Abstract 2189: A spectral flow cytometry panel and workflow for the reproducible assessment of colorectral tumor immunophenotype in the context of immunotherapies

Multiparameter flow cytometry analysis of immune cells within solid tumors has provided insight into the mechanism of action of novel immunotherapies, but challenges exist regarding the interpretation of data stemming from variation in sample processing, selection of optimal combinations of fluorochrome-conjugated antibodies, and population gating strategy. Here we present a 20-color spectral flow cytometry backbone panel and gating scheme that can be applied to syngeneic colorectal tumor samples from different mouse strains with minimal manual adjustment. Using the CT26 and MC38 subcutaneous implant models in BALB/c and C57BL/6 mice, respectively, we tested the ability of our panel and workflow to identify changes in tumor immune cell populations after treatment with the immune checkpoint inhibitors (ICIs) anti-PD-1 and/or anti-CTLA-4 in two independent experiments. Our panel identified differences between the models, largely confined to protein surface expression within the monocyte, macrophage, and dendritic cell compartments. In addition, the panel provided readouts of T cell and NK cell function that correlated with ICI efficacy and with increases in tumor cytokines, including IFNγ, CXCL10, and CCL5, without the need for lengthy ex vivo stimulation and intracellular cytokine staining. The use of autofluorescence extraction and the designation of autofluorescence tags, which is possible on spectral flow cytometers, was essential to properly identify populations. We demonstrate the ability of modern spectral flow cytometers to produce remarkably consistent data over time, without the need for manual detector gain adjustments, which could improve data interpretation between investigators and facilitate the development of novel therapies. Citation Format: Joseph P. Kolb, Kathryn R. Meshaw, Timothy Hutchinson, Karsten Fynboe, Jacob Hauser, William D. Culp, Beth A. Hollister. A spectral flow cytometry panel and workflow for the reproducible assessment of colorectral tumor immunophenotype in the context of immunotherapies [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 2189.