Abstract 1543: 40-color spectral flow cytometry for comprehensive immunophenotyping of murine cancers
Authors: Kare Aris John , Nichols Lisa , Zermeno Ricardo , Raie Marina N. , Tumbale Spencer K. , Ferrara Katherine W. 
Published: March 2024

Abstract
Introduction: High-dimensional immunoprofiling is essential for studying host response to cancer immunotherapy. Synergistic agents often induce systemic changes in leukocyte abundance/function due to targeting, dosage, or delivery, necessitating multi-tissue analyses to decipher immune response mechanisms. We set out to dissect leukocyte composition in lymphoid tissues and tumors by using spectral cytometry, a multiplexed, high-throughput technology.
Methods: We engineered a 40-color spectral flow cytometry panel to immunophenotype all major leukocytes and their functional subsets. Naïve and tumor-burdened C57BL/6 mice treated with combinations of agonist CD40 (aCD40) and checkpoint inhibitors were sacrificed, and tissues were processed. Stained cells were acquired on a 5-laser CyTEK Aurora. Unmixing accuracy and resolution was validated via 780 NxN plots, and high-dimensional analysis was performed.
Results: Spectral cytometry affords resolution of 43+ parameters compared to 26 or less in conventional systems. We first designed a 25-marker backbone panel to identify all immune lineages. Additional channels were assigned to functional markers, allowing for profiling of migration, maturation, and activation in tumor-infiltrating leukocytes (TILs). In naïve secondary lymphoid tissues (SLTs), 35 distinct CD45+ populations were resolved. Germinal center B cells (17% vs. ~0.3% of CD45+ in other SLTs) and T follicular cells (31% vs. ~1-5% of CD4+ T cells in other SLTs) were mostly found in the Peyer’s patches, representing a hypothesized model tissue for studying tertiary immune structures in tumors. Peripheral blood contained the highest frequency of NK cells (4%), monocytes (6%), basophils (0.3%), eosinophils (4%), and neutrophils (7%) as a percentage of CD45+ cells. The spleen held the largest reservoir of CD4/8 central/effector memory cells (5% of CD45+) while ~80% of all T cells in the lymph nodes were naïve. In primary lymphoid tissues (PLTs), 12 distinct thymocyte and 7 B cell populations were resolved. Wanderlust trajectory analysis revealed CD24 and CD127 as key developmental regulators in lymphocyte differentiation. Autofluorescence extraction was essential for myeloid phenotyping. With this approach, we found monocytes comprised ~60% of TILs following aCD40 treatment (~8% in non-treatment and checkpoint only controls) and had altered macrophage differentiation fates. aCD40-treated tumors also contained higher frequencies of activated CD40/80/86+ DCs (~80/86/60% vs. ~60/51/50% of DCs in NTCs).
Conclusion: This work describes a novel tool that efficiently resolves leukocytes on the axes of time, tissue, and treatment. Applications range from serial monitoring of circulating blood, endpoint analysis, validation of multiplexed-IHC, and cell sorting. PLT and SLT data has been made publicly available to serve as a training tool and reference proteomic atlas in future work.
Citation Format: Aris John Kare, Lisa Nichols, Ricardo Zermeno, Marina N. Raie, Spencer K. Tumbale, Katherine W. Ferrara. 40-color spectral flow cytometry for comprehensive immunophenotyping of murine cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1543.