High-Throughput Secretomic Analysis of Single Cells to Assess Functional Cellular Heterogeneity

Abstract

Secreted proteins dictate a range of cellular functions in human health and disease. Due to the high degree of cellular heterogeneity and, more importantly, polyfunctionality of individual cells, there is an unmet need to simultaneously measure an array of proteins from single cells and to rapidly assay a large number of single cells (more than 1000) in parallel. We describe a simple bioanalytical assay platform consisting of a large array of sub-nanoliter microchambers integrated with high-density antibody barcode microarrays for highly multiplexed protein detection from over a thousand single cells in parallel. This platform has been tested for both cell lines and complex biological samples such as primary cells from patients. We observed distinct heterogeneity among the single cell secretomic signatures that, for the first time, can be directly correlated to the cells’ physical behavior such as migration. Compared to the state-of-the-art protein secretion assay such as ELISpot and emerging microtechnology-enabled assays, our approach offers both high throughput and high multiplicity. It also has a number of clinician-friendly features such as ease of operation, low sample consumption and standardized data analysis, representing a potentially transformative tool for informative monitoring of cellular function and immunity in patients.

Analysis of single-cell cytokine secretion reveals a role for paracrine signaling in coordinating macrophage responses to TLR4 stimulation

“…For example, the production of interferon β (IFN-β) in response to viral infection appears to be stochastic, despite a high incidence of infection (9, 11). Microfluidic and nanowell devices that characterize cells in solitary confinement have enabled quantitative and multiplexed measurements of single-cell secretion of cytokines to be made (12, 13), but such assays may not accurately reflect phenotypes that result from the integration of both autocrine and paracrine signals in cell populations over time (14). However, the extent to which cell isolation (and the resulting loss of paracrine signaling) alters cytokine secretion by a population of cells has not been widely explored.…”

Highly multiplexed profiling of single-cell effector functions reveals deep functional heterogeneity in response to pathogenic ligands

“…Multicolor flow-cytometric analysis with rigorous spectral compensation and mass cytometry extended the multiplexing capacity to 5-11 effector functions (7,10). Recently, a spatial encoding mechanism that circumvents the limitation of spectral overlap was demonstrated for codetection of 15 proteins in single cells using highly miniaturized antibody microarrays placed in nanoliter cell-trapping chambers (11)(12)(13). However, this degree of multiplexing is still insufficient to dissect the full functional spectrum of a diverse range of immune cells.…”

Biosensors for Cell Analysis

“…A simpler cytokine assay kit, which did not require sophisticated fluidics, has been developed by Lu et al (97). The secretion profiles were demonstrated for 14 different proteins, including cytokines, chemokines, and growth factors, from single U87 cells (glioblastoma multiforme), U937 monocytes (leukemic lymphoma), and A549 cells (lung carcinoma).…”