Tissue invasion and metastasis are leading causes of death among cancer patients due to cells escaping from the primary tumor and invading distant sites. To better understand these phenomena and develop efficient therapeutic regimens against different types of malignancies, there is a need for exclusive cellular and molecular examination of migrating cells. In this study, aggressive brain cancer cells, G55, migrating through confined microchannels were directly extracted and used for subsequent proteomic analysis via Western Blot and/or immunostaining quantification. The method was based on an engineered Polydimethylsiloxane microchannel platform that facilitated the exclusive extraction of migrating cells and their contents while preventing non-migrating (or proliferatingdenoted as 2D) cell contamination. The migrating cells in physical confinement of the microchannels were exclusively examined for their protein expression. They were found with increased expression of Vimentin, approximately 2.5-fold higher than 2D cells. On the other hand, the migrating cells showed significantly decreased β3-Tubulin and Met signal compared to 2D cells. The differences in biomarker expression between migrating cells and non-migrating cells revealed by this study provided an insight into key features of cancer invasion and metastasis. The successful outcome of this research suggests improved targets for ceasing different types of malignancies. REVISED
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