Abstract 2768: Highly parallel integrated functional and sequencing analysis of single cells using the Beacon® optofluidics platform

Abstract Single-cell RNA sequencing has emerged as a powerful tool for uncovering cellular heterogeneity and its connections to disease progression and treatment efficacy, including immunotherapy. However, many critical phenotypes cannot be deduced solely from gene expression changes. To gain deeper insights, functional readouts of these cells are essential, yet current methods are limited by low throughput and restricted assay capacity. To tackle this challenge, we developed a high-throughput workflow that integrates functional and sequencing analyses at the single-cell level. This workflow combines versatile phenotypic assays with highly parallel single-cell RNA sequencing, leveraging the advanced capabilities of the Beacon® optofluidics platform from Bruker Cellular Analysis. Using this platform, we demonstrated the ability to link cell surface marker staining with gene expression profiling using single cells derived from PBMC samples. We then utilized this platform to analyze the functional and transcriptomic heterogeneity within adoptive cell therapy products. Specifically, we quantified multiple secreted cytokines (IFNγ, TNFα, and IL-2) alongside gene expression in thousands of in vitro-expanded T cells expressing antigen-specific T cell receptors. These non-growth-based, single live-cell functional assays were performed with minimal crosstalk, followed by cDNA capture and synthesis using barcoded beads in a highly parallel manner on the Beacon platform. Single-cell RNA sequencing data were subsequently integrated with functional assay results to uncover the molecular mechanisms underlying the functional heterogeneity of cell therapy products. We discovered that polyfunctional cells—those secreting all three cytokines—exhibit upregulated expression of genes associated with metabolic reprogramming, activation regulation, and DNA replication, hallmarks of T cell activation. Additionally, increased expression of chemokine genes in polyfunctional cells suggests ongoing immune cell recruitment beyond cytokine secretion. Validating these findings could pave the way for strategies to develop enhanced cell therapy products. Beyond uncovering the molecular drivers of functional heterogeneity in single cells, we envision this approach being further applied to advance single-cell functional genomics through integration with high-content CRISPR screening. The Beacon optofluidics platform enables direct functional profiling of gene function and regulation at single-cell resolution, making it uniquely suited to tackle previously intractable questions in cancer research and therapeutic development. Citation Format: Leqian Liu, David Coit, Or Gadish, Julie Perreau, Andrei Rajkovic, Brandon Bruhn, Minha Park, Edward Han, Alireza Salmanzadeh, Vikram Devgan. Highly parallel integrated functional and sequencing analysis of single cells using the Beacon® optofluidics platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2768.