EGF.
3) Breast cancer cells are stimulated by EGF on the cell surface of macrophages.
We will use UV lithography to prepare 3D microenvironments for cell culture and use confocal fluorescence microscopy to assay cell behavior and examine the ECM of macrophages. If EGF is soluble in 3D matrix, breast cancer cells will show increased motility with increased proximity to macrophages. If EGF binds to ECM, then ECM produced by macrophages will be able to stimulate breast cancer cell motility via stimulation of EGFR. If EGF stays on the surface of macrophages, breast cancer cells will not invade the matrix without direct contact with macrophages.
This interdisciplinary project will establish an experimental system to study interactions between different cell types and will help develop novel diagnostic and therapeutic approaches and devices. The researcher will bring the state-of-the-art knowledge and know-how she has acquired in various international institutions to Europe.

We optimized UV lithography for SU-8, PDMS molding using SU-8 masters and fabrication of collagen and matrigel based 3D Controlled in vitro Microenvironments (CivM) using PDMS molds. We showed that breast cancer cells and macrophages proliferated and migrated in the 3D CivMs we fabricated. In addition, we optimized production of macrophage derived extracellular matrices. We tested our hypotheses with 3D invasion assays in hydrogel and 3D CivMs, 2D adhesion and motility assays on macrophage derived extracellular matrices and control surfaces, and EGF content assays of macrophages, macrophage conditioned medium and macrophage derived extracellular matrices. Our data suggest that breast cancer cells are stimulated by EGF through direct contact with macrophages.

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