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- 주제어 : epithelial-to-mesenchymal-like transition (EMT) (검색결과 2 건)
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Lee, Tae Hee Graduate School, Eulji University 2016 국내석사
Radiofrequency ablation (RFA) is widely used for the early stage cancer therapy. However, the residual tumor due to the insufficient heat treatment promotes metastatic potentials, causing recurrence of tumor. Here, we present a cell deformability analysis chip for detecting mesenchymal-like phenotype, which is induced by the insufficient hyperthermia treatment. The current standard laboratory methods to determine whether cells have undergone an epithelial-to-mesenchymal-like transition (EMT), is based on the molecular analysis, such as Western blotting or reverse transcription polymerase chain reaction (RT-PCR). These methods are accompanied by complicated and irreversible preprocesses, such as cell lysis, which makes further examination impossible. Furthermore, large amount of time and equipment is required for the precise analysis. However, deformability of cell can be a label-free marker for demonstrating the changes occurred due to the EMT, since it is known that the epithelial cancer cells are much stiffer than the cells that have undergone an EMT. Thus, simple and rapid microfluidic-based deformability measurement can facilitate phenotype discrimination in cellular level and permits the further analysis, since the method does not require any chemical treatments that give damages to cells. The present chip consists of circular chamber and has five distinct zones with increasing number of constriction channels (10 μm) in radial direction (200, 400, 600, 800, and 1,000 channels for each zone). By applying specific flow rate (1 mL/h) at the center of the chip, pressure profile varies depending on the radial distance, resulting in stiffer cells to be captured at the zones near the inlet and deformable mesenchymal-like cells to move far away from the center. For the experiments, we used the human breast cancer cell line, MCF-7 . The cells were exposed at 42°C for 1 hour in humidified chamber, to mimic the marginal zone of RFA. Under the given condition, 87.6 ± 1.1 % of cells survived, demonstrating that the insufficient RFA can result in residual tumor. To show that the heat treatment enhances the mesenchymal characteristics, the deformability of both heat-treated and untreated cells were measured with the present chip. Remarkable differences were found in both total capture efficiencies (9.6 ± 1.8 % for heat-treated cells versus 19.3 ± 6.5 % for untreated cells) and capture profiles in each zone. In addition, we performed the current standard laboratory molecular analysis, such as RT-PCR, western blot, and immunocytochemistry , to verify the validity of the results obtained from the present chip. All three results showed loss of E-cadherin and upregulation of vimentin, after thermal stress is applied, indicating mesenchymal-like phenotype has been enhanced. As a result, we demonstrated that the present chip was capable of demonstrating EMT; thereby applicable to the epithelial and mesenchymal cell discriminating the epithelial and mesenchymal cell phenotype after thermal stress were induced.
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