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Molecular & Cellular Analysis Technologies
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The tumor microenvironment, and in particular tumor associated inflammation, is a driving force of tumor progression. Immune cell (i.e. leukocyte) infiltration into sites of inflammation requires the coordinate regulation of multiple steps including arrest on endothelium, migration through the endothelial barrier and directed migration through connective tissue. A potential key regulator of leukocyte infiltration is a member of the matrix metalloproteinase family, MMP3. Mice which are null for MMP3 demonstrate reduced infiltration of leukocytes in a variety of model systems, and a number of matrix and non-matrix MMP substrates identified include those known to affect immune cell function. We hypothesize that MMP3, as well as other MMPs, directly enhances leukocyte extravasation during tumor progression Direct analysis of the roles of MMPs is possible through analysis of primary cells isolated from mice with genetic ablation of individual family members. However, current technologies to assay leukocyte extravasation either do not recapitulate key physiological parameters such as the microfluidic shear and apical-basolateral organization of endothelium, or they require extensive tissue samples that excludes their use with primary cells isolated from mice. The goal of this proposal is to exploit the expertise of our collaborative team of Cancer Biologists and Engineers in applying soft-lithography microfabrication technology to the biological challenge of the study of tumor- associated leukocyte infiltration ex vivo. We propose a multidisciplinary approach in the development of planar and multilayer Parallel Capillary Perfused Bioreactors (PCPB) that 1) better approximate the spatial constraints and architecture of blood vasculature, 2) can provide regulated shear flow and 3) are high- throughput in design requiring minimal cell samples for assay conditions. The development of the planar and multi-layer PCPBs for application in studying leukocyte transendothelial migration are detailed below. Completion of these aims will generate novel devices that will provide an ex vivo system that more closely approximates physiological vasculature facilitating novel insights into leukocyte recruitment from circulation. Specific Aim 1: To develop a planar parallel capillary perfused bioreactor (PCPB) A) Design and fabricate planar PCPB with a recirculating nutrient supply system B) Apply the planar PCPB to the assay murine derived leukocyte attachment to endothelial monolayers. Specific Aim 2. To develop a multilayer parallel capillary perfused bioreactor (PCPB) that will support endothelial polarization into apical and basolateral surfaces A) Design and fabricate a multilayer PCPB that will incorporate a filter system; B) establish culture conditions that allow for endothelial polarization across filter of multilayer PCPB and C) define parameters for leukocyte attachment, rolling and transendothelial migration in multilayer PCPB as compared to traditional TEM assay.