Multiplexed analysis of signalling proteins at the single-immune cell level.

High numbers of tumour-associated macrophages (TAMs) in the tumour microenvironment are associated with a poor prognosis. However, the effect of TAMs on tumour progression depends on the proteins secreted by individual TAMs. Here, we developed a microfluidic platform to quantitatively measure the secreted proteins of individual macrophages as well as macrophages polarized by the culture medium derived from breast cancer cells. The macrophages were captured in hydrodynamic traps and isolated with pneumatically activated valves for single-cell analysis. Barcoded and functionalized magnetic beads were captured in specially designed traps to determine the secreted proteins by immunoassay. Individual bead trapping facilitated the recording of the protein concentration since all beads were geometrically constrained in the same focal plane, which is an important requirement for rapid and automated image analysis. By determining three signaling proteins, namely interleuking 10 (IL-10), vascular endothelial growth factor (VEGF), and tumour necrosis factor alpha (TNF-α), we successfully distinguished between differently polarized macrophages. The results indicate a heterogeneous pattern, with M2 macrophages characterized by a higher secretion of IL-10, while M1 macrophages secrete high levels of the inflammatory cytokine TNF-α. The macrophages treated with the supernatant from cancer cells show a similar signalling pattern to M2 macrophages with an increased secretion of the pro-tumoural cytokine VEGF. This microfluidic method resolves correlations in signaling protein expression at the single-cell level. Ultimately, single-macrophage analysis can contribute to the development of novel therapies aimed at reversing M2-like TAMs into M1-like TAMs.