High‐Throughput 3D Matrigel‐based Droplet Microfluidics for Single‐Cell Function‐to‐Omics Analysis of Cytotoxic Immune Cells in Solid Tumor Interactions

ABSTRACT Droplet microfluidics technology provides valuable insights into single‐cell function and interactions through the high‐throughput encapsulation of individual cancer and immune cells to assess cellular heterogeneity. However, these systems fall short when studying solid tumors due to the lack of an extracellular matrix (ECM) that facilitates tumor‐immune cell dynamic interactions. To address these challenges, we have developed an innovative 3D droplet microfluidics platform that incorporates Matrigel, a natural ECM component, for high‐throughput formation and real‐time analysis of tumor‐immune cell interactions and tumor cell death. To achieve a function‐to‐omics approach, our platform integrates Functional Fluorescence‐Activated Droplet Sorting (F‐FADS) to sort 3D Matrigel droplets and isolate immune cell subpopulations based on functional activity. This method enables the selection of highly cytotoxic immune cells by tracking fluorescence‐based functional markers, such as live‐dead staining of tumor targets. By linking functional performance to molecular characterization, F‐FADS allows for high‐quality RNA extraction from sorted immune cells, facilitating downstream omics analyses, such as RNA sequencing. We demonstrated, using a solid tumor cell line for metastatic prostate cancer (PC3 cells), that a 3D Matrigel‐based system enhances PC3 viability and Natural Killer (NK) cell mobility, effectively mimicking the solid tumor ECM for studying tumor‐immune interactions. Next, the droplet sorting based on immune cell functionality was achieved with the 3D Matrigel droplets co‐encapsulated with tumor cells. Following sorting, high‐quality RNA extraction enabled function‐to‐omics analysis, including transcriptional profiling of the sorted immune cells. RNA sequencing revealed distinct gene expression patterns in killer NK cells, including upregulation of genes involved in cytotoxicity, chemotaxis, activation, and metabolism. These results confirm the platform's ability to link immune cell functionality to molecular signatures, providing valuable insights into NK cell heterogeneity and advancing our understanding of immune and cancer cell interactions.