Abstract 1609: Simple and rapid high-plex analysis of RNA and protein from low-frequency sorted T cells
Authors: Fuhrman Kit , Birditt Brian , Ong SuFey , Rosenbloom Alyssa , Hinderfeld Douglas , Geiss Gary , Tam Miguel , Bailey Christina 
Published: August 2018

Abstract
The immune system is increasingly becoming a direct target for cancer therapeutics. These cancer immunotherapy efforts require deep biologic characterization to better understand and characterize rare or low-frequency immune cells, necessitating next-generation methods to expand or complement current flow cytometry or similar methods. Recent advances in flow and mass cytometry have resulted in an improved understanding of immune system heterogeneity. These technologies, however, remain limited in the number of parameters and types of analytes that can be examined in a single sample. To obtain high-plex multi-omic data from rare cells, we utilized a novel workflow, integrating fluorescence-activated cell sorting (FACS) with the NanoString nCounter® Vantage 3D™ RNA:Protein Immune Cell Profiling Assay to deeply characterize multiple flow-sorted immune cell populations. This enabled us to simultaneously interrogate 30 cell surface proteins and 770 immune-related mRNA starting from a heterogenous cell suspension. We employed antibody-conjugated panels from BioLegend that were designed to minimize interference between fluorescently tagged and NanoString oligo-tagged antibodies. To streamline this workflow, cells were co-stained with both BioLegend and NanoString antibodies in a single 30+ antibody stain. Multiple T-cell populations were sorted and profiled from PBMC, including memory and naïve Tregs and CD8 T cells with and without stimulation, allowing for analysis of differentially regulated genes and proteins across samples. Co-stain with both fluorescently and oligo-tagged antibodies did not alter the phenotypic profiles of immune cells, validating the design of the compatible panels used to sort Tregs and CD8 T cells. Demonstrating the value of this workflow in analyzing rare populations, cells were titrated to determine the sensitivity of the workflow, producing concordant protein data from only 500 cells. High-plex RNA data were obtained from 5,000 cells without the requirement for additional molecular biology methods, such as amplification or sequencing library construction, reducing potential for technical biases. Data analysis and visualization was accomplished with nSolver™ and Cytobank software packages to rapidly analyze this high-plex data. Our analysis highlighted the population diversity and activation of key pathways between these different T-cell subtypes. Focused analysis revealed discordance between mRNA and protein levels in key immuno-oncology target genes, including PD-L1. These experiments illustrate the "3D Flow™ Analysis" method as ideally suited for incorporation into cell-sorting workflows, simultaneously producing high-plex, multi-omic data from multiple rare immune cell populations.
Citation Format: Kit Fuhrman, Brian Birditt, SuFey Ong, Alyssa Rosenbloom, Douglas Hinderfeld, Gary Geiss, Miguel Tam, Christina Bailey. Simple and rapid high-plex analysis of RNA and protein from low-frequency sorted T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1609.