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The extracellular matrix (ECM) serves as a complex scaffold with diverse physical dimensions and surface properties influencing NPC cell migration. Polydimethylsiloxane (PDMS), a widely used biocompatible material, is hydrophobic and undesirable for cell seeding. Thus, the establishment of a biomimetic model with varied topographies and surface properties is essential for effective NPC43 cell separation from NP460 cells. This study explored how ECM surface properties influence NP460 and NPC43 cell behaviors via plasma treatments and chemical modifications to alter the platform surface. In addition to the conventional oxygen/nitrogen (O 2 /N 2 ) plasma treatment, O 2 and argon plasma treatments were utilized to modify the platform surface, which increased the hydrophilicity of the PDMS platforms, resulting in enhanced cell adhesion. (3-aminopropyl)triethoxysilane and fibronectin (FN) were used to coat the PDMS platforms uniformly and selectively. The chemical coatings significantly affected cell motility and spreading, as cells exhibited faster migration, elongated cell shapes, and larger spreading areas on FN-coated surfaces. Furthermore, narrower top layer trenches with 5 µm width and a lower concentration of 10 µg/mL FN were coated selectively on the platforms to limit NP460 cell movements and enhance NPC43 cell separation efficiency. A significantly high separation efficiency of 99.4% was achieved on the two-layer scaffold platform with 20/5 µm wide ridge/trench (R/T) as the top layer and 40/10 µm wide R/T as the bottom layer, coupling with 10 µg/mL FN selectively coated on the sidewalls of the top and bottom layers. This work demonstrated an innovative application of selective FN coating to direct cell behavior, offering a new perspective to probe into the subtleties of NPC cell separation efficiency. Moreover, this cost-effective and compact microsystem sets a new benchmark for separating cancer cells.

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.

Immunoassay for detailed analysis of immune-cancer intercellular interactions can achieve more promising diagnosis and treatment strategies for cancers including nasopharyngeal cancer (NPC). In this study, we report a microfluidic live-cell immunoassay integrated with a microtopographic environment to meet the rising demand for monitoring intercellular interactions in different tumor microenvironments. The developed assay allows: (1) coculture of immune cells and cancer cells on tunable (flat or micrograting) substrates, (2) simultaneous detection of different cytokines in a wide working range of 5-5000 pg/mL, and (3) investigation of migration behaviors of mono- and co-cultured cells on flat/grating platforms for revealing the topography-induced intercellular and cytokine responses. Cytokine monitoring was achieved on-chip by implementing a sensitive and selective microbead-based sandwich assay with an antibody on microbeads, target cytokines, and the matching fluorescent-conjugated detection antibody in an array of active peristaltic mixer-assisted cytokine detection microchambers. Moreover, this immunoassay requires a low sample volume down to 0.5 μL and short assay time (30 min) for on-chip cytokine quantifications. We validated the biocompatibility of the co-culture strategy between immune cells and NPC cells and compared the different immunological states of undifferentiated THP-1 monocytic cells or PMA-differentiated THP-1 macrophages co-culturing with NP460 and NPC43 on topographical and planar substrates, respectively. Hence, the integrated microfluidic platform provides an efficient, broad-range and precise on-chip cytokine detection approach, eliminates the manual sampling procedures and allows on-chip continuous cytokine monitoring without perturbing intercellular microenvironments on different topographical ECM substrates, which has the potential of providing clinical significance in early immune diagnosis, personalized immunotherapy, and precision medicine.

One of the greatest unmet needs hindering the successful treatment of nasopharyngeal carcinomas (NPCs) is for representative physiological and cost-effective models. Although Epstein-Barr virus (EBV) infection is consistently present in NPCs, most studies have focused on EBV-negative NPCs. For the first time, we established and analyzed three-dimensional (3D) spheroid models of EBV-positive and EBV-negative NPC cells and compared these to classical two-dimensional (2D) cultures in various aspects of tumor phenotype and drug responses. Compared to 2D monolayers, the 3D spheroids showed significant increases in migration capacity, stemness characteristics, hypoxia and drug resistance. Co-culture with endothelial cells, which mimics essential interactions in the tumor microenvironment, effectively enhanced spheroid dissemination. Furthermore, RNA sequencing revealed significant changes at the transcriptional level in 3D spheroids compared to expression in 2D monolayers. In particular, we identified known (VEGF, AKT and mTOR) and novel (Wnt-β-catenin and Eph-ephrin) cell signaling pathways that are activated in NPC spheroids. Targeting these pathways in 3D spheroids using FDA-approved drugs was effective in monoculture and co-culture. These findings provide the first demonstration of the establishment of EBV-positive and EBV-negative NPC 3D spheroids with features that resemble advanced and metastatic NPCs. Furthermore, we show that NPC spheroids have potential use in identifying new drug targets., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Cell spreading and migration play a pivotal role in many diseases such as tumor metastasis. In particular, nasopharyngeal tumor cells have known of their tendency of migration to pterygoid muscles and further distant metastasis. Although existing studies revealed key characteristics of the nasopharyngeal tumor cells, their migration preference is yet to be thoroughly understood, especially in the physical aspects including the microtopographical factors. Researchers have developed techniques in recent years to study microtopography-related cell behaviors but they are not yet applied in investigating the nasopharyngeal tumor cells. In this work, we elaborate the spreading and migration characteristics of normal and cancerous nasopharyngeal cells on micrograting substrates mimicking the microtopography of myotubes of the pterygoid muscles. We further apply interference reflection microscopy (IRM) to visualize the cell-substrate adhesion dynamics. We are interested in examining the microtopography-related cell spreading and migration behaviors and their correlations, providing insights for deeper understanding and more promising prediction on the nasopharyngeal tumor metastasis.

