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Profiling Immune Cell Populations in the Tumor Microenvironment with Complementary Capillary-Based and Single-Cell Western Assays

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The tumor micro-environment (TME) is a complex mixture of cancerous and non-cancerous cells, including immune cells like T-cells, macrophages, and neutrophils. The TME plays a key role in tumorigenesis and metastasis, and it has recently been recognized that it can dramatically shape a response to therapy. Thus, there is a pressing need to accurately identify and quantify the variety of cell types in any given TME. However, studying the TME presents major challenges. For example, the heterogeneity of the environment requires sensitive and high-resolution techniques to parse subpopulations of different cell types. This challenge is compounded by the severely limited sample size that can be obtained from donor tissues. To address these challenges, we use an in-capillary immunoassay with small sample sizes (3 µL) to identify immune cells commonly found in the TME. We also leverage single-cell Western to uncover trends in population heterogeneity. Human peripheral blood mononuclear cells (PBMCs) were differentiated into dendritic cells (DCs) and regulatory T cells (Tregs), and natural killer (NK) cells were expanded from isolated NK cells. These samples were then analyzed by in-capillary immunoassay and single-cell Western. These analyses revealed the identification and characterization of cell types, at both the single-cell and population level, based on the differential expression of protein biomarkers. Specifically, in-capillary immunoassay analysis identified mature populations by CD209 for DCs, a CD56+/CD3- phenotype for NK cells, and CD25 and Foxp3 expression for Tregs. Analysis of single cells provided further detail within these populations, for example, we observed FoxP3low and FoxP3high subpopulations in Tregs, and an unexpectedly large (81%) CD56-/CD3- subpopulation in undifferentiated PBMCs, suggesting the presence of other cell subtypes. We anticipate that the small sample size, automation, single-cell resolution, and multiplexing ability of these assays collectively will enable a more efficient and deeper characterization of the TME not possible with traditional immunoassays like Western blot and flow cytometry.

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