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Nasopharyngeal carcinoma (NPC) is a highly invasive and metastatic disease occurring in the nasopharynx. For early diagnosis or treatment, it is helpful to separate invasive nasopharyngeal carcinoma (NPC43) cells from epithelial (NP460) cells. However, the lack of proper in vivo models significantly hinders the systematic study of NPC. In this study, an in vitro two-layer scaffold platform was developed to mimic the extracellular matrix of the tissue that allows the investigation of carcinoma and epithelial cell migration behaviors. Three precisely controlled factors were designed to investigate how topographic cues affected migration of NP460 and NPC43 cells: overlay angles between the top and bottom layers, top layer thicknesses, and surface topography of the bottom layer ridges. These designs offered different surface contact areas for cells to probe and form focal adhesions as they migrated on the platforms. As NP460 and NPC43 cells respond differently to the surrounding microenvironments, when the top layers were perpendicular to the bottom layers, 66.3% NPC43 cells could move into the $10~\mu \text{m}$ wide trenches compared to 5.5% for NP460 cells. However, NP460 and NPC43 cells migrating into top layer trenches were hindered when the top layer was too shallow or too thick. Moreover, the gratings added on the bottom layer ridges promoted cells to squeeze into the trenches. These results yielded over 92.3% separation efficiency on platforms with a 15- $\mu \text{m}$ thick top layer being perpendicular to the bottom layer and gratings on the bottom layer ridges.

A polydimethylsiloxane two-layer scaffold platform was designed to provide a three-dimensional biomimetic microsystem that allows the detection of epithelial-to-mesenchymal transition without the use of specific biomarkers. As a proof of concept, a novel microsystem that consisted of two layers of 15 μm thick grating structures was developed. These layers had gratings with 40 μm wide ridges and 10 μm wide trenches, and they were stacked together to form a scaffold platform. To investigate the feasibility of using the engineered platforms for detecting changes in epithelial-to-mesenchymal transition, transforming growth factor beta-1 was added to an untransformed nasopharyngeal epithelial cell line. On flat polydimethylsiloxane surfaces, transforming growth factor beta-1 did not significantly affect nasopharyngeal epithelial size, migration speed, or directionality. However, the effect of transforming growth factor beta-1 treatment on migration speed of nasopharyngeal epithelial cells cultured on the two-layer scaffold platform was significantly different. Furthermore, while almost no untreated nasopharyngeal epithelial cells could squeeze into the 10 μm wide trenches, 21% of the transforming growth factor beta-1 treated nasopharyngeal epithelial cells exhibited traversing behaviors on the two-layer scaffold platforms. Moreover, fibronectin coating on the trenches and bottom layers of the scaffold platforms further enhanced the transforming growth factor beta-1-induced traversing of nasopharyngeal epithelial cells into the narrow trenches. These results demonstrate that the engineered two-layer scaffold microsystem can be used to monitor epithelial-tomesenchymal transition induced changes in cell migration and invasiveness, paving the way of using these platforms in high throughput drug screening.