Abstract 1255: A novel chip-based parallel transfection assay to evaluate paracrine cell interactions
Authors: Michael Sturzl, Elisabeth Naschberger, Andreas Konrad, Roland S. Croner, Nathalie Britzen-Laurent, Helmut Munstedt, Elisabeth Kuhn
Journal: Cancer Research (2012)

Abstract Introduction: The analysis of how genes are regulating interactions between different cell types is of highest importance for the understanding of physiologic and pathologic processes in multi-cellular organisms. We present a novel method for the highly parallel analysis of gene functions in paracrine interactions between different cell types at the microarray level on a chip. The basic idea was to apply a mixture of two different cell types to the technique of reverse transfection with expression plasmids spotted in an ordered array on chips. The goal was to establish conditions that exclusively result in the transfection of one cell type (effector cells) and to use the other cell type as the indicator cells to determine paracrine gene effects (either directly exerted by the secreted product of the transfected gene or induced by the transfected gene). Methods: Reverse transfection was carried out spotting plasmid DNA in Lipofectamine 2000 and 0.2% gelatin on a slide. Subsequently, HEK 293T cells (effector cells) alone or in a mixture with different indicator cells (human fibroblasts, HUVEC, HaCaT, HT29, WiDr) were seeded. After 5 h a pre-warmed diffusion restricting matrix was gently poured onto the slides. The slides were further incubated for 43 h at 37 °C. Then, the matrix was removed, and the slides were washed gently and subjected to immunofluorescence analysis. Results: We successfully solved the two key problems required for this approach: (1) exclusive, differential transfection of the effector cells and not the indicator cells and (2) restriction of diffusion to localize gene effects to the specific transfection area. With the established methodology presented in our study, 192 parallel tests of paracrine gene activations can be conducted on a single chip. Demonstrating the broad applicability of the method, we used different indicator cells such as human primary fibroblasts (of relevance for inflammatory tissue reconstruction and invasion), endothelial cells (of relevance for angiogenesis research), keratinocytes (of relevance for wound healing research) and two different colorectal cancer cell lines (of relevance for cancer research). In addition, we used different indicator genes such as GBP-1 (indicates IFN-gamma like activation), COX-2 (of relevance for tumor research), ICAM-1 and VCAM-1 (of relevance for angiogenesis and inflammation research) and different paracrine inducers by including IFN-γ, TNF-α and IFN-γ. Discussion: Our method provides a significant improvement over current methodologies for the analysis of paracrine gene functions. It is highly relevant to systematically investigate single and combination effects of disease-associated gene clusters in intercellular communication in many different research applications including cancer, inflammation, angiogenesis and infectious diseases research. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1255. doi:1538-7445.AM2012-1255