Nested Nanowell Arrays for High-Throughput Quantitative Analysis of Cytokines from Single Macrophages

Macrophages play a critical role in the development of the tumor microenvironment (TME). Recruited macrophages in the TME differentiate into various phenotypes, each with a distinct profile of secreted cytokines. To describe and understand this large heterogeneity, we developed nested nanowell arrays for the multiplexed analysis of secreted proteins at the single cell level. The array consists of more than 100,000 wells on a cyclic olefin copolymer (COC) substrate. Each well contains seven smaller indents for cocapturing functionalized beads and can be operated with standard laboratory equipment such as pipettes and microscopes. The barcoded beads capture cytokines of interest and allow their quantification via sandwich immunoassays. We developed an image analysis tool and quantified 10 proteins secreted from single macrophages and investigated the effects of stimulation and drug treatment. We found that interleukin (IL)-1β, IL-8, and macrophage inflammatory protein 1α (MIP-1α) were highly secreted by more than 43% of the macrophages, with an increase of MIP-1α secretion under treatment with the chemotherapeutic drugs paclitaxel or docetaxel. Pairwise protein analysis confirmed cosecretion of IL-1β and IL-6 in macrophages stimulated with IL-4/IL-13, which were identified as part of a critical pathway in multiple myeloma before. We also demonstrate further multiplexing with three and four cosecreted proteins, together with assessing the cell viability as an additional parameter important for drug testing. In summary, we have shown that our nested nanowell arrays are an easy-to-use analytical tool for basic research, and we believe that it can be employed for diagnostics and personalized medicine, e.g., for investigation of cancer and immune cells from a biopsy or circulating tumor cells obtained from a liquid biopsy.