Abstract 5624: Characterization of proliferation in multiple T-cell subsets in the CT26 murine colon carcinoma model by multi-color flow cytometry

• Published in: Cancer research (Chicago, Ill.) Ill.), 2017-07, Vol.77 (13_Supplement), p.5624-5624
• Main Authors: Thayer, Matt, Wong, Alden, Draper, David, Saims, Dan, Wise, Scott C.
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2017-5624

The efficacy of immune-modulating anti-cancer therapeutic antibodies that have been FDA-approved in recent years, such as anti-CTLA-4, anti-PD-1 and anti-PD-L1, has altered the paradigm of cancer treatment. Subsequently, the growing interest in the development of new single agent and combination therapies with immune-modulatory effects has generated a need for more powerful immunophenotyping techniques capable of in-depth cell characterization and proliferation assessment. To this end, using the CT26 syngeneic murine colorectal cancer model we have developed an 8 color flow cytometry antibody panel that focuses on the identification of lymphocyte subsets and analysis of proliferation within them utilizing the high-throughput-capable 4-laser, 14-color Attune NxT Flow Cytometer (Thermo Fisher Scientific). Splenocytes from tumor-naïve and CT26 tumor-bearing C57BL/6 mice were stained with CFSE (carboxyfluorescein succinimidyl ester), an amine-reactive fluorescent dye which enables generational tracking for up to seven rounds of cell division, allowing for proliferation to be quantified. Splenocytes from these mice were then stimulated with anti-CD3, anti-CD28, and IL-2. After stimulation for four days, cells were stained with antibodies against CD45, CD3, CD19, CD49b, CD335, CD4, CD8, and FoxP3. We were able to identify differential proliferative capacity between naïve and tumor-bearing mice in CD4+ T-cell, CD8+ T-cell, regulatory T-cell, and B-cell populations. 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 potentially responsive immune compartments, as well as characterizing proliferation, will facilitate identification and development of potential combination therapies by expanding the depth of our ability to provide mechanistic descriptions of drug function and efficacy. Following this proof-of-concept work, we will also present data from tumor-bearing mice treated with immune checkpoint inhibitors in order to identify potential differences in proliferation after treatment with immune-modulating agents. Citation Format: Matt Thayer, Alden Wong, David Draper, Dan Saims, Scott C. Wise. Characterization of proliferation in multiple T-cell subsets in the CT26 murine colon carcinoma model by multi-color flow cytometry [abstract]. In: Proceedings of the American