Advanced Cell Mapping Visualizations for Single Cell Functional Proteomics Enabling Patient Stratification

Highly-multiplexed single-cell functional proteomics has emerged as one of the next generation toolkits for a deeper understanding of functional heterogeneity in cellular populations. Different from the conventional cell population-based bulk and single-cell proteomic and RNA-Seq assays, the microchip-based proteomic measurement at the single-cell resolution enables a unique identification of highly polyfunctional cell subsets that co-secrete many proteins from live single cells and most importantly correlate with patient response to a therapy. For example, the 32-plex IsoCode chip technology has defined a polyfunctional strength index (PSI) of pre-infusion anti-CD19 chimeric antigen receptor (CAR)-T cell products, that is significantly associated with patient response to the CAR-T cell therapy. To complement the clinical relevance of the PSI, we have also developed a comprehensive visualization toolkit by integrating 3D Uniform Manifold Approximation and Projection (UMAP) and t-distributed stochastic neighbor embedding (t-SNE) visualizations into a proteomic analysis pipeline, which provides more advanced analytical algorithms for more intuitive data visualization and interpretation. The functional UMAP and t-SNE visualization of pre-infusion anti-CD19 CAR-T cell products reveals distinct cytokine secretion profiles from groups of specific cells, between patients who either responded or did not respond to anti-CD19 CAR-T cell therapy and demonstrate a marked upregulation of antitumor-associated cytokine signatures in both CD4+ and CD8+ CAR-T cells from responding patients. Using this powerful while user-friendly analytical tool, we can dissect the multi-dimensional single-cell data from complex immune response to immunotherapies and uncover critical underlying mechanisms, which can promote enhanced correlative biomarker discovery, improved bioprocessing, and further personalized treatment development. This article is protected by copyright. All rights reserved.