**1. Introduction**

Nasopharyngeal carcinoma (NPC) is a major health problem for the Southeastern Asian and North African populations [1–3], which can be divided into Epstein-Barr virus

**Citation:** Cui, X.; Liu, L.; Li, J.; Liu, Y.; Liu, Y.; Hu, D.; Zhang, R.; Huang, S.; Jiang, Z.; Wang, Y.; et al. A Microfluidic Platform Revealing Interactions between Leukocytes and Cancer Cells on Topographic Micropatterns. *Biosensors* **2022**, *12*, 963. https://doi.org/10.3390/ bios12110963

Received: 14 October 2022 Accepted: 31 October 2022 Published: 2 November 2022

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(EBV)-positive and negative subgroups. Although current treatments, including radiotherapy, chemotherapy or chemo-radiotherapy, can improve the survival rates, treatment resistance and tumor recurrence remains to date challenging, largely because of the unique and complex intercellular interactions between cancer cells and immune cells, involving various soluble factors released by the tumor microenvironment. The presence of EBVs can alter the biomolecular secretion of NPC cells as well as the immune responses to them. The non-keratinizing Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma represents a unique tumor microenvironment, characterized by dense infiltrating immune cells comprising macrophages [1]. For instance, EBV-infected NPC cells secreted a higher level of IL-1α (1561 pg/mL), IL-1β (16.6 pg/mL) and IL-8 (422.9 pg/mL) as compared to EBV-negative cells [4]. Macrophages secrete cytokines such as TNF (tumor necrosis factor), IL-1, IL-6, IL-8, and IL-12, while the stimulated ones produce more TNF-α, IL-12/IL-23p40, and IL-10 [5,6]. In addition, EBV-infected human monocytes induce the expression of MCP-1 (monocyte chemotactic protein-1) via Toll-like receptor (TLR) 2 [7]; and therefore such MCP-1 further inhibits the IL-12 production by inflammatory macrophages [8]. Clinical studies also highlighted that high serum levels of MCP-1, TNF-α, IL-6 and IL-8 are the most prominent cytokines associated with bone invasion, distant metastasis, and particularly poor outcome in NPC patients [1,7,9]. Hence, precisely investigating and quantifying intercellular interactions and cytokine secretions from immune and cancer cells would help in understanding the NPC developments and identifying potential targets for optimized NPC diagnose and treatments.

The enzyme-linked immunosorbent assay (ELISA) is widely used as a "gold standard" method for cytokine quantifications, which rely on repeated time-consuming sample incubation and washing procedures and cannot be applied for in-situ, real-time and multiplex cytokine profiling. Over the past few decades, microfluidic-based immunoassays have been developed for rapid analysis of cytokine secretion in complex fluidic bio-samples due to the significant advantages of microfluidics in fluid flow controlling and ultra-low reagent consumption [10–13]. For example, a strategy reported by Han et al. is capable of multidimensionally analyzing single cells cytokine secretion frequencies by quantitative micro-engraving [14]. Baraket et al. designed an integrated electrochemical biosensor platform which can perform highly sensitive multi-detection for IL-10 and IL-1β [15]. Choi et al. designed a microfluidic magnetic-beads-based device for protein analysis and bio-molecule detection [16]. Min et al. also developed a microfluidic immunoassay platform for biomolecular quantitative detection, which was based on acridine esterification chemiluminescence [17]. To illustrate the dynamic cytokine profiles of various immune cell subtypes, Junkin et al. developed an automated high-throughput microfluidic chip to rebuild the dynamics of single immune cell [18], which was only supposed to detect the cytokine secretion of single types of immune cells. To achieve the multiple cytokine profiling, our pervious study developed an automated microfluidic microbeads-based device for dynamic immunoassay, which profiled multiple cytokines secretion with a low detection limit and short testing time [19]. However, cells growing in regular culture wells cannot reflect the three-dimensional extracellular biochemical and morphologic environments and more physiological-relevant results [20,21]. Our previous studies showed that nasopharyngeal carcinoma cell migration dynamics and spreading directionality can be regulated by microenvironmental morphology [22], suggesting that the grating-like topography of pterygoid muscles can play a role in nasopharyngeal cancer spreading [23]. Andersson et al. demonstrated the effects of substrate morphology on epithelial cell morphologic behaviors and cytokine secretions [24]. Furthermore, the direct observation of cell behaviors such as migration, together with cytokine measurement, may facilitate the future development of new prognostic tools to reveal cancer-immune cell interactions.

Herein, we report a microfluidic immunoassay device by integrating the cell culture region with microtopographic substrates and our previously reported cytokine dynamics profiling scheme [25,26] by implementing a microbeads-based immunofluorescence assay for achieving more sensitive and parallel detection of multiple cytokines. Co-cultured immune

cells (undifferentiated THP-1 monocytic cells or PMA-differentiated THP-1 macrophages) and immortal cells NP460/NPC43 were cultured in the device, in which cell culture medium was extracted from the cell culture at different time points, transferred and analyzed in the cytokine detection microchambers for quantifying secretions of TNF and IL-12p70 throughout the co-culture period. As IL-12p70 secretion of monocytes can reflect their EBV infection, these results can quantitatively reflect the role of EBV in immune responses upon nasopharyngeal carcinoma.

#### **2. Materials and Methods**
