Abstract 4319: Gene expression profiling of isolated small migratory and invasive breast cancer cells at different times during chemotaxis
Authors: Limame Ridha , Laere Steven Van , Beeck Ken Op De , Camp Guy Van , Vermeulen Peter , Dirix Luc , Peeters Marc , Pauwels Patrick , Wever Olivier De , Lardon Filip 
Published: August 2012

Abstract
Background Chemotactic cell motility and tissue invasion are key processes initiating cancer cell dissemination and metastasis. Adequate molecular profiling of chemotaxis-driven tumor cells is impeded by the transient and stochastic nature of motility occurring in only a small subset of tumor cells. To unveil molecular pathways implicated in breast cancer cell invasion and migration, an in vitro set-up was designed to isolate total RNA for microarray hybridisation from sequestered migratory and invasive breast cancer cell subpopulations at two predefined incubation stages. These target populations were compared to non-migratory and non-invasive cells to obtain a molecular picture of migratory and invasive breast cancer cell behavior. Methods and results A 24-well Transwell set-up was used to perform both migration and Matrigel invasion experiments on MDA-MB-231 breast cancer cells after 24h serum starvation (4x105 cells/insert). A Matrigel coating (20%/15μL) was applied to the bottom-side of insert membranes in both settings. For invasion experiments, a layer of Matrigel (20%/20μL) was added to the top side of the membranes. Initially, an “early” and a “late” timepoint for RNA-extraction have been selected by monitoring migration and invasion using the xCELLigence system (Roche Applied Science). High-quality total RNA was isolated at these predefined timepoints from both migratory / invasive and reference cells and hybridized onto Illumina HumanHT-12 v4 BeadChips in biological triplicates. Differential gene expression analysis and pathway analysis were done using BioConductor in R and Ingenuity Pathway Analysis respectively, considering only FDR-corrected p-values &lt; 0.1 as significant. First, a time-independent comparison was performed between all grouped migratory and invasive samples, revealing a total of 988 differentially expressed genes, of which the majority occurring in the late phase. Further analysis focused on early expression changes as these may be causative to more extended late events. Pathway analysis of differential genes associated with early migratory and early invasive cells in comparison with their reference counterparts identified NF-κB-related (p=0.0034) and TGFβ-related (p=0.04) expression to be closely associated with migration and invasion respectively. Conclusions This work has aimed to determine specific gene expression characteristics of solely migrating and invading breast cancer cells. Preliminary findings suggest strong involvement of NF-κB-based pathways early in migration, but not in invasion. On the other hand, TGFβ-signaling changes were more closely associated with early invasive behaviour. Moreover, a global comparison identified significant expression differences between both states of cell motility.
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 4319. doi:1538-7445.AM2012-4319