Abstract C079: Comprehensive immunophenotypic profiling sheds light on the dynamic interplay between immune cells in the 4T1 breast cancer model upon anti-PD1 and anti-CTLA4 immunotherapy
Authors: Nikolaou Christos , Heller Simon , Kaulfuss Stefan , Stresemann Carlo , Eichten Alexandra , von Ahsen Oliver , Lange Martin 
Published: December 2023

Abstract
Immuno-oncology has revolutionized cancer treatment by harnessing the immune system to target and eliminate tumor cells. In this study, we employed a combination of fluorescence-activated cell sorting (FACS) and Cytek full spectrum flow cytometry to investigate the impact of anti-PD1 (programmed cell death protein 1) and anti-CTLA4 (cytotoxic T-lymphocyte-associated protein 4) treatments on the 4T1 breast cancer model. We comprehensively analyzed 25 surface markers on immune cells to gain insights into the dynamic changes induced by immunotherapy. The 4T1 murine breast cancer model is known for its aggressive and metastatic nature, resembling triple-negative breast cancer in humans. We established a syngeneic orthotopic tumor graft of 4T1 cells in immunocompetent mice and treated them individually with anti-PD1 or anti-CTLA4 antibodies and compared the results to the respective isotype controls. Using FACS, we characterized immune cell populations within the tumor microenvironment and spleenocytes at multiple time points (before treatment, day 5, and day 17) during the treatment course. The Cytek full spectrum flow cytometry platform allowed simultaneous measurement of 25 surface markers, enabling a detailed analysis of immune cell phenotypes and functional states in a single experiment. Our findings revealed significant alterations in the immune landscape following anti-PD1 or anti-CTLA4 treatments. We observed an increase in tumor-infiltrating lymphocytes (TILs) and specifically CD4+ as well as CD8+ T cell infiltration within the tumor, accompanied by upregulation of T cell activation markers, such as CD69, CD38, CD44, PD1 and CD25. The immune cell composition within 4T1 tumors included CD4+ and CD8+ TILs, as well as increased populations of monocytes and macrophages. Additionally, a decrease in regulatory T cells (TRegs) was observed, indicating a potential restoration of immune balance. Moreover, F4/80+ CD206+ tumor-associated macrophages (TAMs) were identified in the 4T1 tumors vs. the spleen. This was associated with increased granzyme B production by CD8+ T cells and a shift towards an effector memory phenotype, suggesting improved cytotoxic function. To gain insights into spatial infiltration patterns of immune cells, we performed Immunohistochemistry (IHC) stainings of various immune cell markers in the 4T1 tumors. In line with the FACS results, we observed increased immune cell infiltration in tumors treated with anti-PD1 and anti-CTLA4 antibodies. Our comprehensive immunophenotypic analysis, utilizing Cytek full spectrum flow cytometry and IHC sheds light on the dynamic interplay between immune cells and the 4T1 breast cancer model during anti-PD1 and anti-CTLA4 immunotherapy. These findings provide valuable insights into the mechanisms underlying the therapeutic efficacy of immune checkpoint blockade in this aggressive cancer model.
Citation Format: Christos Nikolaou, Simon Heller, Stefan Kaulfuss, Carlo Stresemann, Alexandra Eichten, Oliver von Ahsen, Martin Lange. Comprehensive immunophenotypic profiling sheds light on the dynamic interplay between immune cells in the 4T1 breast cancer model upon anti-PD1 and anti-CTLA4 immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C079.