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The three-dimensional (3D) structure of the extracellular matrix and cell-cell contacts are two important cues to altering cell migration behavior and the tumor formation process. In this work, we designed and fabricated microwell arrays with a grating-patterned bottom in polydimethylsiloxane platforms to systematically study the effects of confinement, changes in topography, and cell-cell contacts on the migration behavior of nasopharyngeal carcinoma (NPC43) and immortalized nasopharyngeal epithelial (NP460) cells by time-lapse imaging. When two types of cells were co-cultured in microwells, the migration speed and spreading area of NPC43 cells were significantly increased, which might be attributed to the heterotypic cell-cell contacts with NP460 cells. On a flat surface, NPC43 cells could not form clusters due to the frequent interruptions by the active movements of NP460 cells. However, in 3D microwell arrays, clusters of NPC43 cells formed on the bottom surface while the majority of NP460 cells migrated onto the sidewalls. These cell clusters could be further processed to form spheroids for drug screening. These results also revealed that the 3D microenvironments and cell-cell contacts could have significant implications for NPC cell migration and initiation of tumor formation, which will provide insight for NPC progression and dissemination.

Scaffold microstructures were developed to mimic a three-dimensional extracellular matrix in studying cell migration and invasion. The multiple-layer scaffold platforms were designed to investigate cell migration and separation from top to bottom layer. Two cell lines including immortalized nasopharyngeal epithelial (NP460) cells and nasopharyngeal carcinoma (NPC43) cells with Epstein-Barr virus were compared in this study. On one-layer platforms with trench depth of 15 µm, both NP460 and NPC43 cells were guided to migrate along the 18-µm-wide trenches, and exhibited random migration directions when the trench width was 10 or 50 µm. Nearly no cell was found to migrate in the 10-µm-wide trenches on one-layer platforms. However, the NP460 and NPC43 cells showed very different probability in the narrow trenches on two-layer platforms, making it possible to separate the nasopharyngeal epithelial cells from the carcinoma cells. Moreover, 1-µm deep grating topography on the top layer inhibited NP460 cells to migrate from top ridges to the 10-µm-wide trenches, but promoted such behavior for NPC43 cells. The results demonstrated in This study suggest that the engineered multiple-layer scaffold platforms could be used to separate carcinoma cells in NPC tumor as a potential treatment of NPC., (© 2020 Wiley Periodicals LLC.)

In the present study, 3D biomimetic platforms were fabricated with guiding grating to mimic extracellular matrix topography, porous membrane to resemble the epithelial porous interface and trenches below to represent blood vessels as an in vitro tissue microenvironment. Fabrication technologies were developed to integrate the transparent biocompatible polydimethylsiloxane platforms with preciously controlled dimensions. Cell migration behaviors of an immortalized nasopharyngeal epithelial cell line (NP460) and a nasopharyngeal carcinoma cell line (NPC43) were studied on the 2D and 3D platforms. The NP460 and NPC43 cells traversing through the porous membrane and migrating in the trenches below were studied by time-lapse imaging. Before traversing through the pores, NP460 and NPC43 cells migrated around the pores but NPC43 cells had a lower migration speed with less lamellipodia spreading. After traversing to trenches below, NPC43 cells moved faster with an alternated elongated morphology (mesenchymal migration mode) and round morphology (amoeboid migration mode) compared with only mesenchymal migration mode for NP460 cells. The cell traversing probability through porous membrane on platforms with 30 μm wide trenches below was found to be the highest when the guiding grating was perpendicular to the trenches below and the lowest when the guiding grating was parallel to the trenches below. The present study shows important information on cell migration in complex 3D microenvironment with various dimensions and could provide insight for pathology and treatment of nasopharyngeal carcinoma., (© 2020 The Author(s).)

Invadosomes are invasive protrusions generated by cells which can secrete matrix metalloproteinases for focal digestion of extracellular matrix. They also aid invasive cancer cells in their transmigration through vascular endothelium. However, how the physical and chemical cues in a three-dimensional (3D) system signal the spatial localization of invadosomes remains largely unknown. Here we study the topographic guidance of invadosome formation in invasive nasopharyngeal cells under the stimulation of an inflammatory cytokine, TGF-β1, using engineered gratings with different width and depth. We first report that TGF-β1 can act as an external signal to upregulate the formation of invadosomes with a random distribution on a plane 2D surface. When the cells were seeded on parallel 3D gratings of 5 µm width and 1 µm depth, most of the invadosomes aligned to the edges of the gratings, indicating a topographic cue to the control of invadosome localization. While the number of invadosomes per cell were not upregulated when the cells were seeded on 3D topography, guidance of invadosomes localization to edges is correlated with cell migration directionality on 1 µm deep gratings. Invadosomes preferentially form at edges when the cells move at a lower speed and are guided along narrow gratings. The invadosomes forming at 3D edges also have a longer half-life than those forming on a plane surface. These data suggest that there are integrated biochemical and 3D geometric cues underlying the spatial regulation of invasive structures so as to elicit efficient invasion or metastasis of cells. STATEMENT OF SIGNIFICANCE: Nasopharyngeal cells were integrated with the biological cues and matrix topography to govern the activity and spatial distribution of invadosomes. The biochemical induction of invadosome formation by TGF-β1 in nasopharyngeal cells was observed. When the cells were seeded on parallel 3D gratings, most of the invadosomes aligned to the edges of the gratings due to topographical induced invadosome localization. While the number of invadosomes per cell were not upregulated, guidance of invadosomes localization to edges is correlated with cell migration directionality on 1 µm deep gratings. Invadosomes preferentially form at edges with a higher stability when the cells are guided along narrow gratings. The integrated biochemical and 3D geometric cues could elicit efficient invasion or metastasis of cells., (Copyright © 2019. Published by Elsevier Ltd.)