Abstract 3242: In depth myeloid cell characterization in the murine syngeneic CT26 colon carcinoma model by 10 color flow cytometry
Authors: Thayer Matt , Draper David , Saims Daniel , Rosenfeld-Franklin Maryland , Wise Scott C. 
Published: July 2016

Abstract
The efficacy of immune-modulating anti-cancer therapeutic antibodies that have been FDA-approved in recent years, such as anti-CTLA-4 and anti-PD-1, has driven growing interest in methods that provide a mechanistic understanding of drug function. Development of new mono- and combination therapies with immune-modulatory effects requires more powerful immunophenotyping techniques capable of in depth cell characterization. To this end, using the CT26 murine syngeneic colorectal cancer model we have developed a 10 color flow cytometry antibody panel that focuses on the identification of tumor-infiltrating immune cell subsets derived from myeloid lineage precursors utilizing the high-throughput-capable 4-laser, 14-color Attune NxT Flow Cytometer with autosampler. The panel includes a combination of antibodies against CD45, CD3, CD19, CD49b, CD335, CD11b, CD11c, Ly-6G, Ly-6C, F4/80, and CD115. By excluding cells of lymphoid lineage, we show that this panel facilitates analysis of myeloid derived cells including natural killer (NK) cells, macrophages, neutrophils, dendritic cells (DCs), and monocytic or granulocytic myeloid-derived suppressor cell (mMDSCs and gMDSCs) subsets in tumor and peripheral blood. In addition, this combination of antibodies allows for a more complete analysis of MDSCs which can differentially express several disease-relevant myeloid specific markers including Ly-6G, Ly-6C, F4/80, CD11c, and CD115. In the tumor, the Ly-6G-high population demonstrates differential expression of Ly-6C, 21% Ly-6C-high (granulocytes) and 77% Ly-6C-low (mMDSCs). The majority of the granulocytic population was identified as gMDSCs and the remainder as neutrophils based on CD115 expression. Macrophages constitute 27% of Ly-6G-low cells. In blood, 98% of the Ly-6G-high population was also Ly-6C-high, and this population is predominantly neutrophils. No macrophages (Ly6G-low and F4/80+) were identified in the peripheral blood. These data confirm the expected distribution of myeloid lineages in the tissue types investigated. Finally, we show that the accuracy of analysis is enhanced by the use of fluorescence minus-one (FMO) controls to identify those markers that generate dim signals, as well as a viability dye used to exclude dead cells from analysis. Identification of additional potentially responsive immune compartments will facilitate identification and development of potential combination therapies otherwise overlooked by looking primarily at T-cells. This panel allows for a significant expansion of our ability to provide a complex description of the myeloid subset.
Citation Format: Matt Thayer, David Draper, Daniel Saims, Maryland Rosenfeld-Franklin, Scott C. Wise. In depth myeloid cell characterization in the murine syngeneic CT26 colon carcinoma model by 10 color flow cytometry. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3242.