Abstract 7122: Accelerating immuno-oncology research with flexible workflows using modular flex-fit CyTOF panels
Authors: Shakir Hasan, Ling Wang, Stephen Li, Christina Loh
Journal: Cancer Research (2025)

Abstract Deep functional profiling of immuno-oncology markers and cytokines in whole blood (WB) reveals immune signatures that inform disease prognoses, immunotherapy selection, and outcomes in cancer patients. WB is best analyzed within 24 hours of collection to capture clinically relevant immune signatures; however, WB collection and cytometric analysis are often performed at different sites, which can lead to significant delays. WB stabilizers can overcome this challenge, yet not all antibody panels are compatible with these reagents. To enable flexible experimental workflows for deep profiling of immuno-oncology markers and cytokines in WB, we developed modular Flex-Fit CyTOF™ panels compatible with stabilizers. High-parameter flow cytometry mediated by CyTOF technology enables single-tube staining to identify 50-plus markers at the single-cell level. CyTOF technology provides exceptional resolution without spectral unmixing or compensation, while Flex-Fit CyTOF panels allow simple and quick panel design by combining a customizable selection of pre-validated subpanels. We built two high-parameter antibody panels for analysis of fresh and stabilized WB. The first panel has 21 markers and identifies 12 immuno-oncology markers in 10 major cell subsets. The second panel has 31 markers and identifies 10 cytokines in 32 cell subsets. Stimulated WB samples from two healthy donors were either freshly stained or stabilized with PROT1, RBC lysed and stained. Liquid antibodies in the panel were pooled together and frozen (-80 °C) as single-use aliquots for surface and intracellular staining to reduce technical variability from staining. Optimal panel design ensured that all markers in both panels could be used to accurately identify target cell populations in PROT1-stabilized WB. Following stabilization, nonspecific staining was not observed when compared with freshly stained samples. Cell populations maintained the anticipated co-expression of markers in PROT1-stabilized WB. Prominent cytokines such as IL-17A, TNF-α, IL-10, etc., could be detected in stabilized WB. Overall, we show that PROT1-compatible high-parameter antibody panels enable functional profiling of immune cells by identifying immuno-oncology markers and cytokines. Compatibility with WB stabilization reagents provides workflow flexibility for carrying out functional profiling in longitudinal and multi-center studies in clinical research and healthcare settings. Furthermore, freezing antibody cocktails is a unique feature of CyTOF flow cytometry, ensuring staining consistency. Together, flexible CyTOF workflows enable fast and convenient panel design for high-parameter cytometric analysis of WB and can facilitate biomarker discovery, immunotherapy selection and tracking disease prognoses in cancer. Citation Format: Shakir Hasan, Ling Wang, Stephen Li, Christina Loh. Accelerating immuno-oncology research with flexible workflows using modular flex-fit CyTOF panels [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 7122.