*Article* **A Microfluidic Platform Revealing Interactions between Leukocytes and Cancer Cells on Topographic Micropatterns**

**Xin Cui 1,\*,†, Lelin Liu 2,3,†, Jiyu Li 2,†, Yi Liu 2, Ya Liu 4, Dinglong Hu 4, Ruolin Zhang 5,6, Siping Huang 2, Zhongning Jiang 2, Yuchao Wang 2, Yun Qu 2, Stella W. Pang 5,7 and Raymond H. W. Lam 2,7,8,9,\***

	- <sup>4</sup> BGI-Shenzhen, Shenzhen 518083, China

**Abstract:** 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.

**Keywords:** live-cell immunoassay; topographic micropatterns; on-chip cytokine detection; nasopharyngeal cancer; integrated microfluidics
