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
High-throughput screening (HTS) is a well-established approach for tumor-specific drug development because of its high efficiency and customizable selection of antineoplastic drugs. However, there is still a lack of an appropriate cell-based HTS specific for migratory cancer cells. In the study presented here, we created a novel assay (mHTS): a single-cell-level screening method targeting migratory cancer cells and can be applied in a high-throughput manner. This mHTS platform is based on microchannel devices (providing physical confinement during cell migration and limit migrating cells’ proliferation rate) assembled 96-well plate (fitting to HTS manner). To determine the feasibility of this assay, we quantified the anti-migratory and anti-viability effects of several molecules (Cytochalasin D, Doxorubicin and AZD-6244) on migrating (creeping inside microchannel) glioblastoma multiforme (GBM) cells. After analyzing migration screening data that was collected on a single-cell-level, we were able to compare those drug’s effects on cancer cells’ migration velocity and uncovered the migration inhibiting potential of AZD (500 nM and 1000 nM). Viability data based on single-cell-level screening also allowed us to further understand the same drug’s different lethality toward migrating and normal 2D cultured cancer cells. The Pre-classification of subpopulations enables us to study the heterogeneity of cancer and ensures our method’s feasibility for a high-throughput manner. All these results proved our mHTS platform is suitable for single-cell-level anti-migration drug screening and has potential feasibility in promoting the development of anti-migratory-cancer-drug in a high-throughput manner.
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