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.)

We analyzed the relation of histological typing in late stage nasopharyngeal carcinoma (NPC) with clinical outcome and excision repair cross complementation group 1 protein (ERCC1) expression. The biopsy specimens of 259 patients with NPC were reviewed by two pathologists for classification according to 2005 WHO subtypes. The patients were of stage III to IVB and treated with radiotherapy (RT) alone or concurrent-adjuvant chemoradiotherapy (CRT). Expression of ERCC1 protein detected by immunohistochemistry on paraffin sections was correlated with the histological subtypes. There were 10 cases (3.9%) of differentiated non-keratinizing carcinoma compared with 249 cases of conventional undifferentiated carcinoma. The former exhibited more advanced squamous differentiation with 3 cases belonging to the papillary variant. The degree of ERCC1 expression was generally high compared with the median of the cohort. Clinically, the differentiated group fared poorly compared with the undifferentiated group with respect to loco-regional failure-free rate, distant failure-free rate, disease-free survival and overall survival (p≤0.05). Treatment modality of the 10 patients (5 RT, 5 CRT) was similar to the whole cohort. Contrary to general acceptance that differentiation of non-keratinizing NPC had little bearing on prognosis, we demonstrated that in endemic area differentiation in fact conferred a worse prognosis in stage III to IVB patients. There was positive correlation of differentiation with ERCC1 expression. We advocate precise histological typing of NPC in pathology report for prognostic purpose and outcome correlation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Aims: To study the morphology and immunohistochemical expression of nasopharyngeal intraepithelial lesions and to understand their place in nasopharyngeal carcinogenesis., Methods and Results: Nine cases of nasopharyngeal intraepithelial lesion (NPIL) were diagnosed during nasopharyngeal biopsy screening for nasopharyngeal carcinoma (NPC). Two cases were associated with early invasion. All cases demonstrated specific histological features and consistent positivity on in-situ hybridization for Epstein-Barr virus (EBV)-encoded RNA. Pure NPIL lesions showed low-grade morphology while lesions associated with early invasion were high grade. Immunohistochemical studies showed increased expression of bcl-2 and essentially negative findings for BZLF1 and LMP1. High-grade lesions had relatively stronger expression of bcl-2 and p53., Conclusions: NPIL harbours latent EBV infection and has malignant potential. Multiple steps are involved in its occurrence and progression. Low-grade and high-grade lesions should be managed differently.

Background: The association of Epstein-Barr virus (EBV) with the oncogenesis of nasopharyngeal carcinoma (NPC) is well established. Latent infection by EBV with clonal proliferation has also been demonstrated in preinvasive lesions of NPC. In situ hybridization for EBV-encoded RNA (ISH EBER) now serves as an ancillary test in the definitive diagnosis of these lesions., Methods: Two cases of nasopharyngeal carcinoma in situ (NPCIS) are presented in this study. Their biopsies were studied by ordinary light microscopy, the ISH EBER technique, and immunostaining for bcl-2. Tissue samples from 100 high risk subjects negative for NPC and NPCIS, who served as controls, were also studied using the ISH EBER technique., Results: NPCIS was characterized by abnormal light microscopic appearance as well as positive staining by the ISH EBER technique; these features were not observed in samples from the 100 high risk subjects. Immunostaining for bcl-2 protein was positive but less specific. Postradiotherapy biopsies of the two patients were negative for NPCIS., Conclusions: With the help of the ISH EBER technique, the diagnosis of NPCIS is now possible in routine surgical pathology. As this entity is rare, it is necessary to have a high degree of suspicion when evaluating biopsies from high risk individuals. Radiotherapy for patients with NPCIS is justified in view of the risk of cancer progression and the possibility of a coexisting invasive carcinoma.